XXVI Reunião Anual da FeSBE - FeSBE 2011
Resumo:02-077
AEROBIC EXERCISE TRAINING INDUCED CARDIAC HYPERTROPHY INVOLVES REGULATORY
MICRORNAS, DECREASED ACE-ANG II, AND SYNERGISTIC REGULATION OF ACE2-ANG (1-7)
Fernandes, T. 1; Hashimoto, N. Y. 1; Magalhães, F. C. 1; Fernandes, F. B. 3; Casarini, D. E. 3; Carmona, A.
3
; Krieger, J. E. 2; Phillips, M. I. 4; Oliveira, E. M. 1
1
Laboratory of Biochemistry of the Motor Activity - USP, EEFE-USP
2
Laboratory of Genetics and Molecular Cardiology - InCor , FMUSP
3
Nephrology Division, Kidney and Hypertension Hospital , UNIFESP
4
Laboratory of Stem Cells / Keck Graduate Institute, KGI
Objectives:
Aerobic exercise training (ET) leads to a physiological, non pathological left ventricular hypertrophy (LVH); however, the
underlying biochemical and molecular mechanisms of physiological LVH are unknown. MicroRNAs may be important for LVH,
since they function as negative regulators of gene expression by inhibiting translation or promoting degradation of target mRNAs.
The role of microRNAs regulating the classic and the novel cardiac renin angiotensin system (RAS) was studied in exerciseinduced LVH.
Methods and Results:
Wistar rats (n=30) were assigned to three groups: sedentary (S), Trained 1 (T1) and Trained 2. T1: swimming training consisted
of 60 min 1x/day/10 weeks, with 5% body weight overload. T2 the same as T1 until 8th week, in the 9th week they trained
2x/day and in the 10th week 3x/day. RAS involvement in the left LVH induced by ET was analyzed using Angiotensin II type 1
receptor (AT1) blockade (Losartan- 20 mg/kg/day) during ET protocol. Blood pressure (BP) and heart rate (HR) were evaluated
by direct measurement, LVH by tissue weight/body weight ratio, myocyte diameter by histological method, angiotensinconverting enzyme 1 (ACE) and 2 (ACE2) activity, gene and protein expression by fluorometric method, real-time PCR and
western blot, respectively. Plasma renin activity (PRA) by radioimmunoassay and angiotensin I, II and (1-7) by HPLC. AT1 and
AT2 receptors gene and protein expression also were studied. MicroRNAs profile was analyzed by microarray and it that targeted
RAS genes were validated by real-time PCR. Results were presented as mean±SEM. BP was unchanged while resting HR
decreased in all trained groups. LVH obtained by T1 and T2 training protocol was 17% (p
Conclusions:
LVH induced by ET involves microRNAs regulation and an increase in cardiac AT1 receptor without the participation of Ang II.
Parallel to this, increase in ACE2, Ang (1-7) and AT2 receptor in the heart by exercise suggests that this non classic cardiac RAS
counteracts the classic cardiac RAS activating local vasodilatation pathway. Together these effects might provide the additional
aerobic capacity required by the exercised heart.
Keywords: aerobic exercise training, AT1 receptor blockade, cardiac hypertrophy, microRNAs, renin angiotensin system
Financial Support: FAPESP, CAPES and CNPq
Resumo:02-078
THE ROLE OF TYPE 2 ANGIOTENSIN II RECEPTOR (AT2) IN THE CARDIOPROTECTIVE RESPONSE
INDUCED BY THYROID HORMONE.
Tavares, F. M. ; Barreto-chaves, M. L. M
Department of Anatomy, Institute of Biomedical Sciences, USP
Objectives:
To evaluate the role of AT2 receptor in the recovery of cardiac post-ischemic function in a hyperthyroidism model.
Methods and Results:
Thriodothyronine (T3) (0.7 µg/Kg BW/day, i.p.) was injected to male adult Wistar rats in the presence or absence of AT2R
blocker (PD123319) for 14 days. AT2R blocker (10 mg/Kg/day) was administered subcutaneously through the implantation of
osmotic minipumps (Alzet). Animals were subdivided in four different groups: 1) Control: rats treated with normal saline (n=9);
2) PD (n=6); 3) T3 (n=7) and 4) PD plus T3 (n=4). Animals from each group were used for the assessment of baseline cardiac
performance and to response to ischemia-reperfusion (I-R) protocols. Hearts were rapidly excised and mounted on a Langendorff
apparatus. Isolated hearts were perfused in a retrograde way at a constant flow with oxygenated (95% O2, 5% CO2)
Krebs−Henseleit buffer at a temperature of 37ºC. A water−filled balloon was inserted in the left ventricular cavity to assess the
following parameters: left ventricular pressure, dP/dT max e min, heart rate and perfusion pressure. After 30 min of stabilization,
hearts were subjected to 20 min of zero−flow global ischemia and 45 min of reperfusion. Cardiac hypertrophy was determined
based on heart weight/body weight and heart weight/tibia length ratios. The results demonstrate that T3 induced cardiac
hypertrophy (25% vs control p
Conclusions:
In conclusion, we observed that the blockade of AT2R with PD 123319 induced cardioprotective response, however, it abolished
the cardioprotection induced by T3.
Keywords: AT2 receptor, Cardioprotection, Hyperthyroidism, Ischemia/Reperfusion injury
Financial Support: FAPESP, CNPq.
Resumo:02-079
PLATELET AGGREGATION IS REDUCED DURING EXPERIMENTAL SEPSIS
Gonçalves, M. C. ; Sordi, R. ; Assreuy, J.
Department of Pharmacology / Universidade Federal de Santa C, UFSC
Objectives:
Sepsis is a systemic inflammatory state associated with an infection caused by bacteria, fungi, viruses or parasites. Many
mechanisms are involved at sepsis physiopathology including cytokine release, activation of neutrophils, monocytes, endothelial
cells and also activation of coagulation and fibrinolysis systems. Imbalance between procoagulant and anticoagulant processes
during sepsis has been described. This imbalance is likely to be an important event contributing to multiple organ dysfunctions.
Although there are some reports showing that in human sepsis a reduction in platelet numbers occur, the role of platelets in sepsis
remains unknown. In this study we have attempted to characterize the platelet aggregation in experimental sepsis induced by
cecal ligation and puncture (CLP) in male rats temporally.
Methods and Results:
All procedures have been approved by the institutional animal ethics committee (CEUA/UFSC, Protocol PP00595). About 5 mL
of blood were collected from anaesthetized septic and naïve rats via cardiac puncture with sodium citrate 3.8% (1:9 ratio,
citrate:blood) as anticoagulant. Platelet rich plasma (PRP) was obtained by centrifugation and aggregation assay was performed
in an Infinite Plate Reader (Tecan, Switzerland). About 50.10 6 platelets per well (96-well plate) were assayed during 15 minutes,
at 37°C under moderate and constant shaking. The platelet aggregation was induced by adding 5 µM of adenosine diphosphate
(ADP) or collagen (130 µg/mL) in PRP from naïve and septic animals (6 h, 12 h and 24 h after CLP surgery). The platelet
aggregation was quantified by optical density (OD) at 650 nm. The difference between the basal and peak aggregation OD was
expressed in milliunits of OD (mUOD). The platelet aggregation induced by ADP in septic rats was diminished in all CLP
groups, and it was statistically significant at CLP 12 h group (mUOD: 77.7 ± 0.02; n=6) when compared to naïve group (127.1 ±
0.01; n= 6). Aggregation induced by collagen also decreased substantially 12 h (85.6 ± 0.02; n=6) and 24 h after CLP surgery
(71.1 ± 0.004; n=6) when compared to the naïve group (180.7 ± 0.02; n=6). Nitrate + nitrite (NOX) levels were increased in
plasma samples of septic animals (76.8 ± 14.1, 135.6 ± 18.9 and 90.2 ± 27.4 μM, 6 h, 12 h and 24 h, respectively) when
compared to naïve values (34.8 ± 25).
Conclusions:
Septic animal exhibited lower platelet aggregation when compared to naïve rats. The reduced platelet aggregation ability might
be caused by the large amount of nitric oxide (NO) produced in sepsis. This platelet dysfunction may be contributing to the
hemostatic imbalance of sepsis.
Keywords: Hemostasis, Nitric oxide, Platelet aggregation, Sepsis
Financial Support: CNPq, CAPES and FAPESC.
Resumo:02-080
CHRONIC MALNUTRITION PROMOTED BY THE BRAZILIAN NORTHEAST DIET ALTERS CARDIAC
MORPHOLOGY AND FUNCTION IN RATS.
Mendes, L. V. P. 1; Dias,m. S. 1; Boldrini, L. C1; Takiya, C. M. 1; Oliveira-pinto, L. M 1; Nascimento, J. H.
M. 1; Lamas, M. E. 1; Vieyra, A. 1; Cunha, V. M. N. 1; Lara, L. S1
1
Instituto de Ciências Biomédicas, UFRJ
2
Instituto de Biofísica Carlos Chagas Filho, UFRJ
3
Programa de Pós-Graduação em Ciências Morfológicas, UFRJ
Objectives:
In previous work, we showed that malnutrition promoted for regional basic diet (RBD) from Brazilian northeast in chronic period
of development (RBD-C) modifies the homeostasis of intracellular Ca2+. Such changes suggest the development of a heart
failure process in the RBD-C group. The aims of the present work were: 1- evaluate the contractility of the hearts from RDB-C
rats; 2- assess histologically the heart tissue from RBD-C rats; 3- evaluate the content of free sulfhydryl essential for the catalytic
activity of Ca2+-ATPases
Methods and Results:
Male Wistar rats from healthy mothers after weaning were subjected to the diet for 10 weeks RDB (RDB-C, n = 9), while the rats
from control group were fed a conventional diet (Cont, n = 9). After a period of 13 weeks of age the animals were sacrificed
(CEUA DFBCICB007). After the sacrifice, the heart was used in Langendorff assays, using increasing doses of Isoproterenol.
The values of LVP from hearts of RBD-C group were not different from controls (for 1 µM isoproterenol, 135.4 ± 26.0 vs 133.7
± 25.5 %, respectively, n=5 and n=3, P>0.05). The contractility index calculated for maximum rate of contraction (+dP/dTmax)
was increased in the group RBD-C compared with control group (for 100 nM Isoproterenol, 217.8±84 vs 68.6±14.9, respectively,
P< 0.05). Histological analysis was performed according to Masson's trichrome method, in which atrophy of cardiomiocytes,
inflammatory infiltration, and fat cells were observed in the hearts of rats from RDB-C group. The Ca2+-ATPase activity was
also measured in heart homogenates using gama [32P-ATP]. It was observed a significant increase of the total Ca2+-stimulated
activity in RDB-C group in relation to the control group (2136 ± 25.4 vs 985.6 ± 30 nmolPi / mg.h-1, respectively, n=3, P
Conclusions:
The increase of the contractility index in animals RBD-C indicates an attempt to improve the working heart since the cardiac
output is reduced in this group (reported earlier). Changes in thickness of cardiomiocytes as well as the presence of inflammatory
infiltration indicate the installation of necrosis process in the hearts from RBD-C rats. As reported before using microsomes, the
increased of the total Ca2+-ATPase activity of heart homogenates indicates that somehow the heart cells from RDB-C rats try to
maintain the cardiac contractility and the efficient remove of Ca2+ ions. The loss of reduced sulfhydryls indicates enzymatic
oxidation associated to the loss of catalytic activity. In relation to Ca2+-ATPases, it is possible that the decrease of free
sulfhydryls is associated with SERCA oxidation because it was observed previously a significant reduction of the activity of this
enzyme without any change of protein expression.
Keywords: atrophy, heart, langendorff, malnutrition, Regional Basic Diet
Financial Support: Projeto Casadinho-CNPq; PROCAD-CAPES; FAPERJ Primeiros Projetos, Programa
ALV.
Resumo:02-081
TIME COURSE AND EFFICACY OF MK-467 BLOCKING THE HEMODYNAMIC RESPONSES CAUSED BY THE
SELECTIVE α2 ADRENERGIC AGONIST CLONIDINE.
Dias, L. O. 2,1; Durand, M. T. 1; Castania, J. A. 1; Fazan Jr. , R. 1; Salgado, H. C. 1
1
Department of Physiology, FMRP/USP
2
Universidade Estadual de Santa Cruz, UESC
Objectives:
MK-467, a benzofuroquinolizine derivative, has been considered a selective α2 adrenergic receptor antagonist that can be useful
to examine the role played by α2 adrenergic receptors along with the α1 counterparts in the control of arterial pressure (AP). In
addition, it has not been described, precisely, how long MK-467 blocks α2 adrenergic receptors.In the present study it was
evaluated, in conscious freely moving rats, the time course and the efficacy of MK-467 to block the hypertensive response and
the reflex bradycardia caused by the selective α2 adrenergic agonist clonidine. In addition, it was also investigated a possible
effect of MK-467 on α1 adrenergic receptors by the exam of the hypertensive response and the reflex bradycardia elicited by the
selective α1 adrenergic agonist phenylephrine.
Methods and Results:
Wistar rats (200-300g) were anesthetized (tribromoethanol 250 mg/kg, ip) and the femoral artery and vein were catheterized for
AP recording and drug administration, respectively. Twenty-four hours after the surgical procedure, the experiments were
conducted in conscious freely moving rats. The protocol consisted of basal AP and heart rate (HR) recordings followed by
phenylephrine (4 µg/kg) and clonidine (1.5 mg/kg) injections, before and after MK-467 administration (3 mg/kg). After MK-467
administration clonidine was injected at 5, 15, 30, 45 and 60 minutes while phenylephrine was injected only at 35 minutes. MK467 produced a significant fall in basal AP, which occurred in the first 5 minutes (Basal: 103 ± 4 vs. MK-467: 93 ± 4 mmHg)
eliciting a reflex tachycardia (Basal: 353 ± 13 vs. MK-467: 451 ± 26 bpm). Nevertheless, these responses were not maintained,
and AP returned to basal values within 15 minutes. The hypertensive response (ΔMAP: 40 ± 3 mmHg) and the reflex bradycardia
(ΔHR: -58 ± 9 bpm) to clonidine were blocked after MK-467 at 5 min (1 ± 1 mmHg and -2± 5 bpm), 15 min ( 3 ± 1 mmHg and
+1 ± 7 bpm), 30 min (7 ± 3 mmHg and -6 ± 9 bpm), 45 min ( 8 ± 4 mmHg and -1 ± 6 bpm) and 60 min (5 ± 4 mmHg and -10 ± 5
bpm). On the other hand, MK-467 did not affect the hypertensive response (44 ± 3 vs. 35 ± 5 mmHg after) and the reflex
bradycardia (-58 ± 11 vs. -33 ± 10 bpm after) to phenylephrine.
Conclusions:
The results demonstrate that MK-467 produced a prompt fall in AP combined with reflex tachycardia. In addition, MK-467
promoted peripheral α2 adrenergic receptor blockade which persisted for 60 minutes. Moreover, the administration of MK-467
did not block the response to α1 adrenergic receptor activation with phenylephrine, showing remarkable selectivity to peripheral
α2 adrenergic receptor.
Keywords: α2 adrenergic receptor, MK-467, Clonidine
Financial Support: Capes, CNPq, FAPESP
Resumo:02-082
THERAPEUTIC POTENTIAL OF AMD3100 IN PULMONARY ARTERIAL HYPERTENSION INDUCED BY
MONOCROTALINE IN RATS.
Ferraz, E. F. ; Oliveira, A. P. F. ; Barros, F. ; Riva, D. R. ; Nascimento, J. H. M.
Instituto de Biofísica Carlos Chagas Filho, UFRJ
Objectives:
Pulmonary arterial hypertension (PAH) is a syndrome characterized by increased levels in pulmonary vascular resistance, which
can be experimentally induced by monocrotaline administration, an alkaloid drug that induces toxic effects, leading to endothelial
injury. In other hand, AMD3100 a CXCR4 blocker, is frequently associated with mobilization of endothelial progenitor cells
from bone marrow and possibly to prevent the endothelial dysfunction and PAH. Thus, our aim is evaluate the therapeutic effects
of AMD3100 in PAH model induced by monocrotaline in rats.
Methods and Results:
Twenty male Wistar rats initially weighing approximately 200g were randomized in two groups: monocrotaline (MCT) and
monocrotaline plus AMD3100 (MCT+AMD3100). PAH was induced by one dose of MCT (60 mg/kg, i.p.). Two weeks after
PAH induction, the AMD3100 (1 mg/kg/day) was administered for 14 days. The body weight was measured weekly. At end of
treatment, the hypertrophy index of right ventricle (RV) was determined as the relation between mass of RV and left ventricle
plus septum. The echocardiogram parameters [cardiac output (CO), left ventricular ejection fraction (LVEF), stroke volume (SV),
left ventricular diastolic volume (LVDV)] were assessed using a high-resolution ultrasound (Vevo 770, Visualsonics). The
pulmonary airway pressure was estimated by ventilatory mechanical study. Results are expressed as mean ± SD, with p < 0.05 as
significative. There was no significant difference in body weight, heart weight and hypertrophy index of right ventricle. After 4
weeks, echocardiographic analysis showed decreased values of CO in MCT group compared to pre-MCT treatment (69.56 ±
10.23 ml/min; 53.29 ± 20.07 ml/min, p < 0.05). However, no difference was observed on CO in MCT+AMD3100 group. No
difference was found on LVEF and SV between MCT and MCT+AMD3100. The LVDV was reduced in MCT group compared
to pre-MCT treatment (407.50 ± 86.02 ìl; 322.2 ± 77.47 ìl, p < 0.05), but no significant difference was observed in
MCT+AMD3100 group between initial period and at end of treatment. In other hand, a significant decreased value of pulmonary
airway pressure was observed in MCT+AMD3100 group compared to MCT animals (12.25 ± 2.63 mmHg; 18.80 ± 3.96 mmHg,
respectively). Mortality presented a tendency to be lower in MCT+AMD3100 (37.5 %) than MCT (50%) group, but without
statistical difference.
Conclusions:
The AMD3100 treatment prevented the reduction of CO and LVDV and reduced the pulmonary airway pressure of rats with
monocrotaline-induced pulmonary arterial hypertension.
Keywords: AMD3100, CXCR4, Monocrotaline, Pulmonary arterial hypertension
Financial Support: FAPERJ, CNPq
Resumo:02-083
GHRELIN SIGNALING IN OBESE MICE HEART
Lacerda-miranda, G. ; Vieira, A. K. G. ; Sores, V. M. ; Bernardes, A. F. ; Lessa, J. G. ; Cunha, A. C. S. R. ;
Mattos, A. B. M. ; Cortez, E. ; Garcia-souza, E. P. ; Moura, A. S.
Departamento de Ciências Fisiológicas/LFND/IBRAG, UERJ
Objectives:
Obesity is related to myocardial dysfunction and heart failure. We examined key proteins of cardiomyocyte metabolism in heart
left ventricle from obese (OG) and control (CG) groups from adult mice (180 days) overfed during lactation.
Methods and Results:
Methods: Obesity was induced by litter reduction. Therefore, the study was done in adult mice 180 days old (OG, obese
group(n=10) and CG, control group (n=10). The cardiomyocytes (cmy) of left ventricle were analyzed by light microscopy and
stereology. The content and phosphorylation of cardiac proteins: growth hormone secretagogue receptor 1a (GHSR-1a), protein
kinase B (AKT and pAKT), phosphatidil inositol 3 kinase (PI3K), AMP-activated protein kinase (AMPK and pAMPK) and actin
was achieved by western blotting. Results are expressed as mean ± S.E.M. Statistical significance was determined by Student ttest for unpaired. P < 0.05 was considered statistical significant. Results: Body weight (67.63 ± 8.89g), Blood glucose (160.2 ±
5.91g), liver weight (2.55 ± 0.32g), and visceral fat weight (5.33 ± 0.98g) were higher in OG (n=10) than CG group (n=10)(p
Conclusions:
Early life overnourishment induces in myocardial heart remodeling associated to increase of GHS-R1a, PI3K , AKT but not
AMPK in obese mice heart.
Keywords: ghrelin, heart, cardiomiocity, obesity, hypernutrition
Financial Support: CNPq , FAPERJ, Vital Brasil
Resumo:02-084
MODULATION OF THE UBIQUITIN PROTEASOME SYSTEM IN THYROID HORMONE-INDUCED CARDIAC
HYPERTROPHY.
Lino, C. A. ; Barreto - Chaves, M. L. M.
DEPTO. DE ANATOMIA / INSTITUTO DE CIÊNCIAS BIOMÉDICAS / USP, ICB-USP
Objectives:
To evaluate the expression of proteins involved with the ubiquitin proteasome system (UPS) in thyroid hormone-induce cardiac
hypertrophy.
Methods and Results:
Male Wistar rats at the same age (~235 g) were randomized in two groups: control and hyperthyroid. Experimental
hyperthyroidism was induced by daily intraperitoneal injections of 3,5,3‘-triiodothyronine (7 µg/100 g body weight/day ) for 21
days. Hyperthyroidism was confirmed by the ratio between heart weight (HW) and tibia‘s length (TL). The cardiac mass
increased 31% in hyperthyroid animals compared to control (n=3; p=0,0087). The heart rate (HR) was determined during the
experimental period by tail-cuff plethysmograph (Kent Scientific, Litchfield, CT) and it was increased in the hyperthyroid
animals from 7th day of treatment (n=3; p=0,0003). Preliminary results indicated an increase on gene expression of â5 subunit of
proteasome in hyperthyroid group, evaluated by real time RT-PCR. Besides, MuRF1 expression, an E3 ligase, seems to be
augmented in the myocardium of hyperthyroid animals. Although we have observed an increase on transcription levels of some
UPS components, the expression of ubiquitinated proteins analyzed by Western blot remained unchanged between groups.
Conclusions:
Thyroid hormone exerts several effects on the cardiovascular system. Hyperthyroidism, characterized by high levels of
circulating T3, markedly stimulates the protein synthesis in the heart and leads to a concentric cardiac hypertrophy. The UPS
represents the main mechanism of degradation of intracellular cytosolic and nuclear proteins and its activation has been described
in different models of cardiac hypertrophy. The cardiac cell growth is accompanied by an increase in protein ubiquitination,
proteasome subunit expression, and proteasome activity. For instance, we observed the characteristic cardiac hypertrophy of
hyperthyroidism condition and an increase in gene expression of some components of UPS. Data regarding ubiquitinated proteins
expression and proteasome activity remain to be evaluated yet.
Keywords: SISTEMA UBIQUITINA-PROTEASSOMA, HIPERTROFIA CARDÍACA , HORMÔNIOS TIROIDEANOS
Financial Support: CAPES
Resumo:02-085
INFLUENCE OF HYPERTONIC SALINE SOLUTIONS ON HEART RATE VARIABILITY
Farah, H. M. A. T. 1; Almeida, R. L. 1; Mostarda, C. T. 2; Colombari, D. S. A. 4; Farah, V. M. A. 3; Sato, M.
A. 1; Irigoyen, M. C. 2; Colombari, E. 4
1
Depto de Morfologia e Fisiologia/ Fac. de Medicina do ABC, FMABC
2
INCOR-USP, INCOR
3
Depto de Fisiologia/ Universidade Presbiteriana Mackenzie, Mackenzie
4
Depto Patologia e Fisiologia/ Fac. Odont. Araraquara - UNESP, FOAr-UNESP
Objectives:
Central and peripheral osmoreceptor activation elicits vasopressin secretion and changes in sympathetic activity. It is still
unknown if autonomic changes can be evoked even in the absence of arterial pressure alterations. Indeed, the main objective of
this study was to investigate the acute effect of hypertonic NaCl infusion on blood pressure (BP), heart rate (HR) and
cardiovascular autonomic modulation by spectral analysis.
Methods and Results:
We used male Wistar rats weighting from 280 to 350g. The rats were divided in two groups: 1. Central infusion rats (CIR, n=5),
the rats were anesthetized with ketamine (50 mg/Kg ip) and xylazine (10 mg/Kg im) to implant a guide cannula into the lateral
ventricle (LV). After 5 days of recovery, the right femoral artery was cannulated. The solution of 2 µL of 0.9%, 1.8%, 3.6% and
7.2% NaCl were infused during 2 min in the LV after 1 day of recovery. 2. Peripheral infusion rats (PIR, n=9), the rats were
anesthetized with ketamine (50 mg/Kg ip) and xylazine (10 mg/Kg im) to insert cannulas in the right femoral vein and in the right
femoral artery. Saline solutions of 0.1 mL were infused in the right femoral vein after 1 day of recovery. BP and HR were
evaluated in freely moving conscious animals through a polyethylene tubing inserted in the right femoral artery, using data
acquisition system Windaq, 2000Hz). HR variability was evaluated by spectral analysis using CardioSeries. Animals with
histological confirmation of LV cannulation were considered in this study. Statistical differences were accepted as significant at p
Conclusions:
The solutions administrated in the PIR group altered HR and its modulations while the solutions infused in the LV altered only
the BP sympathetic modulation. The volumes injected were too small (2 µL for LV rats and 0.1 mL for peripheral rats) to cause
any change in HR or BP, therefore we can postulate that repeated salt exposure might induce autonomic changes even without
permanent or maintained hypertension.
Keywords: sodium chloride, heart rate, blood pressure, spectral analysis, lateral ventricle
Financial Support: PIBIC-CNPq, FAPESP and NEPAS.
Resumo:02-086
EFFECTS OF 7-DAY EXPOSURE WITH LOW DOSE OF LEAD ACETATE (PB+2) ON THE CARDIAC FUNCTION
AND MYOCARDIAL MECHANICS OF RATS.
Freire Jr, D1; dos Santos, L. 1; Fiorim, J. 1; Stefanon, I. 1; Vassallo, D. V. 1,2
1
Department of Physiological Sciences., UFES
2
Health Science Center of Vitória, Emescam
Objectives:
Lead (Pb+2) is considered an environmental pollutant of high risk to public health concerning chronic exposure. Although short
term exposures with low Pb+2 doses were rarely analyzed, we recently demonstrated effects on blood pressure and vascular
reactivity of rats. Our objective is to investigate the effects of a short term Pb+2 exposure on the cardiac hemodynamics and in
vitro myocardial mechanics from left (LV) and right (RV) ventricles of rats.
Methods and Results:
Lead acetate (Pb, n=10) or saline (CT, n=10) was injected in Wistar rats during 7 days (1st attack dose 0.4ìg/kg, subsequent
maintenance doses 0.005ìg/kg, i.m. daily). Under anesthesia (urethane 1.2g/kg, i.p.), intraventricular pressures of LV and RV
were assessed by catheterization. Thereafter, hearts were excised and LV papillary muscles and RV stripes were dissected and
placed in an isolated organ bath for in vitro study of myocardial mechanics. In vitro protocols included: steady state contractions
(Lmax); pause-dependent potentiation of force (PPP); and contractile response to isoproterenol (10-4M). For assessment of
transsarcolemmal calcium influx on the contractility, developed force were analyzed in preparations with depleted sarcoplasmic
reticulum (caffeine 10mM) after 10-minute of rest (PRC) by pause of electrical stimulus. P
Conclusions:
Although with low blood concentration, 7-day administration of Pb+2 increased arterial pressure and depressed LV function,
associated with impaired myocardial mechanics of both ventricles. Changes in cardiac muscle may include impaired inotropic
response, and depressed role of sarcoplasmic reticulum and transsarcolemmal calcium influx. Elevated RV pressure with
impaired contractility also suggests some effect on pulmonary vascular resistance.
Keywords: lead , cardiac function, MYOCARDIAL MECHANICS
Financial Support: CNPq,CAPES, FAPES
Resumo:02-087
RALOXIFENE REDUCES BLOOD PRESSURE IN HYPERTENSIVE ANIMALS AFTER OVARIAN HORMONE
DEPRIVATION.
Almeida, S. A. 1; Tiradentes, R. V. 1; Borgo, M. V. 1; Santuzzi, C. H. 1; Moraes, A. N. 1,2; Claudio, E. R. G.
1
; Endlich, P. W. 1; Gonçalves, W. L. S. 1; Abreu, G. R. D. 1; Gouvêa, S. A. 1
1
Depto de Ciências Fisiológicas, UFES
2
Depto de Ciências da Saúde - CEUNES, UFES
Objectives:
Raloxifene displays the profile of a selective estrogen receptor modulator that could be applied as a potential preventative for
osteoporosis but with the additional benefit of preventing breast cancer and coronary heart disease. We investigated the effects of
raloxifene on blood pressure and renal excretion of water and sodium in 2-kidney-1-clip (2K1C) female hypertensive rats that had
previously been ovariectomized and their possible association with changes in endothelial function, as assessed by plasma
nitrate/nitrite determination, which is a surrogate measure of nitric oxide (NO) production.
Methods and Results:
The experimental groups were as follows: (1) hypertensive (2K1C), (2) hypertensive ovariectomized (2K1C+OVX), and (3)
hypertensive ovariectomized treated with raloxifene (2K1C+OVX+R). Seven days after the surgery that produced menopause,
2K1C hypertension was produced in anesthetized animals by applying a silver clip on the left renal artery. Seven days after the
clip application, the rats were put in metabolic cages to allow for the measurement of water ingestion and diuresis, and raloxifene
was administered (2 mg/kg/day i.p., for 7 more days). We found that the treatment with raloxifene promoted a large reduction (P
< 0.01) in blood pressure (197 ± 6 to 164 ± 2 mmHg), an important increase in renal excretion of sodium and water
(2K1C+OVX: 0.755 + 0.030 mEq/day and 8.7 + 0.8 mL/day vs. 2K1C+OVX+R: 1.247 + 0.076 mEq/day and 16 + 2.0 mL/day,
respectively) and an increase in plasma levels of nitrite/nitrate in 2K1C+OVX+R animals, when compared with the 2K1C, (23.4
± 1 vs. 14 ± 0.5 nmol/mL; P < 0.01, respectively).
Conclusions:
These findings suggest that raloxifene exerted its antihypertensive effect, at least in part by increased excretion of sodium and
water in addition to increasing plasma levels of NO, factors that certainly with contribute to the reduction of hypertension.
Keywords: Hypertension, Ovariectomy, Raloxifene
Financial Support: CAPES and CNPq
Resumo:02-088
EFFECTS OF ORAL L-ARGININE ADMINISTRATION ON SERUM LEVELS OF NITRIC OXIDE IN
RENOVASCULAR HYPERTENSION
Tiradentes, R. V. ; Santuzzi, C. H. ; Borgo, M. V. ; Claudio, E. R. G. ; Brum, M. F. ; Endlich, P. W. ;
Abreu, G. R. D. ; Gouvêa, S. A.
Depto de Ciências Fisiológicas , UFES
Objectives:
We have previously shown that L-arginine (L-Arg) has natriuretic and antihypertensive effects in renovascular hypertensive rats.
This decrease in blood pressure can be due to an increase in nitric oxide (NO) production. This work was designed to investigate
the ability of L-Arg to increase NO production in normotensive and renovascular hypertensive rats.
Methods and Results:
Male Wistar rats were anesthetized and a silver clip (0.2-mm) was placed around the left renal artery to produce the 2-kidney,
one-clip renovascular hypertension model. Another group was submitted to similar procedure and treated with L-Arg (10 mg/mL,
p.o.) from the 7th day after surgery. Sham operated rats were used as controls. At the end of the treatment, mean arterial pressure
(MAP) was measured in conscious animals. Serum NO2-/NO3- levels were measured in hemoglobin-free plasma-derived serum
(PDS) by breaking the clot and filtered it through a 10 KDa cut-off membrane and centrifuged on a fixed-angle rotor at 10,000
rpm for 1 hr at 4¢XC. Nitrate reductase was added to the samples to convert NO3- to NO2-. Total NO2- was then analyzed by
reacting the samples with the fluorometric reagent 2,3-diaminonaphthalene and measuring emission at 375 nm and excitation at
415 nm. Nitrite was taken as a reliable index of NO production. We observed a significant reduction in MAP of L-Arg-treated
when compared to untreated rats (120stb5 vs. 170stb8 mm Hg) and, concurrently, occurred an increase in NO production
(25.6¡Ó4.8 vs. 11.8¡Ó1.5 nmol/mL). The treated and untreated sham groups did not differ at the fluorometric assay (14.5¡Ó2 and
13.9¡Ó2.7 nmol/mL and MAP measurements (102¡Ó3 and 104¡Ó3 mm Hg)
Conclusions:
These results suggest that L-Arg treatment has an stimulatory action on the systemic NO formation, which probably explains its
hypotensive and natriuretic effects in renovascular hypertensive rats.
Keywords: L-arginine, Nitric Oxide, Renovascular Hypertension
Financial Support: CAPES and CNPq (Brazil)
Resumo:02-089
EVALUATION OF CARDIOVASCULAR PARAMETERS OF FEMALE RAT OFFSPRING EXPOSED TO THE
EXTRACT OF ST. JOHN'S WORT
Hamada, R. Y. ; Cunha, N. V. D. ; Reis, D. A. S. ; Pinge, M. C. M. ; Gerardin, D. C. C. ; Gomes, G. G. P.
UNIVERSIDADE ESTADUAL DE LONDRINA, UEL
Objectives:
Psychiatric disorders in pregnancy are quite common and the use of psychotropic drugs during these periods is, in most cases,
indispensable. An effective alternative to synthetic antidepressants is the extract of Hypericum perforatum (HP), St. John's Wort,
which can increase levels of serotonin. Studies suggest that antidepressants may interfere with the cardiovascular system of
depressed patients. The widespread use of the extract of HP has attracted attention from the scientific community about its use
during pregnancy and lactation with possible consequences in the offspring, because the herbal drugs are considered safe and
popular self-medicated without professional supervision. Previous study of our laboratory demonstrated that the use of HP in rats
during pregnancy and breastfeeding may increase the basal heart rate in the male offspring in adulthood. In the present work, we
aimed to verify the baseline cardiovascular parameters of female rat offspring exposed to the extract of HP.
Methods and Results:
Wistar rats (n= 7) were treated daily with 100 mg⁄kg of HP or 0.3 ml of 0.9% saline solution (control group) from the first day of
pregnancy (GD1) to the 21st day after birth of pups (PND 21, weaning). After 90 days, immediately prior to starting the
experimental preparation, a vaginal smear was taken from female offspring to determine the stage of the estrous cycle and only
the females presenting estrus phase were used to evaluated cardiovascular parameters. These females underwent surgery to
implant a catheter in the femoral artery for recording of blood pressure and heart rate. The maternal treatment with HP did not
change baseline mean arterial pressure (MAP) and heart rate (HR) of female pups (MAP=110 ± 1.5 mmHg and HR: 400 ± 22
bpm, n=6) when compared with the control group (MAP=113 ± 1.4 mmHg and HR: 393 ± 12 bpm, n=11).
Conclusions:
The results demonstrate that the use of HP in rats during pregnancy and breastfeeding did not modify the basal cardiovascular
parameters in the female offspring in adulthood.
Keywords: CARDIOVASCULAR , EXTRACT OF ST. JOHN'S WORT, HYPERICUM PERFORATUM, FEMALE RAT
Financial Support: PIBIC⁄UEL
Resumo:02-090
INVOLVEMENT OF INOS PATHWAY INTO THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS
(PVN) IN CARDIOVASCULAR AND AUTONOMIC MODULATION DURING LPS ENDOTOXEMIA IN
CONSCIOUS RATS
Matsumoto, A. K. ; Silva, A. M. D. ; Abreu, S. B. D. ; Filhho, P. P. ; Pinge, M. C. M.
Ciências Fisiológicas/Universidade Estadual de Londrina, UEL
Objectives:
During sepsis, areas of the central nervous system are activated and have a role in physiological adaptations to immunity
challenge. One of these areas is the paraventricular nucleus of the hypothalamus (PVN). Data from our laboratory showed the
involvement of PVN in cardiovascular and autonomic modulation during endotoxemia by lipopolysaccharide (LPS). The PVN is
also an important site where nitric oxide (NO) plays a relevant role.The aim of this study was to evaluate the involvement of
nitric oxide from iNOS pathway in the PVN on mean arterial pressure (MAP), heart rate (HR) and spectral analysis of HR
variability (HRV) after administration of LPS in conscious rats.
Methods and Results:
Male Wistar rats were anesthetized for implantation of bilateral guide cannulae to the PVN. After 5 days the rats were
anesthetized for chronic catheterization of the femoral artery and vein. 24 hours later it was performed the bilateral microinjection
of 100 nl of saline or aminoguanidine (250 pmol) into the PVN of conscious rats, followed by LPS administration (iv).
Cardiovascular parameters were analyzed by 2 hours. Saline microinjection into the PVN did not modify the baseline parameters
and after LPS was observed hypotension and tachycardia ( MAP= -24 ± 5 mmHg, HR = 85 ± 15 bpm, p
Conclusions:
Our data suggest an involvement of iNOS pathway into the PVN in cardiovascular and autonomic modulation during the initial
phase of endotoxemia by LPS.
Keywords: AUTONOMIC MODULATION , ENDOTOXEMIA, PARAVENTRICULAR NUCLEUS
Financial Support: PIBIC/UEL.
Resumo:02-091
EFFECTS OF PROTEIN RESTRICTION POST-WEANING ON MORPHOLOGY AND CONTRACTILE
FUNCTIONS IN CARDIOMYOCYTES OF FISCHER RATS
Penitente, A. 1; Novaes, R. D. 1; Natali, A. J. 1; Silva, M. F. D. 1; Ribeiro, M. F. 3; Fernandes, K. M. 1;
Souza, A. M. A. D. 3; Silva, M. E. D. 3; Chianca-jr, D. A. 3; Neves, C. A. 1
1
Departament of General Biology, UFV
4
Departament of Nutrition , UFOP
3
Departament of Biological Science, UFOP
2
Departament of Physical Education , UFV
Objectives:
The present study evaluated the effects of severe protein restriction after weaning on the morphology and contractile function of
isolated left ventricular (LV) cardiomyocytes of rats.
Methods and Results:
Adult Fischer rats were randomized into control (CG = 20) and malnourished (MG = 20) groups. After weaning, the GC and MG
rats received isocaloric diets containing 15% and 6% protein, respectively, during 35 days. Subsequently, the animals were
weighed, sacrificed and the hearts were removed and weighted. LV were dissected, weighed and the cardiomyocytes were
isolated for morphological and contractile function analysis at baseline, in response to changes in extracellular calcium
concentration. Removed hearts were fixed in formalin for 48 hours and the fragments of the LV were dehydrated and embedded
in glycol methacrylate resin (Historesin, Leica) and paraffin. Serial sections were performed two and four µm thick, mounted on
slides and stained with toluidine blue 1% borax, hematoxylin / eosin and subjected to histological and morphometric analysis
using an optical microscope (BX-60, Olympus) connected to a Q-Color 3 digital camera (Olympus). Area and nuclei of
cardiomyocytes were measured using Image-Pro Plus 4.5 (Media Cybernetics). Statistical analysis was performed using mean ±
SEM. The animals in the MG group had reduced body mass, heart and ventricular rate, but higher LV / body weight ratio when
the cardiac mass was normalized in relation to body mass. Regarding the morphological properties of the LV, the protein
restriction reduced cellular morphological parameters of cardiomyocytes analyzed, except the length / width ratio that was greater
in these MG rats. Analysis of cellular contractility demonstrated that protein malnutrition caused a reduction in the amplitude of
ventricular cardiomyocytes contraction, increased the time to peak contraction and to half relaxation of these cells. In addition,
cardiomyocytes from malnourished animals showed changes in contractile properties in response to different variations of
extracellular calcium compared to control animals.
Conclusions:
The severe protein restriction leads to changes in morphology and contractile dysfunction in left ventricular cardiomyocytes of
Fischer rats.
Keywords: cardiomyocytes, Fischer rats, protein restriction
Financial Support: FAPEMIG, CNPq, CAPES, FUNARBE, UFV
Resumo:02-092
BLOCKADE OF 5-HT3 RECEPTORS IN THE MEDIAL SEPTAL AREA CHANGES BAROREFLEX SENSITIVITY.
Urzedo-rodrigues, L. S 1; Ferreira, H. S. 2; Almeida, D. O1; Batista, A. S. 1; Dias, D. P. M. 3; Fregoneze, J.
B. 1
1
Universidade Federal da Bahia, Dpto de Fisiologia , UFBA
2
Universidade do Estado da Bahia, Dpto de Ciências da Saúde, UNEB
3
Universidade de São Paulo, Dpto de Fisiologia, USP
Objectives:
It has been reported that different serotonin receptors are involved in the brain control of cardiovascular function. Recent data
from our laboratory show that 5-HT3 receptors in the medial septal area (MSA) exert a tonic inhibitory influence on blood
pressure (Autonomic Neurosc., 159:51-61, 2011). Among the several serotonin receptors, the 5-HT1A and 5-HT7 receptors seem
to be crucial for parasympathetic control of heart rate. However it is not fully understood the role of serotonin receptors on the
control of baroreflex. In the present study, we investigated whether the blockade of 5-HT3 receptors in the medial septal area
(MSA) modifies baroreflex sensitivity in rats.
Methods and Results:
Male Wistar rats (280-310g) were anaesthetized with Ketamine/Xylazine (80/11.5 mg/kg i.p) for implantation of guide cannulas
in the MSA 5 days before experiments. A catheter was inserted into the left carotid artery 24h before the experiment to allow
blood pressure recording. At the experimental session, after 30 min of baseline blood pressure recording, rats received
microinjection of 5-HT3 receptor antagonist, ondansetron (160 nmol), or 0.9% saline into MSA. At the end of the experiments,
animals were anesthetized and subjected to transcardiac perfusion with saline followed by 10% formalin and had their brain
removed for histological analysis. Only data from animals whose guide cannulas were in the MSA were considered. The
baroreflex sensitivity (BRS) was assessed in the time-domain by means of the Sequence technique. A computer software
(Analyzer v4.4) scanned beat-by-beat time series of systolic blood pressure (SBP) and inter-beat interval (IBI) searching for
sequences of at least 4 consecutive beats in which increases in SPB were followed by IBI lengthing (up sequence) and decreases
in SPB were followed by IBI shortening (down sequence). The slope of the linear regression lines between SPB and IBI was
taken as a measure of BRS. The average of the slopes, number of up and number of down sequences were calculated for each rat
at basal period (before MSA injection), at 0-30 min and 30-60 min period after MSA injections. The data were subjected to oneway analysis of variance (ANOVA) followed by Student-Newman-Keuls post-test. Ondansetron-treated rats showed a significant
reduction in the slope (1.70 ± 0.13 ms/mmHg, n=5) of up sequences as compared to control group (3.25 ± 0.61 ms/mmHg, n=4)
at 30-60 min. The number of up and down sequences was found similar between ondansetron-treated and saline-treated rats.
Conclusions:
The blockade of 5-HT3 receptors in the MSA reduces the average slope of ―up‖ sequences. It has been shown that ―up‖
sequences are better predictors of vagal cardiac activity than ―down‖ sequences. We hypothesize that 5-HT3 serotonin receptors
in the MSA are involved in the modulation of parasympathetic component of baroreflex function.
Keywords: Baroreflex Sensitivity, 5-HT3, Medial Septal Area, Serotonin
Financial Support: Capes, CNPq, FAPESB
Resumo:02-093
CARDIAC ELECTRIC REMODELLING IN RATS CHRONICALLY TREATED WITH ANABOLIC STEROID: LTYPE CALCIUM CURRENT CHARACTERIZATION
Arantes, P. C. ; Rodrigues Jr, L. F. ; Medei, E. ; Nascimento, J. H. M.
Instituto de Biofísica Carlos Chagas Filho, UFRJ
Objectives:
Cardiac abnormalities, arrhythmias and sudden death were reported as consequence of abusive use of anabolic androgen steroids
(AAS). In a previous study, we showed the increase in ventricular action potential duration (APD) in hearts of rats chronically
treated with decanoate nandrolone (DECA). The purpose of present study was to evaluate the contribution of L-type calcium
current (ICa,L) to APD prolongation on AAS-induced cardiac electrical remodelling.
Methods and Results:
Methods: Male wistar rats were treated with nandrolone decanoate (DECA group; dose: 10 mg/kg/week, i. m.) or vehicle
(Control group) during 8 weeks. Ventricular cardiomyocytes were dissociated using colagenase and ionic currents were recorded
using whole cell patch-clamp technic. Voltage protocol to obtain curve I-V consisted of 300 ms pulses, from holding potential of
-50 mV to potentials from -60 to +60 mV potentials, in 10 mV intervals. Protocol to obtain steady-state inactivation voltage
dependency consisted of 1000 ms pre-pulses from -80 mV to +40 mV, in 10 mV intervals, followed by a 300 ms pulse to 0 mV.
Data are presented as mean ± standard error. Results: Current amplitude was normalized by cell capacitance (Control: 108.9 ±
9.75 pF, N = 11; Deca: 103.2 ± 7.75 pF, N = 10). The peak density of ICa,L in Control group was -10,16 ± 0,87 pA/pF (N = 11),
with peak current occurring at 10 mV, activation in -20 mV and reversal potential next to -40 mV. In DECA group, the peak
density of ICa,L was higher (-13,46 ± 1,81 pA/pF; P = 0,0438 vs. Control), with peak at 10 mV. Activation V½ of ICa,L was -8,0
± 0.05 mV in Control group and -7,0 ± 0.05 mV in DECA group, while inactivation V½ of ICa,L was -25 ± 0.05 mV (Control)
and -22 ± 0.33 mV (DECA).
Conclusions:
Our preliminary results suggest an increased density of ICa,L in left ventricle of rats chronically treated with anabolic steroids.
Keywords: Anabolic androgen steroids, Cardiac electric remodelling, L-type calcium current, Rats chronically treated
Financial Support: FAPERJ and CNPq/PIBIC
Resumo:02-094
INFLUENCE OF CRONIC CAROTID AND AORTIC BARORECEPTORS DENERVATION ON AUTONOMIC
MODULANTION IN SPONTANEOUSLY HYPERTENSIVE RATS
Souza, P. R. M. D. 1; Moreira, E. D. 1,1; Mostarda, C. 1; Monteiro-de-moraes, W. M. A. 2; Guimarães, F. 2;
Santos, F. 1; Piratello, A. C. 3; Silva, M. B. 1; Irigoyen, M. C. 1
1
Experimental Hypertension Laboratory - Heart Institute (InCo, FMUSP - InCor
2
University of São Paulo Medical School, São Paulo, Brazil; S, EEFE-USP
3
PPG Nephrology, Department of Medicine – Nephrology Division, UNIFESP
Objectives:
To evaluate the participation of the autonomic nervous system in hemodynamic variables through selective denervation of carotid
and aortic receptors in spontaneously hypertensive rats (SHR) after 10 weeks.
Methods and Results:
SHR (280-320g) were divided into three groups: control (N=12), aortic denervation (SHRAD, N = 13) and carotid denervation
(SHRCD, N =11). Ten weeks after the denervation procedure, the animals were catheterized (right femoral artery and vein) for
direct recording of arterial pressure (AP) and drug administration respectively. The obtained signals were later analysed by
spectral analisys. The results were reported as means ± SD.Results: The blood pressure, heart rate and HRV were similar between
groups. BPV increase in SHRAD when compared with SHR and SHRCD (SHRAD= 135.2±62 vs. SHR=70.7±41 and
SHRCD=61.7±27 mmHg2). LF and HF absolute components of HRV as well as the sympatho-vagal balance were similar
between groups. The absolute LF component of systolic blood pressure increases in SHRAD when compared with SHR
(SHRAD=18.510 vs SHR=9.25 mmHg2) However SHRCD was similar among the groups. (SHRCD=11.1  7 mmHg2)
Conclusions:
The data showed that sympathetic component of heart rate variability are preserved in SHR submitted to chronic selective
denervation. However the sympathetic component acting on the vessels was increased only in aortic denervation, showing the
differential role of the aortic baroreceptors in the homeostasis of blood pressure even in the presence of hypertension
Keywords: selective denervation, autonomic modulantion, hypertension
Financial Support: CAPES
Resumo:02-095
TREATMENT WITH ATENOLOL (B-ADRENERGIC ANTAGONIST) ALTERS THE PROFILE
IMMUNOHISTOCHEMISTRY OF RANK, RANKL, OPG AND MMP-9 OF ALVEOLAR BONE HEALING PROCESS
IN SPONTANEOUSLY HYPERTENSIVE RATS (SHR)
Manrique, N. ; Pereira, C. C. S. ; Okamoto, R. ; Antoniali, C.
Faculdade de Odontologia de Araçatuba-UNESP, FOA-UNESP
Objectives:
Previously results obtained in our laboratory showed that there is a delay in alveolar bone healing process in SHR. This delay was
associated with increased peripheral sympathetic tone of these animals, since treatment with atenolol reduced SBP (systolic blood
pressure) and normalizes alveolar bone formation. The aim of this study was to evaluate the atenolol effect on the expression of
bone metabolism proteins OPG, RANK, RANKL and MMP-9 in dental alveoli of SHR
Methods and Results:
Wistar rats and SHR treated or not with atenolol (100mg/kg/d, po) were subjected to surgical extraction of upper right incisor
tooth. Treatment with atenolol was started one week before surgery and maintained until sacrifice. The alveolar repair was
evaluated in the different phases: initial (7 day), bone formation (14 and 21 days) and remodeling (28 and 42 days). The upper
right maxilla were processed and submitted to immunohistochemical reactions. The middle third of dental alveolus was examined
(20x objective), staining and it was analyzed qualitatively by assigning scores. The results were compared between groups (Mann
Whitney, p
Conclusions:
Our results demonstrated that: 1- RANKL, RANK and MMP-9 but not OPG, are associated with the delay of the alveolar bone
healing process in SHR, 2- treatment with atenolol increases the OPG staining in all phases analyzed and RANKL, RANK and
MMP-9 specifically in 21 e 42 days. These results suggest that in SHR, changes in the initial phase of alveolar bone formation
and remodeling would be a consequence of a differential expression of proteins in bone metabolism such as OPG, RANKL,
RANK and MMP-9, which is modulated by treatment with atenolol.
Keywords: Alveolar process, Atenolol, Hypertension, Immunohistochemistry, Rats, inbread SHR
Financial Support: Fapesp (2008/01893)
Resumo:02-096
REGIONAL EFFECTS OF RESISTANCE-TRAINING ON CELL SIZE IN RAT CARDIAC MYOCYTES
Melo, S. F. S. ; Junior, M. A. C. ; Bozi, L. ; Drummond, L. R. ; Natali, A. J. ; Oliveira, E. M. D.
Escola de Educação Física , USP
Objectives:
The purpose of this study was to test whether the hypertrophic response to resistance-training program for a period of 2 months
was uniformly distributed across the ventricular wall in rats.
Methods and Results:
Fourteen animals were randomly divided into two groups: control (n = 6, CO), and trained (n = 7, TR). The training protocol
consisted of four sets of 10–12 repetitions of the squat exercise performed at 75–80% of one repetition maximum (1RM). Single
ventricular myocytes were isolated from the sub-epicardium (EPI) and sub-endocardium (ENDO) of trained rats and from
sedentary rats for comparison. Cellular hypertrophy (approximately 14% greater cell volume, P
Conclusions:
We have previously shown using this model that resistance training induces the development of concentric cardiac hypertrophy
without ventricular dysfunction or cavity reduction. Here, ours results showed difference between EPI and ENDO
cardiomyocytes size to trained compared to the sedentary rats. Also, to the proteins involved in the maintenance of normal
cardiac Ca2+ homeostasis improving the contractile function in trained group compared to the sedentary.
Keywords: CARDIAC MYOCYTES, RESISTANCE-TRAINING , MYOCYTES
Financial Support: FAPESP
Resumo:02-097
PHOSPHODIESTERASE INHIBITION INCREASES THE POTENCY OF RELAXATION OF THE NEW NO-DONOR
TERPY IN OLD SHR.
Munhoz, F. C. 1; Potje, S. R. 1; Hildebrand, M. C. 1; Pereira, A. C. 2; Ramos, L. C. B. 2; Bendhack, L. M. 2;
Antoniali, C1
1
Dept of Basic Sciences, School of Dentistry of Araçatuba, FOA-UNESP
2
School of Pharmaceutical Sciences, FCFRP-USP
Objectives:
The vasodilatory effect of TERPY, a new NO donor drug, is more potent in old than in young spontaneously hypertensive rats
(SHR). These results suggest that the mechanisms involved in the effect of TERPY could be modulated by age in SHR. So, the
aim of this study was to evaluate possible alterations in the action of soluble guanilate ciclase (sGC), potassium channels (K+),
phosphodiesterase and superoxide anion (O2-) involved in vasodilatory effect of TERPY in old SHR.
Methods and Results:
Animal Research Ethics Committee (CEEA) at the School of Dentistry of Araçatuba approved all the experimental protocol
conduced in the study (proc. number 001776-2010). 4 months old WST and SHR were used in the young group and animals with
16 months were considered old. These animals were killed and had the aorta removed and isolated. Aortic rings were placed
between two stainless-steel stirrups and connected to an isometric force transducer (Letica Scientific Instruments; Barcelona–
Spain) to measure isometric tension in the vessels. The rings were placed in an organ chamber containing Krebs solution
maintained at pH 7.4 gassed with 95% O2 and 5% CO2 at 37ºC. The rings were initially stretched to a tension of 1.5 g and then
they were allowed to equilibrate for 60 min. Denuded aortic rings from SHR (SP> 150mmHg) and WST were pre-contracted
with phenylephrine and concentration-response curves for TERPY (1nM- 100 µM) were constructed in the presence or absence
of ODQ (GCs inhibitor, 1µM), TEA (K+ channel blocker, 1mM), DIPYRIDAMOLE (Phosphodiesterase inhibitor, 1µM),
TIRON (O2- scavenger, 0.1mM) and APOCYNIN (NADPH oxidase inhibitor, 0.1mM). Curves were normalized in percentage of
relaxation, according to the level of contraction. Potency (pD2) and efficacy (maximum effect, ME) of TERPY were evaluated.
The results (mean±SEM) were compared between the groups by Student‘s t test (p
Conclusions:
Taken together, these results show that TERPY induced relaxation is GCs dependent and this dependency is more evident in old
SHR. K+ channels sensitive to TEA and superoxide anions scavenged by TIRON do not to interfere in the effect of the
compound. Besides, in old SHR aorta, NADPH oxidase inhibition doesn‘t increases the potency of TERPY as in young SHR, but
the increased potency in the phosphodiesterase inhibition by DPYRIDAMOLE was more evident in young SHR. These data
suggest that the participation of NADPH oxidase and phosfodiesterase in the relaxing effect of TERPY is differently modulated
by age in SHR.
Keywords: Nitric Oxide donors, SHR, advanced age, phosphodiesterase, NADPH oxidase
Financial Support: CNPQ
Resumo:02-098
EFFECTS OF ADRENOMEDULLIN ALONE OR COMBINED WITH GLUTAMATE IN THE ROSTRAL
VENTROLATERAL MEDULLA OF CONSCIOUS RATS
Barbosa, R. M. ; Barbosa, S. P. ; Freiria-oliveira, A. H. ; Menani, J. V. ; Colombari, E. ; Colombari, D. S.
A.
Dept of Physiology and Pathology, FOAr-UNESP
Objectives:
It has been demonstrated that the peptide adrenomedullin (ADM) microinjected into the rostral ventrolateral medulla (RVLM) of
anesthetized rats produces increases in arterial pressure and in the sympathetic activity, an effect that seems to be mediated by
glutamatergic receptors (Am. J. Physiol., 287: R729, 2004). A previous study from our laboratory demonstrated that the pressor
response induced by glutamate injected into the RVLM was enhanced by the pre-treatment with the peptide angiotensin II also in
the RVLM (Brain Res., 1322: 72, 2010). Due to this interaction, our hypothesis is that ADM in the RVLM could also increase the
pressor response induced by glutamate in the RVLM. Since the anesthesia can alter the response of the substances injected in the
central nervous system and to further study the interaction between ADM and glutamate in the RVLM, in the present study we
investigated the effects of ADM alone or combined with glutamate into the RVLM on the arterial pressure in conscious rats.
Methods and Results:
Male Holtzman rats (280-320 g) with stainless steel guide cannulas unilaterally implanted in the RVLM (n = 5-8/group) were
used. In the day before the experiments, catheters were inserted into the femoral artery for mean arterial pressure (MAP)
recording in conscious animals. In one group of rats glutamate (1 nmol/100 nl) was injected before and 5 min after ADM (100
pmol/100 nl) and in another group of rats glutamate (1 nmol/100 nl) was injected before and 20 min after ADM (100 pmol/100
nl). ADM into the RVLM increases MAP (15 ± 4, vs. saline: -1 ± 1 mmHg, p < 0.05). The pressor responses induced by
glutamate into the RVL before and 5 min after ADM were similar (35 ± 6, vs. after ADM: 36 ± 6 mmHg). In another group of
rats, ADM also induced pressor response (20 ± 5 mmHg, vs. saline: -2 ± 1 mmHg, p < 0.05) and, similarly, there was no
difference between the pressor responses induced by glutamate into the RVLM before (24 ± 7 mmHg) and 20 min after ADM (24
± 5 mmHg). Baseline MAP was 121 ± 4 and 117 ± 5 mmHg, for each group of rats.
Conclusions:
ADM microinjected into the RVLM of conscious rats also induces increases in MAP, as observed in anesthetized rats. Different
from our hypothesis, ADM did not change the pressor response to glutamate into the RVLM, even shortly after ADM, suggesting
that in conscious rats the interaction between ADM and glutamate at the level of RVLM may not occur.
Keywords: blood pressure, RVLM, glutamatergic pathway
Financial Support: PIBIC-CNPq, FAPESP
Resumo:02-099
ROLE OF ANGIOTENSIN II AT1 RECEPTOR IN THE ANGIOTENSIN-(1-7)-INDUCED CORONARY
VASODILATION IN HYPERTROPHIED RAT HEARTS.
Almeida, J. F. Q. ; Sobrinho, D. B. S. ; Alves, G. M. M. ; Dourado, J. G. ; Macedo, L. M. ; Mendes, E. P. ;
Castro, C. H.
Depto. de Ciências Fisiológicas / Uni. Federal de Goiás, ICB
Objectives:
Cardiac hypertrophy is an adaptive response of the heart associated with pathological situations. Pressure overload-induced left
ventricular hypertrophy (LVH) is related with impaired coronary circulation. Our previous study showed that Angiotensin-(1-7)
[Ang-(1-7)] and the receptor Mas are involved in the coronary circulation in physiological conditions. The aim of this study was
to evaluate the involvement of the angiotensin receptors on the coronary effect of Ang-(1-7) in hypertrophied hearts.
Methods and Results:
Subtotal abdominal aortic banding (AB) was performed to induce left ventricular hypertrophy in rats. After 21 days, the hearts
were perfused according to the Langendorff method (constant flow). After a basal period, the hearts were perfused for 15 min
with Ang-(1–7) (20 pmol/L) in presence or absence of receptor Mas antagonist A-779 (2 nmol/L), AT1 antagonist, losartan (1
µmol/L), or in combination. The left ventricles wet weights were recorded, normalized for tibia length and then expressed as
ventricular mass index. Aortic constriction resulted in a significant left ventricular hypertrophy (0,174 ±0,004 vs
0,217±0,012g/cm in AB hearts, p
Conclusions:
These findings indicate that Angiotensin II AT1 receptor is able to modulate Ang-(1-7)-induced coronary vasodilation in
hypertrophied rat hearts.
Keywords: A779, ANGIOTENSIN-(1-7), cardiac hypertrophy, LOSARTAN, receptor MAS
Financial Support: CNPq/INCT-Nanobiofar.
Resumo:02-100
SEROTONIN UPTAKE IN JUGULAR VEIN AND THE INVOLVEMENT OF MONOAMINE TRANSPORTERS.
de Prá, M. A. ; Poli, A. ; Linder, A. E.
FARMACOLOGIA, UFSC
Objectives:
Serotonin (5-hydroxytryptamine; 5-HT) can interact with its receptors causing vascular contractility in a variety of vessels,
including arteries and veins. In the rat jugular vein, there is evidence that the 5-HT2A receptor mediates contraction (JPET 334;
116, 2010). The presence of the serotonergic system in the peripheral vasculature enables the uptake and metabolism of this
monoamine by arteries and veins. 5-HT uptake in veins is 5-HT transporter (SERT) independent (JPET 323; 415, 2007).
Nowadays, substances that interfere in the uptake and metabolism of monoamines are widely used to treat neurological disorders,
although little is known about their influence on the cardiovascular system. The aim of this work is to test the hypothesis that 5HT uptake in veins is mediated by the norepinephrine (NE) transporter and that its function modulates venous reactivity.
Methods and Results:
Jugular veins isolated from male Wistar rats (300-400 g) were placed and maintained in physiological salt solution (PSS) at 370C
containing either vehicle or 5-HT (1 uM) for 15 min. 5-HT (1 uM) was also added for 15 min to vessels that had been previously
exposed for 30 min to the inhibitor of 5-HT uptake, fluoxetine (1 uM), or to the mixed 5-HT and NE uptake inhibitor, imipramine
(1 uM). Tissues were dipped several times in drug-free PSS to avoid extracellular 5-HT contamination, and then processed for
posterior measurements of 5-HT and its metabolite 5-hydroxy indol acetic acid (5-HIAA) by HPLC. Data were expressed as
nanogram of 5-HT or 5-HIAA per milligram of wet tissue. For isometric tension recordings, jugular vein rings were isolated and
mounted in an organ bath. Concentration-effect curves to 5-HT (1 nM – 0.1 uM) were performed in the presence and absence of
imipramine (1 uM) or fluoxetine (1 uM). Experimental protocol approved by the ethical committee (p0036).Results: Basal levels
of 5-HT could be measured in jugular vein (6,12 ± 2,50 ng/mg n=5), whilst the main product of its metabolism 5-HIAA was not
detectable. Whereas no significant changes in 5-HT content (6,28 ± 1,16, n=5; P>0.05) were detected when compared to basal
levels, a significant increase in 5-HIAA content was observed after 15 min (1,88 ± 0,47 ng/mg n=5) of extracellular 5-HT
exposure, indicating 5-HT uptake followed by metabolism. Fluoxetine had no effect in 5-HT and 5-HIAA content in jugular vein
segments exposed to 5-HT, as expected (P > 0.05). Interestingly, when compared to 5-HT exposure for 15 min alone, imipramine
decreased 5-HT levels (2,77 ± 0,73 ng/mg), but not the 5-HIAA levels. The concentration-effect curves to 5-HT were not altered
in the presence of imipramine or fluoxetine (p>0,05).
Conclusions:
Our results support previous findings that 5-HT uptake is SERT-independent in veins. Moreover, we present evidence of a
putative role for the NE transporter in the uptake of 5-HT in the rat jugular vein.
Keywords: SEROTONIN, UPTAKE, VASCULAR REACTIVITY, VEIN
Financial Support: CNPq, FAPESC/CNPq (005/2009), Funpesquisa.
Resumo:02-101
REDUCTION OF VENTRICULAR DYSFUNCTION INDUCEDBY MYOCARDIAL INFARCTION BY HYDRO
ALCOHOLIC EXTRACT OBTAINED FROM FRUITS OF EUTERPE OLERACEA MART. (AÇAI)
Silva, J. S. D. 1; Zapata-sudo, G. 1; Seabra, S. 4; Sousa, P. J. D. C. 2; Resende, A. C. 3; Moura, R. S. 3; Sudo,
R. T. 1
1
Programa de Desenvolvimento de Fármacos - ICB, UFRJ
2
Departamento de Farmacologia, UFPa
3
Departamento de Farmacologia, UERJ
4
Departamento de Farmacologia, UEZO
Objectives:
Delay in the progress of cardiac failure in cardiovascular diseases such as myocardial infarction (MI) could be observed if cardiac
remodeling processes are interrupted. The purpose of this study was to investigate cardiac remodeling and function in rats
submitted to experimental MI and treated with an extract, obtained from seed of açai fruits (Euterpe oleracea Mart) that have
antioxidant, vasodilation and anti inflammatory actions.
Methods and Results:
Protocols were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Wistar male rats (180200 g) were randomly divided in sham-operated (SHAM) and infarcted groups (MI) and each group subdivided in: treatment with
vehicle and with açai (100 mg/kg, p.o.). Under sevoflurane anesthesia, the animals were submitted to a ligature of the anterior
descending coronary artery. After 28 days of treatment, the following hemodynamic parameters were analyzed: mean arterial
pressure (MAP), left ventricular end diastolic pressure (LVEDP), end-systolic pressure (LVESP) and dP/dt. Hypertrophy was
determined using the ratio of heart/body weight. Results- The following table summarizes the results obtained in both SHAM and
MI groups treated or non-treated with the extract. Hemodynamic and heart weight change induced by myocardial infarction (MI)
and treatment with Euterpe oleracea. Mean arterial pressure (MAP), left ventricular end-systolic pressure (LVESP), left
ventricular end diastolic pressure (LVEDP), positive (+) and negative (-) dP/dt. Data were expressed as mean  SEM (n= 6). *P
Conclusions:
Our results demonstrate that açai fruit extract has a significant protection on cardiac dysfunction induced by MI. This beneficial
action of açai is probably dependent on the reduction of myocardial fibrosis due to a protective action against oxidative stress,
reduction of inflammatory reaction and an increase in vascular blood flow to the area of infarction.
Keywords: Açai, Antioxidant, Cardiac failure, Myocardial Infarction, Ventricular dysfunction
Financial Support: FAPERJ, Capes, CNPq, INCT/INOFAR
Resumo:02-102
NADPH OXIDASE INHIBITION POTENTIATES THE RELAXATION INDUCED BY ([RU(TERPY)(BDQ)NO+]3+)
TERPY IN SHR.
Potje, S. R. 1; Munhoz, F. C. 1; Hildebrand, M. C. 1; Ramos, L. C. B. 2; Bendhack, L. M. 2; Antoniali, C. 1
1
Department of Basic Sciences, FOA-UNESP
2
Dept of Physics and Chemistry, FCFRP-USP
Objectives:
Previous experiments show that the hypotensive effect of TERPY, a new NO donor, is increased in SHR, compared to Wistar rats
(WST). Our hypothesis is that this effect could be associated to an increase in the relaxing response of vascular smooth muscle
induced by TERPY. Besides, oxidative stress could be differently modulated in SHR. So, the aim of the present study was to
evaluate the effect of TIRON, a superoxide anion scavenger, and APOCYNIN, a NADPH oxidase inhibitor in the relaxation
induced by TERPY in SHR aorta.
Methods and Results:
Animal Research Ethics Committee (CEEA) at the School of Dentistry of Araçatuba approved all the experimental protocol
conduced in the study (proc. number 001776-2010). 4 months old WST and SHR were killed and had the aorta removed and
isolated. Aortic rings were placed between two stainless-steel stirrups and connected to an isometric force transducer (Letica
Scientific Instruments; Barcelona–Spain) to measure isometric tension in the vessels. The rings were placed in an organ chamber
containing Krebs solution maintained at pH 7.4 gassed with 95% O2 and 5% CO2 at 37C. The rings were initially stretched to a
tension of 1.5 g and then they were allowed to equilibrate for 60 min. Endothelium integrity was assessed by the degree of
relaxation induced by acetylcholine in phenylephrine pre-contracted arteries. Denuded aortic rings from SHR (SP> 150mmHg),
and WST were pre-contracted with phenylephrine and concentration-response curves for TERPY (1nM- 100 μM) were
constructed in the presence or absence of TIRON (0.1mM) and APOCYNIN (0.1mM). Curves were normalized in percentage of
relaxation, according to the level of contraction. Potency (pD2) and efficacy (maximum relaxation, ME) of the vasodilator effects
of TERPY were compared. Differences were considered significant when p
Conclusions:
These data suggest that superoxide anions scavenged by TIRON, in the concentration of 0.1mM, don‘t play a significant role in
the vasorelaxing effect of TERPY in SHR. On the other hand, NADPH oxidase inhibition by APOCYNIN increases the potency
of TERPY in SHR.
Keywords: NITRIC OXIDE DONORS, SHR, OXIDATIVE STRESS, TERPY, SODIUM NITROPRUSSIDE
Financial Support: CNPq, FAPESP
Resumo:02-103
BRADYCARDIA AND PRESSOR RESPONSES TO OBSTRUCTIVE APNEA ARE REDUCED DURING REM SLEEP
IN RATS
Rossi, M. V. ; Schoorlemmer, G. H. ; Cravo, S. L.
Depto. de Fisiologia, Universidade Federal de São Paulo, UNIFESP
Objectives:
Obstructive sleep apnea is a very common condition that leads to several health problems. However, it is still not clear if the
consequences of sleep apnea are due directly to apnea, or to sleep fragmentation. We compared cardiorespiratory responses
induced by obstructive apnea in awake and sleeping rats.
Methods and Results:
We used four Wistar rats with a balloon-tipped catheter implanted in the trachea. Apnea was induced by injection of ~15 µL
water into the balloon. Apnea terminated on withdrawal of the injected fluid. Balloon inflation did not cause tracheal pain
because the balloon was contained in a rigid Teflon tube. Rats also had ECG, EEG and EMG electrodes. Sleep stages were
classified online once a second according to Louis et al. (J Neurosci Methods 133: 71-80, 2004) with Spike2 software. Apnea was
initiated automatically when 4 of the last 5 seconds were classified as REM, and terminated when EMG activity increased to the
awake level. Some rats also had arterial catheters for measurement of arterial pressure, and thoracic balloons for measurement of
respiratory effort. One week after surgery, apneas were induced every time the rat entered REM sleep for 3 h. The same sequence
of apneas was repeated the next day. This way were induced 21 ± 3 apneas/h with average length of 8 s. After apnea termination,
rats usually resumed sleep within 20 s. Apneas longer than 10 s in awake rats induced profound bradycardia (131 ± 14 bpm), a
pressor response (22 ± 3 mmHg), and respiratory effort (pressure swing of > 40 mm Hg). Identical apneas during REM caused a
weak bradycardia (22 ± 3 bpm), and no pressor response (-2 ± 3 mmHg). Respiratory effort also appeared reduced with apneas
during REM
Conclusions:
Responses to apnea in rats are blunted during REM sleep, and resemble those seen in partients with sleep apnea.
Keywords: blood pressure, bradycardia, obstructive apnea, REM, sleep
Resumo:02-104
HYPOXIA INDUCED WITH VASCULAR PROTECTION SOLUTIONS FOR CARDIAC TRANSPLANTATION
INCREASE VASCULAR REACTIVITY.
Simões1; Batista1; Fiorim1; Lima1; Stefanon1; Vassallo1,2
1
Departamento de Ciências Fisiológicas, UFES
2
Escola Superior de Ciências da Santa Casa de Misericórdia, EMESCAM
Objectives:
Previous studies have demonstrated that the existing methods of myocardial protection are not able to avoid the undesirable
effects of ischemia and the reperfusion phenomenon. However, the mechanisms of action of protecting solutions used for cardiac
transplantation on vascular function are not described yet. This study aims to compare the efficiency of vascular protection by
using the protecting solutions, Krebs-Henseleit (KH), Ringer, Bretschneider-HTK (BHTK), St. Thomas (ST) and Celsior
solutions, in preparation of isolated aorta rings of rats submitted to one hour hypoxia.
Methods and Results:
Male Wistar rats, 3 months old, were used. The animals were anesthetized and the thoracic aorta was removed and placed in KH
solution for cleaning of connective tissue and fat, and cut in rings with 3 to 5 mm. The record of isometric tension, as percentage
of KCl-induced contraction, of each ring was obtained as proposed by Marin et al. (1998). Vascular reactivity was used to
investigate pressor responses to phenylephrine (PHE, 10-10 to 10-4 M) before and after 1 hour of hypoxia, at 36.5 OC, in rings
exposed to to KH During hypoxia aortic rings were exposed to the protecting solutions, Ringer, BHTK, ST and Celsior, and KH
used as control. Results showed an increased maximum response (Rmax) (KH: 146 ± 14. 5 % n = 7 vs ST: 184 ± 10.5 %, p <
0.05 n = 10) and sensitivity y (pD2) (KH:-6.02 ± 0.5 n = 7 vs ST-8.07 ± 0.24, p < 0.05 n = 8) in aorta rings exposed to ST in KH
solution. There was no change of these parameters with the other protecting solutions.
Conclusions:
This is the first report to show an increased vascular reactivity in aortic rings exposed to the protective solution ST, when
submitted to 1 hour of hypoxia, showing a pattern that suggests endothelial dysfunction. Results suggested that the ST solution
was the less efficient for vascular protection. inducing endothelial injury due to an increased content of ion Ca2 + and K + in its
composition. Results also suggest that the ST solution might be a risk factor for cardiac protection during transplantation
procedures. Grants:CAPES, FAPES/CNPq.
Keywords: BRETSCHNEIDER-HTK, CELSIOR, ISCHEMIA, SAINT THOMAS , VASCULAR PROTECTION
Financial Support: CAPES / CNPq / FAPES- FUNCITEC
Resumo:02-105
LOW CONCENTRATIONS OF LEAD AFTER 7-DAYS OF EXPOSURE INCREASES BLOOD PRESSURE AND
REDUCES THE PARTICIPATION OF NO IN RESISTANCE ARTERIES
Simões, M. R. 1; Furieri, L. B. 1; Fioresi, M. 1,2; Broseghini, G. B. 1; Vescovi, M. V. A. 3; Faria, T. de O. 1;
Martins, L. R. 1; Stefanon, I. 1; Padilha, A. S. 1; Vassallo, D. V. 1,4
1
Depto. Ciências Fisiológicas/Univ Federal Espírito Santo, UFES
2
Depto. Enfermagem/Univ. Federal Espírito Santo, UFES
3
Depto. Química/Univ. Federal Espírito Santo, UFES
4
Escola Superior de Ciên. Santa Casa Misericórdia de Vitória, EMESCAM
Objectives:
Evaluate the effects of low concentrations of lead after 7 days of exposure on blood pressure, on the reactivity of mesenteric
arteries of rats and the involvement of NO and the renin-angiotensin system in the vasoconstrictor response.
Methods and Results:
Male Wistar rats (250-300 g) were divided into two groups: control (Ct) and Lead (Pb). Treated rats received as drinking water
lead acetate (100 ppm) during 7 days and the Ct only water. After this rats were anesthetized and underwent surgery for
catheterization of the carotid artery for measurement of hemodynamic parameters: Systolic Blood Pressure (SBP), Diastolic
Blood Pressure (DBP), Mean Blood Pressure (MBP) and Heart Rate (HR). Blood samples were collected for determination of
lead, using atomic absorption spectrometry and fluorometry for determination of angiotensin converting enzyme (ACE) activity.
After blood collection the animals were sacrificed and the mesenteric arteries (3rd branch) dissected. The mesenteric artery was
divided into segments of 2 mm and mounted in a small vessel four chamber myograph for measurement of isometric tension.
After stabilization, the functional and endothelial integrity were tested. Then, performed concentration-response curves to
phenylephrine (PHE, 10 nM - 30 ìM). The effects of L-NAME (NOS inhibitor) and enalapril (angiotensin-converting enzyme
inhibitors), on the response to PHE were tested. Statistical analysis: Results were expressed as mean ± SEM. For comparison of
means Student t test were used. p
Conclusions:
Exposure to low concentrations of lead after 7 days increased blood pressure, did not alter the contractile response to PHE.
However, the exposure was able to reduce the role of NO in resistance arteries from Pb-treated rats. These observations also offer
further evidence that lead short time exposure, even at small concentration, is hazardous and it is an environmental risk factor for
cardiovascular disease
Keywords: BLOOD PRESSURE , RESISTANCE ARTERIES , LEAD
Financial Support: CAPES, CNPq e FAPES
Resumo:02-106
OPIOID RECEPTORS IN THE LATERAL PARABRACHIAL NUCLEUS INVOLVED IN THE CONTROL OF
WATER AND SODIUM INTAKE
Pavan, C. G. ; Penasso, M. G. ; Barbosa, S. P. ; de Luca Jr. , L. A. ; Colombari, D. S. A. ; de Paula, P. M. ;
Colombari, E. ; Menani, J. V.
Depto. de Fisiologia e Patologia, unesp
Objectives:
Bilateral injections of the non specific opioid agonist β-endorphin into the lateral parabrachial nucleus increase water and 1.8%
NaCl intake induced by the treatment with the diuretic furosemide (FURO) combined with low doses of the angiotensin
converting enzyme inhibitor captopril (CAP) injected subcutaneously (sc). In the present study, injecting specific agonists and
antagonists into the LPBN, we investigated the participation of µ and κ opioid receptors in the LPBN in the control of FURO +
CAP-induced water and 1.8% NaCl intake.
Methods and Results:
Male Holtzman (290-310 g, n = 8-12) with stainless steel cannulas bilaterally implanted in the LPBN were used. Water and 1.8%
NaCl intake was recorded for 2 to 3 h starting 1 h after the treatment with FURO (10 mg/kg of body weight) + CAP (5 mg/kg of
body weight) sc. Endomorphin-1 (µ opioid agonist), CTAP (µ opioid antagonist), BLR 52537 (κ opioid agonist) or vehicle was
injected into the LPBN 15 to 30 min before starting water and NaCl intake recording. Bilateral injections of endomorphin (1, 2 or
4 nmol/0.2 µL) into the LPBN increased FURO + CAP-induced 1.8% NaCl intake (21.1 ± 2.4, 21.4 ± 2.8 and 21.6 ± 3.0 mL/3 h,
respectively, vs. saline: 8.6 ± 1.5 mL/3 h, p
Conclusions:
The results suggest that µ and κ opioid receptor activation in the LPBN deactivates the inhibitory mechanisms facilitating NaCl
and water intake induced by FURO + CAP treatment.
Keywords: sodium intake, opioid receptors, lateral parabrachial nucleus
Financial Support: FAPESP, CNPq
Resumo:02-107
ACTIVATION OF POTASSIUM CHANNELS BY NITRIC OXIDE PREVENTS ENDOTHELIAL DYSFUNCTION OF
AORTIC ARTERY IN LEAD-TREATED RATS
Fiorim, J. 1; Júnior, R. F. R. 1; Avevedo, B. F. 1; Padilha, A. S. 1; Stefanon, I. 1; Vassallo, D. V. 1,2
1
Department of Physiological Sciences, UFES
2
Health Science Center of Vitória, EMESCAM
Objectives:
Seven days exposure to low lead concentration increased systolic blood pressure (SBP) and reduced vascular reactivity to
phenylephrine. These results were accompanied by increase in the endothelial modulation and nitric oxide (NO) bioavailability
(Plos One, 6: e17117, 2011). Potassium (K+) channels play a key role in regulating membrane potential and vascular tone.
Activation of K+ channels in vascular smooth muscle leads to hyperpolarization, decrease voltage-gated L-type Ca2+ channel
activity, reduce intracellular [Ca2+], and promote vasodilation. The aim of the present study was to evaluate in aortic rings,
whether 7 days lead-treatment affects both nitric oxide-mediated vasodilator responses and NO dependent activation of K+
channels.
Methods and Results:
Methods: Wistar rats were treated with lead (1st dose 4 ìg/100 g, subsequent dose 0.05 ìg/100 g, i.m. to cover daily loss) or
vehicle. Aortic rings from untreated and lead-treated rats were precontracted with phenylephrine (1 µM) or with 60 mmol/l KCl,
and concentration-response curves to acetylcholine (0.1 nM - 300 µM) were performed. The participation of NO in the relaxation
induced by ACh was analyzed incubating the vessels with NG-nitro-L-arginine methyl ester (L-NAME, 100 µM, nonspecific
NOS inhibitor), for 30 min before phenylephrine or KCl administration. The contribution of K+ channels to ACh-induced
relaxation was assessed in aortas previously incubated for 30-min with K+ channel blockers as follows: tetraethylammonium
(TEA, 2 mM, nonselective blocker of K+ channels), 4-aminopyridine (4-AP, 5 mM, blocker Voltage-dependent K+ channels
KV), charybdotoxin (ChTX, 0.1 M, blocker of Ca2+-activated K+ channels - KCa and KV), iberiotoxin (IbTX, 30 nM, selective
blocker of Large-conductance Ca2+-activated K+ channels - BKCa) and apamin (0.5 M, selective blocker of Small-conductance
Ca2+activated K+ channels - SKCa). In some experiments, concentration–response curves to sodium nitroprusside (SNP, 0.01
nM - 0.3 µM) were performed. The participation of Kv and BKCa channels in the SNP induced relaxation was analyzed
incubating the rings with 4-AP and IbTX respectively. Results: In control rats, the basal contraction induced by 4-AP was
remarkably greater than that induced by IbTX suggesting a relevant role of KV channels in controlling the aortic vascular tone.
Moreover, basal contractions induced by 4-AP increased after lead-treatment. In phenylephrine precontracted arteries, ACh
induced a similar relaxant response in aortic arteries from both groups that was abolished by L-NAME. However, when arteries
were contracted with high KCl (60 mmol/l) or preincubated with TEA, 4-AP, IbTX, BKCa and SKCa the relaxation elicited by
ACh was reduced more in lead-treated than in control rats. After IbTX and 4-AP preincubation, the relaxant response to SNP was
reduced more in lead-treated than in control rats.
Conclusions:
Our results indicate that 7 days lead-treatment increases activation of K+ channels by NO and this effect might contribute to
preserve the aortic endothelial function.
Keywords: lead, reactivity vascular, nitric oxide , potassium channels
Financial Support: PRONEX- FAPES/CNPq, CAPES
Resumo:02-108
EFFECT OF ACUTE ACHETYLCOLINESTERASE INHIBITION WITH PYRIDOSTIGMINE ON
ELECTROCARDIOGRAM OF ANESTHETIZED RATS.
Santos, F. M. ; Silva Caa ; Salgado, H. C. ; Fazan Jr. , R
Depto. de Fisiologia/Faculdade de Medicina de Ribeirao Preto, FMRP-USP
Objectives:
Several electrocardiographic indices have been proposed to identify patients at risk of sudden death, including the QT interval
length and/or dispersion. We evaluated the potentially benefic effect of an acute increase in parasympathetic drive to the heart,
obtained by pyridostigmine (PYR), an acetyl cholinesterase inhibitor, in anesthetized rats.
Methods and Results:
After basal recording of electrocardiogram (ECG, lead II), urethane (1 g/kg, ip) anesthetized rats received PYR (0.25 mg/kg) or
isotonic saline intravenously and ECG recording last for 30 min. PYR induced a significant increase in RR (130±1 to 145±2 ms),
PR (48±1 to 51±1 ms) and QT interval (64±1 to 66±1 ms). Nevertheless, corrected QT interval (QTc, Bazett formula) decreased
after PYR (179±3 to 174±2 ms).
Conclusions:
The increase in cholinergic transmission due to the acetycholinesterase inhibition led to an expectable bradycardia with PR
lengthening and a significant shortening in QTc interval. Clinical trials have demonstrated that the prolonged ventricular
repolarization (larger QT interval) is associated with higher incidence of cardiac arrhythmias. The use of PYR as an approach to
enhance vagal drive to the heart might be beneficial in situations of imminent risk of life threatening arrhythmias, such as acute
myocardial infarct.
Keywords: pyridostigmine, electrocardiogram, vagal tone, arrhythmia
Financial Support: FAPESP, CNPq, FAEPA
Resumo:02-109
EFFECTS OF INDAZOL AND RU-INDAZOL IN THE RAT CORPORA CAVERNOSA
Campos, R. M. 1; Sá, D. S. 2; Sousa, E. S. D. 2; Fonteles, M. C. 1; Lopes, L. G. D. F. 2; Lessa, L. M. A. 1;
Nascimento, N. R. F. D. 1
1
Instituto Superior de Ciências Biomédicas/UECE, ISCB
2
Laboratório de Bioinorgânica / UFC, UFC
Objectives:
Erectile dysfunction is a highly prevalent disease. One of the most important pathways involved on the mechanism of penile
erection is the increase of intracelular levels of cyclic guanosine monophosphate due to soluble guanylate cyclase (sGC)
activation. The aim of this present study is to investigate the effects of a metal-based drug, derived from the soluble guanylate
cyclase activator 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), on the relaxation of corpora cavernosa smooth muscle.
The pharmaceutical industry is searching a sGC- stimulating compound that is neither dependent on nitric oxide (NO)
release/activity nor on the redox status of the haem-group of the enzyme. The potential impact of such drugs relies in the fact that
they are able to increase the rate of activity of oxidized sGC a phenotype that is prevalent under disease conditions, especially
where increased oxidative stress is present, such as diabetes. Our group has synthesized a ruthenium–based compound named
Ru–indazol that is probed here for corpora cavernosa relaxation compared to the lead compound, indazol
Methods and Results:
Male Wistar rats (250g-300g) were sacrificed with sodium thiopental (200 mg/Kg;,i.p). Thereafter, the penis was excised out and
careful dissecation was performed in order to remove surrounding tissues. Strips of corpora cavernosa were immersed in KrebsHenseleit solution kept at 37°C and gassed continuously with 95% O2 in 5% CO2. The tissues were fixed in an isometric force
transducer under 0.5g resting tension and kept at equilibration conditions for 60 minutes-period. Modifications of resting tension
were continuously recorded in a 4-channel physiograph ( Narco Biosystems , Houston, Texas, EUA). Tissues were pre-contracted
with phenylephrine (PE; 20 μM) and then concentration-response curves to Indazol (1nM - 100μM) and its derivative compound,
RU-Indazol (1nM - 100μM) were performed. In another set of experiments, the concentration- response curves to both
compounds were repeated in strips treated with a soluble guanylate cyclase inhibitor [1,2,4]oxalodiazolo[4,3]oxonalina (ODQ;
100 μM). These protocols were approved by the local Ethicall Commitee for Animal Experimentation (protocol number:
08628332-4). The data were analyzed by Student‘s t test ( p<0.05).
Conclusions:
RU-Indazol induced a concentration-dependent relaxation of strips of corpora cavernosa with a maximal relaxant response of
100% and IC50 of 8.9 µM (n=5). Indazol promoted a maximal response of 67.6% ± 8.0 (p
Keywords: endothelial dysfunction, nitrergic dysfunction, oxidized soluble guanylate cyclase, soluble guanylate cyclase
activators
Financial Support: This study was supported by CNPq/ Brazil
Resumo:02-110
ROLE OF REDOX-SENSITIVE PROTEINS IN THE ESTABLISHMENT OF CARDIAC HYPERTROPHY
MEDIATED BY RENIN-ANGIOTENSIN SYSTEM IN HYPERTHYROIDISM.
Baraldi, D. D. ; Berger, B. R. ; Conzatti, A. ; Schenckel, P. C. ; Fernandes, T. R. G. ; Carraro, C. C. ; Bellóklein, A. ; Araújo, A. S. R.
Fisiologia, UFRGS
Objectives:
Hyperthyroidism generates important effects on the basal metabolism and cardiovascular system, altering its function and leading
to cardiac hypertrophy. It has been reported the relevant role of renin-angiotensin system (RAS) in the development of cardiac
hypertrophy in hyperthyroidism. In this context, angiotensin II would bind to the AT1 receptor, leading to the activation of
gp91phox (a NADPH oxidase subunit). This enzyme is involved in the reactive oxygen species (ROS) production, such as
superoxide anion and hydrogen peroxide (H2O2). ROS may activate the transcription factor (NrF2), which regulate protein
expression of antioxidants, such as heme-oxygenase-1 (HO-1). Therefore, the aim of this study was to investigate association
amongst H2O2, NrF2, HO-1, and renin-angiotensin system in the establishment of thyroid hormone-induced cardiac hypertrophy.
Methods and Results:
Male Wistar rats (n=5/group) were divided in: control (C), losartan (L) (10mg/Kg/day, intragastric, 28 days), L-thyroxin (T4)
(12mg/L in drinking water, 28 days), and T4+losartan (T4+L). The animals were catheterized to measure the resting heart rate
(HR). After the treatment period, animals were decapitated and its hearts were excised to perform: cardiac hypertrophy index
(CHI) (heart weight/body weight), and H2O2 concentration (nmol/g tissue). The protein expression of AT1 receptor, gp91phox,
NrF-2 and HO was evaluated by Western Blot. To compare groups, two way ANOVA with Student-Newmann-Keuls post hoc
test was used. Differences were considered to be statistically significant at p
Conclusions:
The findings of this study showed that renin-angiotensin system is associated with the development of cardiac hypertrophy
induced by thyroid hormones, being this process possibly modulated by reactive oxygen species. The RAS blockade was able to
reduce hypertrophy development, with the simultaneous reduction in the expression of proteins involved with the cell redox
regulation. These data suggest a connection among cardiac RAS, cellular redox status and its regulators proteins in the
establishment of cardiac hypertrophy in hyperthyroidism.
Keywords: hyperthyroidism, oxidative stress, renin-angiotensin system, cardiac hypetrophy, cell signalling
Financial Support: CNPq, CAPES e FAPERGS.
Resumo:02-111
TOLL-LIKE RECEPTOR 2 IS INCREASED IN RENAL ISCHEMIA/REPERFUSION-INDUCED CARDIAC
HYPERTROPHY IN MICE
Trentin-sonoda M ; Cirino-silva R. ; Carneiro-ramos Ms
Universidade Federal do ABC, UFABC
Objectives:
The innate immune response can exert innumerous effects in vast number of tissues and organs. It‘s well established that
cytokines and Toll-like receptors (TLRs) participate of cardiac trophism modulation. The aim of this study was to investigate the
role of TLRs in heart after renal ischemia/reperfusion.
Methods and Results:
C57bl/6J mice were submitted to unilateral 60 minutes kidney ischemia, followed by reperfusion for 5, 8, 15 and 20 days (n=6
per group). Levels of urea were measured to confirm renal failure as well vimentin mRNA expression. Cardiac ANF gene
expression was used as molecular cardiac hypertrophy marker and all gene expression were analyzed by real time PCR. Urea
levels and mRNA Vimentin were increased in groups 5, 15 and 20 days (P
Conclusions:
First, the results indicate that renal ischemia/reperfusion model is able to induce a transient cardiac hypertrophy in mice.
Moreover, this hypertrophy has a positive correlation with a transient increase of TLR2 mRNA levels, suggesting the possible
involvement of TLR2 cascade activation in cardiac hypertrophy development model. Is well known that TLRs play an important
role in cardiac hypertrophy models, probably through NF-kB or Akt/mTOR activation signaling. Herein, we showed an
experimental model that TLR2 can participate of cardiac hypertrophy process.
Keywords: Renal ischemia/reperfusion, Cardiac hypertrophy, Toll-like receptors
Financial Support: FAPESP, CNPq
Resumo:02-112
VENODILATATION INDUCED BY TERPY INVOLVES ACTIVATION OF SGC, K+ CHANNELS KV, SKCA AND
SARCOPLASMIC RETICULUM CA-ATPASE
Paulo, M. 1; Grando, M. D. 1; Rodrigues, G. J. 2; Dasilva, R. S. 1; Bendhack, L. M. 1
1
Physics and Chemistry, Faculty of Pharmaceutical Sciences , FCFRP-USP
2
Pharmacology,School of Medicine of Ribeirão Preto, FMPR-USP
Objectives:
Nitric oxide (NO) donors have important therapeutic effect for treatment of cardiovascular diseases such as angina pectoris and
hypertension. A major clinical benefit of NO donors is attributed to their venodilator effect, resulting in decreased venous return,
cardiac preload, blood pressure and myocardial oxygen demand. But the most common side effect of these drugs is headache,
which is caused by cerebral vasodilatation. Aim: This study aimed to verify the vasorelaxation induced by Terpy, in basilar artery
and cava vein from 2K (normotensive) and 2K-1C (hypertensive) rats. It was also evaluated the cellular mechanisms involved in
this relaxation.
Methods and Results:
The NO donor Terpy was synthesized in our laboratory. We have analyzed the maximal relaxation (Emax) and potency (pEC50)
of Terpy in cava vein and basilar artery pre-contracted with endothelin-1 (ET-1) after incubation with ODQ (1µM), guanylylcyclase inhibitor (sGC); thapsigargin (1μM) sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor; Rp-8-Br-PET-cGMP 3
µM G-kinase (GK) inhibitor; dipiridamole (3 µM) a phosphodiesterase-5 (PDE5) inhibitor; TEA (1mM) a non-selective K+
blocker and selective K+ blockers: apamin (small-conductance Ca2+-activated-SKca), 4-aminopyridine (voltage-dependent Kv)
and glibenclamide (ATP-sensitive KATP). We evaluated the NO released from Terpy by using confocal microscopy. Vascular
reactivity experiments showed that Terpy did not induce relaxation of the basilar artery from 2K and 2K-1C rats.Terpy did not
release NO in basilar artery. In cava vein, Terpy released NO and induced vascular relaxation. pEC50 and the Emax of Terpy
were lower in 2K-1C (pEC50: 5.42 ±0.22; Emax: 56.7 ±3.1%, n=7) than 2K vein ( pEC50: 6.10 ± 0.12; Emax: 70.3 ± 3.2% n=6,
p
Conclusions:
Taken together, our results demonstrate that Terpy does not release NO and it does not induce relaxation in basilar artery from 2K
and 2K1C rats. Terpy releases NO in cava vein and it induces relaxation in both 2K and 2K-1C rat veins, which was lower in 2K-
1C than in 2K. The venodilatation induced by Terpy involves activation of sGC, K+ channels Kv and SKCa and SERCA.
Keywords: Hypertension, Nitric oxide donor, Potassium channels, SERCA, Venodilatation
Financial Support: FAPESP and CNPq
Resumo:02-113
TREATMENT FOR 15 DAYS WITH TRIBUTYLTIN DECREASE THE VASCULAR REACTIVITY TO
PHENYLEPRINE IN ISOLATE AORTA RINGS OF FEMALE RATS
Rodrigues, S. M. L. ; Ximenes, C. F. ; Batista, P. R. D. ; Simões, F. V. ; Filho, V. S. D. ; Graceli, J. B. ;
Vassalo, D. V. ; Stefanon, I.
Departamento de ciências fisiológicas, UFES
Objectives:
Organotin compounds such as tributyltin (TBT) are used as antifouling paints for shipping companies and as pesticides in
agriculture. It can be trasnsmitted to aquatic organisms and humans by biotransference through the food chain. TBT may cause
endocrine dysfunction by the influence on female gonadal hormones inhibing the aromatase responsible to transforms
testosterone in estrogen. Our hypothesis is that TBT, by inhibiting the estrogen synthesis, could prejudice its cardiovascular
protection. This study aimed to investigate the effects of 15 days exposure to TBT (20 ng/ml, oral administration) on vascular
reactivity to phenylephrine (PHE) in isolated aorta rings of female rats.
Methods and Results:
Female Wistar rats (230-250 g) were divided in control group, treated with the vehicle (CT, n= 8) and the TBT group, treated
with TBT solution (20ng/ml) (TBT, n= 8) by gavage for 15 consecutive days. All the protocols were developed was approved by
the UFES Animals Ethical Committee (nº. 020/2009; vascular reactivity n°. 004/2007). In the end of treatment all animals were
anesthetized with pentobarbital (35mg/kg, i.p.) and the blood was collected for serum levels of 17 β-estradiol (E2) and
progesterone (P4). The thoracic aorta was removed and cutted in rings, immersed in 5 ml of Krebs solution at 37° C, pH 7.4. The
rings were submitted the rings to crescent concentration of phenyleprine (10-10 – 10-4 M). The same curve was held in the
absence of endothelium, in the presence of L-NAME (NOS blocker), tetraetilamônio-TEA (K+ channel blocker) and apocinina
(NADPH inhibitor). The data were expressed as mean ± SEM and analysed by Student's t test non-paired, significant for p < 0.05.
The treatment with TBT decreased levels of E2 (CT:47.2±7.1 vs TBT:32.2±4.3pg/mL, p< 0.05). TBT decreased the maximal
response to PHE (CT: 145.7 vs TBT: 122.4 ± 7.6, p < 0.05). In the abscense of endothelium the TBT group presented a greater
Rmáx than control (CT: 152.6 ± 8.3 vs TBT: 191.1 ± 26.5, p < 0.05). The L-NAME and TEA increment of reactivity was greater
in the TBT group (L-NAME/CT: 149.1 ± 9 vs. TBT: 157.8 ± 3.3, p < 0.05; TEA/CT: 158.1 ± 10.2 vs TBT: 162.5 ± 6.7, p <
0.05). The decrease in the reactivity to PHE during incubation with apocinina was more evident in the TBT group (TC: 70.1 ±
10.1 vs TBT: 59.13 ± 8.9, p < 0.05).
Conclusions:
The treatment of female rats with TBT for 15 days decreased the vascular reactivity to PHE probably for increase dependent on
the NO participation and the K+ channels.
Keywords: Female gonadal hormones, Nitric oxide synthase(NOS), Oxidative stress, Tributyltin, Vascular reactitivity
Financial Support: CAPES / CNPq / FAPES-FACTEC
Resumo:02-114
AUTONOMIC CHANGES AFTER RESISTANCE EXERCISE IN INDIVIDUALS WITH UNTREATED STAGE 1
HYPERTENSION.
Chrispino, T. C. ; Barbosa, T. P. C. ; Neves, F. J. ; Nóbrega, A. C. L.
Fisiologia e Farmacologia, UFF
Objectives:
Subjects with hypertension have impaired autonomic modulation, whereas aerobic exercise training improves this profile.
However, there is no clear evidence of autonomic modulation in response to resistance exercise. Our aim was to evaluate the
autonomic response after one bout of resistance exercise in patients with untreated stage 1 hypertension.
Methods and Results:
Six men with stage 1 hypertension (age 42±8 yrs; systolic blood pressure between 140 and 159 mmHg and/or diastolic blood
pressure between 90 and 99 mmHg) underwent a randomized and crossover study. All of the subjects fulfilled the following
criteria: age between 20 and 60 years, body mass index lower than 35 kg/m², not using medications, absence of any diagnosed
chronic disease, and sedentary. Subjects underwent medical examination and laboratory tests, one repetition maximum test.
Autonomic modulation was assessed by analysis of signals from a digital infrared photoplethysmographer (Finometer®) yielding
both time and frequency domain indices of heart rate variability and arterial baroreflex sensitivity by the alpha index. The
experimental protocol included two days: one with (intervention day) the other and without (control day) one 45-min session of
resistance exercise of 6 body movements composed of 3 series of 20 repetitions at 40% 1RM. Ambulatory blood pressure
monitoring was applied on both days. The protocol was approved by the Ethics Committee (CCM/HUAP 180/08). Systolic blood
pressure was lower after exercise (baseline: 145±6; 0h: 130±3 mmHg, p0.05).
Conclusions:
These findings suggest that individuals with stage 1 hypertension have lower parasympathetic modulation and baroreflex
sensitivity immediately after completion of resistance exercise. The reduced baroreflex sensitivity may be involved in the
mechanisms responsible for post-resistance exercise hypotension.
Keywords: Autonomic Nervous System, Hypertension, Resistance Exercise
Financial Support: Capes, CNPq, FAPERJ, FINEP, Labs D'Or.
Resumo:03-065
EFFECT OF SWIMMING TRAINING ON PLATELET AGGREGATION IN HYPERTENSIVE RATS
Cardoso, A. M. 1; Fiorin, F. S. 1; Dutra, E. J. M. 1; Martins, C. C. 1; Abdalla, F. H. 1; Pereira, L. B. 1;
Bagatini, M. D. 2; Stefanello, N. 1; Ferreira, R. 1; Schetinger, M. R. C. 1
1
Depto de Química/ Universidade Federal de Santa Maria, UFSM
2
Universidade Federal da Fronteira Sul , UFFS
Objectives:
Currently, hypertension affects more than 1 billion adults worldwide and 90–95% of these patients have essential hypertension. It
is well known that in animal models the inhibition of nitric oxide biosynthesis by administration of Nω-Nitro-L-arginine methyl
ester hydrochloride (L-NAME) leads to arterial hypertension. Spontaneous platelet aggregation is largely reported in
hypertension cases and its specific role in the pathophysiology of this disease must be considered since platelets can promote
thrombotic complications. In the other hand exercise training has been associated with several metabolic and cardiovascular
benefits and has been recommended for prevention as well as for treatment of cardiovascular diseases. Thus, the aim of this work
was to investigate the effect of swimming training on platelet aggregation in adult male rats with hypertension induced by LNAME administration.
Methods and Results:
The rats were maintained on a 12:12h light dark photoperiod and fed ad libtum. They were randomly divided in four groups. LNAME (n= 10): Rats were administered nitro-L-arginine methyl ester (30mg/kg/day) orally, by gavage, in the morning for 8
weeks and put on water for 5 min. Exercise L-NAME (n=10): was administered the same L-NAME dose as the first group and
the animals were submitted to swimming effects, from monday to friday, 1h/day, with gradual increase in workload up to it
reaches 5% of body weight in a adapted swimming system for rats with heated water to 31 ± 1ºC. Control (n=10): normotensive
and sedentary animals (put on water for 5 min). Exercise Control (n=10): normotensive animals submitted to an exercise effects.
The animals were euthanized and blood was withdrawn and used for the separation of platelet rich plasma. For platelet
aggregation, the Born (J. Physiol. 95: 168, 1963) technique was performed by turbidimetric measurement with a Chrono-log
optical aggregometer, with AGGRO/LINK® Model 810-CA software for Windows version 5.1. Aggregation was measured at
37°C and expressed as the maximal percent change in light transmittance from baseline at 5 min after the addition of the agonist
adenosine diphosphate (ADP) 10 μM, with platelet poor plasma as a reference. Data were analyzed using 2-way ANOVA,
considering p
Conclusions:
In this study, we observed that platelet aggregation is increased in platelets from L-NAME-treated rats, which can be a risk factor
for atherosclerosis development and thrombotic complications. However, we showed that swimming training has the ability to
modulate platelet aggregation, and, this way, can act as inhibitor of thrombus formation, possessing cardio protector effects that
could be considered as a non-pharmacological treatment for hypertension.
Keywords: Hypertension, Exercise training , L-NAME , Platelet aggregation
Financial Support: Sources of research support: CNPq
Resumo:03-066
INHIBITION OF BRAIN NITRIC OXIDE SYNTHESIS DECREASES RUNNING PERFORMANCE BY
ATTENUATING CUTANEOUS HEAT LOSS MECHANISMS
Lima, P. M. A. ; Coimbra, C. C.
Department of Physiology and Biophysics, ICB / UFMG
Objectives:
To asses the influence of central inhibition of nitric oxide synthesis on physical permorfance and thermoregulatory adjustments in
untrained rats during submaximal-intensity exercise on a treadmill until fatigue.
Methods and Results:
Either 2 μL of 1.43 μmol N-nitro-L-arginine methyl ester (L-NAME, n = 7) or 0.15 M NaCl solution (SAL, n = 7) were injected
into the right lateral cerebral ventricle of male Wistar rats (250-350 g) immediately before the animal was subjected to running
exercise (18 m.min-1, 5% inclination until fatigue). During exercise, tail temperature (Tt) was measured using a thermocouple
taped to the tail, and intraperitoneal temperature, an index of core temperature (Tc), was recorded using a telemetry sensor
implanted into the peritoneal cavity. Heat storage rate (HSR) was calculated. Rats injected with L-NAME showed a 43%
reduction in running performance (33.0 ± 4.5 min L-NAME vs. 57.7 ± 10.8 min SAL, p<0.05).
Conclusions:
Our data showed that central inhibition of the brain nitrergic system decreased the ability to dissipate heat, leading to decreased
physical performance during a submaximal-intensity exercise on the treadmill. We concluded that central nitric oxide
transmission modulates cutaneous heat dissipation.
Keywords: Exercise, Heat storage rate, Heat dissipation, Nitric oxide
Financial Support: CNPq, Capes and Fapemig
Resumo:03-067
LEUCINE SUPPLEMENTATION ENHANCES THE ADAPTATIONS OF ENDURANCE TRAINING ON SKELETAL
MUSCLE IN A GENETIC MODEL OF SYMPATHETIC HYPERACTIVITY-INDUCED HEART FAILURE
Moraes, W. M. A. M. D. 1; Souza, P. R. M. D. 3; Guimarães, F. D. 1; B. Jr. , C. R. 4; Zatz, M. 4; Negrão, C.
E. 1; Brum, P. C. 1; Medeiros, A. 2
1
School of Physical Education and Sport - USP, EEFE - USP
2
Department of Biosciences - UNIFESP , UNIFESP
3
Experimental Hypertension Laboratory , Heart Institute -USP
4
Human Genome Research Center , HGRC- USP
Objectives:
To investigate the effects of leucine supplementation associated with endurance training (ET) on function and morphology of
skeletal muscle in a genetic model of sympathetic hyperactivity-induced heart failure in mice, and whether these effects were
associated with activation of mTOR pathway.
Methods and Results:
Treatment consisted of 4-wk of ET on a motor treadmill, wich consisted in sessions of 60 min based on maximum lactate steady
state (6d/wk), and administration of leucine (1.35g/kg) or placebo (distilled water) intragastrically. We established five groups: a
control without heart failure (WT, n=12 and four groups of mice lacking both á2A and á2C adrenergic receptor subtypes (KO),
which were randomly divided into sedentary receiving placebo (KO, n=12) or leucine (KOL, n=12); trained receiving placebo
(KOT, n=12) or trained receiving leucine (KOLT, n=12). It was analyzed exercise capacity by graded treadmill exercise protocol
performed until exhaustion, cross-sectional area (CSA) by histochemical myosin ATPase, muscle function by ambulation and
grip tests, protein expression levels by western blotting, and mortality curve (logHank). KOT displayed increased exercise
capacity by 14% compared to test performed in the pre-experimental period, increased CSA in type I fibers in soleus muscle (10
and 11% compared to KO and KOL, respectively) and type IIA and IIB in plantaris muscle (13 and 15% compared to KO and
KOL, respectively), and improved performance in ambulation (13%) and grip tests (11%) vs KO and KOL. The leucine
supplementation alone showed no effect on muscle function or the phenotype of the fibers, but associated to ET improved CSA in
IIA and IIB fibers in plantaris muscle, and muscle function, at a rate greater than improve in KOT. The leucine supplementation
associated to ET was the only intervention able to improve survival. Additionally we found that these effects observed in KOLT
were associated with increased protein expression levels of phospho-mTOR:mTOR, phospho-p70S6K:p70S6K and a reduction of
phospho-AMPK:AMPK in 34, 47 and 30% compared to KOT, respectively.
Conclusions:
Supplementation of leucine enhances the effects of ET to improve exercise capacity, to preserve the mass and prevent muscle
disorders. These effects are specific to the glycolytic fibers and are associated with the activation of mTOR pathway and
improved survival.
Keywords: endurance training, leucine, heart failure, skeletal muscle
Financial Support: FAPESP
Resumo:03-068
LACK OF BETA2-ADRENOCEPTORS ACELLERATES SKELETAL MUSCLE ATROPHY IN ISCHEMIAINDUCED HEART FAILURE MICE.
Voltarelli, V. A. ; Bechara, L. R. G. ; Mattos, K. C. ; Bacurau, A. V. N. ; Brum, P. C.
Escola de Educação Física e Esporte da USP, EEFE-USP
Objectives:
Sympathetic hyperactivity is a hallmark of heart failure (HF) and it exerts a toxic effect on cardiac muscle. However, less is
known about the effects of sympathetic hyperactivity on skeletal muscle (SM) in HF. As β2-adrenoceptors mediate the effect of
sympathetic activity in SM being targets of circulating catecholamines, we evaluated SM structure and phenotype of mice with
target disruption of β2-adrenoceptor gene (β2KO) after ischemia-induced HF. Furthermore, we analyzed the expression of key
proteins involved in β2-adrenoceptor signaling related to SM hypertrophy and atrophy.
Methods and Results:
A cohort of male β2KO (KO) mice in a FVB genetic background and their wild-type (WT) controls were submitted to myocardial
infarction (MI) or SHAM surgery at 4 mo of age. After 30 days of surgeries, fractional shortening (FS) was determined by
echocardiography. Exercise tolerance was estimated at 30, 45, 75 and 90 days post-infarction using a graded treadmill exercise
test. Animals were killed at 90 days post-surgeries. Fiber typing, cross-sectional area (CSA) and capillary density were evaluated
by mATPase histochemistry in plantaris and soleus muscles. SM protein expression of Akt1, p-Akt1, Foxo3a, p-Foxo3a, MuRF1
and Atrogin1 was determined by Western Blotting in plantaris muscle. The animals were divided into four groups: CO SHAM,
CO MI, KO SHAM and KO MI. With a mortality rate of 56%, the MI was effective in reducing cardiac function (FS: 23,0 ±
2,3% CO MI vs. 39,0 ± 1,2% CO SHAM and 24,0 ± 3,5% KO MI vs. 38,0 ± 0,6% KO SHAM) and caused hypertrophy and
dilation of left ventricular in infarcted mice. Nineteendays post-surgery, exercise tolerance was significantly reduced in both MI
groups when compared with SHAM groups and this decrease was greater in KO mice (-44% KO MI vs. -32% CO MI). Likewise,
both CO MI and KO MI showed capillary rarefaction associated with decreased type I distribution and increase of glycolytic
fibers in plantaris and soleous. Nevertheless, only KO MI animals displayed reduced CSA of fiber types I, IIA, and IIB in
plantaris and I and IIA in soleous. Among the proteins that were quantified, MI increased the expression of Atrogin1 in both CO
and KO groups when compared to their respective control groups (142% of control for CO MI and 146% of control for KO MI),
but only KO MI mice presented increased expression of MuRF1 in plantaris (210% of control). There were no statistical
differences for Akt1, p-Akt1, Foxo3a and p-Foxo3a proteins between groups.
Conclusions:
Taken together, these results suggest that lack of β2-adrenoceptors accelerates skeletal muscle atrophy in HF, at least at the stage
of HF presently studied, and proteasome ubiquitin system seems to be associated with this finding.
Keywords: Heart failure, Skeletal muscle, Beta2-adrenoceptors
Financial Support: FAPESP
Resumo:03-069
AEROBIC EXERCISE TRAINING IMPROVES CARDIAC MITOCHONDRIAL FUNCTION AND REDOX
BALANCE STATUS IN RATS
Campos, J. C. 1; Queliconi, B. B. 2; Gomes, K. M. S. 1; Dourado, P. M. M. 3; Brum, P. C. 1; Kowaltowski,
A. J. 2; Ferreira, J. C. B. 1
1
Escola de Educação Física e Esporte, EEFE-USP
2
Instituto de Química, IQ-USP
3
Instituto do Coração, InCor-HCFMUSP
Objectives:
Over the last decades, exercise training has been widely recognized as an important and efficient strategy for improving quality of
life. Indeed, exercise training is also an important therapy for preventing and treating a variety of diseases. Resting bradycardia
and increased exercise tolerance are well-known markers of exercise training effectiveness. However, the underlying cellular
mechanisms by which exercise training improves aerobic capacity are not completely understood. In the present study, we aimed
to investigate the effect of eight weeks of aerobic exercise training on cardiac mitochondrial function and redox balance in rats.
Methods and Results:
Male Wistar rats (3 months of age) were randomly assigned into sedentary and trained groups. All rats performed a graded
treadmill exercise test until exhaustion before and after exercise training protocol. Aerobic exercise training consisted of 8 weeks
of running on a motor treadmill, 5 days/week, for 60 min at 60% of the maximal speed achieved in the graded treadmill exercise
test. The animal care and protocols were reviewed and approved by the Ethical Committee of Medical School of University of
São Paulo (2009/56). Trained rats displayed resting bradicardia (from 485±17 before, to 404±16 bpm after training protocol) an
improved running performance (from 451±48 before, to 835±59 meters after training protocol) when compared to sedentary
animals. Eight weeks of exercise training improved mitochondrial function, found by increased state 3 (100±12 vs. 205±58%)
and state 4 (100±13 vs. 166±13%) respiratory rates compared to sedentary animals. Indeed, mitochondria isolated from trained
hearts displayed increased ROS production under either complex I (100±11 vs. 260±62%) or complex III (100±6 vs. 249±69%)
inhibition by rotenone and antimycin A, respectively. Of interest, exercise training reduced ROS production in state 3 (100±15
vs. 64±14%) and state 4 (100±13 vs. 58±12%) respiratory rates when corrected by the total oxygen uptake. Furthermore, trained
rats increased calcium uptake (100±6 vs. 160±25%) and decreased inner membrane potential (100±1 vs. 84±4%) when compared
with sedentary group.
Conclusions:
The recognized effects of aerobic exercise training such as, like enhanced aerobic capacity and decreased resting heart rate, was
associated with improves in cardiac mitochondrial function, presenting increased state 3 and state 4 respiratory rates, maximal
calcium uptake and decreased ROS production in cardiac isolated mitochondria. Our findings support the hypothesis that
enhanced mitochondrial function by increasing respiratory control ratio, improving maximal calcium uptake and reducing ROS
production underlies, at least in part, the positive effects of aerobic exercise training. These findings reinforce the contribution of
exercise training as a non-pharmacological therapy for prevention and treatment of mitochondrial dysfunction-associated
diseases.
Keywords: Exercise training, Cardiac mitochondrial function, Redox Balance
Financial Support: This study was supported by FAPESP (#2009/12349-2, #2010/00028-4).
Resumo:03-070
FUNCTIONAL ASSESSMENT OF INDOOR UNDER-17 SOCCER ATHLETES BELONGING TO THE CITY TEAM
Romero, P. V. S. ; Peserico, C. S. ; Mezzaroba, P. V. ; Esteves, J. V. D. C. ; Andreato, L. V. ; Moraes, S. M.
F. ; Machado, F. A.
Ciências Fisiológicas / Universidade Estadual de Maringá, UEM
Objectives:
To evaluate and quantify the physical fitness along with its motor behavior of indoor soccer athletes from the sub-17 category.
Methods and Results:
The sample was composed of 14 indoor soccer athletes from the sub-17 category, 3 of them being goalkeepers, 2 fixed, 6 wings
and 3 pivots. It was used anthropometric measures of body mass, height and skin folds. To verify the body weight it was used a
Tanita Digital scale to measure the height, we used the stadiometer®. We calculated the body density using Jackson and Pollock
(1978) and for the determination of the fat % it was calculated using the Siri formula (1961). We evaluated parameters related to
the flexibility in sitting and to reach over Wells and Dillon (POLLOCK; WILMORE, 1993), abdominal strength in 1 minute
(AAHPER, 1976), power by the test of grip strength (JOHNSON, NELSON, 1979) and muscle strength in the horizontal
impulsion (JOHNSON;NELSON, 1979), in addition to the speeding tests of 30 meters (POPOV, 1986) and agility of SEMO
(JOHNSON, NELSON, 1979). Tests were carried out to race on a treadmill (Inbrasport, Porto Alegre, RS, Brazil) in airconditioned laboratory, with temperature maintained between 20 °C and 24 °C. The initial velocity was 7 km/h with increments
of 1 km/h every 3 minutes, and it kept and constant 1% inclination. The athletes were strongly encouraged to remain in the test
until the voluntary exhaustion. Ventilatory variables were determined by ergoespirometry, it was used a gas analyzer (Spirometer
VO2000, Inbrasport, Porto Alegre, Brazil), in addition to an electrocardiograph to record the heart rate through the program
ERGO PC (Micromed, Brasilia - Brazil). This study was approved by the Standing Committee on Ethics in Research Involving
Human Beings (COPEP), of the State University of Maringá, under the opinion no. 175/2007. The results were compatible with
the physical tasks required to this sport practice. The athletes were aged (years) 16.79 ± 0.97, height (cm) 175.22 ± 5.18 , BMI
(Kg/m2) 21.56 ± 1.46 . The anthropometric measurements and body composition indicated a low fat % (7.99 ± 3.32 ). The
flexibility of 32.96 ± 6.32 cm was classified as averaged, even though it is not the ideal physical condition required by the sport.
The results obtained for determining the motor suitability, presented: abdominal Strength 45.14 ± 10.45 , lower limbs
strength101.35 ± 40.13 , Speed of 30 meters (s) 4.77 ± 0.23 , Agility SEMO (s) 11.74 ± 0.53 , Horizontal Jump (cm) 220.75 ±
15.35 . The results that showed the cardiorespiratory fitness presented the average values of 59.68 ± 6.71 VO2max (ml/kg min),
193.1 ± 6.08 hrmax (bpm) and Vmax (km/h) 15.40 ± 1.17. These values are presented in avarage ± standard deviation (SD). The
normality of the data was confirmed by the Shapiro-wilk test using the SPSS® 13.0.
Conclusions:
The maringaenses athletes of the sub-17 futsal category showed a morphological profile which is compatible with data of athletes
from other studies involving teams of high-performance, making them suitable to carry out the work-tactical-physical technique
required for sports practice.
Keywords: Futsal, Physical fitness, Motor behavior
Financial Support: CNPq / FADEC.
Resumo:03-071
EFFECT OF THE AEROBIC PHYSICAL CONDITIONING LEVEL ON CARDIAC AUTONOMIC MODULATION
IN HEALTHY SUBJECTS
Dutra, S. G. V. ; Pereira, A. P. M. ; Mazon, J. H. ; Maida, K. D. ; Tezini, G. C. S. V. ; Souza, H. C. D.
Depto Biomecânica e Reabilitação / Universidade de São Paulo, FMRP/USP
Objectives:
The practice of physical exercises periodically promotes beneficial adaptations in the cardiovascular autonomic control, being
largely used for prevention and in association to applied therapy for control of chronic-degenerative diseases (Eur. J. Cardiovasc.
Prev. Rehabil. 14:237, 2007). However, although these adaptations have been widely investigated in several physiopathological
situations, studies are scanty on healthy subjects, mainly regarding the effect of physical conditioning level on cardiac autonomic
control. In this way, the objective of the present study was to investigate the relationship between level of physical conditioning
and cardiac autonomic modulation in healthy subjects.
Methods and Results:
Fifty-three healthy men (aged between 18-45 years old) were submitted to maximum ergoespirometric test (Ellestad) and then
divided into three groups: Sedentary (VO2peak = 22-38 ml.kg-1.min-1, SED, n = 18); Moderate Performance (VO2peak = 38-48
ml.kg-1.min-1, MP, n = 22); and High Performance (VO2peak > 48 ml.kg-1.min-1, HP, n = 13). Analysis of the heart rate variability
(HRV) was performed by using the autoregressive method at R-R intervals of electrocardiographic record (ECG) in three
moments during ergospirometric test: at rest (baseline), 3 (Rec3‘) and 6 minutes (Rec6‘) following the beginning of recovery.
Peripheral blood collection for analysis of lactate was performed at rest, during the peak, and 6 minutes after recovery (Rec6‘).
Data were presented as mean ± standard deviation and Kruskal-Wallis test was used for statistical test (p < 0.05). This study was
approved by the local ethics research committee under the protocol number 8381/2010. The levels of lactate at baseline and peak
moments were lower in group SED (1.9±0.5 and 6.4±1.0 mmol.L -1, respectively) when compared to the other groups (MP:
2.4±0.8 and 7.0±0.9 mmol.L-1; HP: 2.5±0.4 and 7.5±1.2 mmol.L-1). Nevertheless, the latter group (3.9±0.9 mmol.L-1) showed
lower levels of lactate at Rec6‘ compared to group MP (5.8±1.9 mmol.L -1), and this in relation to group SED (7.4±1.1mmol.L-1).
For HRV at rest, no differences were found in any of the spectral parameters. At Rec3‘, groups MP (Variance: 2.4±2.3ms 2; LF:
1.2±1.2ms2; HF: 0.4±0.5ms2) and HP (Variance: 1.9±1.9ms2; LF: 1.2±1.5ms2; HF: 0.6±0.9ms2) showed increases in variance as
well as in LF and HF oscillations compared to group SED (Variance: 0.7±0.7ms 2; LF: 0.3±0.5ms2; HF: 0.1±0.2ms2). At Rec6‘,
group HP (Variance: 4.3±3.5ms2; LF: 2.9±2.6ms2; HF: 1.8±2.6ms2) showed increases in variance as well as in LF and HF
oscillations compared to groups SED (Variance: 1.2±1.4ms 2; LF: 0.9±1.1ms2; HF: 0.2±0.1ms2) and MP (Variance: 2.8±3.1ms2;
LF: 2.4±2.6ms2; HF: 0.6±0.8ms2).
Conclusions:
The level of aerobic physical conditioning does not interfere with cardiac autonomic modulation at rest, but physically
conditioned subjects seem to present a better capacity to resume the baseline values for cardiac autonomic modulation.
Keywords: cardiac autonomic modulation, heart rate variability, physical conditioning, physical exercise, lactate
Financial Support: Source of research support: CAPES.
Resumo:03-072
LEVELS OF CIRCULATING PROGENITOR CELLS AND VASCULAR FUNCTION IN ENDURANCE ATHLETES.
Machado, R. C. ; Baldo, M. P. ; Roelke, L. H. ; Forechi, L. ; Verbeno, B. ; Vassallo, P. F. ; Mill, J. G.
DEPARTAMENTO DE CIÊNCIAS FISIOLÓGICAS, UFES
Objectives:
Recent evidences have shown that vascular function depends not only on resident cells in blood vessel wall but also seems to be
modulated by circulating progenitor cells 〈CPCs〉 derived from bone marrow. Physical training induces mobilization of either
CPCs and the subpopulation of endothelial progenitor cells 〈EPCs〉. Moreover, cell mobilization seems to improve the endothelial
function in presence of established vascular disease and ⁄ or cardiovascular risk factors. However, such effects in healthy subjects
are still poorly studied. Therefore, our aim was investigate the influence of long−term endurance exercise training on the different
subpopulations of CPC 〈CD34+, CD133+, CD34+CD133+ and CD34+KDR+ EPC〉 in amateur runners and healthy sedentary
subjects at rest, and evaluate the artery endothelial function by brachial flow−mediated dilatation 〈FMD〉 in this subjects.
Methods and Results:
The study population was consisted of participants from the Cardiovascular Health Study of Trained Man 〈ESCHOT〉.
Long−term runners 〈TA group, n= 34, ≥ 40km⁄week, for over two years〉, aged 23 to 50 years, were instructed not to exercise for
24 hours before blood collection. Healthy sedentary subjects were included as controls 〈CT group, n= 30〉. Adjustment was made
for age. Populations of CD34+, CD133+ cells and CD34+CD133+ subpopulations 〈CPCs〉, CD34+KDR+ 〈EPCs〉 were
quantified by flow cytometry in the peripheral blood and represented as percentage of positive events in selected mononuclear
cells in the lymphocyte population 〈% CMNL〉. The endothelial function was assessed by FMD of brachial artery using high
resolution vascular ultrasound. Data not normally distributed were compared by the Mann Whitney U test and Spearman′s
correlation coefficient 〈r〉 and expressed as median ± standard error of mean. Data following normal distribution were analyzed
by the Student t−test and the Pearson′s correlation coefficient, expressed as mean ± standard error of mean. P values < 0.05 were
considered significant. Adjustment for age was performed by a multivariate analysis. No statistical difference between groups
was found in the CPC counts: 〈CT: CD34+: 0.0381 ± 0.0036 %CMNL; CD133+: 0.0308 ± 0.0043 %CMNL; CD34+⁄CD133+:
0.0149 ± 0.0024 %CMNL. TA: CD34+: 0.0399 ± 0.0050 %CMNL; CD133+: 0.0194 ± 0.0058 %CMNL; CD34+⁄CD133+:
0.0118 ± 0.0037 %CMNL〉. Higher levels of EPCs 〈p<0.05〉 were observed in the TA group 〈CD34+KDR+: 0.0135 ± 0.0027
%CMNL〉 compared to CT group 〈0.0083 ± 0.0012 %CMNL〉. The FMD in the TA group 〈7.79 ± 0.59%〉 was similar to CT
〈7.90 ± 0.59%〉 with no correlation with EPC percentage.
Conclusions:
The long−term endurance exercise training leads to a recruitment of EPCs to the circulation. This finding was not correlated with
any changes in endothelial function evaluated by FMD, which could be explained by the fact that the studied population is
healthy. More research are needed to elucidate what are the underlying mechanisms involved in the mobilization of EPCs in
response of training in healthy individuals and its function in this context.
Keywords: endurance training, endothelial progenitor cells, endothelial function
Financial Support: CNPq, CAPES and FAPES.
Resumo:03-073
EARLY MODERATE RUNNING EXERCISE BLOCKED OBESITY INDUCED BY LITTER SIZE REDUCTION.
Ribeiro, T. A. D. S. 1; Tófolo, L. P. 2; Mendes, F. C. V. 2; Fabricio, G. S. 2; Malta, A. 2; Oliveira, J. C. D. 1;
Barella, L. F. 2; Martin, J. M. 1; Miranda, R. A. 2; Rinaldi, W. 1
1
Departament of Cellular Biology and Genetic , UEM
2
Departament of Physical Education , UEM
Objectives:
Nutritional insults during perinatal life can cause long-term changes in metabolism, which allow the development of obesity in
adult life. These alterations are associated to a disruption in the central nervous system that leads to an imbalance in the
autonomic nervous system. As the uterine phase, lactation is a sensible period to brain growth and exercise is a potent stimulus to
improve the metabolism. This work studies whether early moderate and low frequency exercise could be a deprogramming factor
in rats that become obese due to overnutrition during lactation.
Methods and Results:
After birth, the litter were divided into two groups: control group of nine pups (NL) and small litter group patterned with three
animals, this Animals were weaned at 21-day-old and were submitted to treadmill training during 70 days, 3 times/week (NLEXE and SL-EXE). The other two groups were formed by sedentary animals (NL-SED and SL-SED). Training was realized in
dark, always at 19h30min, with crescent time and speed (10-60 min and 10-20 m/min, respectively). At 90-day-old, rats were
submitted to a glucose tolerance test and the electrical activity of superior parasympathetic and sympathetic ganglion nerves were
recorded. Results: SL-rats were heavier (13%) and bigger (2%) than NL ones. The SL-rats had a fat tissue accretion of 2-fold.
SL-rats ate 12% more chow than NL-rats. Vagus electrical activity of SL-rats were 35% higher when compared to NL-rats, while,
sympathetic nerve of SL-rats showed 21% decrease in the electrical activity compared to NL-rats. Early exercise autonomic
nervous system in SL-rats.
Conclusions:
Early moderate running training deprograms the obesity induced by litter size reduction. Exercise decreased the fat pads,
improved the glucose intolerance and kept the balance of autonomic nervous system. Exercise reverted the imbalance of training
was able to improve the metabolism, increasing the glucose tolerance and block the obesity onset in SL-rats.
Keywords: MODERATE RUNNING EXERCISE , LITTER REDUCTION, ACTIVITY NERVOUS PARASYMPATHETIC,
ACTIVITY NERVOUS SYMPATHETIC
Financial Support: CNPq, CAPES and Fundação Araucária
Resumo:03-074
SKELETAL MUSCLE BLOOD FLOW AND OXIGENATION DURING MAXIMAL INCREMENTAL DYNAMIC
EXERCISE IN INDIVIDUALS WITH METABOLIC SYNDROME
Sales, A. R. K. ; Silva, B. M. ; Neves, F. J. D. ; Rocha, N. G. ; Medeiros, R. F. ; Pereira, F. D. S. ; Garcia,
V. P. ; Bertoldi, J. ; Manzzano, F. ; Nóbrega, A. C. L. D.
Fisiologia e Farmacologia/Federal Fluminense (Biomédico), UFF
Objectives:
Dynamic exercise has been used to assess integrative cardiovascular, respiratory and muscular function both in healthy
individuals and in patients with exercise intolerance. Individuals with metabolic syndrome (MS), even in the absence of overt
cardiopulmonary disease, have lower peak oxygen uptake (VO2peak) during maximal incremental dynamic exercise when
compared with healthy individuals. In addition, these individuals have endothelial and microvascular dysfunction. Therefore, it is
conceivable that one of the mechanisms leading to their low VO2peak is impairment in muscle blood flow and oxygenation
during exercise. Given this background, the aim of the present study was to compare skeletal muscle blood flow and oxygenation
during maximal incremental dynamic exercise among individuals with SM.
Methods and Results:
Seven individuals with SM (23.0 ± 2.0 years, BMI: 34.1±2.0) and 4 healthy controls (CT) (29.0 ± 3.0 years, BMI: 22.1±0.8) were
enrolled. The individuals with MS had at least three of the following five criteria: waist circumference ≥ 90 cm (man) or ≥ 80 cm
(woman); triglycerides≥ 150mg/dL; HDL cholesterol < 40 mg/dL (man) or < 50 cm (woman); systolic blood pressure ≥ 130
mmHg or diastolic blood pressure ≥ 85mmHg; fasting glucose ≥ 100mg/dL. The individuals of the CT group did not have any of
these criteria. The individuals performed a maximal incremental dynamic exercise test on an electronically braked cycle
ergometer. The protocol consisted on a linear increase of work (watts) every min with constant cycling rate (60 rpm). Ventilation
and expired gases were measured breath-by-breath and VO2peak was considered the highest oxygen uptake value obtained in the
last minute of exercise. Total hemoglobin (THb), an index of muscle blood, deoxyhemoglobin (HHb) and oxyhemoglobin
(HbO2) were measured in the vastus lateralis muscle (VLM) using near-infrared spectroscopy (NIRS). The NIRS probe was
positioned longitudinally on the belly of the VLM (~ 15cm above the patella) and secured with velcro straps around the thigh,
after the skin had been carefully shaved and cleaned. No movement of the probe was observed during exercise. This study was
approved by the Institutional Ethics Committee (013/2010). Individuals with SM had lower VO2peak than CT individuals (SM:
21.9 ± 1.8 vs. CT: 36.0±3.0 mL.Kg-1.min-1, p<0.05).
Conclusions:
Individuals with SM had impaired skeletal muscle blood flow and oxygenation during exercise, which may be partially
responsible for the reduced VO2peak during maximal incremental dynamic exercise in these individuals.
Keywords: dynamic exercise , endothelial dysfunction, metabolic syndrome , peak oxygen, skeletal muscle
Financial Support: CAPES, CNPq, FAPERJ, FINEP, LABS`DOR
Resumo:03-075
THE CONCENTRATION OF A CYSTEINE PROTEASE INHIBITOR INCREASES IN SALIVA FOLLOWING AN
ANAEROBIC POTENCY TEST
Sant'anna,m. L. 1,3; Casimiro-lopes,g2; Sorenson,m. M1; Salerno,vp2
1
Instituto de Bioquímica Médica, UFRJ
2
Escola de Educação Física e Desportos, UFRJ
3
Centro de Instrução Alte Sylvio de Camargo, CIASC
Objectives:
Physical exercise generates multiple physical and biochemical changes in the human body that can be monitored in various
manners. The measurement of these changes can be good indicators of workload, redox state and the state of the immune system.
However, the efficient evaluation of these changes is dependent on the variation measured and the approach applied. A need
exists for a rapid non-invasive approach to evaluate the impact of exercise on an individual. To evaluate the protein composition
of the saliva and identify proteins that display changes between rest and an anaerobic exercise (RAST – running anaerobic sprint
test) identification by mass spectrometry.
Methods and Results:
The procedures in this study were approved by the Ethics Committee of the Hospital Universitario Clementino Fraga Filho. The
subjects were military pentathlon team athletes from the Brazilian Navy. The team consisted of five men and two women (age
27.1 ± 2.3 years, weight 65.2 ± 2.6 kg, BMI 21.9 ± 0.3 kg/m2 and 10.1 ± 2.2% fat). The RAST test was applied with the athletes
performing six 35-m maximal sprints with a 10-second interval between each sprint. Saliva was collected from each team
member before and 5 min after the anaerobic test, then the saliva was centrifuged and the supernatant was stored at – 20° C. The
total protein concentration was determined by a Bradford assay. The concentration of protein in the saliva increased 2 fold
following the RAST (pre-test 0.62 ± 0.03 mg / mL; post-test 1.30 ± 0.08 mg / mL). The samples were normalized for total protein
concentration during sample preparation for SDS-PAGE and separated on an 4-18% gradient gel. Proteins in the gel were
visualized by Coomassie G and scanned for densitometry. Multiple bands displayed consistent differences between the before
and after test samples. A single protein band at 16 kDa displayed a large increase (202 ± 16%, n = 7) in intensity from samples
collected after RAST. The band was excised from a preparative gel, digested with trypsin and analyzed by mass spectrometry
(ESI-Q-TOF). Three isoforms of a cysteine protease inhibitor were identified: cystatin SN (16.5 kDa), cystatin AS (14 kDa) and
cystatin S (14.4 kDa).
Conclusions:
Monitoring of the saliva is a promising rapid non-invasive approach for evaluating the impact of exercise on individuals. The
saliva displayed multiple proteomic variations including three cysteine protease inhibitor isoforms whose concentration increased
following anaerobic exercise.
Keywords: exercise, protease, inhibitor, cystatin, RAST
Financial Support: FAPERJ, CNPq, CIASC, Unit for Mass Spectrometry and Proteomics (CCS-UFRJ)
Resumo:03-076
MAXIMAL POWER OUTPUT ESTIMATES THE MLSS INTENSITY IN CYCLE ERGOMETER BEFORE AND
AFTER AEROBIC TRAINING
Wilke, C. F. ; Ramos, G. P. ; Fonseca, T. R. ; Mortimer, L. Á. C. F. ; Lima, A. M. ; Barros, C. L. M. D. ;
Mendes, T. T. ; Silami-gracia, E.
Federal University of Minas Gerais, UFMG
Objectives:
The maximal blood lactate steady-state intensity (MLSS) is considered the gold standard method for determining lactate
threshold, aerobic capacity evaluation and training prescription. However, the method is expensive and demands practice and
knowledge to data collection and blood handling. Aim: To establish an equation to predict MLSS intensity through a validated
VO2max incremental test protocol in cycle ergometer.
Methods and Results:
Method: The study was approved by UFMG ethic comitee (ETIC 153/08). Twenty-six physically active men (age 24 ± 3y, body
mass 73.4±6.7kg, VO2max 46,9 ± 6,6 mL.kg-1.min-1) were randomly divided into two groups (G1 and G2, n=13). All subjects
performed an incremental test until fatigue to determine the maximum power output (WMAX) and VO2MAX (25W.2min-1)
cycling at 50rpm and two to five constant intensity tests lasting 30 minutes (CT) to determine MLSS. In CT, MLSS was
considered the greater intensity of 30min cycling without blood lactate increase (Δ0.05) (r=0.70; p0.05) (r=0.79; p<0.05).
Conclusions:
Conclusion: The proposed equation (WMFEL-EST = 0.866xWMAX+41.73) was able to estimate the MLSS power output before
and after six weeks of aerobic training. Therefore it would be possible to use only one test to obtain both measured VO2max and
estimated WMFEL in young, physically active individuals before and after an aerobic training period.
Keywords: estimation, lactate threshold, training
Financial Support: CNPq, FAPEMIG, CAPES , Ministério do Esporte, FINEP, FUNDEP/SANTANDER.
Resumo:03-077
ACUTE AND CHRONIC EFFECTS OF EXERCISE ON THE VASCULAR REACTIVITY TO ISCHEMIA IN
HEALTHY WOMEN
Barbosa, T. C. ; Pereira, F. S. ; Rocha, N. G. ; Medeiros, R. F. ; Sales, A. R. K. ; Silva, B. M. ; Neves, F. J. ;
Nobrega, A. C. L.
Depto. Fisio. Farmacologia/Universidade Federal Fluminense, UFF
Objectives:
The endothelium mediates the interplay between the blood and the vascular wall, synthesizing and releasing many vasoactive
substances, which are crucial for vascular biology, including smooth muscle tone regulation. One of the ways that has been
employed to evaluate the endothelial responsiveness is through the temporary arrest of the forearm circulation, followed by a
prompt restoration of blood flow. Such ischemic stimulus leads to a great and sudden increase in shear stress, which increases
nitric oxide production by up to 200% above resting values and provokes a large reactive hyperemia. Previous studies have
shown that the vascular reactivity to ischemia is acutely enhanced after a bout of exercise in healthy subjects. However, it is still
unknown whether the acute enhancement of vascular reactivity after a bout of exercise would be changed by exercise training.
Therefore, the aim of the present study was to investigate the acute and chronic effects of exercise on the vascular reactivity to
ischemia in healthy subjects.
Methods and Results:
Ten healthy, sedentary, non-obese and non-smokers women (age 28±9 years, BMI 23.2±3.6 kg⁄m2) were evaluated to investigate
the acute effect of exercise on the vascular reactivity. Their vascular reactivity was assessed at baseline, 10 and 60 min after an
acute bout of exercise (maximal exercise test) or a control resting period. Other 54 healthy, sedentary, non-obese and nonsmokers women (age 31±8 years, BMI 25.0±3.1 kg⁄m2) were evaluated to investigate the chronic effect of exercise on the
vascular reactivity at baseline, 10 and 60 min after an acute bout of exercise. Thirty-nine of these women were submitted to
exercise training while 15 remained sedentary. The vascular reactivity was considered the change in forearm vascular
conductance [forearm blood flow (measured by venous occlusion plethysmography) divided by mean blood pressure (measured
by auscultation)] provoked by 5 min of circulatory occlusion. The maximal exercise test was performed on a treadmill until
voluntary exhaustion. The exercise training consisted of aerobic and resistance exercises performed 3 times per week, during 12
weeks. Vascular reactivity increased and remained elevated until 10 min after the exercise test [baseline: 7.0±1.2 vs. 10 min:
9.9±0.9 mL⁄(100mL of tissue⋅min⋅mmHg), p0.05 vs. baseline]. There was no change in vascular reactivity during the control
session (p>0.05), in which no exercise was performed. Exercise training increased VO2peak [before: 30.0±0.8 vs. after: 35.0±1.0
mL⁄(kg⋅min), p0.05]. On the other hand, at 10 min after the exercise test the vascular reactivity was enhanced after exercise
training [before: 9.4±0.6 vs. after: 10.7±0.7 mL⁄(100mL of tissue⋅min⋅mmHg), p0.05).
Conclusions:
A single bout of exercise acutely increased the vascular reactivity to ischemia in healthy women, and this phenomenon was
enhanced after a period of exercise training.
Keywords: Acute effects, Aerobic training, Chronic effects, Maximal exercise test, Vascular reactivity
Financial Support: FAPERJ, CNPq, CAPES, FINEP
Resumo:03-078
ANTIOXIDANT RESPONSE IN SALIVA IN FIFTEEN MINUTES BICYCLE TEST
Viana Gomes, D; Rosa, F. L. ; Cardoso dos Santos, G. R. ; Navegantes, C. ; Ramos, K. ; Oliveira, L. T. ;
Real-hohn, A. H. ; Sanntanna, M. D. L. ; Salerno, V. P.
LABMOV/Universidade Federal do Rio de Janeiro, UFRJ
Objectives:
Exercise is known to increase energetic demands and the increased metabolic response can cause oxidative stress. Oxidative
stress can be characterized by an imbalance between the production of free radical and the buffering by antioxidants. Free radical
production has been observed to increase after acute exercise (Int J Dermatol. 40:747, 2001). The monitoring of free radical
production during exercise could be used to prevent a loss in athletic performance by avoiding systemic damage caused by free
radicals. To assess the body‘s response to free radicals produced during a bout of intense exercise, the levels of antioxidants were
measured in saliva, which is easy to collect and requires no invasive procedures. The aims of this study were to assess the use of
saliva for measurements and to determine the antioxidant response during an acute bout of high intensity exercise in healthy
subjects.
Methods and Results:
The cohort consisted of six, healthy men aged 22 ± 3.3 years. The high intensity exercise was consisted of cycling for 15 min
with a workload between 80 and 90% of maximal heart rate (HRmax). Saliva was collected before and after exercise. The levels
of lactate were measured in the rest and immediately after the activity using a lactimeter (Acctrend plus). The reduced glutathione
(GHS) and 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) in saliva were measured following the protocol described by Look (Eur J Clin
Nutr. 51:266, 1997) and Janaszewska (Scand J Clin Lab Invest.62:231, 2002), respectively. The heart rate was measured using a
frequency meter (Garmin Forerunner 305). All experimental procedures were approved by the ethics committee of the
Clementino Fraga Filho Hospital – UFRJ. The mean HR was 173 ± 5.5 bpm, which corresponded to 82% of the maximum HR
for this age group, indicating that the exercise was high-intensity exercise and was confirmed by the concentration of lactate in
the blood. The lactate concentration at rest was 1.6 ± 0.1 mM and immediately after exercise increased approximately 5 fold (9.2
± 3.3 mM), indicating a predominance anaerobic exercise. After the exercise, DPPH was decreased by approximately 13% (P >
0.05). The DPPH reduction could be observed because of an increase of antioxidants in the saliva. GHS, one of the main
antioxidants, increased around 10% in comparison with the rest (P = 0.005).
Conclusions:
The results indicate that this high intensity acute exercise was capable of inducing a change in the redox state in measured in the
saliva. While exercise increases the free radical production, healthy subjects are capable of buffering these reactive compounds
by increases in antioxidants system. Furthermore, the use of saliva is a viable system for measuring antioxidant capacity.
Keywords: Oxidative-stress, exercise, ANTIOXIDANT capacity, Free radical, Lactate
Financial Support: FAPERJ, CNPq
Resumo:03-079
EFFECT OF HIGH NUTRIENT AVAILABILITY ON REDOX BALANCE AND INSULIN RESISTANCE IN MUSCLE
CELLS
Sampaio, I. H. 1,2; Barbosa, M. R. 2; Silveira, L. R. 1,2
Escola de Educação Física e Esporte de Ribeirão Preto, EEFERP - USP
2
Bioquímica e Imunologia da Faculdade de Medicinada Rib Preto, FMRP - USP
1
Objectives:
Aim: We investigated the effect of elevated nutrient availability in the redox state of cells from skeletal muscle and relation with
insulin resistance.
Methods and Results:
Methods and Results: The skeletal muscle cells were isolated from quadriceps muscle from Wistar rats and cultured in DMEM
medium. After differentiation, the cells were exposed to different concentration of nutrients including glucose (5 mM), glucose
(10 mM), glucose (25 mM) and fatty acid (palmitic acid 700 µM) with or without insulin (10.000 µU/mL). The muscle cells were
also previously treated with N-Acetylcysteine (NAC) (10 mM) or electrically submitted to moderate muscle contraction (60 min).
The oxygen consumption was measured using a Clark-type electrode (Hansatech Instruments). The intracellular reactive oxygen
species (ROS) production, NAD+/NADH ratio and GSH/GSSG ratio were determined by fluorimetric assay. In addition, the
mRNA level of PGC1α was determined by PCR-Realtime and uptake of 2-desoxy-[2,6 3H]-glucose (0.1 µCi/mL) by
radioactivity assay (Scintillator Beckman-LS 5000TD). The oxygen consumption was significantly reduced at presence of
palmitate plus insulin (p<0.05).
Conclusions:
Conclusion: Our results showed that elevated nutrient availability reduces mitochondrial oxygen consumption favoring ROS
production and consequently insulin resistance. The effect of antioxidant and muscle cell contraction shifting the intracellular
redox state suggest that ROS are important regulator of glucose metabolism in skeletal muscle cells.
Keywords: High Nutrient, Redox Balance, Insulin Resistance, Muscle Cells
Financial Support: Support: FAPESP, PIBIC (USP) and CNPq.
Resumo:03-080
EVALUATION OF THE EFFECT OF MODERATE PHYSICAL TRAINING ON THE BEHAVIOR OF THE
VELOCITY OF CONDUCTION IN DIABETIC RATS
Silva, R. T. B. 1; Lira, K. D. S. 1,2; Figueiredo, T. G. 1; Maia, J. N. 1,3; Moraes, S. R. A. 1,2,3
1
Department of Anatomy , UFPE
2
Program of Post-Gradation in Physiotherapy , UFPE
3
Program of Post-Graduation in Neurosciences and Behavioral S, UFPE
Objectives:
This study investigated if a regular moderate aerobic physical training could influence the development of diabetic neuropathy
peripheral.
Methods and Results:
We used 18 male rats, albino, Wistar, with body weight ranging between 230-270g (animals were induced to diabetes). They
were separated into: Sedentary control group (SCG, n=4); Sedentary diabetic group (SDG, n=4); Trained control group (TCG,
n=5) and Trained diabetic group (TDG, n=5). After fasting of 12 hours at 60 days of life the animals of the diabetic groups were
induced to diabetes (streptozotocin solution). The trained groups were submitted to a protocol of moderate physical training,
swimming 5 days/week for 6 weeks, and duration from 10 minutes (initially) to 60 minutes (the end of the protocol). The sensory
nerve conduction velocity (SNCV) was checked at the beginning and end of the study. Weekly we checked body weight and
serum glucose of the animals. The independent samples were submitted to analysis of variance - ANOVA (one way) followed by
Bonferroni test, and the paired samples were submitted to Student t test. The results were expressed as mean ± standard deviation
with a confidence level of 95%. At the end of the experiment, there wasn‘t difference in SNCV between the experimental groups
(SCG = 43,22±2,50; SDG = 38,61±0,87; TCG = 40,14±3,95; TDG = 39,20±4,25), but there was a decrease in body weight of
diabetic groups (SDG = 216,00a±23,21 and TDG = 193,20b±41,25) in comparison to other two groups (SCG = 355,00a±55,73 e
TCG = 256,00b±57,57), and serum glucose of TDG (303,40c±86,70) in comparison with the SDG (369,50c±44,49), but this
reduction was not sufficient to normalize glucose levels.
Conclusions:
The results of this study suggest that exercise training may protect the peripheral nervous system by lowering blood glucose
levels, thereby reducing nervous exposition to high glucose levels.
Keywords: NERVE CONDUCTION VELOCITY, DIABETES MELLITUS, SWIMMING
Financial Support: CNPq/PIBIC/UFPE e CNPq/Universal nº 477096/2008-5.
Resumo:03-081
HYPOTENSION AFTER RESISTANCE EXERCISE OF DIFFERENT INTENSITIES IN HYPERTENSIVE WOMEN:
RESPONSE OF THE FOREARM BLOOD FLOW
4
Brito, A. F. 1,2,3,4; Santos, M. S. B. 1,2,3,4; Santos, A. D. C. 1,2,3,4
Programa Associado de mestrado em Educação Física UPE/UFPB, UPE/UFPB
2
Lab of Research in Physical Training, Performance and Health, LETFADS
3
University of Pernambuco/Escola Superior de Educação Física, UPE/ ESEF
1
Dept of Physical Education/ University Federal of Paraíba, DEF/ UFPB
Objectives:
The physiological responses promoted by resistance exercise minutes after their execution are controversial and have not been
sufficiently clarified, especially in hypertensive public, considering that it is treated by different drug classes. For this, we
evaluated the effect of one session of resistance training with different intensities in the post-exercise hypotension (PEH) and
forearm blood flow (FBF).
Methods and Results:
The study was conducted with ten women with hypertension (55 ± 3 years; 25.7 ± 3 kg/m2), who regularly participated in a
program of resistance exercise at the UFPB‘s gym. To participate in the study, they should be carrying only hypertension as
cardiometabolic disease and use only the drug class of inhibitors of angiotensin converting enzyme and diuretics. To determine
the sample size used the 3.1.0 software Gpower a statistical power of 0.80, an alpha error of 0.05, a difference of PEH for systolic
blood pressure of 2 mmHg and residual standard deviation of 2 mmHg. The women were subjected to three experimental
sessions: session control (SC), exercise 50% (EX50%), and 80% (EX80%) of 1 RM, ever held between seven and nine o'clock in
the morning. The order was determined randomly and individually so that each subject has its own order. Before the study, all
were instructed to avoid foods high in caffeine 12 hours before the collections and do not perform physical activities 48 hours
before the experimental sessions. For each session, subjects were assessed pre-and post-intervention. In the pre-intervention,
women were at rest in the supine position for 10 minutes and at the end of this was recorded BP (by Finometer) and FSA
(through the plethysmograph, Hokanson AG 201, Washington, USA). Later they were taken to the gym, where they remained for
about 20 minutes to the sessions of exercise (EX50% and EX80%) or remained at rest in each of the equipment during the same
time in the session SC. EX50% and EX80% were composed for a set of 10 repetitions in 10 exercises, with an interval of 90
seconds between exercises. Then, the women returned to the laboratory, were placed in the supine position for the FSA and BP
measurements were again performed at 10, 30, 50, 70 and 90 minutes of recovery (post-intervention). The PEH was more evident
in EX80% compared to EX50%, where its magnitude was -28mmHg versus -18mmHg for SBP, -13mmHg versus -8mmHg for
DBP and -27mmHg versus -18mmHg for MAP, respectively. As a result, the FSA has increased significantly in both sessions,
but this increase was most evident in the EX80% at 10, 30, 50, 70 and 90 minutes of recovery (4.97 ± 0.28, 4.73 ± 0.28, 4.88 ±
0.29; 4.81 ± 0.50; ml/min/100ml 4.80 ± 0.25) compared with EX50% in the same moments, respectively (4.22 ± 0.24, 4.10 ±
0.38; 4:36 ± 0.27, 4.28 ± 0.28, 4.10 ± 0.39 ml/min/100ml).
Conclusions:
Thus, we conclude that resistance exercise was effective in causing PEH, this phenomenon is accompanied by increases in FSA
within the first minute of recovery.
Keywords: hypotension, resistance exercise, blood pressure, hypertension, forearm blood flow
Resumo:03-082
CHRONIC ABSENCE OF TAIL ARTERY INNERVATION ATTENUATES CUTANEOUS HEAT LOSS AND
INCREASES BLOOD PRESSURE DURING PHYSICAL EXERCISE UNTIL FATIGUE.
Lima, M. R. M. 1; Pires, W. 1; Fonseca, I. A. T. 1; Wanner, S. P. 1,2; Coimbra, C. C. 1,2; Lima, N. R. V. 1
1
Department of Physical Education, UFMG
2
Department of Physiology and Biophysics, UFMG
Objectives:
Tail skin vasodilation is the principal mechanism of heat loss during physical exercise in rats, and cutaneous blood flow is
modulated by vasoconstrictor sympathetic activity. Denervation of tail artery acutely increases heat loss, a response that is
blocked after a few days as a consequence of morphological and functional adaptations of the tail vasculature. So far, there are no
published reports of the effects evoked by chronic absence of tail artery innervation on exercise-induced thermoregulatory and
cardiovascular responses. Therefore, the present study was aimed at investigating the effects of chronic absence of the tail artery
innervation on cutaneous heat loss and cardiovascular adjustments during physical exercise until fatigue.
Methods and Results:
All experimental procedures were approved by the Ethics Committee for Animal Experimentation of the Federal University of
Minas Gerais (protocol 109/09). Adult male Wistar rats were used in the experiments (210-240 g; at the time of first surgery).
Under anesthesia, the animals were submitted to tail artery denervation (TAD) or Sham-TAD surgery as control procedure. After
3 weeks of recovering, temperature sensors were implanted into the peritoneal cavity and polyethylene catheters were inserted
into the ascending aorta. Each rat had two days to recover and was then submitted to two experimental situations: (1) exercise on
a treadmill at 18m/min until fatigued (Sham-TAD: n=6 and TAD: n=10) or (2) resting on the treadmill belt (Sham-TAD and
TAD: n=5). Both experimental situations were performed at the same time of day with an interval of two days. During the
experiments, core body temperature (Tb), tail skin temperature (Ttail), mean arterial pressure (MAP), and heart rate (HR) were
measured. Glyoxylic acid-induced fluorescence was used to confirm the effectiveness of denervation. Data are expressed as
means ± S.E.M. Differences between groups were assessed by a multiple factor analysis of variance followed by the least
significant difference test. Paired Student‘s t-test was used to compare physical performance. Significance level: P < 0.05. In
resting rats, TAD did not change the thermoregulatory and cardiovascular parameters. In contrast, TAD attenuated the exerciseinduced increase in Ttail (Sham-TAD: 33.3 ± 0.6º C vs. TAD: 31.5 ± 0.4º C; fatigue point; p=0.03). Despite this change in
cutaneous heat loss, neither Tb nor time to fatigue was different between groups. Furthermore, TAD rats presented a greater
exercise-induced increase in MAP (Sham-TAD: 124 ± 3 mmHg vs. TAD: 133 ± 2 mmHg; fatigue point; p=0.03), although HR
was not different compared to Sham-TAD animals.
Conclusions:
Our results indicate that the chronic absence of tail artery innervation is determinant for cutaneous heat loss and cardiovascular
adjustments during physical exercise until fatigue. Furthermore, we suggest that the attenuation of heat loss in denervated-rats
may have resulted from morphological or/and functional adaptations in tail vasculature, and that the absence of reflex bradycardia
response was possibly due to changes in the baroreflex sensitivity.
Keywords: cardiovascular system, performance, thermoregulation
Financial Support: CNPq, CAPES, and FAPEMIG
Resumo:03-083
CHARACTERIZATION OF S-NITROSOGLUTATHIONE REDUCTASE IN SKELETAL MUSCLE AND
PHARMACOLOGICAL INHIBITORS
Yamashita, A. M. S. ; Mousinho, V. C. ; Figueiredo-freitas, C. ; Sorenson, M. M.
Instituto de Bioquímica Médica, UFRJ
Objectives:
Objectives: The purpose of this research is to characterize the expression and activity of GSNO-R in skeletal muscle and to
examine its role in the regulation of muscle contraction.
Methods and Results:
Methods: The enzymatic activity of GSNOR was measured at 340 nm by consumption of GSNO in homogenates of rat liver,
kidney, brain, heart, soleus (type I fibers) and extensor digitorum longus (EDL, type IIb fibers). Activity was normalized to total
protein concentration (Pierce BCA protein assay kit). Expression of GSNOR was characterized using electrophoresis followed by
Western blot with a polyclonal antibody. Results: The activity of GSNO-R was detected in all tissues tested. Activity found in
skeletal muscle was similar to that in other tissues such as heart and brain. The GSNO-R inhibitor C1 (3 - [1 - (4-acetylphenyl)-5phenyl-1H-pyrrole-2yl] propanoic acid) was tested at 30 µM, pre-incubated with the homogenate for 2 min. It inhibited GSNO-R
activity in all tissues.
Conclusions:
Conclusions: The enzyme GSNO-R has enzymatic activity in skeletal muscle similar to tissues such as heart and brain, with
minor variations depending on the major types of skeletal muscle fibers in soleus and EDL.
Keywords: glutathione, muscle, Nitric oxide, S-nitrosoglutathione reductase, S-nitrosylation
Financial Support: CNPq, FAPERJ
Resumo:03-084
MITOCHONDRIAL COMPLEXES ALTERATION IN ANIMALS EXERCISED AT HIGH INTENSITY INTERVAL
TRAINING
Ricardo, J. C. ; Martins, E. L; Ramos D. ; Casimiro-lopes, G; Jardim-messeder, D; Salerno, V. P; Sorenson,
M. M; Galina, A.
Universidade Federal do Rio de Janeiro, UFRJ
Objectives:
Previous studies suggested that the benefits of high intensity interval training (HIIT) compared with endurance exercises are
related to increased in expression of oxidative enzymes and mitochondrial biogenesis. In addition, rats subjective to HIIT showed
decreased insulin resistance and increased GLUT4 in cell membrane. However, analyses of mitochondrial physiology in
glycolytic and oxidative muscles were not described. The objective was evaluate the mitochondrial physiology in oxidative and
glicolitic muscles from animals subjective to HIIT.
Methods and Results:
One group of 5 male Wistar rats (Rattus norvegicus) was sedentary (S) and the other trained by swimming to non-exhaustion for
3 days for 2 weeks (T). The animals were sacrificed 48 hours after of the last training session. Soleus and tibialis anterior muscles
were isolated and the fibers were permeabilized with saponin to evaluate mitochondrial oxidative activity with different
substrates. There were no changes in physical capacity, in total body mass and in visceral fat mass. However, contrary to
expectations, there were observed a 1.5 fold decrease in the maximal oxygen consumption for both tibialis anterior (glycolytic)
and soleus (oxidative) muscles.
Conclusions:
Contrary to expectations proposed for HIIT we observed that the mitochondrial maximal respiration capacity in muscle is
decreased. This result suggests a muscle adaptation to anaerobic exercise.
Keywords: mitochondrial complexes , exercised , high intensity training
Financial Support: CNPq and FAPERJ
Resumo:03-085
EFFECTS OF CHRONIC ABSENCE OF ARTERIAL BARORECEPTORS ON BLOOD PRESSURE AND HEART
RATE VARIABILITY DURING PHYSICAL EXERCISE PERFORMED IN THE HEAT.
Pires, W. ; Lima, M. R. M. ; Wanner, S. P. ; Fonseca, I. A. ; Vaz, G. F. ; Fumega, U. ; Coimbra, C. C. ;
Lima, N. R. V.
Educação Física / Universidade Federal de Minas Gerais, UFMG
Objectives:
There is evidence showing that chronic absence of arterial baroreceptors disrupts the sympathetic-cardiovascular responses to
heat stress. The spectral power analysis of blood pressure (BPV) and heart rate (HRV) variability has been used to evaluate the
modulation of the autonomic nervous system during several physiological conditions, including the physical exercise. Thus, the
present study was aimed at investigating the effects of chronic absence of arterial baroreceptors on blood pressure and heart rate
variability during exercise performed in a warm environment.
Methods and Results:
Adult male Wistar rats weighing 250-350 g were used in all experiments. Animals were housed in individual cages under
controlled light (0500-1900 hours) and temperature conditions, with water and rat chow provided ad libitum. All experimental
procedures were approved by the Ethics Committee of the Federal University of Minas Gerais for the Care and Use of Laboratory
Animals (protocol 178/10). The animals were submitted to sinoaortic denervation (SAD; n=5) or sham denervation (SHAM; n=5)
as a control procedure. After two weeks, a polyethylene catheter was implanted into the ascending aorta, and a temperature sensor
was implanted into the peritoneal cavity. Rats were allowed to recover during two days and were then submitted to the exercise
trials on a treadmill at 18 m/min until fatigued. Each animal ran in both cool (25º C) and hot (35º C) conditions on separated
days. Pulsatile arterial pressure was recorded (sample rate = 2 KHz) by the aortic catheter and intraperitoneal temperature (Tb) by
telemetry. Power spectral density was obtained using the fast Fourier transformation method and Hanning windows (512) with
50% overlap. Spectral power components for very low- (VLF, < 0.05. During exercise in the heat, SHAM rats showed higher
BPV (LF; 11.4 ± 2.6 mmHg2 SHAM-Hot vs. 5.0 ± 0.3 mmHg2 SHAM-Cool; p<0.05).
Conclusions:
The effects evoked by the chronic absence of arterial baroreflex suggest that the mechanism plays an important role modulating
autonomic nervous system and core temperature adjustments induced by physical exercise performed in the heat.
Keywords: baroreceptors, blood pressure, exercise, spectral analysis, temperature
Financial Support: CAPES, CNPq and FAPEMIG.
Resumo:03-086
A NEW METHOD TO STUDY FATIGUE DURING EXERCISE: RAT WHEEL RUNNING DURING A
PROGRESSIVE DISTANCE/FOOD SYNCHRONIZED PROTOCOL.
Fonseca, I. A. T. 1; Araújo, F. D. A. 2; Lima, M. R. M. 1; Pires, W. 1; Young, R. J. 3; Rodrigues, L. O. C. 1
1
Department of Physical Education , EEFFTO/UFMG
2
Physiologic Science, ARFIS/UFU
3
Zoology, PUC/MG
Objectives:
In wild environment, animals exercise for food, reproduction and to escape from predators. In laboratory rats, exercise has only
been obtained by the unnatural electrical stimulation. The rat daily food achievement synchronized to an exercise bout has not
been investigated yet. Therefore, the aim of this study was to measure the rat wheel activity pattern during a progressive
distance/food ratio (DFR) daily protocol, to simulate a natural variability in food resources in the wild environment.
Methods and Results:
Five male Wistar rats at 6 weeks of age (228 ± 1.4 g, mean ± SEM) were housed individually in a metal cage (GAUSTEC
LTDA) (40 x 40 x 40 cm) with a metal wheel (diameter 30 cm, circumference 942 mm). They had free access to wheel, food and
water for 10 days (baseline wheel running situation). In baseline situation the spontaneous running activity was 1422 ± 484.9
m/day and the ad libitum food ingestion was 23 ± 0.7 g/day. Then the running wheel was connected to an electronic food
dispenser (distance/food situation) that released a food pellet (about 4.1 ± 0.1g) synchronized to the running distance. Within this
device, the animal must exercise to receive food. Initially, the first synchronized distance/food ratio (DFR in m/g) was the
average distance run and food intake in baseline situation (66 m/g). From this situation the DFR was arbitrarily increased
progressively (15% every three days for 72 days). The total distance run, average speed, delivered pellets were recorded each
hour during the whole experiment. Body mass and food intake were measured every day between 7 am to 11 am. The food intake
was calculated by the difference between delivered and eaten pellets. The animals were kept in a controlled light (14 - 10 h lightdark cycle) and temperature (24 - 26 ºC) environment. Data are expressed as means ± SEM. Running distance, food intake and
body mass were compared between situations using ANOVA followed by the Tukey‘s test. The correlation between rat running
distance and DFR was done using Pearson´s correlation coefficient. The significance level was set at p < 0.05. The rats mean
running distance during exercise synchronized with food dispenser situations increased progressively according to the increase in
DFR (114 ± 35.4 m/g in baseline situation to 309 ± 11.7 m/g in the last situation; p < 0.05). The animals increased their running
distance to obtain the daily food amount increasing their time spent in exercise for food. The average speed was not different
between the DFR situations but increased when the food was synchronized in the first situation in comparison to baseline (17 ±
2.1 vs. 27 ± 0.4 m/min; p< 0.05) and then there were no further differences.
Conclusions:
The new method created a rat voluntary exercise that seems to be closer to its natural activities in wild conditions.
Keywords: distance, fatigue, food, voluntary exercise, wheel running
Financial Support: FAPEMIG; CNPq; CAPES.
Resumo:03-087
THE EFFECT OF SIBERIAN GINSENG STANDARDIZED EXTRACT ON BIOCHEMICAL PARAMETERS,
WEIGHT AND PERFORMANCE IN SEDENTARY RATS TESTED FOR TREADMILL RUNNING.
Arouca, A. B. 1; Crege, D. R. X. D. O. 1,1; Ramos, L. A. F. 1; Ishizu, L. Y. 1; Faria, L. O. M. D. 2; Areas, M.
A. 2; Costa, K. G. D. 8; Silva, F. O. C. D. 8; Macedo, D. V. D. 9; Grassi-kassisse, D. M. 1
1
Depto. de Anatomia, Biologia Celular, Fisiologia e Biofísica, UNICAMP
2
Depto. de Anatomia, Biologia Celular, Fisiologia e Biofísica, UNICAMP
3
Depto de Anatomia, Biologia Celular, Fisiologia e Biofísica, UNICAMP
4
Depto de Anatomia, Biologia Celular, Fisiologia e Biofísica, UNICAMP
5
Depto de Anatomia, Biologia Celular, Fisiologia e Biofísica, UNICAMP
6
Depto de Anatomia, Biologia Celular, Fisiologia e Biofísica, UNICAMP
7
Depto. de Bioquímica, UNICAMP
8
Depto. de Bioquímica, UNICAMP
9
Depto. de Bioquímica, UNICAMP
10
Depto de Anatomia, Biologia Celular, Fisiologia e Biofísica, UNICAMP
Objectives:
This study aimed to examine whether Siberian ginseng standardized extract (Eleutherococcus senticosus) promotes effect on
weight, biochemical parameters such as glucose, lactate, triacylglycerol, cholesterol and performance in sedentary rats submitted
to an incremental treadmill Performance Test (PT).
Methods and Results:
Methods and materials: Adult male Wistar rats (n = 12) were divided into two groups (control, C and supplemented, S). Animals
were adapted to treadmill running test (10 m/min) for 15 days through 10 minutes/day. After this period, animals were submitted
to the first PT (Test 1): 3 minutes in a speed of 10m/min; after that, increase 1m/min every 2 minutes until speed 20m/min; after
that, increase 2m/min every 3 minutes until exhaustion. The S group received oral Siberian ginseng standardized extract diluted
in 1mL of 0.9% saline solution (100mg/kg, 5 days per week) while the rats in group C received only 1mL 0.9% saline solution
for 8 weeks. Weight and food intake were controlled. The groups were submitted to other two PT: after the fourth week (Test 2)
and after the eighth week (Test 3). Through a small incision at the tail, we analyzed the parameters of lactate in Accutrend
Performance® test strips (Roche) and blood glucose Active® test strips (Roche) before, immediately after and 5 minutes after the
end of PT, considering Test 1 and Test 3. The animals were sacrificed 48h after Test 3. Biochemical parameters of blood glucose,
triglyceride and cholesterol were analyzed before sacrifice by Accutrend Plus® strip (Roche) and Active® strip (Roche).
Performance analysis was evaluated by MATLAB program, in which rat mass and running time inside PT were considered and
the results were expressed as rat mass x meter (kg x m). One-way ANOVA and Tukey-Kramer test for multiple comparisons
were used for statistical analysis. Student t test was used for additional analysis (blood glucose, cholesterol and triacylglycerol).
Significantly results were considered when p 0.05). There were no significant differences in biochemical parameters like blood
glucose (C, 129.8±3.40 mg/dL), cholesterol (C, 158.0±2.30 mg/dL) and triacylglycerol (C, 210.5±21.66 mg/dL) (p>0.05).
Conclusions:
The present data indicated that the oral administration of Siberian ginseng standardized extract in sedentary rats was effective in
improving performance in a treadmill PT along 8 weeks even though no metabolic changes occurred.
Keywords: Performance, Siberian ginseng, Treadmill running, Wistar rats
Financial Support: CAPES
Resumo:03-088
DIFFERENT CHONIC EXERCISE PROGRAMS AFFECT THE BONE MINERAL CONTENT AND DENSITY IN
OVARIECTOMIZED FEMALE RATS
Fernandes, B. B. 1; Drummond, L. R. 1; Peluzio, M. C. G. 1; Del Carlo, R. J. 1; Silva, C. H. O. 1; Castro, C.
A. 1; Júnior, J. F. Q. 1; Ramos, R. M. S. 1; Lavorato, V. N. 1; Natali, A. J. 1
1
Depto. de Educação Física/Universidade Federal de Viçosa, UFV
2
Depto Nutrição e Saúde Humana/Universidade Federal de Viçosa, UFV
3
Depto de Medicina Veterinária/Universidade Federal de Viçosa, UFV
4
Depto. de Estatística/Universidade Federal de Viçosa, UFV
Objectives:
The deficiency secretion of estrogen in women in the post-menopausal state is related to bone mass loss. This study investigated
the influence of chronic treadmill running and swimming on the bone mineral content (BMC) and density (BMD) in
ovariectomized female rats.
Methods and Results:
Female Wistar rats (age: 20 wks; body weight: 271,42 ±17,6g) underwent surgical ovariectomy (OVX) or laparotomy (SHAM)
and were allocated to OVX running (OR), SHAM running (SR), OVX swimming (OS), swimming SHAM (SS), OVX control
(OCON), or SHAM control (SCON). Animals were housed in individual cages in a room with temperature of 22 ± 2ºC and
light/dark cycle of 12 hours. They received 20g of chow daily had water ad libitum. Animals in the running groups underwent a
program of treadmill running (16m/min, 60min/day, 0º grade, 5 days/week) for 10 weeks. Animals in the swimming groups
swam in a tank filled with water (30 ± 2 ºC) with an overload of up to 3% of body weight (60min/day, 5 days/week) for 10
weeks. Animals in OCON and SCON groups remained their cages without scheduled exercise for 10 weeks. After euthanasia the
right femur was removed, cut free of soft tissues for further. The BMD and BMC analysis were performed using DEXA
equipment. ANOVA three way was used to compare BMC and BMD among groups and a significance level of p< 0,05) body
mass than that of SHAM animals by the end of the experimental period. The SR group had higher BMD compared to SS (0.237 ±
0.015 g/cm2 vs. 0.219± 0.018 g/cm2, respectively). The animals in the OS group exhibited a higher BMD than those in the SS
group (0.241 ± 0.029 g/cm2 vs. 0.219 ± 0.018 g/cm2, respectively). The OR group showed higher values of BMC than OCON
group (0,308 ± 0,026 g vs. 0,272 ± 0,036 g, respectively).The OS group presented higher BMC as compared to OCON (0,301 ±
0,054 g vs. 0,272 ± 0,036 g, respectively). The values for BMC in the SCON group were higher than those in the OCON group
(0.297 ± 0.032 g vs. 0.272 ± 0.036 g, respectively).
Conclusions:
Chronic swimming increased the BMD and BMC in OVX female rats whereas chronic treadmill running increased the BMC in
OVX and the BMD in SHAM female rats.
Keywords: Physical activity, Menopause, Bone Health, Rats, Osteoporosis
Financial Support: FAPEMIG
Resumo:03-089
RESISTANCE TRAINING REGULATES IL-10 AND SREBP-1C EXPRESSION, SIZE AND WEIGHT OF
MESENTERIC FAT MASS IN OVARIECTOMIZED RATS
Stotzer, U. S. ; Duarte, F. O. ; Gatto, C. D. V. G. ; Silva, G. H. G. D. ; Domingues, M. M. ; Rodrigues, M.
F. C. ; Perez, S. E. A. ; Araújo, H. S. S. D.
Ciências Fisiológicas/ Universidade Federal São Carlos, UFSCar
Objectives:
The exercise has prophylactic effect upon the pathogenesis triggered by chronic inflammation. This benefic effect seems to be, in
part, result of the reduction of visceral fat promoted by regular exercise and/or an induction of anti-inflammatory cytokines after
each exercise session. Visceral fat mass is an important source of interleukin 10 (IL-10), an anti-inflammatory cytokine which is
increased in ovariectomized rats. However, the anti-inflammatory role of resistance training is poorly explored and is unknown in
ovariectomized rats. Therefore, we investigated the effect of both one bout and chronic resistance training on the IL-10 mRNA
levels in ovariectomized rats. In addition, we investigated the expression of SREBP-1C mRNA levels, a transcription factor
involved in lipogenesis and the size and weight of mesenteric fat mass.
Methods and Results:
Adult female Sprague-Dawley rats were randomly divided into six groups (n=6 per group): sham-sedentary (S-SED),
ovariectomized-sed (OVX-SED), S-trained (S-T), OVX-T, S-acute (S-A) and OVX-A groups. Sham or ovariectomy procedure
was performed at 10 weeks of age. Three weeks after the surgery, trained groups started 10 weeks of climbing in a 1.1-m vertical
ladder with progressive load of 65%, 85%, 95% and 100% of rat‘s previous maximal overload, plus 30g attached to their tails
until exhaustion. S and OVX acute groups performed one bout session. All animals were killed 92 days after ovariectomized.
Trained animals were killed 48 hours after the last training session. Immediately, mesenteric adipose tissue was weight and part
of this tissue was immediately frozen in liquid nitrogen for mRNA analyses and 100mg was prepared for morphological analyses
in colidina and osmium tetroxide. The IL-10 mRNA expression was significantly lower (64%) and SREBP-1C was higher (36%)
in OVX compared to sham groups (p
Conclusions:
These data highlight that resistance training can help in reducing the usual state of chronic inflammation in ovariectomy-induced
obesity by decreasing SREBP-1C expression and visceral fat area as well by increasing the gene expression of the antiinflammatory cytokine, IL-10. These results may be clinically relevant to the menopausal population.
Keywords: Estrogen, Exercise, Gene expression, Adipose cells
Financial Support: CNPq and CAPES
Resumo:03-090
INFLUENCE OF EXERCISE ORDER ON MUSCLE DAMAGE IN TRAINED MEN
Gattai, P. P. ; Ferreira, J. C. ; Wurtele, M. ; Araújo, R. C. ; Foschini, D.
Biofísica, UNIFESP
Objectives:
Analyze the effects of change in the usual order of strength training exercises on muscle damage, number of repetitions and
delayed onset muscle soreness (DOMS) in young trained adults.
Methods and Results:
We selected 10 healthy young adults (age= 22.14±2.67 years) with at least 6 months experience in strength training leading to
muscle hypertrophy (BMI= 25.62±2.88 kg⁄m²). Subjects were divided randomly into two groups: A control group (CG), which
performed the exercises in the usual order of training (Chest→Triceps) and an experimental group (EG), which performed the
exercises in the reverse order. The study design was initiated by a session of strength tests (triceps machine and bench press
machine) to determine the load of maximum voluntary contraction (MVC) in both exercises. The following week, the volunteers
performed eight sets of each exercise at 75% MVC. Subjects were allowed to pause for 1 minute between the bouts and for 3
minutes between exercises. We considered increases in plasma creatine kinase (CK) and a reduction in the number of repetitions
(RNR) between subsequent sets as an indicative of muscle damage. Muscle soreness was assessed using a subjective perception
of pain scale. 3 blood samples, the first 30 minutes before the training session, another immediately after the session and a third
24 hours after the same session were taken. For statistical analysis we used the Student t test and ANOVA. The CK levels
increased 24 hours after the training session only in the CG. The total volume of training conducted by the CG was higher than
that undertaken by EG. The RNR between sets was higher in the EG when compared to the CG for both exercises.
Conclusions:
After data analysis, we reach the conclusion that the execution order of strength training exercises influences the volume of work
performed as well as the muscle damage, but not the DOMS, and that furthermore the group that initiated with the smaller muscle
group exercise had a lower workload during the training session.
Keywords: Delayed onset muscle soreness, Exercise Order, Muscle damage, Strength training
Financial Support: Conselho Nacional de Desenvolvimento Científico e Tecnológico
Resumo:03-091
RECOVERY PROCESS OF THE ILEUM CONTRACTILE RESPONSE AND REDOX STATUS AFTER ONE
SESSION OF MODERATE EXERCISE IN C57BL/6 MICE
Mendonça, K. M. ; Berro, L. F. ; Martinez Jr, G. ; Alves, G. A. ; Silva, L. R. ; Ribeiro, R. F. ; Aboulafia, J. ;
Nouailhetas, V. L. A.
Biofísica, UNIFESP
Objectives:
Exercise is an important tool in prevention and treatment of chronic-degenerative diseases. Despite several evidence of the
beneficial effects of exercise, it has been shown that an acute session of moderate aerobic exercise causes increased production of
free radicals, which may induce oxidative stress, with consequent damage to many organs and tissues. The aim of this work was
to study the effects of an acute moderate aerobic exercise on the contractility and level of oxidative stress in the intestinal muscle
of C57BL/6 mice mainly by focusing on the recovery process.
Methods and Results:
Three-month old, male C57BL/6 mice were submitted to an acute session of treadmill running at 55-65% Vmax after 5-days of
acclimatation period. Animals were ascribed to the following groups: control (CT), exercised and killed immediately after the end
of the exercise session (EX), exercised and killed after 12, 24, 48, 72, 96 and 120 hours after the end of the exercise session
(REC-12, REC-24, REC-48, REC-72, REC-96 and REC-120, respectively). Data are presented as percentages in relation to
control group. We performed Student‘s t test and one-way ANOVA (followed by Bonferroni post-test) to compare groups. P <
0.05 was considered statistically significant. Ileum isometric contractions were recorded in the presence of Tyrode solution at
37°C, pH adjusted to 7.4, bubbled with air. Tissue responsiveness was evaluated determining the potency (EC50) and the efficacy
(Emax) from non-cumulative concentration-contractile response curves built up in response to either carbachol (CCh), bradykinin
(BK) or KCl-induced membrane depolarization. EC50 was determined as the concentration of the stimulant that induced 50% of
the maximum contraction (Emax). Emax values from KCl-induced contractions were significantly decreased in REC-12 (40%),
REC-24 (33%), REC-48 (33%), REC-72 (39%), REC-96 (42%), REC-120 (36%) relative to CT group. Also, Emax values from
CCh-induced contractions were significantly decreased in REC-12 (27%), REC-24 (29%), REC-48 (30%), REC-72 (48%), REC96 (52%), REC-120 (53%) relative to CT group. Finally, Emax values from BK-induced contractions were significantly
decreased only in REC-24 (54%), REC-96 (53%), REC-120 (48%) relative to CT group. Oxidative damage to whole ileum
proteins was spectrophotometrically quantified by determining tissue carbonyl content according to the method described by
Reznick and Packer (1994).and spectrophotometrically assessed at 370 nm. Protein content of the last pellet was evaluated
through Bradford (1976) technique. We observed a significantly greater carbonil quantity in REC-72 (56%) in comparison with
CT, and EX, REC-96 and REC-120 groups did not differ from the control group.
Conclusions:
It is shown that one acute session of moderate exercise impaired both the eletro- and pharmacomechanical couplings in C57Bl/6
intestine. Interestingly, even with a recovery time raging from 12 to 120 hours, this altered contractility is not recovered. The
increased level of protein oxidation was evident only 72 hours after the end of exercise session suggesting that ileum may suffer
oxidative stress, despite the moderate intensity of the exercise protocol. Shorter recovery periods should be analysed in order to
better characterize the recovery process.
Keywords: isometric contraction, moderate exercise, oxidative stress, protein oxidation
Financial Support: Fapesp
Resumo:03-092
PRIOR AEROBIC EXERCISE TRAINING PARTIALLY PREVENTS SCIATIC NERVE INJURY-INDUCED
SKELETAL MUSCLE DYSFUNCTION IN RATS
Gomes, K. M. S. ; Bechara, L. R. G. ; Vessoni, A. T. ; Campos, J. C. ; Moreira, J. B. N. ; Voltarelli, V. A. ;
Mattos, K. C. ; Monteiro, A. W. A. ; Brum. , P. C. ; Ferreira, J. C. B.
Escola de Educacao Fisica e do Esporte/ USP, EEFE-USP
Objectives:
Introduction and objective: Sciatic nerve injuries are known to quickly induce severe skeletal muscle atrophy and dysfunction,
while aerobic exercise training (AET) has been shown to improve skeletal muscle function in healthy and sick individuals.
Considering that potential protective effect of AET against sciatic nerve injuries have never been described before, we aim in the
present study to characterize the effects of previous AET on skeletal muscle mass and ex vivo function in rats underwent to
sciatic nerve injury (SNI).
Methods and Results:
Methods: Twenty-nine male Wistar rats were randomly assigned into the following groups: sedentary control submitted to fake
surgery (Sham; n=5), sedentary submitted to SNI (S-SNI; n=10) and aerobically trained submitted to SNI (T-SNI; n=14). It is
worth highlighting that AET was performed prior to SNI surgery. All rats performed a graded treadmill exercise test until
exhaustion before and after AET protocol. Moderate-intensity AET was performed during four weeks (60% of maximal running
speed; five days/week). Rats were killed 14 days after Sham or SNC surgery and EDL muscle was carefully harvested and
weighted. Ex vivo EDL muscle function was evaluated in an organ tissue bath equipped with a force transducer, where muscles
were mounted and electrically stimulated while incubated with Krebs‘ Heinselet solution. Supra-maximal stimulation current and
optimal muscle length were determined (1 Hz) with a constant resting period (60s) between subsequent contractions. Following a
10-min recovery period, force–frequency characteristics (10, 20, 30, 50, 80, 100 and 150Hz; 350 ms) were recorded. Peak
isometric force during electrical stimulation was registered. Data are presented as mean ± standard error from mean. Statistical
significance was considered achieved when P was
Conclusions:
Conclusion: Taken together, our data suggest that previous moderate-intensity AET reduces EDL muscle atrophy and contractile
dysfunction in rats submitted to SNI.
Keywords: AEROBIC EXERCISE TRAINING, SCIATIC NERVE INJURY, SKELETAL MUSCLE
Resumo:03-093
FAT PARTITIONING EFFECTS OF HIGH INTENSITY INTERVAL TRAINING IN DEXAMETHASONE TREATED
RATS.
Martins, E. L. 1; Marinho, G. M. 1; Souza, F. 1; Chicaybam, G. 2; Barreto, A. 2; Pires, L. 2; Ramos, D. M. 1,2;
Casimiro-lopes, G. 1,2
1
Instituto de Bioquímica Médica, UFRJ
2
Instituto de Nutrição, UERJ
Objectives:
High intensity interval training (HIIT) is a training method whose main characteristic is based on short periods of exercise bouts
with small rest intervals between sets completed in few minutes of duration when compared with traditional training protocols.
HIIT method is able to promote many health benefits, however this protocol is exhaustive and not adequate as a nonpharmacological therapy. However non-exhaustive bouts of HIIT also showed some results control rats suggesting that this could
be a promising strategy for health promotion. Dexamethasone (DEXA) treatment can induce insulin resistance, altered lipid
profile and other associated diseases in rats. The reproducibility of this model could be an adequate opportunity to evaluate the
effects of HIIT method in some alterations induced by DEXA. The objective of this study was to evaluate the effects of nonexhaustive bouts of HIIT in rats treated with dexamethasone.
Methods and Results:
Adult male Wistar rats (Rattus norvegicus) were divided in four groups: Sedentary (S; n=5), Exercised (EX; n=5),
Dexamethasone (D; n=5) and Dexamethasone exercised (D-Ex; n=5). The treatment with DEXA was done during eight days.
Following that period EX and D-Ex rats were subjected to three non-exhaustive bouts of HIIT protocol. The exercise sessions
were done in a swimming pool and consisted of 3 bouts of 20 seconds with 10 seconds of rest while bearing a weight equivalent
to 10% of their body weight. The training during 8 days alternated. The treatment with DEXA was maintained during the exercise
training and 48 hours after the last training session all the animals were sacrificed with a lethal dose of sodium thiopental (5mg/
100g). Statistical analysis was done with ANOVA one-way with Newman-Keuls post test, with p value set at 0.05. Both DEXA
treated rats (D and D-Ex) presented significantly lower total body mass (-34% and -26% respectively). Visceral fat mass (-61% in
both groups; p
Conclusions:
Non-exhausive bouts of HIIT protocol were efficient to reverse TAG accumulation in liver of DEXA treated rats. Longer periods
of training could offer additional benefits in another health parameters. Our results suggest that this king of training could be a
promising strategy as a non-pharmacological therapy in fat altering conditions as a less time consuming alternative.
Keywords: Exercise, Dexamethasone, Triacylglycerol in tissue
Financial Support: CNPq and FAPERJ
Resumo:03-094
ACUTE AEROBIC SWIMMING EXERCISE INCREASE NUCLEOTIDE CATABOLISM IN RAT BLOOD SERUM
Senger, M. R. 1,2; Pedrazza, E. L. 3; Oliveira, Á. R. D. 2; Bonan, C. D. 3
2
ESEF, UFRGS
1
Instituto Oswaldo Cruz, FIOCRUZ
3
Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS
Objectives:
Besides its consolidated role as an energetic molecule, ATP has many functions in extracellular space. At the cardiovascular
system this molecule and its catabolics, mainly ADP and adenosine, are involved in the blood coagulation and in the vascular
tone, functions that are strongly altered during physical activity. The sequential hydrolysis of ATP to adenosine it‘s realized by a
group of enzymes called nucleotidases. This group of enzymes is constituted by NTPDases, 5‘-nucleotidase and NPP. The
purpose of this investigation was to examine the effect of an acute swimming exercise session on nucleotide catabolism on rat
blood serum.
Methods and Results:
Male wistar rats were divides in two groups (n=6 each group): exercise (E) and sedentary (S). The group (E) the rats was
submitted to one swimming session (60 min) with a constant load of 4% of body weight in the tail. The rats of the (E) group
presented a significant increase (student t-test, P≥0.05) on the activity of the three enzymes tested when compared to (S) group.
The ATP and ADP hydrolysis were increased 55.3% and 43.1%, respectively. The 5‘-nucleotidase was activated 57.4%on the (E)
group. The NPP activity was activated 24.2%.
Conclusions:
Our results has shown that the ectonucleotidases are activated after one session of aerobic swimming exercise in rat blood serum,
suggesting that this enzymes are involved in an increased adenosine production and consequently an exercise-mediated
vasodilatation.
Keywords: Exercise, Nucleotidases, Serum, ATP, Adenosine
Financial Support: CAPES, FAPERJ, CNPq.
Resumo:03-095
ROLE OF NAD(P)H OXIDASE ON TENSION PRODUCTION CAPACITY AND FATIGABILITY OF ISOLATED
RAT SKELETAL MUSCLE
Vessoni, A. T. ; Bechara, L. R. G. ; Guimarães, F. L. ; Negrão, C. E. ; Brum, P. C. ; Ramires, P. R.
Escola de Educação Física e Esporte da Univ. de São Paulo, EEFEUSP
Objectives:
To investigate, in an in vitro model, the involvement of the enzymatic complex NAD(P)H oxidase on contractile function of
skeletal muscle tissue at rest and during contractile activity.
Methods and Results:
After being sacrificed by decapitation, the right and left soleus muscles of 7 male Wistar rats (14 month old, 485 ± 39g) were
carefully removed and attached to an isometric force transducer within an in vitro system containing aerated (95% O2, 5% CO2)
Krebs-Ringer solution (in mM: 137NaCl, 5KCl, 2CaCl2, 1KH2PO4, 1MgSO4, 24NaHCO3, 11C6H12O6; 22oC; pH 7.4).
Following a 30-minute stabilization period, in which individual optimal lengths for isometric contraction were determined (L0,
184.4 ± 24.3 mm), the muscles were electrically stimulated (0.2 ms and 80 v pulses) 3 times, with increasing train durations and
pulse frequencies (1 ms at 1 Hz; 300 ms at 30 Hz; 1200 ms at 100 Hz). Then, they randomly underwent one of two possible
pharmacological treatments: incubation with 1mM apocynin – a NAD(P)H oxidase inhibitor (APO, n=7) or distilled H2O (CON,
n=7) during 20 minutes, before being re-stimulated (as described above) to determine the effects of inhibition of enzymatic
complex NAD(P)H oxidase on the capability of skeletal muscle to generate isometric tension under basal conditions. The results
presented as the mean ± standard deviation of range (%) of tensions generated by each group before and after incubation were
compared using the Student‘s t-test with p < 0,05. After 30 minutes of incubation, the muscles were then submitted to a low
frequency fatigue-inducing protocol, in which they were stimulated during 8 minutes with 300 ms trains at 30 hz, separated by
900ms intervals, in order to determine the effects of inhibition of enzymatic complex NAD(P)H oxidase on the tissue fatigability.
The results presented as mean ± standard deviation of variation (%) of the initial tension generated by each group after 60
second-intervals were compared using a 2-factor (time, treatment) variance analysis (ANOVA) with p < 0.05. Under basal
conditions, apocynin-treated muscles showed a greater ability to generate isometric tension under submaximal electical
stimulations of 1ms at 1Hz (0.18 ± 0.08% vs. 0.09 ± 0.07%, p=0.05) and 300ms at 30Hz (0.17 ± 0.10% vs. 0.05 ± 0.08%, p =
0.03), when compared to H2O-treated group. However, when submitted to a maximal electrical stimulation (1200 ms at 100 Hz),
the isometric tension generated in both groups was not influenced by apocynin (0.01 ± 0.02% vs. 0.02 ± 0.04%, p=0.30).
Regarding fatigue, the muscles treated with apocynin showed greater losses of capacity to generate isometric tension after 6 (0.48
± 0.12% vs. 0.59 ± 0.08%, p < 0.05), 7 (0.42 ± 0.12% vs. 0.54 ± 0.10%, p < 0.05) and 8 (0.36 ± 0.11% vs. 0.50 ± 0.10%, p
Conclusions:
The inhibition of NAD(P)H oxidase by apocynin promoted an increase in the ability of isolated skeletal muscle to produce
submaximal isometric tension under rest, although the same treatment accelerated the fatigue process of the tissue when subjected
to an increased contractile activity.
Keywords: apocynin, fatigue, isometric force, skeletal muscle, oxidative stress
Financial Support: CNPq and FAPESP.
Resumo:03-096
EFFECTS OF WESTERN DIET AND MODERATE-INTENSITY EXERCISE ON THE COLONIC OXIDATIVE
STRESS.
Haddad, M. T. 1; Guimarães, C. B. D. 1; Papineli, R. 1; Ribeiro, R. F. 2; Nouailhetas, V. L. A. 2; Rosa, E. F.
1,2
1
2
Centro Universitário São Camilo, CUSC
Departamento de Biofísica/Universidade Federal de São Paulo, UNIFESP
Objectives:
A Western diet (WD), defined by high-fat, low-calcium and vitamin D content has been shown to induce colonic oxidative stress.
Recently we observed that moderate exercise practiced throughout life could prevent the increase of intestine oxidative stress.
Since these factors have been related to the development of colon cancer and the effects of moderate exercise and western diet on
the colonic redox status are poorly known, the aim of this study was to evaluate their impact on colonic oxidative stress.
Methods and Results:
Male C57BL/6J mice, 4 wk old, were ascribed into four groups, two of them fed with AIN-76A standard diet: sedentary (CTSED, n=4) and exercised (CT-EX, n=5); and two others fed with western diet: sedentary (WD-SED, n=5) and exercised (WDEX, n=10). Animals in WD-SED and WD-EX groups had access to the AIN-76A standard diet from weaning until the 4th week
of life, followed by free access to the western diet ad libitum, until animal death, while animals from CT-SED and CT-EX groups
had free access ad libitum to the standard AIN-76A diet. The amount of feed ingested by mice from WD-SED and WD-EX was
measured daily throughout the procedure, and body weight control was done at 7 days interval throughout the procedure. Body
weight from CT-EX group was measured before the onset of exercise. Animals from exercised groups were submitted to 5 days
of adaption which consisted of a daily session of treadmill running at 10m/min for 15 min. Animals from CT-EX and DI-EX
groups performed a moderate exercise protocol consisiting of treadmill running at 13m/min, which started at 12th week of the
animal life and terminated 48 hour before the animal death Sacrifice of the animals in all groups has occurred by cervical
dislocation at 16 weeks of life. Heart and gastrocnemius muscle from each animal were isolated, weighed, and expressed as g/g
body weight for determining the level of cardiac and gastrocnemius hypertrophy. The level of colon oxidative stress was
determinate by the level of protein oxidation, measuring carbonyl radicals by the technique of Reznick. Animals submitted to a
Western Diet presented a mortality rate around 37,5 %, during the treatment, while control animals mortality rate was around 1%.
Animal body weight from CT-EX group increased of 6.6% at the onset of moderate exercise, while the body weight in WD-SED
and WD-EX groups increased of 29,2% and 22,2%, respectively after the initiation of the diet. Food intake varied over the weeks.
Animals in WD-SED group start eating 10.3 ± 0.5 g/animal in the first week, and ended up ingesting 15.7 ± 0.0 g in the last
week; animals from WD-EX group ingested 11.0 ± 0.2 g/animal in the first week and 16.2 g in the last one. No influence of either
the moderate exercise or the western diet was detected in the level of cardiac and gastrocnemius hypertrophy and in the level of
carbonyl radicals formed.
Conclusions:
The Western diet increased the mortality rate of animals. However, Western Diet and physical exercise have no impact on
intestinal protein oxidation of colon.
Keywords: Intestine, Moderate-exercise, Oxidative-stress, Western Diet
Financial Support: FAPESP, Centro Universitário São Camilo
Resumo:04-025
EFFECT OF PH ON GLUCOSE AND ELECTROLYTE JEJUNAL ABSORPTION IN RATS
Viana, M. P. ; Borges, E. L.
Dpto de Biofísica e Fisiologia/Instituto Ciências Biológicas, ICB-UFMG
Objectives:
Aim: Studies have shown that glucose is able to decrease the pH of the surface epithelium jejunal preparations when added in
vitro and the existence of a high concentration of protons in the immediate area of the mucosa could be of considerable
significance for absorption of electrolytes (J. Nutr. 116: 768, 1986). The aim of this study is to assess whether the change in pH
of Tyrode (solution used for perfusion of the jejunum) interferes with the absorption of glucose and electrolytes.
Methods and Results:
Methods and Results: Male Wistar rats weighing 200 to 220g (n=6) were utilized. All experiments were carried out in compliance
with the local Ethical Principles in Animal Experimentation (CETEA/UFMG protocol no 230/2010). Jejunal absorption of
glucose and electrolytes was investigated in rats. A Tyrode solution containing twice glucose, sodium and potassium
concentration (pH 7.0, 7.4, 8.0 and 8.5) was infused through the jejunal loops during 40 minutes. The glucose absorption was not
significantly affected by Tyrode pH. However, there was significantly decrease in sodium absorption at pH 7.0 and 8.5 (41.13 ±
2.79 mM and 41.37 ± 1.71 mM, respectively; p<0.05).
Conclusions:
Conclusion: These data indicate that the pH of Tyrode has no influence on glucose absorption. However, the major potassium
uptake occurs at pH 8.0, while the absorption of sodium is impaired at pH 7.0 and 8.5.
Keywords: Absorption, Glucose, pH, Potassium, Sodium
Resumo:04-026
THE INFLUENCE OF CORTICOSTERONE IN THE DIFFERENTIATION OF MUCOUS NECK CELLS IN RAT
GASTRIC MUCOSA DURING EARLY WEANING
Zulian, J. G. ; Osaki, L. H. ; Rigonatti, C. A. M. ; Gama, P.
Instituto de Ciências Biomédicas, USP
Objectives:
Recent studies suggest that mucous neck cells (MNC) are markers of cellular differentiation in the gastric epithelium. Early
weaning (EW), that means the abrupt interruption of suckling, increases differentiation in the rat gastric mucosa, in parallel with
high corticosterone levels. This hormone can direct or indirectly regulate gastric growth and maturation. Thus, our aim was to
investigate whether corticosterone is involved in MNC differentiation.
Methods and Results:
Wistar rats were separated into four groups on the 15th postnatal day: suckling control (S), suckling treated with RU-486 (SRU),
early weaning (EW) and early weaning treated with RU-486 (EWRU). (Protocol approved by CEEA – ICB nº 86/08). In the EW
groups, the pups were separated from their dams and milk was substituted by semi-solid and solid diet. After the onset of early
weaning, pups were i.p. injected with vehicle (corn oil) or RU-486, an antagonist of glucocorticoid receptor. MNC population
was evaluated on day 18, in samples submitted to histochemical reactions with lectin Griffonia simplicifolia II (lectin GSII) and
also Periodic Acid-Schiff combined with Alcian Blue (PAS-AB). Early weaning increased the number of MNC evidenced by
PAS-AB compared to S group (P
Conclusions:
Our results showed that the blockage of corticosterone action by RU-486 administration impairs mucous neck cell differentiation
stimulus triggered by early weaning. We conclude that besides the dietary pattern, corticosterone also has a role in the control of
gastric cell differentiation.
Keywords: corticosterone, dietary pattern, differentiation, stomach, weaning
Financial Support: CAPES e FAPESP
Resumo:04-027
EFFECTS OF ANTI-CD3 ANTIBODY IN SALIVARY GLANDS OF SPONTANEOUSLY DIABETIC MICE
Metidieri, H. T. ; Mayoral, É. E. ; Rojas, F. A. ; Peroni, L. A. ; Mâncio, R. D. ; Picardi, P. K. ; Caldeira, E.
J.
Tissue Morphology Laboratory-Morphology and Basic Pathology, FMJ
Objectives:
Diabetes mellitus results in various complications, also compromising the salivary glands. The current treatment for this
condition should be a substituting method to exogenous insulin, which, in spite of considerable advances, is still associated to
constraints and lack of long-term effectiveness in relation the recovery of the tissue injuries. Thus, the aim of this study was to
evaluate the anti-CD3 antibody as alternative therapy in the recovery of salivary glands of spontaneously diabetic mice.
Methods and Results:
Ten spontaneously diabetic mice (NOD) were divided into two groups of 5 animals: group I (untreated diabetic mice) and group
II (anti-CD3/treated diabetic mice). The animals were kept under standard conditions of housing, feeding and treatment at the
Sector of Laboratory Animal Experimentation, Department of Morphology and Basic Pathology, Faculty of Medicine of Jundiaí,
FMJ. Group II received the anti-CD3 antibody (IMUNY BIOTECHNOLOGY) administered intravenously (5 µg/day) by the
period of 5 days. Group I received daily intravenous injections of saline to simulate the experimental conditions of treated group.
Glucose level was monitored during the treatment and salivary gland samples were collected at the end of experimental period for
morphometrical analysis. Treated animals consumed less liquid (ml) (0.85 ± 0.09 P≤ 0.05) and solid (g) (-0.48 ± 0.67 P≤ 0.05),
when compared with the animals of the group I (1.9 ± 0.45 and 0.63 ± 0.13 respectively). The weight (g) was increased in
animals of group II (+ 0.32 ± 0.22 P≤ 0.05). High levels of glucose (mg/dl) were observed in untreated animals, while in treated
animals, the reduction was observed (≤165.6 ± 29.87). Tissue restructuring (Statistical Results: 0.0209 P≤ 0.05) was observed in
animals submitted to immune therapy.
Conclusions:
The immune treatment promoted the recovery of salivary acinar cells, demonstrating that this immunoregulatory function of antiCD3 might be important for the reversal of hyperglycemia-induced tissue injury.
Keywords: salivary glands, structure, diabetes, treatment
Financial Support: NAPED and FAPESP (grant number: 2010/05455-8 and 2010/51619-2)
Resumo:04-028
PROTECTION OF PORTAL HYPERTENSIVE GASTROPATHY BY ESTROGEN IN MODEL PARTIAL PORTAL
VEIN LIGADURE (PPVL)
Morgan-martins, M. I. 1; Hartmann, R. M. 2; Schemitt, E. G. 1; Marroni, N. A. P. 1
1
Universidade Luterana do Brasil, ULBRA
2
Hospital de Clínicas de Porto Alegre, HCPA
Objectives:
Portal hypertension (PH) is a complication secondary to cirrhosis that is characterized by an increased blood flow and / or
vascular resistance in the portal system, causing the appearance of a collateral circulation hyperdynamic. The partial portal vein
ligation (PPVL) is the experimental model used in rats to study the pathophysiological mechanisms involved in pre-hepatic portal
hypertension. Estrogen is an antioxidant molecule with various physiological actions. The aim of this study was to evaluate the
antioxidant activity of endogenous estrogen in experimental rats PPVL compared with intact rats castrated.
Methods and Results:
We used 20 Wistar rats, weighing on average 250g were divided into four groups: sham-operated (SO); intact with partial portal
vein ligation (IL), castrated (C) and castrated with partial portal vein ligation (CL). On Day 1: castration or sham-operated, on the
7th day of surgery PPVL, the 15th day after the LPVP was observed portal pressure (PP) in the mesenteric vein of rats on the
polygraph Letica. Blood was withdrawn by retro-orbital plexus to measure the level of estradiol an diferents groups. Was
evaluated oxidative stress, were the lipid peroxidation (LPO) in the stomach was assessed using the technique of thiobarbituric
acid reactive substances (TBARS) and activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT). For
histological evaluation the sections of the stomach were stained with haematoxylin and eosin. Statistical analysis was ANOVA Student-Newman-Keuls, (Mean ± SE) were considered significant at p
Conclusions:
In this experimental model, we observed that estrogen a contributed to preserve the gastric tissue and portal pressure maintained
at normal levels. Possibly by stimulating nitric oxide synthesis and decreased production of vasoconstrictor agents and reactive
oxygen species, decreasing lipid peroxidation.
Keywords: oxidative stress, estrogen, portal hypertension
Financial Support: FIPE
Resumo:04-029
ACTIVITY OF DIGESTIVE ENZYMES FROM THE HEPATOPANCREAS OF LITOPENAEUS VANNAMEI
SUBMITTED TO DIFFERENT DIETARY LEVELS IN HETEROTROPHIC CULTURE
Nascimento Jr, C. R. 1; Silva, J. F. 1; Andrade, D. H. H. 1; Souza, K. S. 1; Melo, F. P. 2; Veríssimo, T. 2; de
Araújo, R. R. 2; Silva, J. F. X. 1; Correia, E. S. 2; Bezerra, R. S. 1
1
Universidade Federal de Pernambuco, UFPE
2
Universidade Federal Rural de Pernambuco, UFRPE
Objectives:
Carciniculture has been the main sector in the Brazilian production of aquatic organisms, especially in rearing Litopenaeus
vannamei. However, among the problems encountered by producers stand out spending with feed that is based on application of
fishmeal. In order to reducing costs and increase the shrimp production, various farming techniques are being researched such as
heterotrophic system that has shown promising results. Despite the success of this system, little is known of its effect on digestive
enzymes that have a significant influence on the determination of the nutritional requirements of animals.
Methods and Results:
the aim this work was to evaluate the activity of digestive enzymes of L. vannamei submitted to different dietary levels in
heterotrophic system. Specimens of L. vannamei weighing 1.10±0.23 g were reared in heterotrophic for a period of 12 weeks on
the Base de Pesca e Aquicultura (UFRPE). The food consisted of a commercial feed containing 35% crude protein and was
offered twice daily in the levels of 8% (control group T1), 6% (T2) and 4% (T3) of the total biomass of each tank. After the
cultivation period, were collected ten hepatopancreas of the shrimp of each treatment and homogenized in a solution of 0.01 M
Tris-HCl pH 8.0 and 0.15 M NaCl at a concentration of 40 mg tissue/mL and centrifuged at 10,000xg at 4 °C for 25 min to obtain
the enzyme extract. The proteolytic activities were determined using the nonspecific substrate (1% azocasein) and specific
(BApNA, SApNA e Leu-p-Nan). The electrophoretic patterns (SDS-PAGE) and protease zymogram were carried out with
separation gels at a concentration of 12.5%. The use of 1% azocasein showed a higher total proteolytic activity in T2
(5.2±0.4U.mg-1). It was found higher trypsin activity to T2 (25.1±0.1U.mg-1) and T3 (24.5±1.7U.mg-1) using BApNA as
substrate. Analyzing the enzymatic action of chymotrypsin through the use of SApNA, it was found that the greatest activity was
in T3 (0.44±0.05U.mg-1). Regarding the activity of leucine aminopeptidase in the presence of the Leu-p-Nan, enzymatic activity
was higher for T1 (1.14±0.09U.mg-1) and T3 (1.08±0.04U.mg-1). The electrophoretic profile of the enzymes was 15, 17 and 18
protein bands, for the treatments T1, T2 and T3, respectively, with molecular masses of between 7 and 200 kDa. The proteolytic
activities of these bands were demonstrated in zymograms and animals of treatments T1 and T3 showed 10 bands with activity,
while T2 presented 13. In all treatments, it was noted bands that showed activity below 29 kDa.
Conclusions:
According to the results, it was observed that the heterotrophic culture positively affected the activity of digestive enzymes,
suggesting an improvement in digestion and assimilation of nutrients by L. vannamei. Also, show the ability of shrimp to adapt to
different growing conditions, especially in reducing the food supply, enabling it to feed on microorganisms in the water.
Keywords: heterotrophic, protease, Litopenaeus vannamei
Financial Support: CNPq, SEAP/PR, FINEP/RECARCINE, FACEPE e EMBRAPA
Resumo:04-030
EFFECTS OF FOOD RESTRICTION ON THE MYENTERIC PLEXUS AND MORPHOLOGY OF ILEUM OF
WISTAR RATS IN AGING PROCESS
Cirilo, C. P. ; Rampazzo, A. P. D. S. ; Schoffen, J. P. F. ; Zapater, M. C. V. U. ; Vicentini, F. A. ; Natali, M.
R. M.
Dep. Ciências Morfológicas/ Universidade Estadual de Maringá, UEM
Objectives:
The myenteric plexus is composed of neurons responsible for integrating the motor activity of the gastrointestinal tract and by
glial cells that play important roles in the maintenance of the neuronal homeostasis and regulation of intestinal epithelial barrier.
The objective of this work was to evaluate the total neuronal population, the glial cells and the elements of the ileum wall of rats
in aging process submitted to food restriction.
Methods and Results:
For this study, male Wistar rats (Rattus norvegicus) were divided into 3 groups (n=5), C7 and C12, euthanized at 7 and 12
months of age, respectively, and RA12 that received 50% of the feed given to the C12 group for 5 months (from 7 months of
age), being killed at 12 months of age. After anesthesia of the animals were collected the blood for biochemical analysis, and
samples of ileum, for histological study of the intestinal wall and obtaining whole-mount preparations subjected to HuC/D and
S100 immunohistochemical techniques for total population neuronal and glial cells analysis, respectively. The quantification was
performed in 64 images/animal/technique (intermediate and antimesenteric regions) and measurements made at 100 cellular
bodies/animal/ technique. For histological analysis, samples of ileum were fixed in Bouin and embedded in paraffin (sectioned to
7-µm-thick) for the total wall, tunica mucosa and muscle analysis and in historesin (sectioned to 2.5-µm-thick) for evaluate the
villous, crypts, goblet cells and metaphase index. The sections were stained by the histochemical reaction of periodic acid-Schiff
(PAS) to evidence the goblet cells and using hematoxylin-eosin for the other analysis. The determination of metaphase index and
quantification of goblet cells were accomplished in 2500 cells/animal. Morphoquantitative analyses of myenteric plexus and
morphometry of ileal wall were accomplished using the software Image Pro-Plus 4.5. The data were submitted to analysis of
variance (ANOVA) and Tukey's post test with a significance level of 5%. Comparing the C7 and C12 groups was possible to
detect that the aging process didn‘t altered blood components analyzed, the number and cellular profile of HuC/D and S100
immunostained cells. Histological analysis showed a reduction of muscular layer and metaphase index (p
Conclusions:
The food restriction was beneficial for reducing cholesterol and triglycerides levels. However, we considered that the model of
food restriction caused morphoquantitatives alterations in the myenteric plexus and in the histology of the ileum, interfering
negatively in intestinal function.
Keywords: aging, enteric glia, food restriction, intestinal wall, myenteric plexus
Financial Support: CNPq, Fundação Araucária
Resumo:04-031
THE EFFECT OF 1,8-CINEOLE ON GASTRIC EMPTYING RATE AND ON THE CONTRACTILE BEHAVIOR OF
GASTRIC OR DUODENAL ISOLATED STRIPS OF RATS.
Veras, H. R. F. ; Jucá, D. M. ; Silva, M. T. B. ; Santos, A. A. ; Magalhães, P. J. C.
Departamento de Fisiologia e Farmacologia, UFC
Objectives:
1, 8-cineole is a monoterpene found in essential oils of plants found in the Northeast Brazil such as Croton nepetaefolius and
Eucalyptus tereticornis, which are commonly used in folk medicine for treating respiratory and gastrointestinal disorders. In this
study, we intended to evaluate the effects of 1,8-cineole on gastric emptying (GE) rate of a liquid test meal, as well as to
determine its effects on the contractile behavior of gastrointestinal smooth muscle in vitro.
Methods and Results:
Evaluation of the GE rate was performed using male Wistar rats (200-250g) that were divided in 3 groups: control (C; vehicle
0.2% Tween 80, p.o.) and 1,8-cineole-treated rats that received by gavage 10 (CIN10) or 100 mg/kg (CIN100). After 30 min, rats
received perorally a liquid test meal (Glucose 5%, Phenol Red 0.75 mg/ml). Ten minutes later, the animals were euthanized
following ethical guidelines, and visceral exeresis was performed to determine the fractional dye retention in rat stomach. The
amount of dye recovered in C group was 53 ± 2% (n = 8), values significantly higher (p < 0.05, ANOVA) than those observed in
CIN10 (44 ± 5%; n = 8) or CIN 100 (38 ± 4%; n = 8) groups. In another set of animals, isolated strips of gastric fundus or
duodenum were maintained in bath chambers containing Tyrode solution (resting tension = 1 g; pH 7.4; 37 °C, continuously
aerated). Recordings of the contractile activity were obtained through force transducers connected to data acquisition system
(Windaq, PM-1000, USA). Solutions of 1,8-cineole were prepared by adding Tween 80 (0.2% v/v). In a concentration-dependent
manner (p < 0.001, ANOVA), 1,8-cineole (0.1 to 3 mM) relaxed the basal tone of the strips of gastric fundus or duodenum with
EC50 of 1.09 [0.89 - 1.34] and 0.58 [0.25 -1.36] mM (n = 6), respectively. In preparations of gastrointestinal strips under Ca2+free conditions and in presence of EGTA (0.2 mM) and K+ (60 mM), 1,8-cineole inhibited the concentration-effect curve induced
by Ca2+ (0.1, 1 and 2mM) addition, reducing the contractile force induced by the addition of Ca2+ at 2 mM to 24.0 ± 3.4% of a
reference contraction induced by 60 mM K+, values significantly lower (p < 0.05, Paired t-test) than those obtained in control
preparations (80.0 ± 10.2%).
Conclusions:
1,8-cineole accelerates gastric emptying rate of liquids in rats and it possesses myorelaxant properties on isolated strips of gastric
fundus or duodenum. Such myorelaxant actions are putatively due to its ability in reducing Ca2+ influx through plasma
membrane in smooth muscle cells as previously reported (Clin Exp Pharmacol Physiol, 36(11):1120, 2009) and they are probably
involved in the 1,8-cineole ability to produce increased GE rate in rats.
Keywords: 1,8-cineole, gastric emptying, gastrointestinal smooth muscle, gastric fundus and duodenum, rats
Financial Support: CNPq
Resumo:04-032
THE SEROTONERGIC/OXYTOCINERGIC PATHWAY IS INVOLVED IN DECREASED GASTRIC EMPTYING
INDUCED BY ACUTE EXERCISE IN AWAKE RATS.
M. T. B da Silva1; W Okoba1; C. P. S. Campos 1; F. G. V. Oliveira1; A. D. N Pinheiro1; R. C. Palheta-junior
2
; A. A. dos Santos1
1
Department of Physiology and Pharmacology-UFC/Fortaleza-CE-, FISFAR
2
School of Veterinary Medicine-UNIVASF/Petrolina-PE,, CMVET-UNIVASF
Objectives:
In our previous publication, we showed that acute exercise exploiting the lactate threshold decreases gastric emptying of liquids
in rats. Thus, we decided to investigate the involvement of the serotonergic/oxytocinergic pathway on cardiovascular parameters
and gastric retention (GR) induced by acute exercise (AE) in rats.
Methods and Results:
After approval by the local ethics committee, N°82/10, 48 male Wistar rats (230-280g)were equally divided into respective
groups Sham (S), Exercise (E), Sham+Ondansetron (SO), Exercise+Ondansetron (EO), Sham+Atosiban (Sa) and
Exercise+Atosiban (Ea). Initially, all rats were subjected to a non-weight aquatic adaptation (a scaled 10 to 40min exercise, for 5
days). After 24 hours, they were anesthetized for the cannulation of femoral blood vessels and thereafter fasted for over 24 hours
with free access to an oral rehydration solution (ORS). On the day of experiment, all rats underwent an exercise session, EA
(swimming for 15-min, 5% BW) with the groups SO, EO, Sa and Ea pretreated (15min.) with (Ondansetron 50μg/kg or Atosiban
10μg/kg via iv). Rats in group S were placed in shallow waters over 5 min. Consequently, we monitored the MAP and HR
parameters over the next 10-min. Shortly afterwards, the animals were fed to a liquid test meal (SG 5% phenol red) to assess the
GR, 10-min postprandial (J Physiol, 131:452-62, 1956). Data was expressed as mean±SEM and compared by ANOVA and
Tukey test. Results: When compared to E, the S, Sa and Ea groups manifested hypotension (110.4 ±1.7, 106.26 ± 3.6 and
87.1±1.2 vs 121.7±1.9 mmHg, p <0.001).
Conclusions:
AE provoked adjustments in cardiovascular parameters, thus increasing GR of liquids. This was prevented by prior treatment of
the animals with Ondansetron and Atosiban, confirming the involvement of the serotonergic/oxytocinergic pathway in this
phenomenon.
Keywords: gastric retention, acute exercise, serotonin, oxytocin
Financial Support: CNPq, CAPES and FUNCAP
Resumo:04-033
HEMODYNAMIC, ENDOCRINE AND GASTRINTESTINAL ACTIVITY INDUCED FROM RIGHT ATRIAL
STRETCH IN AWAKE RATS.
Junior, R. C. P. 1; Pinheiro, A. D. N. 2; Okoba, W. 2; Oliveira, F. G. V. 2; Silva, M. T. B. 2; Elias, L. L. K. 3;
Antunes-rodrigues, J. 3; Santos, A. A. 2
1
Universidade do Vale do São Francisco, UNIVASF
2
Fisiologia e Farmacologia/Universidade Federal do Ceara, UFC
3
Faculdade de medicina de Ribeirão Preto, USP-RP
Objectives:
Isotonic hypovolemia increases the gastric retention (GR) of a liquid meal, the central venous pressure (CVP) and plasmatic
oxytocin (OT) levels (Neurogastroenteroly 11:93, 1999; Exp Neurol 206:192, 2007). Thus, we decided to determine GR, plasma
levels of OT, as well as hemodynamic responses due to mechanical atrial stretch (AS).
Methods and Results:
After approval by the mandated Research Ethics Committee 2009/02, male Albino rats, (250-280g, n=56), were anesthetized and
submitted to right auricle appendectomy or not (control) before performing the cannulation of the carotid artery and a via right
jugular vein cannulation up to the right atrium for recording hemodynamic parameters and executing mechanical atrial stretch,
respectively. (Pesquisa Médica 2:11, 2008). After 24-h, we continuously monitored the mean arterial pressure (MAP), central
venous pressure (CVP), heart rate (HR), as well as the cardiac output (CO) of all the rats which were subsequently distributed
into two groups randomly: rats submitted to mechanical stretching of the right atrium with a 50µL intra atrial balloon over 5-min
(AS) or with a deflated balloon (sham stretch) until the end of study. In a group of rats, we studied the effect of AS on GR, by
feeding the animals with a test meal (1.5 mL, 0.5mg.ml-1 of phenol red in 5% glucose solution) 15 min after initial monitoring.
After a 10 min post-prandial interval, the rats were sacrificed by a thiopental overdose for the determination of GR using the dye
dilution retention method (Adv Physiol Educ. 33: 343, 2009). We determined OT plasmatic levels by radioimmunoassay in AS,
as well as sham stretch groups. In another set of rats, we verified the effect of AS on baroreflex responsiveness where after one
minute of AS, we administrated Phenylefrine (FE, 10 ug.Kg-1) or Sodium Nitroprusside (SNP, 10 mg.Kg-1) AND subsequently
analyzed the ∆HR/∆MAP index. Data (mean ±SE) were analyzed using ANOVA followed by Student Newman-Keuls test,
considering values with p < 0.05 significant. When compared to the sham stretch group, AS promoted the increase of GR (54.1±
3.0 vs 69.9 ± 3.5%), CVP (1.2 ± 0.5 vs 5.0 ± 1.0 cmH2O), plasmatic levels of OT (3.6 ± 0.5 vs. 6.8 ± 0.5 pg.mL-1), as well as
tachycardia (371 ± 7 vs. 421± 22 b.p.m), but neither MAP (118.3 ± 3.0 vs. 119.9 ± 4.0 mmHg) nor values of CO (136.0 ± 6.8 vs.
151.6 ± 16.4 mL.min-1). Furthermore, we did not note significant alterations in the ∆HR/∆MAP index after FE (-5.0 ± 0.4 vs. 5.2 ± 0.4 beats.min-1.mmHg-1) neither after prior SNP treatment (-2.2±0.4 vs. -2.2±0.5 beats.min-1.mmHg-1). However, right
atrium appendectomy prevented the effects induce by AS on GR (52.5 ± 5.2 vs. 53.3 ± 4.6 %), on CVP levels (12.4 ± 3.2 vs. 14.2
± 3.0 cmH2O), OT plasmatic secretion (3.8 ± 0.7 vs. 4.4 ± 0.8 pg.mL-1) and on the reflexive tachycardia (475 ±14 vs. 501 ± 6
b.p.m). Therefore, the MAP levels in this group remained within stable levels up to the end of studies (107.6 ± 2.6 vs 109.4 ±
3.1mmHg).
Conclusions:
The increases in gastric retention, as well as the hemodynamic and hormonal alterations due to right atrial stretch are mediated by
a neuroendocrine mechanism dependent on the integrity of the right atrial appendix, but not on the responsiveness of arterial
baroreceptors.
Keywords: ATRIAL STRETCH , HEMODYNAMIC, ENDOCRINE, GASTRINTESTINAL ACTIVITY , OXCYTOCIN
Financial Support: CAPES, FUNCAP, FAPESP e CNPq.
Resumo:04-034
GASTROPROTECTIVE ACTIVITY OF RIPARIN II ON ETHANOL-INDUCED GASTRIC LESIONS IN MICE:
INVESTIGATION OF POSSIBLE MECHANISMS OF ACTION
Feitosa, M. L. 1; Vasconcelos, L. F. 1; Ocarvalh, A. M. R. D. 1; Rocha, N. F. M. 1; Dias, M. L. 1; Fernandes,
M. L. 1; Silva, M. I. G. 1; Filho, J. M. B. 2; Gutierrez, S. J. C. 3; Sousa, F. C. F. D. 1
1
Fisiologia e Farmacologia/Faculdade de Medicina, UFC
2
Laboratório de Tecnologia Farmacêutica. , UFPB
3
Departamento de Bioquimica e Farmacologia / Farmácia, UFPI
Objectives:
The gastric injury by ethanol is a model widely used to experimental evaluation of antiulcerogenic drugs. This model has
fundamental importance for scientific research concerning the fact that we can evaluate possible mechanisms by which
substances may act promoting gastroprotection. In this study, we decide to evaluate the gastroprotective effect of riparin II
against ethanol-induced lesions and verify the role of nitric oxide, ATP-dependent K+channel and prostaglandins in this action.
Methods and Results:
Male Swiss mice (20-30g) were divided in four groups: vehicle (3% Tween 80 in distilled water; p.o.), ripII-25 (riparin II 25
mg/kg; p.o.), ripII-50 (riparin II 50 mg/kg; p.o) and CYP (Cyproheptadine 10 mg/kg; p.o.), used as a gastroprotective reference
drug. One hour later, absolute ethanol (0.2 mL/animal) was administrated orally to all groups. Thirty minutes after the
administration of ethanol, the mice were killed and their stomachs were removed and opened along the greater curvature for
examination. The total and injured stomach areas were measured by computer program and expressed in terms of percent (%) of
ulcerated gastric area. To study the possible mechanism of action, separate experiments were conducted using the following
drugs: L-NAME, an inhibitor of the NO synthase activity (10 mg/kg; i.p.), glibenclamide, an antagonist of KATP+ channels (10
mg/kg; i.p.) and indomethacin, a nonselective cyclooxygenase inhibitor (10 mg/kg; p.o.). L-NAME and glibenclamide were
administered 30 min before animals receiving riparin II (50 mg/kg; p.o.), while indomethacin (10 mg/kg; p.o.) was administrated
2 h before the drug test. One hour later, absolute ethanol 0.2 mL was applied in each animal. Thirty minutes after the
administration of ethanol, the mice were killed and their stomachs were removed for examination as previously described. Data
are presented by mean ± S.E.M and analyzed by ANOVA followed by Student Newman Keuls as the post hoc test. RESULTS:
The administration of absolute ethanol produced lesions in the gastric mucosa (17.84±0.482), which were reduced in the animals
pretreated with ripII-25 mg/kg (8.675±0.816; p
Conclusions:
Our study conclude that riparin II has an gastroprotective effect and this effect appears to be involved, at least in part, with
endogenous NO and KATP+ channels opening.
Keywords: ulcer, ethaol, riparin II
Financial Support: CNPq/ CAPES
Resumo:04-035
EVALUATION OF GASTROPROTECTIVE ACTIVITY OF CHLOROGENIC ACID ON ETHANOL/HCL-INDUCED
ULCER MODEL
Shimoyama, A. T. ; Santin, J. R. ; Machado, I. D. ; Müller, G. G. ; Farsky, S. H. P.
Faculdade de Ciências Farmacêuticas/USP, FCF/USP
Objectives:
The pathophysiology of gastric ulcer has focused on an imbalance between aggressive and protective factors in the stomach, such
as acid secretion, pepsin, mucus secretion, blood flow, cellular regeneration, prostaglandins and epidermal growth factors.
Chlorogenic acid (CGA) is a class of substances contained in a vast amount of plants, food and beverages. It's known to have
antioxidant effects, anti-inflammatory, hypotensive and anti-diabetic, but the literature lacks information regarding this acid to be
effective as a gastroprotective agent. Thus, the aim of the present study was to evaluate the activity of CGA using ethanol/HClinduced ulcer model in mice.
Methods and Results:
Swiss male mice (n=5) were orally treated with CGA (5, 25 or 50 mg/kg), Omeprazole (30 mg/kg) or vehicle (control). One hour
later, gastric ulcer was induced by administration of ethanol/HCl (1.0mL of ethanol 60% + HCl 0.03M, gavage). One hour after
the ulcer induction, animals were anesthetized and sacrificed, and gastric ulcers were quantified by measuring the percentage of
the injured area. Nitric oxide (NO) was measured in stomach homogenate, by Griess reaction. CGA or omeprazole treatments
significantly decreased (p
Conclusions:
Results obtained suggest that CGA may be a gastroprotective agent in the ethanol/HCl-induced ulcer model, which supports
further experiments to investigate the mechanisms involved in this protective action.
Keywords: Chlorogenic Acid, Ethanol, Gastroprotective, Ulcer
Financial Support: FAPESP (2010/18069-9)
Resumo:04-036
EFFECTS OF PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR GAMMA (PPARGAMMA) PARTIAL
AGONIST LYSO-07 ON ETHANOL/HCL-INDUCED ULCER MODEL.
Santin, J. R. 1; Machado, I. D. 1; Shimoyama, A. T. 1; Müller, G. G. 1; Galdino, S. L. 2; Pitta, I. R. 2; Farsky,
S. H. P. 1
1
Faculdade de Ciências Farmacêuticas, FCF/USP
2
Universidade Federal de Pernambuco, UFPE
Objectives:
Gastric ulcer is characterized by lesion formation and gastric tissue necrosis. This process is also marked by intense neutrophil
migration which contributes to the damaging process. Recently, it has been proposed that of peroxisome proliferator activated
receptor (PPARs) agonists can be used as anti-ulcer agents. Thus, this study aimed to evaluate the effects of PPARγ partial
agonist Lyso-07 in ethanol/HCl-induced ulcer model.
Methods and Results:
Swiss male mice (n=5) were orally treated with Lyso-07 (5, 25 or 50 mg/kg), pioglitazone (40 mg/kg, control PPARγ),
omeprazole (30 mg/kg) or vehicle (control) one hour before inducing gastric ulcer by oral administration of the damaging agent
(0.5mL of ethanol 60% + HCl 0.03M). One hour later, animals were anesthetized, blood was collected by puncturing the
abdominal aorta and the femurs exudates were removed for total leukocyte counting, using a Neubauer chamber. The subtypes of
leukocytes in the blood and in the bone marrow were quantified in May-Grunwald stained blood smears using optical microscope
and by flow cytometry, respectively. The stomachs were removed to measure the percentage of the injured area. Lyso-07
treatments (25 or 50 mg/kg) significantly decreased (p
Conclusions:
Data herein obtained suggest that the Lyso-07 presented gastroprotective activity in the ethanol/HCl-induced ulcer. In addition,
Lyso-07 affect the neutrophil traffic from bone marrow into blood, which may contribute to the gastroprotective effect, inhibiting
the migration of inflammatory cells into the injured tissue.
Keywords: gastric ulcer, PPAR , neutrophil, ethanol, gastroprotective
Financial Support: Fapesp (2010/17175-0), CNPq and Capes.
Resumo:05-021
IDENTIFICATION AND CYTOCHEMISTRY ANALYSIS OF NUCLEAR ANNULUS OF BOVINE SPERM
Lemos, M. S. ; Moreira, P. F. S. ; Moraes, A. S. ; Beletti, M. E.
Área de Ciências Morfológicas-Univ. Federal de Uberlandia, UFU
Objectives:
The objective of this study was to isolate and identify the biochemical nature of the nuclear structure of the annulus, a component
of the nuclear matrix of bovine spermatozoa.
Methods and Results:
Semen samples were washed with PBS and subjected to ultracentrifugation in the concentration gradient of cesium chloride and
sucrose. The pellet obtained was treated with Triton X-100 at 10% for 24 hours and then treated with a solution of DTT
(dithiothreitol) 40 mM for 24 hours. After all steps several glass slides have been prepared for viewing by scanning electron
microscopy and for the testing cytochemistry. Three cytochemical stains were done: "Xylidine ponceau (XP) for protein, PAS
(periodic acid-Schiff staining) for neutral sugars and glycoproteins, and toluidine blue pH 4.0 for DNA. The first staining was
done with 0.1% XP solution placed on the blade by 15minutes; the PAS staining was performed with 0.5% periodic acid for 10
minutes and Schiff's reagent for 25 minutes and the third staining was done with 0.025% toluidine blue pH 4.0 for 15minutes.
After staining, the slides were examined under a light microscope coupled with a system for image capturing. Using the scanning
electron microscope, the nuclear annulus appeared as an elliptical ring with a membrane partially or completely occluding the
interior. Cytochemistrically the annulus was positive for XP and PAS staining, demonstrating to have a glycoprotein constitution.
The toluidine blue staining showed an elliptical ring with long filaments attached, demonstrating that DNA strands are firmly
adhered to the nuclear annulus.
Conclusions:
The study allowed the cytochemical characterization of nuclear annulus structure as a glycoprotein complex with attached DNA.
Keywords: nuclear annulus, bovine sperm, cytochemical analysis
Financial Support: CNPq and FAPEMIG
Resumo:05-022
COULD THE MATERNAL EXPOSURE TO ANTIDEPRESSIVE FLUOXETINE ALTER THE FERTILITY IN MALE
RAT OFFSPRINGS?
Vieira, M. L. ; Hamada, R. Y. ; Ferreira, P. C. L. ; Barbieri, M. ; Mesquita, S. F. P. ; Gerardin, D. C. C.
UNIVERSIDADE ESTADUAL DE LONDRINA, UEL
Objectives:
Evidence suggests that maternal depression during pregnancy is at least as common as postpartum depression. The use of
psychotropic drugs during these periods is in most cases essential. Fluoxetine (FLX), a selective serotonin (5−HT) reuptake
inhibitor drug, has been widely prescribed for depression during pregnancy and lactation. 5−HT hypothalamic concentration is
reduced in critical periods of brain masculinization on male rats, this probably happens to allow adequate testosterone action. In
this sense, levels of 5−HT changes could influence the hypothalamic sexual differentiation that occurs in the perinatal period. It is
known that 5−HT antagonizes testosterone masculinizing effects on size of sexually dimorphic hypothalamic nuclei and
gonadotropin release. In this way, the use of FLX by mothers could disrupt brain development resulting in reproductive
alterations in their progeny. The objective was to investigate possible changes on male offspring through reproductive
parameters, resulting from maternal exposure to FLX.
Methods and Results:
Wistar rats received FLX 7.5 mg⁄kg or saline solution (CT) 0.9% (by oral gavage) from gestational day one to postnatal day
(PND) 21. On PND 90, the following parameters were observed in male pups: body and reproductive organs (testis and
epididymis) weights, sperm counts in the testis and epididymis. The other testis was submitted to the usual histological routine
for analysis in light microscopy and morphometric analyses were realized. Data ± SEM were compared by Student´s t test with
Welch´s correction (∗p0.05).
Conclusions:
On basis of above observations, the results suggest that maternal exposure to FLX may have diminished endogenous testosterone
in male pups, which is crucial for brain sexual differentiation. This reduction has probably led to alteration on the
hypothalamus−pituitary−gonad axis in adulthood.
Keywords: ANTIDEPRESSIVE, ENDOGENOUS TESTOSTERONE, FERTILITY, MALE RAT, MATERNAL EXPOSURE
Financial Support: PIBIC/UEL.
Resumo:05-023
RAT SPERMATOGENESIS IN VITRO: GERM CELL DIFFERENTIATION IN A CO-CULTURE MODEL.
Pimenta, M. T. ; Jasiulionis, M. G. ; Porto, C. S. ; Lazari, M. F. M.
Department of Pharmacology - Experimental Endocrinology, UNIFESP - EPM
Objectives:
Spermatogenesis is a complex process that involves the interaction of germ and somatic cells. Mechanisms mediated by Sertoli
cells can regulate the proliferation, differentiation, and development of germ cells. The in vitro production of meiotic and
postmeiotic germ cells would indicate an optimal culture condition for survival and differentiation. However in vitro systems for
the study of mammalian spermatogenesis have been severely limited since co-cultures of testicular cells fail to allow
differentiation of germ cells. The aim of this study was to establish and characterize a new co-culture model to improve the
proliferation and differentiation of germ cells.
Methods and Results:
All experimental procedures were approved by the Ethical Committee from UNIFESP (CEP 0937/10). Primary co-culture was
obtained from 7-day old male Wistar rats, whose testes have higher percentage of gonocytes and type A spermatogonia. Testes
were removed, decapsulated and subjected to three-step enzymatic digestion under shaking conditions: the first using trypsin and
DNase; the second using glycine and DNase and the third using collagenase and DNase. The cells were cultured on Matrigel
(Becton Dickinson) covered dishes, in DMEM/F12 (phenol red-free) containing 0.02 g/L of gentamicin, for different periods (4,
7, 10 and 15 days), at 35ºC, 5% CO2. After several attempts, the best supplementation condition was obtained using 0.5% fetal
bovine serum (FBS). Proliferation and differentiation of the cells was analyzed by several methodologies: 1) histochemistry and
morphological analysis by optical microscopy; 2) immunocytochemistry using markers for undifferentiated (PLZF,
promyelocytic leukemia zinc-finger) and differentiated (c-KIT) germ cells; 3) Real time PCR with Sybr Green reagent (Applied
Biosystems) to determine the mRNA levels for specific markers of cell proliferation and differentiation (proliferating cell nuclear
antigen, Pcna, and histone H1-like protein in spermatids 1, Hils1); and 4) flow citometry using nucleic acid staining with
propidium iodide (PI) to evaluate cell cycle and the cell ploidy. Germ cells expressing PLZF and c-KIT could still be detected
after 7 days of culture, but morphological analysis revealed the presence of elongated spermatids already by culture day 5. The
quantitative PCR confirmed the increase of Pcna and Hils1 transcripts at specific stages during culture. Flow cytometry allowed
identification of three populations of cells, diploid, tetraploid and haploid, and the influence of medium supplementation on the
relative proportion of these populations. It was noticeable the appearance of additional hypofluorescent haploid peaks during
culture, reflecting different degrees of chromatin condensation.
Conclusions:
We developed a co-culture system of Sertoli and germ cells with appropriate conditions to allow cell proliferation and
differentiation. This model can now provide an important tool to investigate critical points in the regulation of proliferation and
differentiation of germ cells.
Keywords: co-culture, differentiation, germ cell, proliferation, spermatogenesis
Financial Support: FAPESP, CAPES, CNPq.
Resumo:05-024
RESIDUAL OIL FLY ASH (ROFA) EXPOSURE PROMOTES TESTICULAR OXIDATIVE STRESS IN WISTAR
RATS
Motta, G. A. ; Halmenschlager, G. ; Damiani, R. M. ; Piva, M. O. ; Rhoden, C. R. ; Rhoden, E. L.
Universidade Federal de Ciências da Saúde de Porto Alegre, UFCSPA
Objectives:
Epidemiologic studies have demonstrated increased human morbidity and mortality due to elevations in the concentration of
ambient air particulate matter (PM) (N Engl J Med 343:1742–1749, 2000). In addition, recent studies have shown that PM can
lead to male reproductive toxicity (Inhal Toxicol 19:275–281, 2007). Therefore, the aim of this study was to analyze the response
of testicular tissue of rats after a chronic exposure to different concentrations of ROFA.
Methods and Results:
Methods: A total of 35 male Wistar rats were divided into four distinct groups: G1 (Saline, 10µL; n=8), G2 (ROFA 50µg/10µL,
n=9), G3 (ROFA 250µg/10µL; n=9) and G4 (ROFA 500µg/10µL; n=9). They were exposed to ROFA during 90 days by
intranasal instillation. After this period, the animals were euthanized and the testicles were removed and frozen at -80C°.
Oxidative analyses were performed on testicles through the Thiobarbituric Acid Reactive Substances test (TBARS), which
measures malondialdehyde (MDA) concentration, and through the activity of the antioxidant enzymes catalase (CAT) and
Superoxide Dismutase (SOD). Data are demonstrated as mean ± standard deviation and were statistically analyzed by one-way
ANOVA, followed by Tukey post-hoc test (P
Conclusions:
The exposure of testicular tissue under different doses of ROFA promoted an increase of SOD activity, the first line of
antioxidant defense, but decreased CAT activity, causing testicular lipid peroxidation.
Keywords: exposure, oxidative stress, ROFA, testicles
Financial Support: CAPES, CNPq and UFCSPA
Resumo:05-025
TRIBUTILTIN AS A POTENTIAL RAT REPRODUCTIVE CICLE DISRUPTOR
Filho, V. S. D. 1; Lopes, P. F. I. 1; Podratz, P. L. 1; Costa, M. B. D. 2; Matsumoto, S. T. 2; Samoto, V. Y. 3;
Silva, I. V. 1; Takiya, C. M. 3; Graceli, J. B. 1
3
INSTITUTO DE CIÊNCIAS BIOMÉDICAS/UFRJ, UFRJ
1
DEPARTAMENTO DE MORFOLOGIA/UFES, UFES
2
DEPARTAMENTO DE BIOLOGIA/UFES, UFES
Objectives:
Organotin compounds (OT), such as tributiltin (TBT), have been widely used as biocides and disinfecting agents in circulating
industrial cooling waters, as well as antifouling paints for marine vessels. There are many reports of the biological effects of OT,
which vary in their toxic effects on eukaryotes. These OT are potent endocrine disrupters in gastropods, through the inhibition of
aromatase, changed reproductive parameters, and induced mollusks sterile by the development of a syndrome called imposex.
The importance of OT as environmental endocrine disrupters and their potential to adversely affect human health, was previously
demonstrated in several comprehensive studies of the reproductive toxicity of TBT in rodent models, however the toxic effects in
reproductive cycle not much is understood. We evaluated the potential toxicity of TBT as reproductive disruptor in female rats, as
impairer of regular reproductive cycling correlated with changes in sexual steroids.
Methods and Results:
Wistar female rats presenting normal estrus cycle (±230g, aged ±3 months, R. norgegicus) were randomly divided in: 1) Control
(CON, treated with vehicle by gavage, n=8); 2) Treated with TBT (TBT, 100ng/Kg, treated with TBT by gavage, n=8) . After 20
days of daily treatment, we analyzed the estrous cycle (EC), percentage (%) of cycling, phases of the estrous cycle, serum level of
17β-estraiol (E2), and progesterone (P4), weight of uterus, weight of ovaries, comet and micronucleus analysis in erythrocytes
and, micronucleus analysis in ovarian cells of hamster (CHO-K1) line. Also, the uterine and ovarian tissues were analyzed with
histological sections stained with hematoxylin and eosin. Results are presented as mean±SEM. One-way analysis of variance
(ANOVA) followed by unpaired Student t-test). Differences were assumed to be significant when p
Conclusions:
These data suggest that serum changes in female sexual hormones levels, induced by TBT , impairs cyclic reproductive function,
causes morphofunctional damage in reproductive organs and possesses a toxic potencial over female rats reproductive system.
Keywords: endocrine disrupter, estrous cycle, female reproductive organs, triorganotins
Financial Support: UFES e FAPES (nº 45446121/09)
Resumo:05-026
QUALITATIVE VALIDATION OF AN ARTISAN HOUSE FOR EXPOSURE OF RATS PARTICULATE MATTER.
Campos Junior, O. ; Dias, F. C. R. ; Melo, T. N. T. ; Neves, E. M .
departamento anatomia/universidade federal de pernambuco, UFPE
Objectives:
Several studies have linked the continued exposure to ambient levels of particulate matter (PM) and reduced life expectancy and
increased mortality and morbidity. The objective of this study was to develop and validate a craftsmanship exposure chamber,
where rats were exposed to dust from diesel (MP) via nebulization.
Methods and Results:
The analysis of the temperature, humidity and pressure of the chamber of handicraft exhibition were taken using a digital thermohygrometer and a monomer of 60psi/4Kgf/cm2. The powder used for diesel exposure, kindly provided by the Laboratory of
Experimental Air Pollution, University of São Paulo (IPL / USP), was diluted in distilled water at three different concentrations:
1μg, 10 mg and 50 mg. They used eight rats Rattus norvegicus albinos with 90 days of age were divided into four groups (n = 2):
Control Group (CG), Group 1 (G1) and Group 10 (G10) and Group 50 (G50), respectively exposed nebulization of 3 ml of
distilled water (GC), 1μg (G1), 10 mg (G10), 50 mg (G50) of diesel dust suspended in 3ml of distilled water. The four
experimental groups were subjected to the nebulizer for 1 hour daily for a period of 8 consecutive days. The animals were
weighed before exposure and 1 second after the last exposure (8th exposure). After the last day of exposure and weighing, the
animals were anesthetized (ketamine and xylazine) and lungs were collected and fixed by immersion in Bouin fixative. After
routine histological processing, the slides were examined in bright field microscope. The exposure chamber had a craft outlet
pressure of 5psi, the same business model presented by INALTEC PLUS 6600, which has an air flow of 8L/min and generation
of aerosols in the range 0.3 to 0.8 micrometers. Qualitative assessment of lung histology revealed an inflammatory process in
early G1, progressing to pictures of emphysema and hyperplasia in groups G10 and G50, respectively, with the presence of
particulate matter in the cluster lung parenchyma and macrophages.
Conclusions:
The qualitative assessment of the lungs of animals exposed to dust from diesel exposure chamber validates the craft as a system
of exposure to particulate matter.
Keywords: pollution , particulate matter, inhalation
Financial Support: National Institute for Integrated Analysis of Environmental Risk - INAIRA
Resumo:05-027
EFFECT OF AGENTS FOUND IN AIRPOLLUITION OF THE CITY OF RECIFE-PE ON THE IGS WISTAR RATS.
Melo, T. N. T. ; Dias, F. C. R. ; Campos Jr. O. . ; Lima Jr. M. J. M; Neves, E. M.
departamento anatomia/universidade federal de pernambuco, UFPE
Objectives:
With growing concern about the adverse effects of air pollution on health, developed a project that aims to find out if air pollution
is affecting the city of Recife the testicle, and thereby compromising the reproduction. In this context, we present biometric
parameters testis of adult rats subjected to air pollution.
Methods and Results:
Methods: Wistar male rats formed three groups: polluted F1 (F1, n = 10); polluted group F2 (F1, n = 13) and control (C, n = 6)
consisting of mice generated, breastfed and kept in the vivarium until euthanasia. Animals in groups F1 and F2 were kept in a
room of pollution (area = 4.8 m2; 14.500L volume) with constant flow of air polluted city of Recife, directed by a conduit with
PVC 100mm diameter. Rats with F1 were generated and maintained in room breastfed pollution with two objectives: 1)
Formation of the F1 group (n = 10), and 2) Playing for the formation of F2 (n = 13). All experimental animals received water and
food ad libitum and were subjected to a reversed cycle of daily 12h light and 12h dark, temperature between 20-240C, with
humidity between 45-75% and air exhaust system installed. At 90 days old mice of experimental groups (F1, F2 and C) were
weighed, anesthetized, fixed with Bouin, orchiectomized, and testes were weighed. After obtaining the body and testicular
weights, we calculated the gonadosomatic index (GSI): ratio between the testicular mass and body weight for all rats of three
experimental groups (F1, F2 and C).Statistical analysis used the Student t test. The level of statistical significance was considered
p ≤ 0.05. ts: The average body weights obtained at the end of exposure showed no statistical difference between experimental
groups (365 ± 44g = F1, F2 = 354 ± 30g, C = 369 ± 16g). The average testis weight was significantly greater in the F2 group,
when compared with groups C and F1 (F1 = 1.68 ± 0.09 g; GF2 = 1.78 ± 0.09 g C = 1.65 ± 0.10 g) .The GSI Group F2 was
significantly higher compared to the IGS group C (C = 0.89 ± 0.05%, F1 = 0.93 ± 0.11%, F2 = 1.01 ± 0.076%).
Conclusions:
In Recife, the average annual PM2, 5 was 11μg / m³, staying within the estimate by the WHO (25μg / m³) (17. International
Symposium on Undergraduate Research, University of São Paulo, 844, 2009), but through these results we suggest that the level
of air pollution in the city of Recife was enough to promote change in testis size, especially in the F2, F1 whose parents had been
created under the effect of air pollution.
Keywords: polluition, particulete matter, spermatogenesis
Financial Support: National Institute for Integrated Analysis of Environmental Risk - INAIRA
Resumo:05-028
EFFECT OF CHRONIC MALNUTRITION IN RAT VAS DEFERENS: MORPHOLOGYCAL AND BIOCHEMICAL
ANALYSIS
Bezerra, C. G. P. 1; Souza, A. M. 1; Muzi-filho, H. 1; Boldrini, L. C. 2; Takiya, C. M. 2; Nesi, R. T. 2;
Valença, S. S. 1; Einicker-lamas, M. 3; Vieyra, A. 3; Lara, L. S. 1; Cunha, V. M. N. 1
1
Programa de Farmacologia Celular e Molecular - ICB, UFRJ
2
Programa de Ciências Morfológicas - ICB, UFRJ
3
Instituto de Biofísica Carlos Chagas Filho, UFRJ
Objectives:
Malnutrition observed during pregnancy and the first days of life promotes adaptive changes related to a higher risk of disease in
adulthood. In this context, we previously demonstrated changes in calcium-dependent processes of rat vas deferens (RVD) that
seems to compromise male reproductive capacity. The aims of the present work were: 1-investigate the RVD architecture; 2evaluate the Ca2+ sensitivity of the Ca2+-ATPases from different types (SERCA and PMCA); 3- investigate if protein kinase A
(PKA) or protein kinase C (PKC) can phosphorylate the Ca2+-ATPases; 4- evaluate a possible oxidative damage caused by
malnutrition.
Methods and Results:
Animals were chronically malnourished from weaning until 13 weeks of age (CM group) using the model described as Regional
Basic Diet (Arch. Latinoam. Nutr. 40; 533, 1990). After this period, control and CM male rats were sacrificed (CEUA
DFCBICB007). Histomorphometric analysis were performed according to Masson's trichrome method, in which atrophy was
observed in the prostatic portion of male rats from CM group (59% of control muscle mass; n=3). The measurement of enzymatic
hydrolysis of gama-32P[ATP] showed that Ca2+-ATPases present in the RVD homogenate exhibited an increased of Vmax
values – 653% (SERCA) and 603% (PMCA), n=5 – and a reduced Km value for SERCA (27%; n=5) in CM group. The
phosphorylation of the Ca2+-ATPases of different types was seen in a specific film (phosphor screen) after the 32P-proteins be
separated by SDS-PAGE electrophoresis. Chronic malnutrition promotes increase in the PKAi-sensitive phosphorylation (related
to PKA) of the 140 and 110 kDa bands, related to PMCA (487%; n=3) and SERCA (186%; n=3), respectively. By the other hand,
an increase in the calphostin C-sensitive phosphorylation (related to PKC) was detected only in the 140 kDa band, corresponding
to PMCA (186%; n=3). In CM group, it was also observed a significant increase of lipid peroxidation by TBARS method (659%;
n=3) as well as protein carbonylation (292%; n=6).
Conclusions:
Taken together, the present data help to understand the Ca2+-dependent molecular events that affect reproductive capacity of
adult male rats chronically malnourished. It seems that the oxidative damage of membranes and protein constituents in vas
deferens cells alters calcium homeostasis. In this context, the Ca2+-ATPases show high hydrolytic activity and elevated levels of
phosphorylation. However, it seems that all of these efforts (and others) are not enough to reestablish the normal contractility of
vas deferens as it is observed muscle atrophy, and as previously reported, it be may related to a deficient transport and raise of
death of sperm cells observed in this organ.
Keywords: Rat vas deferens, chronic malnutrition, regulatory phosphorylation, oxidative damage, kinetics of calcium pumps
Financial Support: Projeto Casadinho-CNPq; PROCAD-CAPES; FAPERJ Primeiros Projetos, Programa
ALV
Resumo:05-029
EFFECTS OF CHRONIC NITRIC OXIDE SYNTASE INHIBITION ON RAT PROSTATE SMOOTH MUSCLE
REACTIVITY.
Calmasini, F. B. ; Leiria, L. O. S. ; Pissinatti, L. ; Antunes, E.
Department of Pharmacology / Faculty of Medical Sciences , UNICAMP
Objectives:
The prostate is a gland innervated mainly by adrenergic and cholinergic fibers that act synergistically controlling the smooth
muscle reactivity. Non-adrenergic non-cholinergic (NANC) purinergic fibers, through the release of adenosine triphosphate
(ATP), also play a role in the smooth muscle contraction tone regulation. Moreover, signal transduction pathways using NOcGMP regulates diverse physiological functions such as smooth muscle contractility, neurotransmission and cell
growth/proliferation. However, the NO-cGMP signaling system in prostate smooth muscle regulation is still poorly investigated.
Chronic NO inhibition in different tissues lead to functional, molecular and biochemical alterations, and therefore has been
widely used as a pharmacological strategy to study the role of NO in several tissues. Therefore, the aim of this study was to
evaluate the effects of chronic NO blockade in the rat isolated prostate smooth muscle.
Methods and Results:
The experimental protocols were approved by the Animal Ethical Committee of UNICAMP. Male Wistar rats were treated or not
with the NO synthesis inhibitor L-NAME (20 mg/rat/day) given in the drinking water for 4 weeks. After treatment, animals were
sacrificed through isoflurane overdose, and the prostate was isolated and weighed. Concentration-response curves to
phenylephine (PE, 1 nM-100 µM), carbachol, (CCh, 1 nM - 100 µM), and á,â-methylene ATP (1, 3 and 10 µM) were obtained.
The neurogenic contractile responses induced by electrical-field stimulation (EFS, 1-32Hz) were also obtained. Values of potency
(pEC50) and maximal responses (Emax) were calculated. Histological analyses in prostates from control and L-NAME-treated
rats were performed. Chronic L-NAME administration promoted a significant increase the prostate weight (503 ± 27.6 mg, n=5, p
Conclusions:
Our findings show that long-term NO deficiency in rats causes in vitro functional alterations characterized by increased
contractile responses to á-adrenoceptor and muscarinic receptor activation, but not to P2X1 activation. Whether chronic NOdeficiency in rats mimics the benign prostatic hyperplasia is under investigation.
Keywords: prostate, L-NAME, nitric oxide
Financial Support: CAPES
Resumo:05-030
COMPARATIVE STEREOLOGICAL STUDY ON THE MORPHOLOGY OF THE SEMINIFEROUS TUBULES OF
RATS FED CANOLA AND SOYBEAN OIL DIETS.
Almeida, A. F. G1; Costa, C. A. S. 3; Nascimento - Saba, C. C. A. 3; Rueff-barroso, C. R. 1; Santos, R. M.
M. 2; Lancetta, C. F. F. 1
1
Departamento de morfologia , UFF
2
Departamento de fisiologia e farmacologia, UFF
3
Departamento de ciências fisiológicas, UERJ
Objectives:
The intake of polyunsaturated fatty acids (PUFAs), especially omega 3 (n-3) and omega 6 (n-6), is essential to promote the proper
development and maintenance of various physiological systems. In Brazil, the soybean oil (composed of 8% of n-3 and 54% of n6) is the most consumed oil in all social classes, corresponding to an average of 82% of total calories obtained from vegetable oils
and fats, while canola oil (comprising 11% of n-3 and 21% of n-6) is not much consumed, corresponding to an average of 4.2%
(Rev Saúde Pública. 2005 Aug;39(4):530-40). The human body has a preference to metabolize n-3 fatty acids, and it is believed
that the ideal proportional intake of n-3 and n-6 is 5 molecules of n-6 for 1 molecule of n-3. However, there are reports in which
the proportion vary from 3:1 to 10:1. Based on this information, the purpose of this study was to evaluate the best ratio of intake
of these fatty acids, comparing diets with soybean and canola oils, on the developmental of seminiferous tubules.
Methods and Results:
10 male Wistar rats were divided into two groups: 5 animals were fed diets containing 7% soybean oil (control group) and 5
others received diets containing 7% canola oil (canola group). At 60 days these animals were sacrificed and testicles processed
following routine techniques for paraffin embedding. The volume density (Vv), the length density (Lv), surface density (Sv) and
the transverse area of testicular tubules were determined by stereological methods. Data were analyzed using t test considering p
Conclusions:
Animals fed canola oil had lower testicular growth than those fed with soybean oil, showing that the highest proportions of n-6
compared to n-3, present in soybean oil, are probably more beneficial to testicular development.
Keywords: canola oil, Essential fatty acids, seminiferous tubules, soybean oil, testicle
Resumo:07-011
EFFECT OF UREA ON THE ACTIVITY OF ABCC1 AND CELL VIABILITY OF THE MA104 CELL LINE
Santos, D. H. F. 1; Fonseca, L. M. 2; Capella, M. A M. 1,2; Lopes, A. G. 1
1
Instituto de Biofísica carlos Chagas Filho, IBCCF
2
Instituto de Bioquímica Médica, IBqM
Objectives:
The accumulation of urea in the renal medulla is due to the action of easily regulated transporters and is a critical factor for the
mechanism of urinary concentration. It‘s known that high concentrations of urea activate signaling pathways in kidney epithelial
cells. Urea itself is a known denaturant agent, but kidney cells are capable of surviving in the presence of high urea
concentrations.Previous data from our group showed that Ma104 cells are resistant to urea and that this molecule increased the
viability of NaCl treated cells. We have also shown that both NaCl and urea are capable of modulating the ATPase activity of
ABCC1. In this work we aim to evaluate the cellular responses of Ma104 cell line to hyperosmolar conditions due to urea.
Methods and Results:
Cells were incubated in transwell plates (5x105 cells/ well) for Five days until the monolayer was completely established. After
that, 9 mg of urea were added to the upper medium, lower medium, both mediums, or none, creating three treatment conditions
and one control condition. Urea dosage was performed using a commercial kit based on urease activity after 6, 24 and 48 hours of
incubation. After 6 hours of incubation the values measured of urea in the three ocnditions were: Urea top: 5,350±0,1919 mg,
Urea bottom: 2,218±0,1640 mg. (P
Conclusions:
The results suggest that the treatment with urea does not affect the viability of both the Ma104 cells and the MDCK cell line. It
does, however reduce the transport activity of ABCC1, which might be linked to an attempt of preserving intracellular levels of
GSH, which is paramount to cell survival in hyperosmotic environments. It‘s also important to note that the Ma104 cells show a
bidirectional transport of urea, indicating the presence of urea transporters in both the basolateral and apical membranes.
Keywords: urea, Ma104, hyperosmolar, activity, viability
Financial Support: CAPES, CNPq, FAPERJ, Ary Frauzino Foundation (FAF-ONCO II)
Resumo:07-012
QUALITY OF EVOLUATION OF DIABETIC PATIENTS ON HEMOLIADILYSIS IN THE CITY OF PELOTAS/RS
2010, ACADEMIC WORK – NURSING COLLEGE. FEDERAL UNIVERSITY OF PELOTAS, PELOTAS/RS.
Noschang, J. 2; Vidales-braz, B. M. 1; Carvalho. M. P. 3; Peres, W. 4; Guanilo, M. E. E. 1
1
Faculdade de Enfermagem, UFPEL
2
Faculdade de Medicina, UCPEL
3
Faculdade de Fisioterapia, UCPEL
4
Centro de Ciências Químicas Farmacêuticas e de Alimentos, UFPEL
Objectives:
The chronic renal failure nowadays represents an important public health problem in the world, being considered as an
“epidemic” of alarming growth. In Brazil, currently, it is estimated that there are over two million Brazilians bearing
some degree of renal dysfunction. The diabetic nephropathy (DN) is the main cause of chronic renal failure, 40% of DN
bearers need dialysis. Purpose: Evaluating the Quality of life related to health (QLRH) of diabetic patients with
nephropathy submitted to hemodialysis in two specialized centers in the city of Pelotas/RS.
Methods and Results:
Observational study of transversal type quantitative delineation and of descriptive character which used as collection
instruments a socio-demographic questionnaire and the Kidney Disease and Quality of Life Short Form (KDQOLSFTM), as it consists of a specific instrument to be applied in individuals with chronicle renal disease on some type of
dialysis program. The data obtained were processed and analyzed in the SPSS program - Statistical Package for the
Social Sciences - version 15.0. Results: The sample had a total of 35 patients, in which 23 (65,7%) were males. The general
age ranged from 38 to 78 years (M: 61,3; a DP: 13,8) and the time of hemodialysis varied from 1 to 144 months. The
dimensions which presented higher average rates were a stimulus from the team (82,12), quality of social interaction
(76,00) and list of symptoms and problems (75,89). The lower rates were for emotional function (28,57), physical function
(30,00), work situation (31,32) and burden of kidney disease (37,50).
Conclusions:
It is believed that this study has positively contributed for the identification of important matters concerning the health of
these patients, as well as for the implementation of new strategies to care for diabetic patients with nephropathy on
hemodialysis in the city of Pelotas/RS, as it provides scientific subsidies which indicate a committed QLRH.
Keywords: Diabetic, diabetic nephropathy, Evaluating the Quality of Life, hemodialysis
Resumo:07-013
PROGRESSION OF DIABETIC NEPHROPATHY IN RATS
Júdice, M. N. ; Castiglione, R. C. ; Barbosa, C. M. ; Sousa-menezes, J. ; Morales, M. M.
Universidade Federal do Rio de Janeiro, UFRJ
Objectives:
Diabetic nephropathy is one of the most serious complications of diabetes mellitus (DM) and the most common cause of endstage renal failure. The main feature of early functional renal changes in diabetes is glomerular hyperfiltration. In the present
study we investigated the temporal evolution of renal dysfunction in rats submitted to streptozotocin induced diabetes.
Methods and Results:
Diabetes was induced by a single dose of streptozotocin (45mg/Kg, i.p.) in 8 weeks-old male Wistar rats (180-200 g) (Protocol
approved by Animal Ethics Comittee). The animals were divided in two groups: controls (CTRL, n = 4-6) and diabetics (DM, n =
4-5). Glycemia was determined twice a week (blood tail), using the glucometer OneTouch Ultra2. DM animals remained
hyperglycemic until 28 weeks after diabetes induction (glycemia higher than 200 mg/dL). Glomerular filtration rate (GFR), water
and food ingestion, weight gain/loss, glycemic levels and renal excretion of total proteins, glucose, and albumin were analyzed in
4, 8, 12, 16, 20, 24 and 28 weeks after diabetes induction. For urine collection and analyses of food and water intake, the animals
were placed in metabolic cages for 24 hours and then were subjected to ketamine (75mg/Kg) and xylazine (10mg/Kg) anesthesia
(i.p.) for blood collection. DM animals did not show a normal increase in body weight, as observed for CTRL animals, despite
the higher ingestion of water and food observed in DM compared to CTRL animals. DM animals showed an increase in urinary
flow 4, 8, 12, 16, 20, 24 and 28 weeks after the diabetes induction (37.41±1.01, 50.28±4.69, 39.93±8.79, 46.79±6.14, 50.88±7.44,
28.83±8.21, 45.22±3.34 µL/min respectively) compared to CTRL groups (7.76±1.33, 5.90±0.99, 6.95±1.16, 5.25±0.49,
5.66±2.05, 6.23±0.26, 4.83±0.52 µL/min respectively). The same pattern of increase was observed in GFR in DM groups
(10.18±1.17, 5.13±1.09, 5.52±1.07, 9.90±2.90, 5.34±1.99, 4.38± 0.96, 5.76±0.65µL/min/g respectively) compared to CTRL
groups (5.30±0.59, 3.29±0.54, 1.71±0.48, 3.70±0.24, 0.29±0.11, 1.64±0.59, 1.480±0.06 µL/min/g, respectively). Proteinuria was
higher in DM groups 4, 8, 12, 16 and 20 weeks after diabetes induction (18.10±4.72, 10.45±2.70, 23.15±3.83, 26.13±2.92 and
44.82±4.03 mg/day, respectively) compared to CTRL groups (10.01±1.84, 5.18±1.07, 10.80±1.33, 10.27±0.86 and 13.79±1.02
mg/day respectively). Osmolar clearance was increased in DM groups 4, 8, 12, 16, 20, 24 and 28 weeks (0.07±0.01, 0.08±0.020,
0.1±0.032, 0.13±0.019, 0.22±0.021, 0.29±0.013, 0.33±0.01mL/min, respectively) compared to CTRL groups (0.024±0.00017,
0.025±0.002, 0.03±0.003, 0.032±0.0017, 0.032±0.0022, 0.035±0.0011, 0,034±0,002 mL/min, respectively). Statistical analysis
was performed by t-tests and One-Way ANOVA (Newman-Keuls post-test). Differences were considered significant when
p<0.05.
Conclusions:
GFR and urinary flow were higher in DM compared to CTRL animals showing a change in the renal function, consequently,
producing a higher excretion of protein in the urine of DM animal model at 4, 8, 12, 16 and 20 weeks after induction of diabetes.
Keywords: DIABETIC NEPHROPATHY , glomerular filtration rate, hyperglycemia, rats, streptozotocin
Financial Support: FAPERJ, CNPq, CAPES, FUNEMAC, PRONEX
Resumo:07-014
HISTOLOGICAL AND ULTRASTRUCTURAL ANALYSIS OF THE RENAL PELVIS IN NORMAL AND DIABETIC
WISTAR RATS.
Lima, V. M. 1; Chopard, R. P. 2
1
DEPTO. DE CIRURGIA/FACULDADE DE MED. VETERINÁRIA E ZOOTECNIA, FMVZ/USP
2
DEPTO. DE ANATOMIA/INSTITUTO DE CIÊNCIAS BIOMÉDICAS, ICBIII/USP
Objectives:
This project aims to describe and analyze the collagen, elastic and smooth muscle fibers of the renal pelvis of Wistar rats by
analyzing structural changes in experimental groups compared with the control group.
Methods and Results:
It was used male adult Wistar rats (Rattus norvegicus albinos) divided into two different groups (A and B), normal Wistar rats
and diabetic rats. The rats in group B were induced diabetes by Alloxan dissolved in physiological solution of sodium chloride
0.9% (135mg Alloxan in 6ml Saline), was then injected 0.2 ml of solution per 100g of body weight. The region of the renal pelvis
that contains representation of fibers was collected and reduced into small fragments. The cuts obtained are stained by the
following methods: Iron hematoxylin (Verhoeff) for demonstration of elastic fibers; Resorcin fuchsin (Weigert) for
demonstration of elastic and elauninic fibers; Azan for disclosure of the component collagen and smooth muscle; Picrosirius to
observe the component collagen, specifically collagens type I and III. The results were analyzed according to each technique.
Verified by light microscopy, the group of normal rats showed a higher area of collagen type I, if compared to the diabetic group.
We also observed that the collagen type III are not present in the two groups. Normal rats have two layers of collagen in the
submucosa and another one in the adventitia, with muscle fibers between them. The elastic fibers are well organized in lamellae
in the submucosa and sparce in the adventitia. Already in diabetic rats the layers of collagen are continuous, but disorganized.
The elastic fibers are sparse in the adventitia and interrupted in the submucosa, without lamellae. Ultrastructural results
demonstrated that in animals without induction of diabetes smooth muscle cells that form the renal pelvis are evenly arranged,
with slightly rounded nuclei and the presence of organelles such as mitochondria is constant, but not in large quantity. In the
diabetic animals we noticed that the nuclei of muscle cells are elongated, cytoplasmic organelles such as endoplasmic reticulum
and others are arranged unordered, also observed a significant increase of mitochondria and the presence of vilosity facing the
light of the renal pelvis.
Conclusions:
From the methodology and results, we infer that diabetes causes structural and ultrastructural differences renal pelvis of rats,
mainly in the organization of collagen and elastic fibers and also by the considerable increase of mitochondria in these rats, which
can demonstrate that they have a more intense energy expenditure.
Keywords: RENAL PELVIS, WISTAR RATS, ELASTIC FIBERS, COLLAGEN FIBERS, MUSCLE FIBERS
Financial Support: FAPESP
Resumo:07-015
GLUCAGON-LIKE PEPTIDE-1 RECEPTOR ACTIVATION INCRESES GLOMERULAR FILTRATION RATE AND
PROXIMAL TUBULAR SODIUM REABSORPTION
Crajoinas, R. O. 1; Oricchio, F. T. 1; Pessoa, T. D. 2; Pacheco, B. P. M. 1; Lessa, L. M. A. 2; Malnic, G. 2;
Girardi, A. C. C. 1
1
Instituto do Coração, HC-FMUSP
2
Fisiologia e Biofísica, ICB-USP
Objectives:
Glucagon-like peptide 1 (GLP-1) is a gut incretin hormone considered a promising therapeutic agent for type 2 diabetes because
it stimulates beta cell proliferation and insulin secretion in a glucose-dependent manner. Cumulative evidence supports a role for
GLP-1 in modulating renal function; however the mechanisms by which GLP-1 induces diuresis and natriuresis have not been
completely established. This study aimed to define the cellular and molecular mechanisms mediating the renal effects of GLP-1.
Methods and Results:
GLP-1 (1 μg/kg×min) was intravenously administered in rats for the period of 60 minutes. Continuous infusion of GLP-1 induced
an increase in the urinary excretion of cAMP when compared to controls (121 ± 9 vs. 8 ± 3 pmol/min, P = 0.003). GLP-1 infusedrats displayed increased urine flow, fractional excretion of sodium, potassium and bicarbonate compared to those rats which
received vehicle (1% BSA/saline). GLP-1-induced diuresis and natriuresis were also accompanied by increases in renal plasma
flow and glomerular filtration rate. Real time RT-PCR in microdissected rat nephron segments revealed that GLP-1 receptormRNA expression was restricted to glomerulus and proximal convoluted tubule. In rat renal proximal tubule, GLP-1 significantly
reduced NHE3-mediated bicarbonate reabsorption via a protein kinase A (PKA)-dependent mechanism. Reduced proximal
tubular bicarbonate flux rate was associated with a significant increase of NHE3 phosphorylation at the PKA consensus sites in
microvillus membrane vesicles.
Conclusions:
Taken together, these data suggest that GLP-1 has diuretic and natriuretic effects which are mediated by changes in renal
hemodynamics and by downregulation of NHE3 activity in the renal proximal tubule. Moreover, our findings support the view
that GLP-1-based agents may have a potential therapeutic use not only as antidiabetic drugs but also in hypertension and other
disorders of sodium retention.
Keywords: incretin, protein kinase A, proximal tubule, renal hemodynamics, sodium reabsorption
Financial Support: FAPESP
Resumo:07-016
WATER AND SODIUM TRANSPORT INHIBITED BY THE ANTIMALARIAL DRUG ARTESUNATE IN RAT IMCD
PERFUSED IN VITRO.
Yano, Y. ; Rouch, L. K. ; Magaldi, A. J.
Hospital das Clinicas da Faculdade de Medicina da USP, HCFMUSP-LIM 12
Objectives:
Artesunate (Art) is a new effective antimalarial drug but a reversible polyuria and natriuresis has been related with its use
(Campos S.B., Kidney Int 2001,59:1044). This study was designed to determine the effect of Art on water and Na+ absorption in
rat IMCD.
Methods and Results:
The osmotic water permeability (Pf, &mum./sec) and Na+ lumem-to-bath flux (Jlb pEqcm2/sec) were determined in normal rats
IMCD(n=16) isolated and perfused in vitro by the standart methods. Art (10-5M) added to the bath in Vasopressin(Vp) absence
decreased Pf from 4.0stb1.2 to -18.3&plusmn1.4 (p
Conclusions:
In summary, Art in IMCD: 1) inhibits partially the basal Pf and the Vp-stimulated Pf by 50% and totally the Vp-stimulated Na+
Jlb flux ; 2) is blocked by Anti-V2, and by Indo, leading us to suppose that its action could be by PGE2 stimulation synthesis by
mean of Vp receptors, explaining, at least in part, the observed polyuria and natriuresis during the use of this new antimalarial
drug .
Keywords: Artesunate, Sodium Transport, Vasopressin, Water Transport
Financial Support: FFM, LIMHCFMUSP, FMUSP,FAPESP,CNPq
Resumo:07-017
THE MATERNAL AMPLIFIED PERINATAL ESSENTIAL FATTY ACID DEFICIENCY DISCLOSES RENAL
DISFUNCTION IN THE ADULT OFFSPRING
Ribeiro, V. S. ; Silva, A. R. ; Pereira, S. F. J. ; Castro-chaves, C. ; Paixao, A. D. O.
DEPARTAMENTO DE FISIOLOGIA E FARMACOLOGIA, UFPE
Objectives:
The maternal amplified perinatal period in an essential fatty acid deficient diet (EFAD) effects on renal function and blood
pressure in the adult offspring were investigated.
Methods and Results:
All rats were maintained with controlled conditions and with free access to food and water. The male reproducers were on
commercial diet until mating, while the females were either on a control diet (CON) or on an EFAD since 3 weeks before mating
until end of lactation. CON (soy oil) had 2.7% of linoleic acid (LA) and 0.05% of &ALPHA;-linolenic acid (LNA) while EFAD
(coconut oil) had 0.01% of LA, being practically free of LNA. The offspring were reduced to 8 males/mother on Day 3 of life
being weaned on commercial diet until adults. They were separated according to the maternal diet in CON (n=11) and EFAD
group (n=15). As adults, the offspring were housed for 24h in metabolic cages (MC) with free access to food and water, in order
to calculate: the food (FI) and water (WI) ingestions; the urine volume (V), density (D), Na + , K+, protein, and creatinine
excretions by 100g/body weight. Indirect Systolic Blood Pressure (SBP) was taken 5 times per day in the adult offspring.
Glomerular filtration and proximal tubule Na + reabsorption were measured respectively by creatinine and Li + clearances for 3h in
the MC, in the adult offspring previously water expanded at 5 mL/100g. Blood and urine samples were collected, in order to
calculate the excretions and clearances. Heart, lungs, liver, spleen, kidneys and testicles were removed and weighed (wet weight),
dried for 48h at 70oC and then, they were subsequently weighed (dry weight) with values being corrected to 100g/body weight.
Results are in mean±SEM. Offspring from the EFAD group weighed less than CON respectively at: birth (6.1±0.1 vs 5.6±0.1* g;
CON vs EFAD), weaning (52±0.8 vs 46±1.1* g; CON vs EFAD), growth (203.5±1.5 vs 193.7±2.5* g; CON vs EFAD) and adult
(320±5.0 vs 298.7±8.0* g; CON vs EFAD). At the 24h adult offspring studies: FI (6.8±0.2 vs 7.1±0.2 g/100g; CON vs EFAD);
WI (11.5±0.5 vs 10.9±0.3 mL/100g; CON vs EFAD); V (4.8±0.3 vs 4.6±0.3 mL/100g; CON vs EFAD), a D (1.046±0.001 vs
1.049±0.0005 g/mL; CON vs EFAD), K+ (5499±463.6 vs 5242±512.9 ìEq/mL/100g; CON vs EFAD) and creatinine urinary
excretions (4.1±0.3 vs 4.1±0.2 mg/mL/24h; CON vs EFAD) were similar between groups but Na+ (416.5±21.5 vs 348.3±21.6*
ìEq/mL/100g; CON vs EFAD) and protein excretions (17.9±1.3 vs 12.3±0.8* mg/100g/24h; CON, n= 10 vs CAGE, n= 8) were
decreased in the EFAD. PAS measured in adult offspring (149±0.64 vs 147±0.9 mmHg; CON, n= 12 vs EFAD, n= 9) was similar
between groups. The lack of difference on creatinine clearance (220.5±12.6 vs 212.8±15.7 µL/min/100g; CON, n= 9 vs EFAD,
n= 8) stands for an unaltered glomerular filtration by the diet. The decrement in Li+ clearance (62.0±3.2 vs 37.3±7.9*
µL/min/100g; CON, n= 8 vs EFAD, n=7) indicates increased Na + proximal tubule reabsorption. Organ wet and dry weights were
similar among CON and EFAD adult offspring.
Conclusions:
Maternal amplified perinatal EFAD reduced the weight gain from birth and altered renal functional aspects in the adult offspring.
Keywords: ESSENTIAL FATTY ACID DEFICIENCY , PROXIMAL TUBULE REABSORPTION, RENAL FUNCTION
Financial Support: PROCAD 8052 and FACEPE/IBPG-0143-2.07/09
Resumo:07-018
EARLY GLOMERULAR CHANGES INDUCED BY HIGH-FAT DIET IN OVARIECTOMIZED RATS.
Amaral, L. S. B. 1; Silva, F. A. 1; Ribas, W. B. D. 1; Silva, J. A. 1; Souza, S. I. 1; Magalhães, A. C. M. 1;
Macedo, C. L. 2; Coimbra, T. M. 3; Soares, T. J. 1
1
Instituto Multidisciplinar em Saúde/UFBA , IMS/UFBA
2
Departamento de Ciências Naturais/UESB, DCN/UESB
3
Departamento de Fisiologia, FMRP/USP
Objectives:
The association between obesity and estrogen deficiency may contribute to the changes of renal function and structure. Previous
studies in our laboratory have demonstrated increased blood pressure and visceral fat depot, decreased fractional excretion of
sodium and tubulointerstitial lesions in ovariectomized rats fed a high-fat diet. The aim of this study was to evaluate the effects of
high-fat diet on urinary protein and albumin excretion and glomerular changes in ovariectomized rats.
Methods and Results:
Twenty-four ten-week-old female Wistar rats were ovariectomized (OV) or sham-operated and divided into four groups: SSD
(sham operated rats with standard diet, n=6); OSD (OV rats with standard diet, n=6); SHFD (sham operated rats with high-fat
diet, n=6); OHFD (OV rats with high-fat diet – 54.4% of total calories from fat, n=6). Monthly urine samples were collected to
quantify albumin and protein excretion. The quantification of urinary protein excretion was performed by colorimetry. Urinary
albumin excretion was quantified in ovariectomized and sham animals by electroimmunoassay, using a specific antibody against
rat albumin. The animals were killed 24 weeks after the diet, at which time the kidneys were removed for histological and
morphometric analysis. The slices of renal tissue were stained with Masson's trichrome and examined under light microscopy.
Glomerulosclerosis was evaluated semiquantitatively in 100 glomeruli per animal by counting the number of quadrants showing
sclerosis. The tissue was also used for morphometric studies of glomerular size. Measurements of the glomerular tuft area were
done on 30 consecutive cortical glomeruli and 15 corticomedullary glomeruli of each animal from different experimental groups.
Data concerning albuminuria and proteinuria were submitted to the Kruskal–Wallis nonparametric test. Other data were
submitted to the Newman-Keuls test. The level of significance considered was p
Conclusions:
Our results suggest that high-fat diet and ovariectomy may be contributing to the proteinuria and glomerular sclerosis and
hypertrophy in these animals.
Keywords: Glomeruloesclerose, Proteinúria, Dieta hiperlipídica, Ovariectomia
Financial Support: CNPQ and FAPESB
Resumo:07-019
INFLUENCE OF HAEMODIALYSIS AND HEPATITIS C ON BLOOD OXIDATIVE STATUS IN PATIENTS OF
DIALYSIS UNITS IN RIO GRANDE/RS-BRAZIL.
Kalb, A. C. 1; Silva, N. M. O. D. 2; Martínez, P. E. 1; Martínez, A. M. B. 2
1
Instituto de Ciências Biológicas, FURG
2
Faculdade de Medicina, FURG
Objectives:
The uremic state and the bio-incompatibility of haemodialysis (HD) are associated with oxidative stress in HD patients. High
prevalence of hepatitis C virus (HCV) has been found in many dialysis units around the world, giving to HD patients an increased
probability to acquire this infection. This study aimed verifies the influence of haemodialysis and HCV infection on blood
oxidative status in patients of dialysis units in Rio Grande⁄RS⁄Brazil.
Methods and Results:
It was measured oxidative stress markers in blood cells and plasma, and blood collection were performed before haemodialysis
procedure. The following groups were considered: 1.Control subjects (CTRL); 2.Haemodialysis−maintened patients (HD);
3.HCV−infected patients (HCV) and 4.Haemodialysis−maintened patients infected by HCV (HCVD) (n=14 per group). In
erithrocytes, was verified non−protein sulfhydryl (SH) content (which include the antioxidant GSH), and lipid peroxidation. In
leucocytes, it was measured total antioxidant capacity against peroxyl radicals. It was also verified lipid damage in plasma
samples. Statistical analysis was performed through non−parametric Kruskal−Wallis test where data was expressed in medians
and interquartile range (25−75). Results: 1. In erithrocytes, SH content of all groups of patients presented absence (p>0.05) of
statistical differences when compared to CTRL group (CTRL: 0.00067 (0.000521−0.000809); HD: 0.000691
(0.000530−0.001016); HCV: 0.000462 (0.000292−0.000508); HCVD: 0.000587 (0.000334−0.000890)); however, there was a
significant difference (p
Conclusions:
Probably due to successive stimuli caused by dialysis, HD patients had slightly higher basal levels of GSH in erithrocytes, which
although subtle, were sufficient to decrease the observed values of lipid peroxidation. These stimuli could also provide an
improvement in antioxidant competence in leucocytes. However, it seems that HCV in some way blocks this compensatory
response, once HCVD patients presented high content of lipid damage both in red cells and plasma. The results suggest that
HCV−infected patients submitted to dialysis in Rio Grande are quite more susceptible to oxidative damage than only
HCV−infected or only haemodialysis−maintened patients, and this fact implicates a more careful clinical approach.
Keywords: Haemodialysis, Hepatitis C, Oxidative status
Financial Support: CNPq
Resumo:07-020
QUANTITATIVE EVALUATION OF LEUKOCYTES AND PHYSICAL PARAMETERS IN RATS WITH
NEPHROTIC SYNDROME INDUCED BY DOXORUBICIN HYDROCHLORIDE
Almeida. 1; Melo1; Jardim. 1; Domingues1; Moreira 1; Santos1; Melo1; Silva2; Pereira1
1
Universidade Federal dos Vales do Jequitinhonha e Mucuri, UFVJM
2
Universidade Federal de Minas Gerais, UFMG
Objectives:
The present study intended achieve a quantitative analysis of rat peripheral blood leukocytes with NS induced by doxorubicin as
well as evaluate some physical parameters.
Methods and Results:
It was used 40 male Wistar rats, average weight of 300 grams. The animals were housed in individual cages, and have received a
standard commercial diet and water ad libitum. We share the animals into two groups: DOXO group (n=20), underwent
intravenous injection of doxorubicin (Doxolem®/7.5mg/kg body weight) and SAL group (n=20), which received intravenous
injection of saline (D. Boonsanit et al, 2006). To confirmation of renal disease, microalbuminuria tests were performed - cobas
mira plus ® device with use of kits BIOCLIN /Quibasa ®. To comparison between groups test t student was performed.
Significant level of *p < 0,05 was established. The animals in each group were analyzed 7, 14, 21 and 28 days after the injections.
Urine was collected for 24 hours (metabolic cage) on day before sacrifice. On the sacrifice day, animals were weighed and
underwent thoracotomy under anesthesia (ketamine / Xylazin - 70/10 mg / kg). Blood samples were collected by cardiac
puncture. After specific lyses of the red cells, the absolute leukocytes count was performed using Newbaeur Chamber. The
organs were perfused with buffered saline (PBS) and the kidneys were removed and immediately weighed. The weight of the
kidneys was normalized using body weight. According to our results, the DOXO group presented a decrease of the absolute
number of leukocytes 7 days (2309 ± 322,5 vs. 3740 ± 423.4 cells number, p < 0,001) after the injection however on 14th after
injection the same group presented an increased of this parameter (6475 ± 249,8 vs. 4270 ± 250.1mL, p < 0,0001). In the first
three weeks the animals from DOXO presented a reduction in water consumption, but no statistical differences was observed.
Similar results were observed in relation to urine volume. Regarding body weight, it was observed that DOXO group kept or
reduced weight, differing from the controls which gained weight over weeks, with significant statistical difference between
groups for days 14 (277.9 ± 11.61 vs. 343.2 ± 13.98g, p < 0,007), 21 (296.1 ± 11.49 vs. 389.4 ± 15.27g, p < 0,0005) and 28
(308.2 ± 14.69 vs. 379.0 ± 8.895g, p < 0,004). About kidney weight in the DOXO group there was an increased value in weight
after the second week of injection, 14 (5.354 ± 0.16 vs. 4.388 ± 0.14g, p < 0,005), 21 (6.408 ± 0.17 vs. 4.309 ± 0.10g, p <
0,0001), 28 days (6.921 ± 0.29 vs. 4.413 ± 0.18g, p < 0,0001).
Conclusions:
We believe that increasing the number of leukocytes observed in the 14 th day of injury is possibly related to the inflammatory
response established in renal tissue. We confirmed some changes in the body, as reported in the literature, indicating the
progressive nature of the disease. Other immunophenotyping experiments are being conducted to confirm or clarify the behavior
of the immune system in different stages of the disease.
Keywords: Doxorubicin, Nephrotic Syndrome, Proteinuria
Financial Support: CAPES
Resumo:08-043
DISTRIBUTION OF O2 CHEMORECEPTORS AND CONTROL OF CARDIORESPIRATORY RESPONSES TO
HYPOXIA IN NILE TILAPIA (OREOCHROMIS NILOTICUS)
Zeraik, V. M. ; Belão, T. C. ; Sanches, J. R. ; Kalinin, A. L. ; Rantin, F. T.
Departamento de Ciências Fisiológicas, UFSCar
Objectives:
In fish the O2 chemoreceptors are mainly found in the gill arches and monitor the O2 tensions of the inspired water (externally
oriented) and/or the arterial blood (internally oriented). During hypoxia these receptors trigger cardiorespiratory adjustments in
order to maintain an adequate O2 transfer to the tissues. The aim of the present study was to determine the O2 chemoreceptors
distribution through the gill arches of Nile tilapia and the role of these receptors on the cardiorespiratory responses to hypoxia in
this species.
Methods and Results:
One buccal and two opercular canullae, and a pair of ECG electrodes were implanted in a group of intact fish (control group, n =
10; Wt = 258,46 ± 7,42 g). This procedure served to measure the following cardiorespiratory variables: respiratory frequency fR, ventilatory tidal volume VT, gill ventilation - VG, O2 extraction from the ventilatory current – EO2, oxygen uptake - VO2,
and heart rate – fH. After an overnight recovery period from surgery fish were subjected to the following O2 tensions during 40
min each: 140 (normoxia), 100, 70, 50, 30 e 20 mmHg. To verify the O2 receptors distribution in the gills, the first pair of gill
arches was surgically extirpated (operated group, n=10; Wt = 232,53± 8,78 g). After a recovery period of 4 days, operated fish
were subjected to the same procedures described above for the intact ones. VO2 of intact fish was significantly higher than that of
operated in all experimental O2 tensions. In normoxia the fR of the operated fish was 30% higher than that recorded for the
control group. During graded hypoxia this variable was even higher (44% at 50 mmHg) in the operated group. No significant
differences were observed in VT and VG of both groups, except in the most hypoxic tension of 20 mmHg, when the operated
group presented a significant lower VG. The ventilarory requirement (VG/VO2) was significantly higher in operated fish during
normoxia, at 100 mmHg and 20 mmHg (more hypoxic tension). The fH of operated fish was significantly higher in normoxia and
at 100 mmHg. Below these tensions both groups presented similar fH. In both groups EO2 was maintained within a range of 70 to
80%. Intact fish presented significantly higher EO2 only during normoxia.
Conclusions:
In Nile tilapia the O2 chemoreceptors are distributed beyond the first gill arch, probably through the four gill arches. These
receptors trigger the cardiorespiratory adjustments necessary to face the hypoxic conditions at which the species is frequently
subjected in its natural environmental conditions.
Keywords: brânquias, hipóxia, quimiorreceptores, respostas cardiorrespiratórias, Tilápia-do-Nilo
Financial Support: FAPESP
Resumo:08-044
OXIDATIVE DAMAGE INDUCED BY CIGARETTE SMOKE EXPOSURE AFFECTS SKELETAL MUSCLE
BEFORE LUNG TISSUE
Alves, E. C. 1; Nesi, R. T. 6; Carlos, S. P. 1; Silva, L. A. 1; Effting, P. S. 1; Valença, S. V. 6; Pinho, R. A. 1;
Tuon, L. 1; Chiappa, G. R. 1,10
1
Universidade do Extremo Sul Catarinense, UNESC
6
Universidade Federal do Rio de Janeiro, UFRJ
10
Hospital das Clinicas de Porto Alegre, HCPA
Objectives:
Studies have shown that the cigarette smoke (CS) is responsible for pathogenesis of several pulmonary diseases including chronic
obstructive pulmonary disease (COPD). It is well known that cigarette smoke is responsible for lung damage, inflammation and
oxidative stress in pulmonary emphysema. In addition, COPD is frequently associated with muscle loss and skeletal muscle
dysfunction. For this reason, the present study investigated the inflammation and oxidative stress in mice exposed to cigarette
smoke for 7, 15, 30, 45 and 60 consecutive days to know when the oxidative damage in skeletal muscle happened, with acute or
chronic CS exposure.
Methods and Results:
Thirty-six C57BL-6 mice were divided into six groups (n = 6): control group, 7-, 15-, 30-, 45- and 60-day exposure to CS,
respectively. The CS groups were exposed to cigarette smoke 3 times/day (4 cigarettes/session). Twenty-four hours after the last
cigarette exposure, the animals were sacrificed and used for subsequent assays. In addition to the analysis of lipid and protein
oxidation (TBARS and Carbonylation), sulfhydryl assay, histological and morphological studies were performed. The data
demonstrated the relationship between the oxidative damage measured through lung, diaphragm and quadriceps tissues.
Conclusions:
Our data demonstrated the influence of CS in oxidative damage not only in lung, but in skeletal muscle, showing the diffusion
effect of CS and that skeletal muscle are affected before the lung tissue.
Keywords: cigarette smoke, mice, Oxidative damage, skeletal muscle
Financial Support: Universidade do Extremo Sul Catarinense- UNESC
Resumo:08-045
ESTRADIOL MEDIATES THE ACUTE LUNG INFLAMMATION AFTER INTESTINAL ISCHEMIAREPERFUSION (I/R) IN FEMALE AND MALE RATS.
Fantozzi, E. T.
Farmacologia/ICB, USP
Objectives:
Sexual dimorphism interferes with Th1 and Th2 lymphocyte functions, and accordingly estradiol (e2) plays a role on developing
acute lung injury (ali) caused by intestinal I/R. Female rats are more resistent to organ injury caused by hemorrhagic shock. Nitric
oxide (NO) exerts pro and antinflammatory effects in gut trauma-induced lung injury, but its interaction with estradiol is still not
clear. Here we assessed the effect of E2 on the lung and intestinal inflammation after intestinal I/R and investigated the putative
involvement of inducible NOS (iNOS) pathway.
Methods and Results:
Anesthetized wistar male and female rats (60 days, 200g) were subjected to occlusion of the superior mesenteric artery (SMA) for
45 min, followed by 2 hr of reperfusion. Groups of rats (n=5) were treated with E2 (17β estradiol, 280μg/kg, s.c. 24 h before or
30 min (i.v.) after induction of ischemia. In parallel, rats were treated with selective iNOS inhibitor, aminoguanidine (50 mg/kg
iv) or with L-arginine (300mg/kg, i.p.) 1h before arterial occlusion. Lung and intestinal vascular permeability (LVP and IVP)
were assessed by Evans blue dye extravasation and neutrophil recruitment to the tissues by the myeloperoxidase (MPO) method.
Lung generation of IL-10 was also assessed by ELISA in cultured lung tissue. 7-days-ovariectomized (OVx) rats had LVP, IVP
and MPO increased after I/R whereas IL-10 levels were significantly reduced. E2 treatment reverted the increased LVP, but not
IVP nor MPO. Aminoguanidine alone reduced LVP in OVX rats, but failed to do so when associated to E2. Aminoguanidine in
non-OVx rats markedly exacerbated LVP and IVP. Male rats treated with E2 were protected with a significant reduction of LVP.
Conclusions:
Endogenous NO from iNOS, likely reduces the vascular effects of intestinal I/R. In addition, L-arginine imitated E2 since it
reduced ALI after intestinal I/R. In conclusion, estradiol reduces lung inflammation after intestinal I/R, but the concomitant
blockade of NOS suppresses this protection.
Keywords: Intestinal isquemia-reperfusion , Pulmonary inflammation, Rats, Estradiol, Nitric oxide
Financial Support: CNPq and FAPESP.
Resumo:08-046
INFLUENCE OF DIFFERENT PHASES OF ESTROUS CYCLE IN THE VENTILATORY RESPONSES TO
HYPERCAPNIA AND HYPOXIA IN RATS
Marques, D. A. 1,1; Gargaglioni, L. H. 1; de Carvalho, D. . 1; Szawka, R. E. 2; Franci, J. A. 3; Bícego, K. C. 1
1
Morfologia e Fisiologia Animal, UNESP, FCAVJ
3
Laboratório de Neuroendocrinologia I, USP
2
Departamento de Fisiologia e Biofísica, UFMG
Objectives:
Determine the respiratory and thermoregulatory responses to hypoxia and hypercapnia in rats at each stage of the estrous cycle
Methods and Results:
The present study examined the hypoxic (HVR) and hypercapnic (HCVR) respiratory responses of female rats weighing 250300g, in different phases of estrous cycle: estrus, diestrus, proestrus and also in ovariectomized rats. Ventilation was measured
using a whole-body plethysmograph (Bartlett and Tenney, 1970). Vaginal smears were taken daily to verify estrous cycle
regularity and only rats showing at least five consecutive regular 4-day estrous cycles were included in this study. Pulmonary
ventilation (VE) and body temperature (Tb) were measured followed by 30 min of hypercapnic exposure (7% CO2 in air) and
hypoxia (5% O2) in unanesthetized animals. Under resting conditions, there is no difference in VE between animals. Tb was
greater in estrus phase compared to other groups during air and 7%CO2. Hypoxia caused an increase in VE and a drop in Tb in
all groups, but the HVR was smaller in diestrus (717.8 ± 41.5 mL/Kg/min), proestrus (712.1 ± 34.1 mL/Kg/min), and in
ovariectomized (516.6 ± 13.4 mL/Kg/min) rats compared to estrous phase (1213.2 ± 284 mL/Kg/min), due to a reduced tidal
volume. Hypoxia-induced drop in Tb was not different between groups. HCVR was also reduced in diestrus (978.6 ± 88.9
mL/Kg/min), proestrus (1056.7 ± 108.9 mL/Kg/min), and in ovariectomized rats (717.3 ± 71.2 mL/Kg/min), due to a decreased
tidal volume. Thus, the different phases of the estrous cycle influence the respiratory responses to hypercapnia and hypoxia in
rats, acting on tidal volume. Additionally, Tb is increased in estrous cycle during normoxia, normocapnia and hypercapnia but is
not different in hypoxic challenge.
Conclusions:
The different phases of the estrous cycle influence the respiratory responses to hypercapnia and hypoxia in rats, acting on tidal
volume.
Keywords: estrous cycle, ventilatory responses, hypercapnia, hypoxia
Financial Support: FAPESP and CNPq
Resumo:08-047
EFFECTS OF OLEANOLIC ACID ON PULMONARY MORPHOFUNCTIONAL AND BIOCHEMICAL VARIABLES
IN EXPERIMENTAL SEPSIS
Ramos, M. B. D. A. 1; Santos, R. S. 1; Silva, P. L. 1; Oliveira, G. P. D. 1; Cruz, F. F. 1; Assis, E. F. D. 2;
Castro-faria-neto, H. C. D. 2; Gattass, C. R. 1; Rocco. , P. R. M. 1
1
Instituto de Biofísica Carlos Chagas Filho, IBCCF
2
Instituto Oswaldo Cruz , IOC
Objectives:
Sepsis and its associated complications, such as acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and multiple
organ failure, present high morbidity and mortality despite recent advances in therapeutic strategies. Recently, natural products
derived from plant extracts and their synthetic derivatives are being increasingly used to treat a wide range of diseases due to
their anti-inflammatory property. In this line, oleanolic acid (OA), a triterpenoid compound present in a great variety of plants
and food products, modulates the production and activity of pro-inflammatory cytokines and enzymatic antioxidant defense. The
aim of the present study was to test the hypothesis that OA may curtail the inflammatory process, improving lung morphology
and function in experimental sepsis.
Methods and Results:
36 BALB/c mice were randomly assigned into two groups: a) Sepsis was induced by cecal ligation and puncture (CLP) surgery,
and b) Sham operated group was used as control. One-hour after surgery, Sham and CLP groups were further randomized into
subgroups receiving saline (SAL), OA (10 mg/kg ip), or dexamethasone (DEXA, 1 mg/kg ip). After 24 hours, animals were
anesthetized, and static lung elastance (Est, L), lung histology (light microscopy), levels of interleukin (IL)-6, and IL-8 in the
bronchoalveolar lavage fluid and the degree of cell apoptosis in lung and kidney were analyzed. Est, L (55%) and alveolar
collapse (75%) were higher in CLP-SAL group compared to Sham-SAL (p< 0.05). Furthermore, CLP-SAL group compared
showed interstitial edema, polymorphonuclear cell infiltration, and high degree of epithelial cell apoptosis in lung and kidney
compared to Sham-SAL. The treatment with OA and DEXA reduced Est, L (35%, and 42%, respectively) and alveolar collapse
(70%, and 64%) in relation to CLP-SAL group. The OA and DEXA treatment minimized the polymorphonuclear cell infiltration
and the degree of apoptosis in lung and kidney compared to CLP-SAL group. However, DEXA, but not OA reduced the levels of
IL-6, and IL-8 (p<0.05).
Conclusions:
In the present model of sepsis, both OA and DEXA improved lung morpho-function, as well as acted on distal organs.
Nevertheless, the mechanisms related to these beneficial effects differ in OA and DEXA. Therefore further studies are required to
better elucidate the mechanism of action of OA in sepsis.
Keywords: Acute Lung Injury, Interleucin, Histology, Lung mechanics, Oleanolic Acid
Financial Support: CNPq, PRONEX-FAPERJ, FAPERJ, CAPES, INCT-INOFAR
Resumo:08-048
FREQUENCY OF TIME-CYCLED CONTROL BREATHS DURING BIPHASIC POSITIVE AIRWAY PRESSURE
MODIFIES THE EXPRESSION OF LUNG INFLAMMATORY AND FIBROGENIC MEDIATORS IN PULMONARY
AND EXTRAPULMONARY ACUTE LUNG INJURY.
Saddy, F. ; Moraes, L. ; Oliveira, G. P. ; Santos, C. L. ; Cruz, F. C. ; Morales, M. M. ; Garcia, C. S. ; Pelosi,
P. ; Rocco, P. R.
Laboratório de Investigação Pulmonar/IBCCF, UFRJ
Objectives:
To compare the effects of different frequencies of time-cycled control breaths during biphasic positive airway pressure (BiVent)
with pressure controlled ventilation (PCV) on lung histology, arterial blood gases, inflammatory and fibrogenic mediators in
models of pulmonary (p) and extrapulmonary (exp) acute lung injury (ALI) with similar transpulmonary pressures.
Methods and Results:
Wistar rats were randomly divided into two groups. In ALI groups, animals received Escherichia coli lipopolysaccharide
intratracheally (100 microg, ALIp) or intraperitoneally (1 mg, ALIexp). After 24 h, animals were anesthetized and further
randomized as follows: 1) pressure controlled ventilation mode (PCV) with tidal volume (VT)=6 ml/kg and inspiratory to
expiratory ratio (I:E)=1:2; 2) BiVent with three different time cycles (controlled/spontaneous breath cycles 50, 75 and 100).
BiVent was set with two levels of CPAP [inspiratory pressure (PHigh=10cmH2O) and positive end-expiratory pressure
(PLow=5cmH2O)] and the inspiratory time was sustained as THigh=0.3s. All rats were mechanically ventilated with FiO2=0.4
and PEEP=5cmH2O for 1 hour. BiVent yielded better functional improvement and less lung injury compared to PCV
independent of ALI etiology. BiVent 50 improved gas exchange and reduced atelectasis compared to PCV, BiVent75 and
BiVent100 in both ALI groups. Interleukin (IL)-6, IL-1 beta, and type III procollagen (PCIII) expressions were lower in BiVent
compared to PCV. BiVent 50 presented lower mRNA expression of IL-6, IL-1 beta, and PCIII compared to PCV, BiVent75, and
BiVent100 in both ALI groups.
Conclusions:
BiVent presented greater beneficial effects on respiratory function and a reduction in lung injury compared to PCV. Among the
different frequencies of BiVent, BiVent50 demonstrated better functional results with less lung damage and expression of
inflammatory mediators independent of ALI etiology.
Keywords: Mechanical ventilation, Ventilator induced lung injury, Acute lung injury, Inflamatory mediators
Financial Support: PRONEX, FAPERJ, CNPq
Resumo:08-049
PULMONARY AND LIVER INJURY AFTER CHRONIC EXPOSURE TO SUBLETHAL DOSES OF MICROCYSTINLR.
Carvalho, G. M. C. 1; Oliveira, V. R. D. 1; Casquilho, N. V. 1; Soares, R. M. 1; Azevedo, S. 1; Pires, K. M. 2;
Valença, S. S. 2; Zin, W. A. 1
1
Universidade Federal do Rio de Janeiro, UFRJ
2
Universidade do Estado do Rio de Janeiro, UERJ
Objectives:
Biological pollution caused by cyanotoxins leads to respiratory function impairment. Thus, we aimed to study pulmonary
mechanics, lung and liver histology in mice chronically submitted to sublethal doses of microcystin-LR (MCLR) and evaluate
whether the results depended on the doses.
Methods and Results:
Male Swiss mice received ten doses of distillated water intraperitoneally (ip, 100 mL, CTRL n=6) or sublethal doses of MCLR
(5, 10, 15 and 20 ìg/kg ip in 100 mL of distilled water, TOX n=24) on alternate days. 24 h after the last injection pulmonary
mechanics [static elastance (Est), viscoelastic component of elastance (ÄE), resistive (ÄP1), viscoelastic (ÄP2), and total (ÄPtot)
pressures] were determined, and lungs and livers were prepared for histopathology. ANOVA was used to test differences among
the groups. Est, ÄE, ÄP1, ÄP2 and ÄPtot were significantly higher than CTRL [25.7 ± 0.60 cmH2O/mL; 3.73 ± 0.14
cmH2O/mL; 0.61 ± 0.04 cmH2O; 0.75 ± 0.03 cmH2O; 1.36 ± 0.07 cmH2O (mean ± SEM)] in TOX10 (30.9 ± 1.40 cmH2O/mL;
5.1 ± 0.41 cmH2O/mL; 0.9 ± 0,12 cmH2O; 1.1 ± 0.09 cmH2O; 2.0 ± 0.17 cmH2O), TOX15 (33.6 ± 1.59 cmH2O/mL; 4.9 ± 0.36
cmH2O/mL; 0.8 ± 0.02 cmH2O; 1.0 ± 0.06 cmH2O; 1.8 ± 0,06 cmH2O) and TOX20 (38.9 ± 4.8 cmH2O/mL; 5.4 ± 0.74
cmH2O/mL; 0.9 ± 0.07 cmH2O; 1.1 ± 0.13 cmH2O; 2.0 ± 0.21 cmH2O), but did not differ among them. TOX5 (30.4 ± 2.25
cmH2O/mL; 4.3 ± 0.23 cmH2O/mL; 0.7 ± 0.01 cmH2O; 0.9 ±0.03 cmH2O; 1.5 ± 0.04 cmH2O) was similar to CTRL in all
parameters. Alveolar collapse was higher in TOX15 (22.2%) and TOX20 (32.6%) than in CTRL (10.8%). The lung inflammatory
cell content (cells/ìm2) increased in all MCLR doses, but did not differ among them: TOX5=1.88 x 10-3, TOX10=2.07 x 10-3,
TOX15=2.19 x 10-3 and TOX20= 2.17 x 10-3 in relation to CTRL= 0.79 x 10-3. The liver weight was significantly higher than
CTRL= 1.75 g in TOX5= 2.06 g, TOX10= 2.12 g, TOX15= 2.19 g and TOX20= 2.15 g, but did not differ among them. All TOX
mice showed a complete loss of liver architecture with hepatomegaly, steatosis, dilated sinusoidal spaces, necrosis, inflammatory
foci and a high degree of binucleated hepatocytes.
Conclusions:
Chronic exposure to MCLR impaired pulmonary mechanics, lung and liver histology. These findings did not depend on the
degree of exposure.
Keywords: Chronic Exposition, Liver Histology, Lung Histology, Microcystin-LR, Respiratory Mechanics
Financial Support: FAPERJ, CNPq, MCT.
Resumo:08-050
ANTIOXIDANT TREATMENT PROTECTS AGAINST MATRIX METALLOPROTEINASES ACTIVATION AND
CARDIOMYOCYTE INJURY DURING ACUTE PULMONARY THROMBOEMBOLISM.
Santos-o. S1; Neves-e. M. N. 1; Ferraz, K. C. 2; Ceron, C. S. 1; Rizzi, E. 1; Gerlach, R. F. 3; Santos- J. E. T. 1
3
Department of Morphology, Estomatology and Physiology,, FORP-USP
2
Department of Pharmacology, State University of Campinas, UNICAMP
1
Department of Pharmacology, Faculty of Medicine of Ribeirao , FMRP-USP
Objectives:
Increased reactive oxygen species (ROS) promote matrix metalloproteinases (MMPs) activities and may underlie cardiomyocyte
injury and degradation of cardiac troponin I (cTI) during acute pulmonary thromboembolism (APT). We examined whether
pretreatment with tempol (a general ROS scavenger) prevents MMPs activation and protects against cardiomyocyte injury of
APT. We have also examined the therapeutic effects of tempol administration after APT.
Methods and Results:
Hemodynamic evaluations were performed in non-embolized sheep treated with saline (n=4) and in sheep that were embolized
with autologous blood clots (n=8) and that received continuous infusion of tempol (1.0 mg/kg/min) before (n=7) and after
induction of APT (n=7). The activity of MMPs and reactive oxygen species in right ventricle, as well as the levels of serum TnIc
were measured by zymography, DHE and enzyme immunoassay, respectively. APT increased the mean pulmonary arterial
pressure and pulmonary vascular resistance by approximately 146% and 164%, respectively (both P
Conclusions:
We found evidence supporting the idea that RV oxidative stress after APT increases MMPs activities in the RV leading to
degradation of sarcomeric proteins including cTI. Antioxidant treatment may prevent MMPs activation and protect against
cardiomyocyte injury after APT.
Keywords: acute pulmonary thromboembolism, matrix metalloproteinases, oxidative stress, pulmonary hypertension, troponin
Financial Support: FAPESP, CAPES and CNPq
Resumo:08-051
PULMONARY AND RENAL EFFECTS OF INTRAVASCULAR VOLUME REPLACEMENT IN A NON-SEPTIC
MODEL OF ACUTE LUNG INJURY
Silva, P. L. 2,1; Güldner, A. 1; Uhlig, C. 1; Carvalho, N. R. 1; Beda, A. 1; Rentzsch, I. 1; Kasper, M. 3; Pelosi,
P. 4; Rocco, P. R. M. 2; Abreu, M. G. D. 1
2
Laboratory of Pulmonary Investigation, IBCCFº, UFRJ
1
1Department of Anesthesiology and Intensive Care Therapy, TU Dresden
3
Institute of Anatomy, TU Dresden
4
Dipartimento di scienze chirurgiche e diagnostiche integrat, University of Genua
Objectives:
Intravascular volume replacement may be necessary during acute lung injury (ALI). The aim of the present study was to compare
the effects of Ringer‘s acetate (RA), gelafundin (GEL), and hydroxyethyl starch (HES) on lung and renal damage in experimental
ALI.
Methods and Results:
Non-septic ALI was induced in pigs (36±3 kg) by lung saline lung lavage followed by ventilation with high tidal volumes.
Protective ventilation was then initiated and 25% of the estimated blood volume was drawn. The intravascular volume was
partially replaced with RA, GEL or HES (n=10/group) to achieve 90% of the intrathoracic blood volume index (ITBVi) before
blood drainage. The amount of fluid to achieve the target ITBVi was higher with RA (2250±764 ml) than GEL (704±159 ml) and
HES (837±82 ml). Gas exchange and hemodynamic variables did not differ among groups. Lung elastance was higher in RA and
GEL than HES. The W/D ratio was more elevated in RA (7.9±1.0) than GEL (6.5±0.5) and HES (6.5±0.7), (p
Conclusions:
In this non-septic ALI model, volume replacement with RA worsened lung mechanics and histology as well as pro-inflammatory
lung response and edema compared to GEL and HES. However, GEL led to higher acute kidney injury score than other groups.
Keywords: Acute Lung Injury, Intravascular Volume, Lung Histology, Mechanical Ventilation, Wet-to-Dry Ratio
Financial Support: DFG, CAPES-DAAD, CNPq, PRONEX, FAPERJ
Resumo:08-052
EFFECTS OF DIFFERENT TYPES OF POLYMERIC LIPOSOMES USED FOR GENE DELIVERY ON LUNG
MECHANICS AND HISTOLOGY
Xisto, D. G. 1,2; Abreu, S. C. 1; Silva, J. D. 1; Silva, A. L. 1; Crossetti, J. 1; Martini, S. V. 2; Temprana, C. F.
3
; Alonso, S. V. 3; Rocco, P. R. M. 1; Morales, M. M. 2
1
Laboratory of Pulmonary Investigation, IBCCF, UFRJ
2
Laboratory of Cellular and Molecular Physiology, IBCCF, UFRJ
3
LBM, Department of Science and Technology, UNQ
Objectives:
The benefits of gene therapy for the treatment of respiratory diseases have been disappointing due to viral vectors toxicity and
immunogenicity. Consequently, the need for safer alternatives has led to the development of polymeric liposomes. However,
some particles may also lead to toxicity limiting their use. The aim of this study was to analyze the impact of different polymeric
liposomes on lung mechanics and histology.
Methods and Results:
48 BALB/c mice were randomly divided into 6 groups, which were intratracheally instilled with 50 ul of 5 mM of the following
suspensions: DMPC:DC8,9PC:DOTAP, polymerized (A), DMPC:DC8,9PC:SA, polymerized (B), DMPC:DC8,9PC: miristoyl
choline chloride (MCl), polymerized (C), DMPC:DC8,9PC:DOTAP, non-polymerized (D), DMPC:DC8,9PC:SA, nonpolymerized (E) and DMPC:DC8,9PC: MCl, non-polymerized (F). At twenty-four hours, the animals were anesthetized,
tracheotomized and mechanically ventilated. Lung static elastance, resistive and viscoelastic/inhomogeneous pressures were
analyzed by end-inflation occlusion method. Lungs were fixed and stained for histopathological analysis. Resistive and
viscoelastic pressures and static elastance were similar in all groups, as well as tissue cellularity. However, animals treated with
non-polymerized particles presented higher mortality rate (60%).
Conclusions:
The present polymeric liposomes particles did not impair lung mechanics, histology or increase tissue cellularity. However, nonpolymerized liposomes particle presented higher systemic toxicity with greater mortality.
Keywords: liposomes, lung mechanics, lung histology
Financial Support: FAPERJ, CAPES, CNPq, PRONEX-FAPERJ
Resumo:08-053
DIAPHRAGMATIC MOTION STUDIED BY B-MODE ULTRASONOGRAPHY IN MORBID OBESE SUBJECTS
Tenório, L. H. S. 1; Amaral, F. J. 2; Neto, J. B. C. 3; Passos, V. M. M. 1; Santos, A. C. 5; Lima, A. M. J. 4;
Brasileiro-santos, M. S. 5
1
Fisioterapia, UFPE
Radiologia/Hospital Barão de Lucena, HBL
3
Cirurgia geral/Hospital Agamenon Magalhães, HAM
4
Departamento de Morfologia e Fisiologia Animal, UFRPE
5
Educação Física, UFPB
2
Objectives:
Diaphragm mobility can be assessed by several images techniques, however there is no data about the assessment of diaphragm
mobility by ultrasound in morbid obese patients. Therefore, the aim of this study was to evaluate diaphragm mobility by
ultrasound B-mode in individuals with morbid obesity.
Methods and Results:
Thirty-one patients were evaluated of both genders, with mean age of 35.5± 9.7 years, body mass index of 43.8±3.3 Kg/m2 and
with no previous history of cardiac or respiratory illness. The diaphragm mobility (DM) was assessed by the ultrasound Philips
HD7 (Philips Medical Systems, Bothell, WA, USA) with a convex transducer of 2.-5MHz of high resolution in B-mode. The DM
was observed in three moments: quiet breath (QB), voluntary sniffing (VS) and deep breathing (DB) as we used the liver window
on the right side to assess these three moments. Data distribution was analyzed by the Kolmogorov-Smirnov test and analysis of
variance (ANOVA) of one factor. Results are expressed as means ± standard deviation and as 95% confidence limits. The DM
was assessed with success during the QB, VS and DB on the right hemidiaphragmatic side in all obese subjects. After inferential
analysis it was observed significant differences at DM during the QB and VS (1.87±0.49cm vs. 3.49±1.17 cm, p<0,001).
Conclusions:
According to the results of this work was shown that B-mode ultrasonography is a reproducible method to assess the hemi
diaphragmatic motion in the morbidly obese.
Keywords: Ultrasonography , Morbid Obesity, Diaphragm
Resumo:08-054
IMPACT OF EXERCISE ON INFLAMATORY PROCESS AND REMODELING IN A MURINE MODEL OF
CHRONIC ALLERGIC INFLAMMATION.
Marques, P. S. ; Araújo, C. C. ; Samary, C. S. ; Guimarães, I. H. ; Crosseti, J. C. ; Xisto, D. G. ; Rocco, P.
R. M.
Universidade Federal do Rio de Janeiro, UFRJ / IBCCF
Objectives:
Asthma is a common disease associated with inflammatory and remodeling changes in airway and lung parenchyma. Regular and
moderate aerobic exercise has been used as an adjunct therapy to minimize the inflammatory process. However, so far, no study
has analyzed whether exercise may minimize or even reverse airway remodeling in asthma. The aim of this study was to
investigate the effects of regular and moderate aerobic exercise on the inflammatory and remodeling processes in a murine model
of chronic allergic inflammation.
Methods and Results:
28 BALB/c mice (female, 20-25g) were randomly assigned into 2 groups: in OVA group, mice were immunized by
intraperitoneal injection of ovalbumin (OVA, 10 µg, ip) on 7 alternate days. After day 40, they were challenged with three
intratracheal instillations of OVA (20 µg, it) at 3-days intervals. Control group (C) received saline using the same protocol.
Twenty-four hours after the last challenge animals were further subdivided into 2 groups: sedentary (S) and trained (T). T group
ran on a motorized treadmill at moderate intensity (8-12 m/min), 5% grade, 30 min/day, 3 times a week for 4 weeks. Twenty-four
hours after the last training session, lung mechanics and histology, as well as collagen fiber content in the airways were
evaluated. OVA-S group presented higher static lung elastance (63%), airway resistance (100%) and viscoelastic pressure (78%),
as well as fraction area of alveolar collapse (36%), mononuclear cells (50%), polymorphonuclear cells (37%), and collagen fiber
content (23%) compared to C-S group. OVA-T group showed a reduced static lung elastance (22%), airway resistance (65%),
viscoelastic pressure (37%), fraction area of alveolar collapse (36%), number of mononuclear cells (34%) and collagen fiber
(16%) content compared to OVA-S.
Conclusions:
In the present model of chronic allergic inflammation, regular and moderate aerobic exercise attenuated the inflammatory and
remodeling processes, improving lung mechanics.
Keywords: exercise, asthma, process inflamatory, female, remodeling
Financial Support: CNPq, FAPERJ, PRONEX, FAPERJ
Resumo:08-055
THE IMPACT OF TWO DIFFERENT SOURCES OF MESENCHYMAL STEM CELLS IN A MURINE MODEL OF
ELASTASE-INDUCED EMPHYSEMA
Antunes, M. A. 1; Abreu, S. C. 1; Cruz, F. F. 1; Oliveira, A. C. R. 1; Xisto, D. G. 1; Paz, A. H. R. 2; Cirnelima, E. O. 2; Morales, M. M. 1; Rocco, P. R. M. 1
1
Instituto de Biofísica Carlos Chagas Filho/CCS, UFRJ
2
Laboratório de Embriologia e Diferenciação Celular, HCPA
Objectives:
Mesenchymal stem cells (MSCs) represent a promising tool for respiratory diseases. Bone marrow (BM) was the first source
reported to contain MSCs. However, for clinical use, BM may be detrimental due to the highly invasive donation procedure and
the decline in MSC number and differentiation potential with increasing age. More recently, adipose tissue (AD) was introduced
as an alternative source for MSCs. We compared the effects of MSCs derived from these sources on lung mechanics and
histology in a murine model of elastase-induced emphysema.
Methods and Results:
Thirty female C57BL/6 mice (20-25 g) were randomly assigned into 2 groups. In control (C) animals, saline was intratracheally
(it, 50 ìL) injected, whereas emphysema mice received porcine pancreatic elastase (ELA, 0.1 UI, 50 ìL, it). Saline and ELA were
intratracheally injected once a week during 4 weeks. Three hours following the last instillation, C and emphysema groups were
further randomized into subgroups receiving saline (SAL, 50 ìL), BM-MSC, and AD-MSC (1x105) intravenously. Adipose tissue
was harvested from mouse epididymal fat. After one week, lung mechanics (static elastance, resistive and viscoelastic pressures),
and histology (total and differential cell count in lung tissue, and fraction area of normal, collapsed and hyperinflated alveoli)
were analyzed. Lung static elastance, viscoelastic pressure, the number of polymorphonuclear cells in the tissue, and the fraction
area of alveolar hyperinflation were higher in ELA-SAL compared to C-SAL. Both BM-MSCs and AD-MSCs reduced similarly
lung static elastance (ELA-SAL: 39±3; ELA-BM-MSC: 31±3; ELA-AD-MSC: 28±0.5 cmH2O/ml, Mean±SD; p
Conclusions:
MSCs from bone marrow and adipose tissue reduced the inflammatory process improving lung mechanics. However, at day 7 no
significant repair was observed.
Keywords: adipose, bone-marrow, elastase, emphysema, stem cell
Financial Support: PRONEX-FAPERJ, CNPq, FAPERJ, CAPES
Resumo:08-056
ESTABLISHMENT OF A CONTROLLED SWINE MODEL OF THE ACUTE RESPIRATORY DISTRESS
SYNDROME
Gomes, S. 1; Hirota, A. 1; Costa, Elv 1; Barbeiro, Df 3; Tucci, Mr 1; Beraldo, Ma 1; Nascimento, Ec2;
Belmino, R. 1; Carvalho, Crr1; Amato, Mbp 1
1
Divisão de Pneumologia - INCOR, HCFMUSP
2
Depto de Patologia, FMUSP
3
Lab de Emergências Clínicas, FMUSP
Objectives:
The Acute Respiratory Distress Syndrome (ARDS) is defined in humans, but there is no agreement on the main characteristics of
acute lung injury in animals. The aim of this study is to establish an animal model of lung injury, which is stable and similar to
human ARDS
Methods and Results:
Methods: We tested 32 pigs (Landrace) in 4 groups (Sham n=5, Injury with histology n=7, Injury without histology n=20, and
Long term protocol ARDSNET n=10) applying a two-hit injury: first, massive lung lavage till PaO2/FiO2
Conclusions:
This model of severe ARDS mimics human ARDS, exhibiting most of its functional/inflammatory features, also presenting
stability for long periods of time.
Keywords: lung injury, experimental model, ards
Financial Support: FINEP, CNPq, CAPES e HCFMUSP
Resumo:08-057
EFFECTS OF DIFFERENT POSITIVE END EXPIRATORY PRESSURE LEVELS ON LUNG AND DISTAL ORGANS
IN MODELS OF PULMONARY AND EXTRAPULMONARY ACUTE LUNG INJURY WITH OR WITHOUT INTRAABDOMINAL HYPERTENSION.
Miranda, P. J. B. 2; Santos, C. L. 2; Oliveira, M. G. 2; Moraes, L. 2; Santos, R. S. 2; Silva, J. D. 2; Morales,
M. M. 3; Capelozzi, V. L. 4; Rocco, P. R. M. 2; Garcia, C. S. N. B. 2
2
Laboratory of Pulmonary Investigation, IBCCF, UFRJ
3
Laboratory of Cellular and Molecular Physiology, IBCCF, UFRJ
4
Department of Pathology, School of Medicine, , USP
Objectives:
Pulmonary (p) and extra-pulmonary (exp) acute lung injury (ALI) differ according to their pathophysiology, even though ALI
patients undergo similar ventilatory therapy. This study investigated the impact of different positive end-expiratory pressure
(PEEP) levels in ALIp and ALIexp in the presence or absence of intra-abdominal hypertension (IAH).
Methods and Results:
Wistar rats (n=72) were randomly allocated to receive Escherichia coli lipopolysaccharide intratracheally (200 µg, ALIp) or
intraperitoneally (1 mg, ALIexp). After 24 hours, they were randomized into subgroups with or without IAH. Animals developed
IAH by inserting gauze into abdominal cavity reaching a maximum stabilized intra-abdominal pressure equal to 15 mmHg. They
were then ventilated with FiO2=0.4, VT = 6 ml/kg, and PEEP = 5, 7 or 10 cmH2O during 1 hour (End). Respiratory system (rs),
lung (L) and chest wall (w) static elastances (Est), arterial blood gases, lung histology (light and electron microscopy), interleukin
(IL)-1&beta, IL-6, caspase-3 and type III procollagen (PCIII) mRNA expressions in lung tissue, as well as the number of lung,
liver, kidney and villi cell apoptosis were analyzed. Est,rs, Est,L, Est,w, PaO2, PaCO2, and pHa were comparable at Baseline in
all groups. In the presence of IAH, we found that: 1) PEEP of 5, 7 or 10 cmH2O led to oxygenation improvement independent of
ALI etiology (30%); 2) Est,w increased after surgery (38%) and remained increased until End; 3) PEEP7 led to lower Est,L and
less alveolar collapse in ALIexp, but yielded reduced alveolar-capillary membrane, epithelial and endothelial damage, cell
apoptosis in lung tissue, as well as mRNA expression of IL-1β, IL-6, PCIII, and caspase-3 in both ALI groups; 4) PEEP10
resulted in hyperinflation in both ALI groups; 5) IL-1&beta, IL-6 PCIII, and caspase-3 mRNA expressions were higher only in
ALIexp ventilated with PEEP10 as well as in ALIp with PEEP5; and 6)liver, kidney, and villi cell apoptosis increased
independent of PEEP level.
Conclusions:
In the presence of IAH, PEEP7 improved lung mechanics and morphology and induced less epithelial, endothelial, and alveolar
capillary membrane damage as well as cell apoptosis in lung tissue independent of ALI etiology. However, in ALIexp, PEEP10
led to increased expression of IL-1β, IL-6 PCIII, and caspase-3 in lung tissue while lower PEEP resulted in higher expression of
these mediators in ALIp. Therefore, PEEP should be set not only according to IAH but also to the etiology of ALI.
Keywords: acute lung injury, intra-abdominal hypertension, cytokines, type III procollagen, apoptosis
Financial Support: CNPq, PRONEX-FAPERJ, FAPERJ, CAPES
Resumo:08-058
ACTIVITY OF THE COMPOUNDS IQG-607 AND IQG-639 IN A MURINE MODEL OF TUBERCULOSIS
Rodrigues-junior, V. S. 1,2; Santos, A. A. Jr. 1,3; Jader, A. S. 1; Ritter, E. 1; Souto, A. A. 1; Calixto, J. B. 4,1;
Basso, L. A. 1,3; Santos, D. S. 1,3; Campos, M. M. 1,2,5
1
Instituto Nacional de Ciência e Tecnologia em Tuberculose, PUC RS
5
Instituto de Toxicologia, InTox, PUC RS
2
Programa de Pós-Graduação em Medicina e Ciências da Saúde, PPGMCS, PUC RS
3
Programa de Pós-Graduação em Biologia Celular e Molecular, PPGBCM, PUC RS
4
Departamento de Farmacologia, UFSC
Objectives:
Tuberculosis (TB) continues to be one of the deadliest diseases in the world. The emergence of multi-drug-resistant strains of
Mycobacterium tuberculosis, the unbearable side effects of the available drugs, and the frequent patient non-compliance in
completing the therapy have increased the need for development of new effective agents. Isoniazid (INH) is the most prescribed
drug for active TB and prophylaxis, and requires activation by the enzyme KatG. The M. tuberculosis enoyl-ACP reductase
(InhA) has been shown to be the primary target for INH. Our group has published the rational design and synthesis of new
isoniazid-derived compounds with possible anti-TB activity. Importantly, it was shown that these compounds do not require
activation by KatG to bind its molecular target InhA. We have also demonstrated that INH analogs are able to inhibit the activity
of wild-type and INH-resistant M. tuberculosis InhA in vitro. This work describes the pre-clinical in vivo evaluation of two
pentacyano(isoniazid)ferrateII complexes, named IQG-607 and IQG-639, in a mouse model of TB.
Methods and Results:
All the experimental protocols were approved by the local Animal Ethics Committee (CEUA 09/00094). Male Swiss mice (N=5,
25-30 g) were intravenously infected, with 1 x 107 viable M. tuberculosis bacilli suspended in 0.2 ml of Middlebrook 7H9
medium. Treatment was started 5 days post-infection, and the compounds IQG-607 and IQG-639 (250 mg/kg) were administered
orally, once a day, during 28 or 56 days. Separate groups of mice were treated with the reference drug INH (25 mg/kg; positive
control) or saline solution (negative control). At the end of treatments, mice were euthanized by isoflurane inhalation. The
spleens and lungs were aseptically removed, and the spleen weight (in grams) was determined. These organs were homogenized
and the colony-forming units (CFU) were determined after 30 days of incubation in solid medium, at 37 °C. After 28 days of
treatment, either IQG-607 or INH significantly reduced M. tuberculosis-induced splenomegaly, with inhibition percentages of 50
and 58 %, respectively. Interestingly, after 56 days of treatment, the reduction of the splenomegaly by either IQG-607 or INH
was increased, with inhibition percentages of 67 and 66 %, respectively. Moreover, after 28 or 56 days of treatment, CFU
numbers were significantly reduced in either spleens or lungs of IQG-607- and INH-treated groups. Otherwise, IQG-639 was not
capable of significantly modifying any evaluated parameter.
Conclusions:
The promising activity of IQG-607 in M. tuberculosis-infected mice suggests that it is a good candidate for clinical development
as a new anti-tuberculosis agent. Additional experiments are being currently performed to determine the pharmacokinetic and
toxicological parameters of this compound.
Keywords: Tuberculosis, InhA inhibitors, Preclinical Test, Drug Development
Financial Support: INCT-TB CNPq, BNDES, FINEP, CAPES
Resumo:08-059
ELECTROPHYSIOLOGICAL STUDIES OF THE DIAPHRAGM OF ELASTASE-INDUCED EMPHYSEMA IN
MICE.
Cã¢ndido, S. C. O; Teodoro, L. C. ; Zavan, B. ; Bento, A. C. ; Paiva, A. G. ; Junior, V. A. P. ; Soncini, R.
Ciências Biomédicas - Universidade Federal de Alfenas, UNIFAL - MG
Objectives:
Emphysema, a type of obstructive disease (COPD), has morphological and functional changes of the whole lung tissue. The
changes are generally in respiratory mechanics, increased air spaces, alteration of histological parameters and airway extracellular
matrix of the. The aim of this study was to evaluate electrophysiological and histochemical and imunohistochemical changes of
the diaphragm of elastase-induced emphysema in mice.
Methods and Results:
Materials and methods: Thirty-two Swiss male mice (25-30g) were randomly divided into control group and emphysema. The
control group received intratracheal instillation of saline, single dose (Sal x 1) and double dose (Sal x 2). The emphysema group
received intratracheal instillation of porcine pancreatic elastase, dose 0,3 U/mouse, single dose (0,3 U x 1) and double dose (0,3
U x 2). Twenty days after the first instillation, the mice were decapitated to isolate from the left hemi-diaphragm fragment of 5
mm width . The fragments were suspended in an isolated organ bath filled with Krebs with d-tubocurarine. After 30 min, was
started the electrical stimulation to construct the curve force-frequency, using a voltage to maximal stimulus. Power Lab system
was used to register the Force (g). For morphological analysis, the diaphragm and lungs were removed and embedded in paraffin.
In the study of the diaphragm was used picrosirius for colagen fibers and hematoxylin eosin for immunocytochemistry of αactin. The lungs were prepared for histology and morphometry. For statistical analysis, the results were expressed as mean ïſt±
SEM. For comparison of two means was applied to t-test of Student. For comparison of three or more means applied - the
analysis of variance (One-way) followed by Tukey post-test when appropriate. The significance level for rejection of the null
hypothesis was set at 5% (p
Conclusions:
Conclusion: The data show that the strength of the diaphragm is not altered in emphysema group, indicating that its function is
preserved. However, the morphology of the diaphragm indicates that there is increased collagen and actin. Then, the results
indicate that there may be a relationship between morphological settings and preservation of function of the diaphragm of
elastase-induced emphysema in mice.
Keywords: collagens fibers, Diaphragm, Elastase, Mice, Pulmonary Emphysema
Financial Support: FAPEMIG
Resumo:08-060
COMPARISON OF TWO DIFFERENT ROUTES OF DELIVERING ADIPOSE-DERIVED STEM CELLS IN A
MURINE MODEL OF EMPHYSEMA.
Oliveira, A. C. R. D. 1; Antunes, M. A. 1; Abreu, S. C. 1; Cruz, F. F. 1; Xisto, D. G. 1,2; Morales, M. M. 1,2;
Rocco, P. R. M. 1,2
1
Laboratório de Investigação Pulmonar - IBCCF, UFRJ
2
Laboratório de Fisiologia Celular e Molecular - IBCCF, UFRJ
Objectives:
To investigate the effects of two different routes of adipose-derived stem cell (ASC) administration on lung mechanics and
histology in a murine model of elastase-induced emphysema.
Methods and Results:
Thirty female C57BL/6 mice were randomly divided into control (C) and emphysema (E) groups. E groups were intratracheally
instilled with 0.1U of porcine pancreatic elastase (PPE) in 50 µL of saline once a week during four weeks, and C groups received
only 50 µL of saline per instillation. Three hours following the last instillation, saline or ASC, which was obtained from
epididymis of male mice, were intravenously (i.v.) or intratracheally (i.t.) administrated in C (C-ASC i.v. and C-ASC i.t.) and E
(E-ASC i.v. and E-ASC i.t.) groups. After one week, lung static elastance (Est), resistive (ΔP1) and viscoelastic (ΔP2) pressures,
total and differential cell count in lung tissue, and fraction area of normal, collapsed and hyperinflated alveoli were analyzed. ESAL group presented higher Est (+23.1%), ΔP2 (+11.2%), and fraction area of alveolar hyperinflation (23.9 ± 3.3%) compared to
C-SAL group. ASC reduced lung elastance (E-ASC i.v.: -28.6% and E-ASC i.t.: -26.6%) and viscoelastic (E-ASC i.v.: -28.8%
and E-ASC i.t.: -24.7%) similarly independent of administration route. However, ASC was unable to revert hyperinflated areas,
but was effective in reducing inflammatory cell infiltration in lung tissue in both ASC routes.
Conclusions:
ASC modulates the inflammatory processes, improving lung mechanics in the present murine model of elastase-induce
emphysema.
Keywords: DIFFERENT ROUTES, ADIPOSE-DERIVED STEM CELLS, EMPHYSEMA
Financial Support: FAPERJ, CNPq, CAPES, PRONEX
Resumo:08-061
APOCYNIN AND TEMPOL PROMOTE INCREASE OF NO PRODUCTION IN OVALBUMIN-INDUCED ASTHMA
IN BALB/C MICE.
Nesi, R. T. 1; Lanzetti, M. 2,2; Santos-silva, M. A. 2; Pires, K. M. 2; Guilherme, R. 1; Neves, J. S. 1;
Benjamim, C. F. 1; Valença, S. D. S. 1
1
FEDERAL UNIVERSITY OF RIO DE JANEIRO , UFRJ
2
State University of Rio de Janeiro, UERJ
Objectives:
The events that contribute to both oxidative and nitrosative stresses in asthma are well described. However, the effects of
superoxide on oxidative damage and NO production in asthma are poorly understood. The present study investigated the role of
superoxide in ovalbumin-induced asthma.
Methods and Results:
Male BALB/c mice were assigned into six groups: Control (CTR), CTR tempol, CTR apocynin, ovalbumin (OVA), OVA tempol
and OVA apocynin group (n=30; five animals per group). The CTR groups were sensitized with 200 µL of phosphate buffer and
the OVA groups were sensitized with 200 µL of solution containing 50μg of OVA and 5 mg of adjuvant. All of animals were
sensitizated subcutaneously on 0 day and through intraperitoneal injection (i.p.) on 14st day. After 4 days, both groups were
challenged with 2% nebulized OVA (30 minutes/day for 3 consecutive days). Before each challenge, the animals from CTR
tempol, CTR apocynin, OVA tempol and OVA apocynin were submitted to administration of dismutase superoxide (SOD)
mimetic called tempol (100mg/Kg weight) and NADPH oxidase inhibitor known as apocynin (14mg/Kg weight) by i.p. On the
23st day each animal was sacrificed and the half of the lung was homogenized and the other half was fixed for histological
analysis and bronchoalveolar lavage (BALF) was done for the following biochemical dosages: the total content of ROS; catalase
(CAT), eosinophil peroxidase (EPO) activities; nitrite levels; hydroperoxides levels. The total content of ROS was increased in
OVA group (0.26 ± 0.08) when compared to CTR group (0.03 ± 0.01; p
Conclusions:
Both drugs promoted the increased of NO production OVA-induced asthma. We suggest that, although superoxide pathway may
not influence oxidative damage, NO production may contribute to nitrosative damage in experimental asthma.
Keywords: superoxide, nitric oxide, asthma, apocynin, tempol
Financial Support: CNPQ, Capes and FAPERJ
Resumo:08-062
BONE MARROW-DERIVED MONONUCLEAR CELL THERAPY MODULATES THE INFLAMMATORY AND
REMODELING PROCESSES INDEPENDENT OF THE ADMINISTRATION ROUTE IN EXPERIMENTAL
CHRONIC ALLERGIC ASTHMA
Abreu, S. C. 1; Antunes, M. A. 1; Cruz, F. F. 1; Maron-gutierrez, T. 1,2; Ornellas, D. S. 1,2; Teodoro, W. R. 3;
Capelozzi, V. L. 3; Morales, M. M. 2; Rocco, P. R. M. 1
1
Laboratório de Investigação Pulmonar - IBCCF/UFRJ, UFRJ
2
Laboratório de Fisiologia Celular e Molecular - IIBCCF/UFRJ, UFRJ
3
Departamento de patologia, Universidade de São Paulo, USP
Objectives:
This study investigated the best administration route of bone marrow-derived mononuclear cell in a murine model of chronic
allergic asthma, based on the analysis of the inflammatory and remodeling processes.
Methods and Results:
Thirty-six female C57BL/6 mice were randomly assigned into six groups. In the OVA group, mice were sensitized and
challenged with ovalbumin while control group (C) received saline using the same protocol. C and OVA groups were further
randomized into subgroups receiving saline (50 L, SAL) or bone marrow-derived mononuclear cell (BMDMC, 2x106, CELL)
intravenously or intratracheally, 24 hours before the first challenge. Airway and lung parenchyma inflammation and remodeling
were evaluated using light, confocal and electron microscopy. In addition, airway resistance, viscoelastic pressure, static
elastance were analyzed. BMDMC therapy led to a reduction, independent of the route of BMDMC administration, in alveolar
collapse, eosinophil infiltration, broncoconstriction index, smooth-muscle actin expression, subepithelial fibrosis, myocyte
hypertrophy and hyperplasia, and the amount of myofibroblasts in airways and lung parenchyma compared to OVA-SAL. Airway
resistance and viscoelastic pressure were significantly more reduced after cell therapy with intratracheal instillation (38% and
24%, respectively) compared to intravenous (40% and 25%).
Conclusions:
In the present model of chronic allergic asthma, BMDMC therapy was effective at modulating the inflammatory and fibrogenic
processes independent of the route of cell administration.
Keywords: asthma, remodeling , stem cells, administration route
Financial Support: PRONEX-FAPERJ, CNPq, FAPERJ, CAPES, INCT-INOFAR.
Resumo:08-063
DOES CHRONIC INTERMITTENT HYPOXIA PRODUCE CHANGES IN THE RESPIRATORY CONTROL OF
UPPER AIRWAY RESISTANCE? CELLULAR AND SYSTEMIC EVIDENCES.
Moraes, D. J. A. ; Bonagamba, L. G. H. ; Zoccal, D. B. ; Machado, B. H.
Dpto of Physiology/School of Medicine of Ribeirão Preto, FMRP/USP
Objectives:
Introduction: During eupneic breathing, the control of upper airway resistance is characterized by pre-inspiratory (pre-I)
abduction and post-inspiratory (post-I) adduction causing decreases during inspiration and increases during expiration
respectively. This dynamic coordination by central mechanisms over the breathing cycle has been suggested to be vital for the
maintenance of blood gases homeostasis in various respiratory-related behaviors, including during conditions of hypoxia. Aims:
To evaluate if chronic intermittent hypoxia (CIH) produces changes in the respiratory control of upper airway resistance in the
awake rats and in the working heart-brainstem preparation (WHBP) and to characterize the electrophysiological mechanisms
involved in these changes.
Methods and Results:
CIH paradigm consisted of 6% O2 for 40 s every 9 min, 8 h/day. On the 11th day, the EMG of diaphragm (DIA) and tongue
protrudor genioglossus (GG) muscles were evaluated simultaneous with plethysmographic recordings in awake rats breathing
room air. In the WHBP, phrenic (PN), cervical vagus (cVN), hypoglossal (HN) and recurrent laryngeal (RLN) nerves activities
were recorded. In addition, intracellular recordings of pre-I and post-I neurons of pre-Bötzinger (pre-BötC) and Bötzinger
Complex (BötC) were made in presence or not in presence or not of antagonists of excitatory and inhibitory synaptic
neurotransmission (KYN: 1-6 mM; Biccuculine: 20 μM and Strychnine: 1μM). By these recordings, the membrane input
resistance and the neuronal excitability (the threshold current required to evoke an action potential) were determined. In awake
CIH rats (n=7) the pre-I abduction of GG was greater than in control (n=4) (15±3 vs 2±0.1%; p<0.05).
Conclusions:
We suggest that the CIH reduces upper airway resistance in unanesthetized rats during inspiration and expiration, by mechanisms
involving electrophysiological changes in the BötC post-I and pre-BötC pre-I neurons.
Keywords: Chronic Intermittent Hypoxia, Respiratory Control, Upper Airway Resistance, Bötzinger Complex, pre-Bötzinger
Complex
Financial Support: CAPES, CNPQ and FAPESP
Resumo:09-026
EFFECT OF THE ESTROGEN IN MODULATION OF THE HEXOKINASE MITOCHONDRIAL AS POSSIBLE
ANTIOXIDANT PREVENTIVE AGENT OF THE ROS GENERATION
Ferreira, C. R. ; Filho, A. G.
Instituto de Bioquímica Médica, UFRJ
Objectives:
The brain is a tissue that demands of lot of energy, being the mitochondrial oxidative phosphorylation essential to the neurons to
maintain the high demands of ATP. In addition, in situations of bioenergetic crisis, the decrease mitochondrial respiration may
produce reactive oxygen species (ROS) generation. Among many mechanisms suggested in literature to the decrease of
mitochondrial ROS, the estrogen appears as the physiological sex-specific regulator of ROS production. Estrogen induces
association of the Hexokinase-II (HK-II) to voltage dependent anionic channel (VDAC) in neuron cells. Our group had
demostraded that rat and mice brain mitochondrial hexokinase (HK) activity play a critical role ADP/ATP re-cycling decreasing
ROS production (J Biol Chem., 38, 39846, 2004). The aim of this study was to analyze in rats whether the gender and ages could
affect the ROS generation and HK activities in rat brain mitochondria. In addition ovarioctemized rats were used in order to
challenge the hypothesis of estrogen prevention of ROS formation.
Methods and Results:
The brain mitochondria were isolated of rats (200-350 g) by percoll gradient. The detailed analyses of ETC by oxygen uptake
were performed using high-resolution respirometry (Oroboros Oxygraph-O2K). The ROS production was measured by
fluorescence method Amplex Red. The results showed that when males e females (4 months age) were compared for respiratory
substrates (2 mM pyruvate/malate, 5 mM glutamate and 10 mM succinate) no differences were observed.. There was a trend to
stimulate more respiration by activating of the HK with addition of 20 mM 2-DOG in females than in males. No differences were
observed in stimulated ROS generation in both males and females. However, in females younger (2 months) were observed
differences in the function mitochondrial of, ATP synthesis (state 3) and stimulated respiration by HK activation using 2-DOG
increases by 1.5 folds when compared to females older (4 months). Despite of these observations, sham operated rats presented
bigger respiration in state 3, induced by 2-DOG and FCCP than ovarioctemized rats of same age (2 months). In addition, the
respiration of state 3 and FCCP (maximum respiration) were almost the same in younger females. Whereas, in older females, the
respiration induced by state 3 were only 1/3 of that induced FCCP. The ROS production showed a tendency to be higher in
ovarioctemizades than sham rats.
Conclusions:
Our data suggested that estrogens could play a role modulating HK contributing to decrease ROS production. Female rats of the
different ages showed different respiratory profiles and ROS production. Despite of apparent lack differences in rats older of the
different sex, more studies are necessary to a better evaluation of estrogen effects in brain mitochondria.
Keywords: Electron transport chain, Estrogen, Hexokinase, Mitochondria, Species oxygen reactive
Financial Support: Capes, CNPq, FAPERJ, INCTEN
Resumo:09-027
CROSS-TALK BETWEEN G-PROTEIN-COUPLED AND P2X RECEPTORS IN MICE LEYDIG CELLS
Costa, R. R. ; Aguiar, J. F. ; Varanda, W. A.
Depto. Fisiologia- Faculdade de Medicina , FMRP-USP
Objectives:
Ca2+ influx and intracellular Ca2+ increase are essential to testosterone production and secretion in Leydig cells. In these cells Ttype Ca2+ channels are activated by luteinizing hormone (LH) and are modulated by protein kinases. In adittion to T-type
channels, P2X receptors are also present in the plasma membrane of Leydig cells and may serve as another pathway for Ca2+
entry. Consistent with this ATP effectively increases testosterone secretion. As LH leads to the activation of adenylate ciclase and
phospholipase C, increasing intracellular Ca2+ concentration [Ca2+]i, our goal was to investigate the effects of LH on ATP
currents and on [Ca2+]i stimulated by ATP.
Methods and Results:
Whole-cell currents were measured in Leydig cells in response to 100 μM ATP before and after LH and H89 (blocker of PKA).
Fluo-3 and mag-fluo4 were used in order to simultaneously monitor the cytosolic and endoplasmic reticulum Ca2+ activity,
respectively. Fluorescence was measured on a Leica SP-5 confocal microscope. We have found that inhibition of PKA
significantly reduced the amplitude of ATP activated currents at -60 mV, from -179.7 ± 9 to -89.4 ± 25 pA (n= 13 cells) (H89
applied 5 min before testing the ATP). However, application of LH between ATP pulses did not alter the ATP activated currents.
ATP (100 μM), induced a fast 3.7 ± 0.1 fold increase in fluorescence in relation to basal level, that did not occur in the absence of
external Ca2+ or in the presence of 300 μM suramine This response was significantly reduced by 1.9 ± 0.1 fold by the PKA
inhibitor H89 (1 μM) and by 2.5 ± 0.2 fold following a 5 minutes incubation with LH. Ryanodine (100 μM) did not modify the
amplitude and the time to peak of the [Ca2+]i transients induced by ATP, but the half-duration (1/2D) is significantly larger.
Interestingly, a previous stimulation of the cell with ATP impaired the [Ca2+]i typically induced by a subsequent application of
LH. A line scan analysis of the fluorescence indicated that the increase in [Ca2+]i starts close to the plasma membrane.
Simultaneously with the increase in [Ca2+]i stimulated by ATP on the cytosol there is a depletion of Ca2+ in the endoplasmic
reticulum. Comparing the fluorescence transients we see larger amplitudes in the case of ATP ( 3.6 ±0.1) than with LH (2.2 ±
0.2). The time to peak is faster for ATP (9.2 ± 0.4 s) than for LH (14 ± 1.1 s), and the half-duration is shorter for LH (14.3 ± 1.8
s) than for ATP (25 ± 1.7 s).
Conclusions:
In these experiments ATP activated cation currents were reduced by PKA inhibition, but are not modified by LH. In terms of
Ca2+ signaling, we confirmed the presence of P2XR and we observed that RYR inhibition did not abolish the purinergic Ca2+
transient. In contrast, the Ca2+ transients in response to ATP were reduced by the inhibition of PKA. This suggests a possible
role of PKA as modulator of the purinergic induced Ca2+ transients. These results also imply that ATP-mediated P2X receptor
activity exerts an inhibitory influence on the final effect of LH on Leydig cells and therefore on testosterone secretion.
Keywords: Leydig cells , P2x receptorts, calcium signaling
Financial Support: FAPESP, FAEPA
Resumo:09-028
EFFECT OF EBSELEN ON MICE BRAIN MITOCHONDRIA METABOLISM
Pereira, P. R. P. ; Camacho-pereira, J. ; Galina, A.
Instituto de Bioquímica Médica, IBqM
Objectives:
Ebselen (EBS), also called PZ51, is a selenium drug with a variety of pharmacological properties and therapeutic which have
been associated as a mimetic of glutathione peroxidase and thioredoxin reductase . It has been demonstrated that EBS protect
against deterioration of brain function in patients with cerebral infarction and subarachnoid hemorrhage. EBS underwent several
clinical trials and is now considered as a potential antioxidant drug to treat diseases associated with excessive oxidative stress, but
further studies are needed to establish the threshold doses that decrease reactive oxygen species (ROS) formation without causing
further cellular stresses. In this paper, we evaluate the effect of EBS in a central enzyme of glucose metabolism, hexokinase, and
the antioxidant effect of EBS in mitochondria ROS generation from mice brain.
Methods and Results:
EBS was able to inhibit a key metabolic enzyme such as hexokinase, which phosphorylates glucose to glucose-6-phosphate.
Measuring the activity of hexokinase by spectrophotometric assay in isolated mitochondria of mouse brain, we observed that their
activity was reduced by almost 50% with 5μM EBS. It was evaluated the effect of EBS on mitochondrial generation of ROS
measured with the Amplex Red oxidation method. We observed that EBS has a biphasic effect in which with EBS in the range of
1 μM to 3 μM there was an increase in brain mitochondria ROS production and above 5 μM until 10 μM, EBS promoted a
reduction in ROS formation.
Conclusions:
It was found that EBS is able to inhibit the hexokinase activity, an enzyme that is involved in mitochondrial ROS homeostasis. In
addition, EBS has a dual effect depending on the dose of antioxidant. We do not know whether both effects are correlated. These
data suggest that EBS could be an inhibitor of cell glucose metabolism, even at low doses.This data is important in relation to the
compound toxicity might cause when used for clinical treatment.
Keywords: BRAIN, EBSELEN, METABOLISM, MITOCHONDRIA
Financial Support: CNPQ FAPERJ
Resumo:09-029
REGULATION OF WILSON DISEASE ATPASE (ATP7B) ACTIVITY BY PROTEIN KINASE C (PKC).
Cardoso, L. H. D. ; Hilário-souza, E. ; Vieyra, A. ; Lowe, J.
Instituto de Biofísica Carlos Chagas Filho, UFRJ, IBCCF
Objectives:
Copper is an essential metal in all organisms, although in high concentrations it becomes toxic. Therefore, copper level in the
organisms must be highly regulated. ATP7B is a copper transporting ATPase expressed in liver that is essential in copper
homeostasis, and it is known that Wilson disease is caused by mutations in this protein. In previous studies we have already
shown that its ATPase activity is inhibited when phosphorylated by protein kinase A (Int. J. Biochem. Cell Biol. 43: 358, 2010).
In the present work we investigated whether protein kinase C signaling pathway might also modulate this Cu(I)-ATPase activity.
Methods and Results:
In a porcine liver preparation (J. Biol. Chem. 267: 12016, 1992), different isoforms of protein kinase C (α, ε, δ) were detected by
Western Blotting. A colorimetric assay was used to detect Cu(I)-ATPase activity in the preparation (J. Biol. Chem. 66: 375,
1925), and the results were given in nmol x mg-1 x min-1 (means ± SE). Phorbol 12-myristate-13-acetate (PMA), a PKC
activator, increased ATP7B activity (control: 16.95 ± 0.86; 10 nM PMA: 26.44 ± 1.84; n≥4), while calphostin C, a PKC inhibitor,
decreased its activity (control: 36.94 ± 0.54; 10 nM calphostin C: 23.21 ± 6.76; n≥6). Addition of phosphatase λ after preincubation with PMA also decreased its activity to the same level of addition of phosphatase λ without PMA (control: 22.84 ±
1.07; phosphatase λ: 10.07 ± 3.00; 10 nM PMA /phosphatase λ: 10.25 ± 2.38; n≥4). No changes were observed when Ca2+ 2 μM
or PMA/Ca2+ 2 μM were added, however PMA/EGTA increased the Cu(I)-ATPase activity (control: 17.47 ± 1.71; 10 nM PMA
/Ca2+ 2 μM: 19.11 ± 2.72; Ca2+ 2 μM: 21.49 ± 2.30; 10 nM PMA/ 2 mM EGTA: 35.70 ± 1.97; n≥6), indicating the involvement
of a PKC isoform which is not stimulated by calcium. The activation of the Cu(I)-ATPase activity is reverted by the PKCε
specific inhibitor (control: 30.66 ± 1.69; 0.5 μM iPKCε: 12.24 ± 0.59; 0.5 μM iPKCδ: 26.77 ± 2.05; n≥4), showing that this novel
PKC isoform is responsible for the stimulation of ATP7B activity.
Conclusions:
ATP7B activity from porcine liver is regulated by PKCε, which leads to a stimulation of the copper transporting activity.
Different signaling pathways involving phospholipase C in hepatocytes can activate PKCε, which in turn could increase ATP7B
activity and therefore be an important element for copper homeostasis.
Keywords: ATP7B, PKC, Wilson disease, protein kinases
Financial Support: FAPERJ, CNPq, PIBIC-CNPq.
Resumo:09-030
CHARACTERIZATION OF THE ACTIVITY OF NA+-ATPASE IN HUMAN BREAST CANCER CELLS MCF-7
Dias, M. A. ; Fonseca, M. M. ; Capella, M. A. M. ; Lopes, A. G.
Instituto de Biofísica Carlos Chagas Filho, UFRJ
Objectives:
Recently, the ouabain insensitive, K+-independent Na+-ATPase was identified, isolated and characterized in guinea-pig small
intestinal cells (Biochim. Biophys. Acta (Biomembranes) 1808; 1684, 2011). This protein was previously characterized in a
number of kidney and intestinal cells, but there is no study on its expression or activity in tumor cells. The objective of this study
was to characterize the presence of Na+-ATPase activity in human breast cancer MCF-7 cells, comparing with that already
described for MDCK cells.
Methods and Results:
To observe the sensitivity of MCF-7 and MDCK cells to furosemide, the best Na+-ATPase inhibitor known, the cells were
seeded in 96 wells plates and incubated for 48 h with furosemide concentrations varying from 100 nM to 2 mM and the cellular
viability was measured by MTT. Both cell lines were sensitive to furosemide concentrations above 250 uM (p
Conclusions:
MCF-7 cells present a very low, undetected activity of Na+-ATPase in basal condition which can be increased after exposure to
hormones related to body volume regulation, suggesting that altered body volume (for example, hypertension) may alter the
expression/activity of this protein in tumor cells.
Keywords: Na+-ATPase, Breast Cancer, Ionic Transporters, MCF-7 Cells
Financial Support: CNPq, CAPES, FAPERJ, PRONEX
Resumo:09-031
REGULATION OF SODIUM INDEPENDENT GLUTAMATE TRANSPORT BY HYDROCORTISONE IN
CULTURED CHICK RETINAL CELLS
Garza, . D. R. 1; Oliveira, K. R. M. 1; Sá, L. L. C. 2; Garcia, T. B. 1; Crespo-lopez, M. H. C. 1; Herculano, A.
M. 1
1
Universidade Federal do Pará , UFPA/ICB
2
Instituto Evandro Chagas - Seção de Meio Ambiente, IEC
Objectives:
Aim: Transport of glutamate in the vertebrate central nervous system is crucial to assure a high signal-to-noise synaptic
transmission and to prevent excitotoxicity. In retinal tissue a family of sodium dependent and independent transporters are
responsible for the majority of the uptake of glutamate released at the synaptic cleft. Recently we demonstrated that about 40% of
the glutamate transport in retinal tissue is mediated by sodium-independent transporters (Neurochem int. 56:59, 2010). The aim
of the present study is to evaluate the neuroedocrine regulation of the sodium independent glutamate/cysteine (-SH) transport in
retinal cells. Hydrocortisone was used in our analysis since previous reports have demonstrated its involvement in the regulation
of two glial proteins crucial for the glutamate transport phenotype: the sodium-dependent transporter EAAT1 (Neurochem int.
37:179, 2000) and the cytoplasmatic Glutamine Sinthethase that cycles glutamate to glutamine (Proc. Natl. Acad. Sci USA.
91:4786, 1994). Thus, using primary retinal cell cultures as experimental model, we investigated if hydrocortisone is able to
regulate the sodium independent transport of glutamate in the retina.
Methods and Results:
Methods and Results: primary cultures where established from retinal cells of E8 chick embryos and cultivated at 37⁰C with a
humid 5% CO₂ atmosphere in a DMEM medium containing 10% FBS for 7 days. Glutamate transport was evaluated by high
performance liquid chromatography as described previously. Extracellular –SH content was determinated by DTNB reduction.
The experiments in medium without sodium, NaCl was replaced by equimolar concentration of LiCl. Our results have
demontrated that retinal cell cultures treated with hydrocortisone (0,33 μg/ml) showed a significant increase of glutamate uptake
(39 ± 5,88% over the control). Similar increase was observed in the cultures incubated in a sodium medium free ( 29 ± 6,7% over
control). This result suggests that hydrocortisone evokes a glutamate uptake mediated by sodium independent transporter. To
ratify this hypothesis we evaluate the effect of hydrocortisone in the –SH release in a medium without sodium as described
above. Our data showed an increase of –SH content in a sodium-independent manner (76,81 ± 5,57% over control).
Conclusions:
Conclusions: Hydrocortisone treatment induces an increase in the glutamate uptake mediated by sodium independent glutamate
transporters and this effect was followed by an intense release of –SH in the medium suggesting a possible role of
glutamate/cystine antiporter. hydrocortisone treatment of chick retinal cultured cells increases the total extracellular sulfidryl
when stimulated with L-glutamate in a sodium-independent manner and increases the uptake of glutamate in both sodium
independent and dependent systems.
Keywords: Cystine/glutamate antiporter, Glutamate uptake, Hydrocortisone, Retina, Sulfidryl
Financial Support: CNPQ/ FAPESPA/MAKARÚ
Resumo:09-032
CHANGES IN BROWN ADIPOSE TISSUE THERMOGENESIS INDUCED BY THYROID HORMONE
Curty G. 1; Salvatte, K. 1; Carvalho, D. P. 2; de Meis, L. 1; Ketzer, L. A. 1,3
1
INSTITUTO DE BIOQUÍMICA MÉDICA, RIO DE JANEIRO , UFRJ
2
INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO, RIO DE JANEIRO, UFRJ
3
PÓLO AVANÇADO DE XERÉM, RIO DE JANEIRO / XERÉM, UFRJ
Objectives:
Homeothermic animals control body temperature by using endogenous mechanisms to produce and dissipate heat. These
mechanisms comprise thermogenesis, which can be divided into obligatory or adaptive (facultative). The obligatory
thermogenesis is the basal metabolic rate and occurs when the organism is at rest. Already the adaptive thermogenesis occurs in
response to diet and environmental factors as cold exposure. Thyroid hormones increase the basal metabolic rate and are
considered essential for adaptive thermogenesis. The skeletal muscle and brown adipose tissue (BAT) are important sites for
adaptive thermogenesis. It is suggested that the adaptive response in muscle is promoted by shivering and the activity of some
enzymes, such as sarco(endo)plasmic reticulum Ca2+-ATPase(SERCA 1).In skeletal muscle, SERCA 1 is an enzyme able to
pump Ca2+ from the cytosol into the sarcoplasmic reticulum lumen using the energy derived from ATP hydrolysis. However,
SERCA 1 is able to hydrolyze ATP without pumping Ca2+, producing more heat per ATP cleaved than that released during Ca2+
transport. In the BAT, the uncoupling protein 1 (UCP 1) contributes significantly to adaptive thermogenesis. Recently was
identified the expression of SERCA 1 in the mitochondria and endoplasmic reticulum of BAT through immunoelectron
microscopy. However, the role of this enzyme in this tissue is not clear. The purpose of this study is to investigate the
thermogenic role of SERCA 1 in BAT in response to increased thyroid hormone, as well as expression and activity of UCP1 in
these animals.
Methods and Results:
Hyperthyroidism was induced in Wistar rats by injection of T4 (100 μg/kg body weight), subcutaneously, for 10 days.
Hyperthyroid rats had a decrease of body weight gain (29.6 ± 3.5 g versus 40.1 ± 4.2 g in control, n=8) and an increase of BAT
weight (56 %). The endoplasmic reticulum vesicles from BAT were prepared by differential centrifugation and stored at –70ºC.
The mitochondrial fraction was obtained by Percoll gradient. The thermogenic parameters were measured using an isothermal
titration calorimeter and high-resolution oxygraph. Thyroid hormone induced SERCA 1 expression and promoted an increase in
heat released by ATP hydrolysis in the endoplasmic reticulum, suggesting an increase in thermogenesis. Citrate synthase activity,
an enzyme of the Krebs cycle, was used as an indicator of mitochondrial biogenesis. Although the citrate synthase activity to
show an increase in hyperthyroidism (1.79 ± 0.2 μmol/mg.min versus 1.26 ± 0.2 μmol/mg.min in control), the UCP1 expression
was unchanged. Hyperthyroidism promoted a rise in oxygen consumption (0.65 ± 0.1 μmol O2/mg.min versus 0.3 ± 0.05 μmol
O2/mg.min in control) and heat production (7 fold) by mitochondria.
Conclusions:
These data suggest that thyroid hormones modulate not only the UCP1 activity, but also the SERCA 1 expression and the activity
of in BAT. Here we show the importance of the thyroid hormones in adaptive thermogenesis promoted by BAT.
Keywords: Brown adipose tissue, Thermogenesis, Thyroid hormones, Ca2+-ATPase of sarcoplasmic reticulum, uncoupling
protein 1
Financial Support: CNPq, FAPERJ, FINEP
Resumo:09-033
VALINOMYCIN AND OUABAIN PREVENT VERAPAMIL INHIBITORY EFFECT ON EMBRIONIC
DEVELOPMENT OF THE SEA URCHIN ECHINOMETRA LUCUNTER.
Leite, J. C. A. ; Marques-santos, L. F.
Deptartamento de Biologia Molecular, UFPB
Objectives:
Ca2+ is an intracellular messenger that controls a wide range of physiological functions. The increase in cytosolic calcium
concentration is derived from intracellular stores mobilization, e.g. endoplasmatic reticulum (ER), or influx through channels,
especially voltage−gated channels (Cav) present on cell surface. According to scientific literature, sea urchin embryogenesis is a
process induced exclusively by Ca2+ mobilization from ER, as Ca2+ influx is not necessary for this process (PNAS 75:1915,
1974). However, our group previously showed that verapamil (VP), an unspecific calcium channel blocker, induced a significant
blockage on early embryonic development of the sea urchin Echinometra lucunter. Similar results were obtained with diltiazem
and nifedipine. These data suggest calcium influx dependence on embryogenesis of this species. In the present work we
investigate the effect of the potassium ionophore valinomycin (VAL) and the Na+⁄K+ ATPase inhibitor ouabain (OUA) on VP
inhibitory effect over E. lucunter early embryonic development. K+ efflux mediated by VAL alters membrane potential, which
can influence the opening of some types of Cav and its pharmacological regulation (J. Cell Sci. 98: 343, 1991). Alternatively,
Na+⁄K+ ATPase inhibition by OUA activates the reverse mode of the Na+⁄Ca2+ exchanger, promoting Ca2+ influx (J.
Pharmacol. Sci. 110: 111,2009).
Methods and Results:
Animals were collected at Cabo Branco Beach (João Pessoa, Brazil). Eggs and spermatozoa were collected by KCl intracoelomic
injection. Fertilization was induced by the addition of activated sperm suspension to eggs suspension (1:5000). Embryos were
treated with the VAL or OUA, before or after VP (100 μM) treatment. One hundred embryos were analyzed to each treatment
and early embryonic development (2−4 cell embryo and morula) was monitored under optical microscopy. Pretreatment with
VAL and OUA blocked VP inhibitory effect. Although, VAL and OUA did not prevent VP inhibitory effect when added after the
calcium channel blocker.
Conclusions:
This set of data strongly suggests that VP inhibitory effect on E. lucunter embryonic development was induced by the impairment
of Ca2+ influx through Cav and this influx is crucial in the first minutes of the embryogenesis. Our work highlighted the
involvement of extracellular Ca2+ on embryonic development of this specie.
Keywords: Verapamil, Valinomycin, Ouabain , Embrionic Development, Echinometra lucunter
Financial Support: CNPq /CAPES
Resumo:09-034
ELECTROTHERAPY STUDY OF THE HEALING PROCESS IN ANIMALS: THE USE OF SODIUM DICLOFENACIONTOPHORESIS ADMINISTRATION
Xavier, A. F. 1; Medeiros, M. A. M. B. 2; Nascimento, R. F. 2; Oliveira, J. M. 3; Silva, B. A. 2; Souza, E. P. 2
3
Faculdades Integradas do Ceará, FIC
2
Universidade Federal da Paraíba, UFPB
1
Universidade Federal do Ceará, UFC
Objectives:
Iontophoresis is a noninvasive technique that uses a power (
Methods and Results:
We used 20 animals of the Wistar male adult, average weight of 175g, placed into four groups with five animals each group.
Trichotomy, antisepsis and skin lesion were carried in all animals using a disposable microtome blade, previously anesthetized.
The first group (saline control group) and second group (sodium diclofenac control group) received no iontophoresis and third
group (saline experimental group) and fourth group (experimental group sodium diclofenac) received the application of
iontophoresis. All groups received 6 therapy applications, being removed the skin of each animal and evaluated the resulting scar
tissue by the scoring system 0-3 with observation of the epithelium, the presence of hemato-fibrinous and collagen fibers through
the light microscopy by Mallory‘s stain. The control group animals showed a great lack of epithelial lining in 40%, hematofibrinous present in 100% of the animals and the collagen fibers had 40% the formation of fibers, but in a disorderly fashion.
About 60% of sodium diclofenac experimental group showed a marked formation of the epithelium, with 100% showing the
absence of hemato-fibrinous and 100% with the formation of arranged and orderly collagen fibers.
Conclusions:
Based on our results, we conclude that the efficiency and effectiveness of the technique associated with drug actions promote the
remission of the healing of animals involved in this experiment.
Keywords: Electrotherapy, Iontophoresis, Healing, Sodium diclofenac
Financial Support: FIC, PIBIC-CNPq
Resumo:09-035
BRADYKININ B2 RECEPTOR INVOLVEMENT IN HUMAN RED BLOOD CELLS INVASION BY PLASMODIUM
FALCIPARUM
Souza-silva, L. 1; Ferreira-dasilva, C. T. 1; Caruso-neves, C. 1,2; Pinheiro,a. A. S. 1,3
1
Instituto de Biofísica Carlos Chagas Filho, IBCCF/UFRJ
2
Institutos Nacionais de Ciência e Tecnologia , INCT/INBEB/CNPq/MCT
3
Institutos Nacionais de Ciência e Tecnologia, INCT/INPeTAm/CNPqMCT
Objectives:
P. falciparum is a protozoan parasite that causes the most virulent form of human malaria. It infects both liver and red blood
cells, but the erythrocytic stage of infection is responsible for all symptoms and pathologies associated with the disease. We have
recently shown that components of the renin-angiotensin system (RAS), specifically Angiotensin II (Ang II), through its
conversion into Ang-(1–7), decreases infection of new erythrocytes during P. falciparum blood stage (PLos One; 6, e17174,
2011). The kallikrein-kinin system (KKS) is another complex pathway linked to RAS by a number of molecules including the
angiotensin converting enzyme (ACE). Studies showing a direct relationship between plasmatic level of bradykinin, an effector
molecule of KKS, and severe malaria were first demonstrated in 1966 (Ann. Trop. Med. Parasitol. 60; 304, 1966). More recently,
a new KKS inhibitor, designated anophensin, was identified in the salivary glands of the malaria vector mosquito, Anopheles
stephensi, indicating a possible role of KKS components in the life cycle of malaria parasite (Ins. Bioc. Mol. Biol. 35; 466, 2007).
In the present work, we demonstrate the role of bradykinin in the modulation of P. falciparum erythrocytic cycle.
Methods and Results:
Erythrocytic asexual stages of P. falciparum, W2 strain, were maintained in continuous culture in RPMI 1640 medium in the
presence of A-type human blood and 10% A-positive human serum, 50 μg⁄ml gentamicin at 4–5% hematocrit. Cultures
synchronized at the schizont form were treated or not with bradykinin or other drugs and parasitemia was measured by counting
of the ring forms in blood smear, after 24h. Bradykinin (10‾14–10‾8M) reduced erythrocyte invasion by parasite in a dose
dependent manner with maximum effect of 51% inhibition observed at 10‾8 M, when compared to control (n=8). This effect was
completely reversed by 10‾8M HOE-140, an antagonist of B2 bradykinin receptor (n=5). The same phenomenon was observed
when cultures were treated with 10‾7M A779, a specific Ang-(1-7) receptor antagonist (n=8). The presence of B2 receptor on the
surface of erythrocytes was evaluated by immunoblotting, using membrane fraction of normal cells and was detected at the
molecular weight of 50kDa. Captopril (10‾6M), a well-known ACE inhibitor, responsible to increase the plasmatic level of
bradykinin, reduced invasion in 40% (n=5). The concomitant addition of increasing concentrations of Ang-(1-7) (10‾12-10‾6M)
together with decreasing concentrations of bradykinin (10‾8-10‾14M), showed an additive effect of both peptides, with inhibition
levels close to 50%, in relation to control (n=5).
Conclusions:
Taken together, our results suggest a cooperative role of bradykinin and Ang-(1-7) in the impairment of P. falciparum
erythrocytic cycle. The bradykinin effect depended on B2 and AT(1-7) receptors activation, indicating a possible
heterodimerization of both receptors in the erythrocyte membrane.
Keywords: Plasmodium falciparum, Red blood cells invasion, kallikrein-kinin system, Bradykinin B2 receptor
Financial Support: FAPERJ, CAPES, INBEB, INPETAm and CNPq
Resumo:10-031
EFFECTS OF N-ACETYLCYSTEINE AND/OR DEFEROXAMINE ON MYONECROSIS IN DYSTROPHINDEFICIENT MUSCLE FIBERS OF MDX MICE.
Moraes, L. H. R. ; Dias, P. ; Burgos, R. R. ; Neto, H. S. ; Marques, M. J. ; Minatel, E.
Anatomia, Biologia Celular, Fisiologia e Biofísica / IB, Unicamp
Objectives:
Duchenne muscular dystrophy (DMD) is caused by deficiency of the cytoskeletal protein dystrophin. The mdx mouse is an
animal model of DMD that also lacks dystrophin. In mdx mice and in DMD, oxidative stress appears to contribute substantially to
the skeletal muscle damage. The current study investigates the role of the antioxidant N-acetylcysteine (NAC) and the iron
chelator deferoxamine (DFX) or NAC-DFX in combination on myonecrosis in dystrophin-deficient muscle fibers of mdx mice.
Methods and Results:
Mdx mice (14 days old) received daily intraperitoneal injections of saline, NAC (150mg⁄kg), DFX (150mg⁄kg) or NAC plus DFX
for 14 days. C57BL⁄10 mice were used as controls. Forelimb muscle strength was evaluated at the beginning and end of treatment
by grip strength meter. After the treatment, the sternomastoid (STN), diaphragm (DIA) and tibialis anterior (TA) muscles were
removed and used for the quantification of necrotic fibers (labeled with Evans blue dye)(EBD), regenerated muscle fibers with
central nuclei and inflammation area. Creatine kinase (CK) levels were analyzed for biochemical evaluation of muscle fiber
degeneration. There was a significant increase in strength in mdx mice treated with NAC plus DFX compared to saline-treated
mdx mice (2,56±0,53g⁄g in NAC plus DFX treated mdx mice vs 2,03±0,46 g⁄g in saline mdx mice, p≤0.02; Student′s t-test.). In
contrast, NAC or DFX treatments did not change forelimb muscle strength in mdx mice. NAC, DFX and NAC plus DFX
treatments caused a significant decrease in EBD staining in DIA, STN and TA muscles in mdx mice (p≤0.05; Student′s t-test)
compared to saline-treated mdx mice. In STN muscle, a significant decrease of fibers with central nuclei and inflammatory area
was observed after treatment with NAC, DFX and NAC plus DFX (p≤0.05; Student′s t-test). In TA muscle, NAC and NAC plus
DFX treatments significantly reduced the percentage of fibers with central nuclei in mdx mice compared to saline-treated mdx
mice (p≤0.05; Student′s t-test). Although was observed a reduction in inflammatory area of TA mdx muscle after all treatments,
the decrease was not statistically significant (p≥0.05; Student′s t-test). In DIA muscle, NAC treatment reduced significantly the
percentage of fibers with central nuclei in mdx mice compared to saline-treated mdx mice (p≤0.05; Student′s t-test). Furthermore,
the inflammatory area decreased in DIA muscle of mdx mice after treatments with NAC, DFX and NAC plus DFX (p≤0.05;
Student′s t-test).
Conclusions:
These results suggest that NAC, DFX and NAC plus DFX treatments protects STN, TA e DIA muscles against myonecrosis in
mdx mice.
Keywords: mdx mouse, oxidative stress, antioxidants
Financial Support: CAPES‾Proex; FAPESP (#2010/01087‾4; #2008/50731‾3); FAEPEX.
Resumo:10-032
EVALUATION OF LIVER TISSUE IN DIABETIC RATS INDUCED BY STREPTOZOTOCIN AND TREATED WITH
AQUEOUS EXTRACT OF EQUISETUM PYRAMIDALE.
Santos, S. E. 1; Aquino, J. P. 1; Silva, M. B. D. 1; Dourado, D. M. 2; Pereira, D. M. 4; Silva, B. A. K. D. 4;
Coelho, R. M. 3; Bento, L. M. A. 2
1
LABORATÓRIO DE FISIOLOGIA EXPERIMENTAL, UAU
2
LABORATÓRIO DE TOXINOLOGIA E PLANTAS MEDICINAIS, UAU
3
LABORATÓRIO DE PRODUTOS NATURAIS, UAU
4
DOCENTE DO CURSO DE FISIOTERAPIA, UAU
Objectives:
This study has as aim to evaluate the effect of plant extract E. pyramidale em the liver of diabetic rats.
Methods and Results:
Wistar Mice with body weight of 250g in average, inducted to Diabetes Melitus(55 mg/kg STZ). They were divided into 4
groups: Extract control(EC,n=5), saline control(CSn=5), diabetc extract(DE,n=6) and diabetc saline(DS,N=5).By DE and CE
Gavage they received extract of E. pyramidale and CS and DS received saline, both at the dose of 1ml/for a day. After 40 days
the animals were euthanized and samples of the liver removed, fixed in 10% buffered formalin, processed, embrdded in paraffin
to be sectioned to 5mm and evaluated by histochemical staining reaction of schiff. By capturing 10 fields per individual
ramdomly, chooosing the field hepatocytes with cytoplasm filed with glycogen in the 40 x objective for statistical analysis. The
test used for comparison of glycogen in hepatocytes was between groups analysis of variance ANOVA with Tukey post-test(p
Conclusions:
In the measurement of glycogen in hepatocytes controls had lower numbeer compared to diabetcs, demostrating that glycogen
accumulates in the liver, preventing its proper functioning. There were no histological changes in the controls, but tissue necrosis
in diabetcs patients. Morphologically there is no significant difference between the groups. Futher studies are needed to evaluate
the liver tissue in diabetc rats treated with E. pyramidale.
Keywords: diabetic, plants, Equisetum pyramidale, streptozotocin, liver
Financial Support: pic aesa: universidade anhanguera uniderp
Resumo:10-033
EVALUATION OF HEMATOLOGIC AND BIOCHEMICAL PARAMETERS OF RATS SUBJECTED TO SECONDDEGREE THERMAL BURN
Pereira, D. S. T. 1; Lima-ribeiro, M. H. M. 2; Cunha, C. R. A. 1; Carneiro-leão, A. M. A. 2; Correia, M. T. S.
3
1
Programa de Pós-Graduação em Ciências Biológicas, UFPE
Programa de Pós-Graduação em Biociência Animal, UFRPE
3
Departamento de Bioquímica, Centro de Ciências Biológicas., UFPE
2
Objectives:
Determine the biochemical and hematological parameters of rats subjected to second-degree thermal burn.
Methods and Results:
Seven rats (Rattus norvegicus), albino Wistar male were obtained from the Vivarium Creation of the Department of Nutrition,
Federal University of Pernambuco and the experiments conducted in the Animal Experimental Center of Experimental Surgery at
that institution. All procedures related to animal use were performed according to the standards recommended by the Brazilian
College of Animal Experimentation (COBEA) and approved by the Ethics Committee on Animal Experiments of UFPE
(Protocol: 23076.015015/2009-31). The animals were between 9 and 10 weeks old, weighing on average 268 to 283 g, were preanesthetized with atropine sulfate at a dose of 0.04 mg / kg intramuscularly. After 10 minutes we used an anesthetic combination
of ketamine 10 % (90 mg / kg) with xylazine 2 % (10 mg / kg) intramuscularly and underwent shaving of the back, by the pull of
the direct and antisepsis with polyvinylpyrrolidone-iodine 1 %. The second-degree thermal injury was produced with a solid
aluminum bar 1 cm in diameter. On the 7º, 14º, 21º and 28º days after surgery, the animals were anesthetized for blood collection
by cardiac puncture for hematological and biochemical, followed by euthanasia with sodium pentobarbital (100 mg / kg
intraperitoneally). During the study period the lesions showed no signs of infection. The bleeding, when this was related to
damage caused by the animal itself. The hematological values obtained in this study showed no significant changes as a function
of induction of the burn during the period analyzed (Erythrocytes: 7.6 ± 0.48; Hemoglobin: 13.65 ± 0.5; Platelets: 846400 ± 0.71,
leukocytes: 7980 ± 0.71; Basophils: 0.2 ± 0.05; Eosinophils: 1.38 ± 0.18; Lymphocytes: 82.37 ± 0.83; Monocytes: 1.9 ± 0.2),
revealing themselves but normal values in rats (Medicina veterinária, 3(2):1-8, 2009). The rats, like other mammals, have to
maintain strict control of the internal environment thus ensuring the homeostasis (COBEA, 2003). It is known that rats can
produce changes in these parameters as a result of pathological processes or external factors such as sex, ancestry, age, diet,
handling and environment (Ceres, 56(1):051-057, 2009). However, average values of biochemical parameters analyzed in this
study were consistent with previously reported data specific to normal animals (Calcium: 10.04 ± 0.42; Pro-Thrombin: 9.94 ±
0.16; Fibrinogen: 457.32 ± 0.25; Alkaline Phosphatase: 212.68 ± 0.52; Glutamic Oxalic Transaminase: 180.02 ± 0.35; Glutamic
Pyruvic Transaminase: 53.28 ± 0.41; Gamma-Glutamyl Transpeptidase: 5.76 ± 0.23, Creatine: 0.54 ± 0.04; urea: 46.34 ± 0.04
and Amylase: 842.06 ± 0.48).
Conclusions:
Based on the results obtained did not observe significant changes in hematological and biochemical parameters evaluated, not just
differences occurring due to thermal burn injury in the second degree.
Keywords: Biochemical parameters, Burn, Hematologic parameters, Rats
Financial Support: FACEPE
Resumo:10-034
ORGANIZATION OF COLLAGEN AND MT1-MMP EXPRESSION IN THE PERIODONTAL LIGAMENT OF RAT
INCISORS UNDER EXPERIMENTALLY ALTERED ERUPTION.
Omar, N. F. 1; Gomes, J. R. 2; Neves, J. S. 1; Narvaes, E. A. O. 1; Novaes, P. D. 1
1
Departamento de Morfologia, FOP/UNICAMP
2
Departamento de Biologia Estrutural, Molecular e Genética , UEPG
Objectives:
In the rodent teeth have been demonstrated that the hypofunction condition increases the rate of tooth eruption. The constant
movement that occurs in rat incisor eruption is accompanied by intense activity in periodontal ligament remodeling. The ligament
extracellular matrix remodeling occurs by direct action of enzymes known as metalloproteinases (MMPs). In this sense, this
study investigated: the eruption rate of rat incisors; the periodontal ligament resistance strength in eruption moviment; the
collagen organization and the MT1-MMP expression in periodontal ligament.
Methods and Results:
Twenty male Wistar rats had their lower left incisors cut, every two days, at the interdental papilla level, using high-drill rotation,
after anesthesia by halothane, producing hypofunction condition (HP) during 7 days. Other twenty rats were kept in normal
condition of eruption were used as control (N). The eruption rate was measured with an ocular millimeter from the gingival
margin to the top of the tooth in group HP. In the N, the measure was made from the gingival margin to a mark made in the tooth,
previously. After 7 days, ten animals of each group were killed by cervical dislocation under anesthesia, and the tensile strength
of the periodontal ligament was measured using an algometry. After extracting, the periodontal ligament of the animals was
scraping with a periodontal curette, collected in microtubes containing Tris buffer and used to assess expression of MT1-MMP by
western blotting. It was applied 20µg of samples total protein in poliacrylamide 12% gel. For polarization analyses 10 animals of
each group were killed by perfusion through the heart, the mandibles were removed included in paraffin and the periodontal
ligament of mesial, lingual and distal faces were analyzed in polarized light microscopy. In the hypofunctional group (HP) the
eruption rate (p
Conclusions:
In this study, we conclude that in hypofunction condition there is an increase in rate eruption with degradation of collagen in the
periodontal ligament added to an increase in MT1-MMP expression that leads to a lower resistance in this tissue.
Keywords: Rat, Periodontal ligament, Collagen, Metalloproteinases
Financial Support: FAPESP
Resumo:10-035
CHANGES IN THE COLLAGEN CONTAINING TISSUE FROM RATS CHRONICALLY TREATED WITH
SUPRAPHYSIOLOGICAL DOSE OF GLUCOCORTICOID
Munhoz, A. L. B. 1; Zavan, B. 1; Paffaro Jr, V. A. 1; Pimentel, E. R. 2; Vilela, A. S. B. 1
1
Biomedical Sciences Institut, UNIFAL
2
Institut of Biology, Unicamp
Objectives:
To investigate the changes related to non collagenous proteins, extracellular matrix proteoglycans and glycosaminoglycans ,
fibroblasts in the calcaneal tendon and cornea from male and female rats after prolonged treatment with supraphysiological doses
of glucocorticoid.
Methods and Results:
We have used 20 Wistar rats, divided into four groups of 5 animals each group, control males, treated males, control females and
treated females. The treated group received during five weeks intraperitoneal injection of betamethasone dipropionate +
betamethasone disodium phosphate (0,075 mg/kg). The control group animals received weekly equivalent volume of saline. After
the sixth week, animals were sacrificed by injection of 10% chloral hydrate (0.3 mL/100 g body weight) and their corneas and
Achilles tendons were collected. For morphological study, histological sections of 7ìm thickness were stained with hematoxylin
and eosin. These slides were used for counting the tendon fibroblasts, using the NIS-Elements software. The biochemical analysis
was performed extracting extracellular matrix components and these extracts were analyzed by SDS-PAGE as well as dosage of
non collagenous proteins and glycosaminoglycan (GAG) sulfate. The analysis of GAGs was undertaken on tissue with papain
digestion and subsequent agarose gel electrophoresis in propylenediamine buffer. Statistical analysis was performed by analysis
of variance (ANAVA) followed by the comparison test of Tukey. When the assumptions of normality and homoscedasticity did
not met it was applied the nonparametric Mann-Whitney-Wilcoxon test. The treatment increased the amount of tendon non
collagenous proteins in female, but did not change it in males. The cornea has the amount of non collagenous proteins decreased
significantly in both groups. The quantification of tendon fibroblasts nuclei has not revealed significant differences between
experimental groups. In these experimental conditions, both males and females had not altered the amount of glycosaminoglycans
present in tendon and cornea. However, the results suggest that there are less of these compounds in the cornea of females
compared with males. In tendon, the GAG found in control and treated groups was dermatan sulfate. The treatment seems to
change the type of GAG in cornea, from keratan sulfate to dermatan sulfate.
Conclusions:
The glucocorticoid treatment appears to increase the amount of tendon non collagenous proteins in females, however, it does not
change in males, whereas in cornea of treated groups a significant decrease in the amount of non collagenous proteins are
presented in extracellular matrix. In both males and females, the treatment did not seem to change the amount of
glycosaminoglycans present in tendon and cornea. However, the obtained amount of glycosaminoglycans in cornea suggests that
females have less of these compounds compared to males.
Keywords: glucocorticoid, extracellular matrix, tendon, cornea, rats
Financial Support: FAPEMIG
Resumo:10-036
DIFFERENT RESPONSES OF INTRAMEMBRANOUS AND ENDOCHONDRAL AUTOGENOUS ONLAY BONE
GRAFTS TO LOW LEVEL LASER THERAPY
Biguetti, C. C. 1; Caviquioli, G. 1; Holgado, L. D. A. 1; Marquardt Filho, E. J. 1; Moreschi, E. 1; Ribeiro
Junior, P. D. 1; Duarte, M. A. H. 2; Matsumoto, M. A. 1
1
Depto. Ciências da Saúde/ Universidade Sagrado Coração, USC
2
Faculdade de Odontologia de Bauru/Universidade de São Paulo, FOB/USP
Objectives:
Some doubts are still raised upon the behavior of different types of autogenous bone used for craniomaxillofacial reconstruction
in relation to their embryologic origin. The aim of this study was to evaluate the process of incorporation of intramembranous
(IM) and endochondral (EC) bone grafts, associated or not to low level laser therapy (LLLT).
Methods and Results:
Thirty-two male New Zealand rabbits underwent onlay autogenous bone grafts retrieved from the calvaria and the iliac crest and
were divided in four groups: Control groups – Calvaria (CC) and Iliac (CI) negative controls, and Experimental groups – Calvaria
(ExC) and Iliac (ExI)- exposed to LLLT at 16 J/cm2. After 7, 14, 30, and 60 days the grafts retrieved for microscopic
morphological analysis of the interface, measurement of the grafts thickness, and histochemical analysis for the detection of
osteoclasts in the surface of the grafts. Bone grafts incorporation occurred uneventfully in both IM and EC grafts. Differences in
the resorption process were evident in iliac graft, presenting resorption level of 40% in CI against 8% in ExI at the 14th day.
After 30 days, the resorption level was maintained at 41%, rising to 70% at the 60th day period in CI, while ExI presented 15%
and 45% of resorption after 30 and 60 days, respectively. No significant differences were noted in the calvaria graft. A significant
higher number of osteoclasts was observed after 7 days.
Conclusions:
It was concluded that LTB exerts a more efficient biomodulative activity on EC graft, preventing the resorption process in an
important level.
Keywords: Low level laser therapy, Rabbits, Bone grafts, Intramembranous, Endochondral
Financial Support: FAPESP 2008/11485-7; 2009/14989-9
Resumo:10-037
THE REPAIR OF RABBITS CALVARIAL BONE DEFECTS USING BIOACTIVE VITROCERAMIC
BIOSILICATO®
Caviquioli, G. 1; Biguetti, C. C. 1; Holgado, L. D. A. 1; Saraiva, P. P. 1; Kawakami, R. Y. 1; Renno, A. C. M.
1
; Matsumoto, M. A. 1
1
Universiade Sagrado Coração, USC
2
Universidade Federal de São Paulo, Campus Baixada Santista, UNIFESP
Objectives:
A number of biomaterials have been developed in order to act as bone substitutes. In the present study, the behavior of a new
granulate bioactive vitroceramic in bone repair defects was evaluated. Thirty rabbits underwent surgical confections of defects in
parietal bones, distributed into 4 groups, according to the filling material: 1 – blood clot (Control), 2 – particulate autogenous
bone, 3 – bioactive vitroceramic (Biosilicato®), and 4 - bioactive vitroceramic and particulate autogenous bone.
Methods and Results:
Specimens were retrieved after 7, 14, and 30 days for microscopic morphological analysis. Similar patterns of new bone
formation were observed amongst experimental groups, with a more evident woven bone deposition at day 14, directly upon the
non-viable bone grafts particles, as well as on the biomaterial granules, and bone maturation at day 30. Despite the foreign body
reaction induced in the presence of the vitroceramic, evident from the 14th day, no deficit on bone formation and maturation
occurred during the process. The presence of both biomaterial and bone graft offered a greater volume in the region of the defect
until the last period, differently from the Control Group where small trabeculas surrounded by connective tissue could be seen,
resulting in a repaired defect thinner than the original bone.
Conclusions:
Biosilicato® presented a satisfactory behavior during the repair of bone defects, permitting new bone formation directly upon its
granules surfaces when used associated with bone graft or alone.
Keywords: Bone repair, Rabbit, Bioactive glass, Biomaterial
Financial Support: FAPESP 2009/17294-1
Resumo:10-038
BONE REPAIR INVESTIGATION IN DEFECTS GRAFTED WITH HYDROXYAPATITE COMBINED WITH
LASER THERAPY IN RATS SUBMITTED TO PASSIVE TOBACCO EXPOSURE
Franco, G. R. ; Figueira, L. A. ; Ribeiro, D. C. P. ; Miguel, N. M. ; Cunha, M. R.
Depto Morfologia e Patologia/Faculdade de Medicina de Jundia, FMJ
Objectives:
Defects with bone mass loss are frequently treated with bone autografts. Endografts of bones using biomaterials, such as
hydroxyapatite also have been used for the same purpose, replacing autografts. In addition to these biomaterials that represent an
excellent alternative and coadjuvant in bone reconstruction, laser therapy has been shown to contribute to accelerate the fracture
repair by improving local microcirculation and increasing collagen synthesis However, bone tissue health conditions are basic for
osteointegration of the implant. Thus, excessive tobacco consumption, either as an active or as a passive smoker, may harm the
process of regeneration due to its deleterious effects to bone tissue. The objective of this study was to evaluate the process of
bone regeneration stimuled with laser therapy when hydroxyapatite granules are implanted in bone defects of the femur of rats
submitted to passive tobacco exposure.
Methods and Results:
Porous hydroxyapatite granules were implanted in bone defects produced in the left distal femoral epiphysis of twenty rats Wistar
(Rattus norvegicus) subjected to prolonged passive tobacco exposure during eight months. Immediately after implantation of the
biomaterials, gallium-arsenide laser irradiation was applied to the recipient area at an intensity of 5.0 J/cm2. The rats were
divided into four groups: animals control receiving hydroxyapatite implants without (group G1) and with (group G2) laser
therapy, animals treated (passive tobacco) receiving hydroxyapatite implants without (group G3) and with (group G4) laser
therapy. After eight weeks of the bone implant with the biomaterial, the animals were sacrificed and the specimens of the implant
region were submitted to routine histological testing, and maintained in paraffin blocks for histological analysis. In the results,
was observed good radiopacity of the implant recipient area and of the hydroxyapatite granules in all of studied groups.
Formation of new bone around the hydroxyapatite granules showing characteristics of trabecular and cortical bone was observed
in the studied groups controls (G1 and G2). In the groups submitted to passive tobacco exposure (G3 and G4), the hydroxyapatite
granules were involved by connective tissue without bone neoformation.
Conclusions:
The passive tobacco exposure injures the bone neoformation in skeletal defect and the laser therapy protocol used was not
adequate to stimulated the osteogenic process.
Keywords: HYDROXYAPATITE , LASER, TOBACCO, BONE, REGENERATION
Financial Support: FAPESP/ 2010/ 05769-2
Resumo:10-039
QUANTIFICATION OF OSTEOCLASTS INTO DENTAL ALVEOLI OF WISTAR RATS TREATED WITH
EXTRACTS OF PROPOLIS PURE WITH AND WITHOUT CONTAMINATION BACTERIAL
LIPOPOLYSACCHARIDE
Pereira, Y. C. L. 1; Iyomasa, M. M. 2; Issa, J. P. M. 2; Ervolino, E. 4; Mishima, F. 2
1
Faculdade de Odontologia de Piracicaba, FOP UNICAMP
2
Faculdade de Odontologia de Ribeirão Preto, FORP USP
3
Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, FCFRP
4
Faculdade de Odontologia de Araçatuba, FOAR
5
6
Faculdade de Medicina de Ribeirão Preto, FMRP
Faculdade de Odontologia de Ribeirão Preto , FORP USP
Objectives:
Aim: Propolis is a resinous substance, which antibacterial, antiinflammatory, antiviral, antifungal, immunostimulating healing
and local anesthetic has been considered for clinical use. The lipopolysaccharide (LPS) is recognized as an endotoxin and may
induce inflammatory processes. Our objectives were to analyze, in vivo, the action of Pure Propolis Extract (EPP) in dental
alveoli or not contaminated with lipopolysaccharide (LPS) bacteria.
Methods and Results:
Methods and Results: For in vivo study, 14 rats were submitted to extractions of the superior first molars, right and left, which
immediately had the right dental socket contaminated with 0.1 mL of lipopolysaccharide (LPS) (100ìg/kg) and maintained
without such contamination. The groups (n=7) for each treatment after 2 weeks were analyzed: GI-Negative Control (NC) - no
treatment, GII-Treated with Pure Propolis Extract (EPP). The bone samples were removed, demineralized, processed by routine
histologic technique, submitted to systematic sections (6 µm of thick) for TRAP immunohistochemical staining. The number of
osteoclastic cells stained were counted. The relationship between bone formation and the number of clastic cells was obtained
and submitted to statistical analysis. Observing the normality of data distribution, we proceeded to the factorial ANOVA and
Tukey-Kramer test (p
Conclusions:
Conclusion: We conclude that at 14 days of treatment, it was observed statistical difference between groups with green propolis
contaminated with LPS.
Keywords: TRAP, Bone, osteoclasts, lipopolysaccharide, wistar
Resumo:10-040
INCIDENCE OF DENTAL CALCULUS (DC) AND PERIODONTAL DISEASE (PD) IN BEAGLE DOGS FROM THE
CENTRAL ANIMAL FACILITY (BIC) OF THE FEDERAL UNIVERSITY OF SANTA CATARINA (UFSC), BRAZIL.
Lorenzo, M. A. 1; Santos, A. C. 1; Bello, L. F. C. O. 2; Santos, G. A. 1; Rothstein, J. M. J. 1
1
Biotério Central/Universidade Federal de Santa Catarina, UFSC
2
Médico Veterinário Autônomo, VetBello
Objectives:
Periodontal disease affects 85% of adult dogs (Clin. Vet. 1; .6-7, 1996), one of the most common infirmities in small animal
clinics. It results of the inflammatory response to dental plaque, which is the accumulation by bacteria on the tooth surface, which
is subsequently mineralized, forming the DC (tartar) (Odont. Peq. An., 2010). This disease can generate clinical signs such as bad
breath, drooling, tooth mobility, and unusual signs like dysphagia, anorexia, fractures and osteomyelitis (Comp. Cont. Educ.
Pract. Vet. 12; 951-960, 1990). There is evidence that this illness can also determine the involvement of vital organs (heart, liver,
kidneys) and joints (Clin. Vet. 1; .6-7, 1996). This study determined the incidence of DC and PD for dental and age group in
beagle dogs reared under controlled management and feeding at the BIC/UFSC.
Methods and Results:
A total of 25 beagle dogs of the breeding herd kennel BIC/UFSC, 19 females and 6 males with a mean weight of 17 kg and age
1.5-9 years. This animals were anesthetized with tiletamine/zolazepam 5% - 10 mg/kg, xylazine hydrochloride 2% - 0.5 mg/kg
and atropine sulfate 0.05% - 0.04 mg/kg, IM and underwent a clinical examination of oral cavity for measuring the degree of DC
and PD through the table of scores LASCALA and MOUSSALLI (Period. clín.,1980) and BEARD & BEARD (Vet. Clin. of
North América 19, 1,1989), respectively. Were observed that the incisors (I) were the least affected tooth by the DC (score
0:14,17±1,76/n=300) and the molars (M) had a higher incidence of score 1(12,9±1,18/n=250); already, canines (C) and premolars
(PM) were severely affected with prevalence of scores 2 (13,5±0,95/n=100) and 1 (13,94±1,25/n=400), respectively. In the
presentation of PD, the results were as follows: ‗I‘ predominated the score 0 (17,92±1,04/n=300), ‗C‘ and ‗PM‘ score 1
(1,25±0,75/n=100;7,31±0,86/n=400, respectively) and ‗M‘ scores 0 and 1 (10,5±1,91 and 11,8±1,33/n=250,respectively). In the
evaluation of DC and PD, associated to the animal age, it was observed in both the trend rise in scores with increasing age. The
‗I‘ have a significant increase in the score 1(DC:5,16±0,51;PD:3,75±0,39/n=132), at the age of 3-6 years. The ‗M‘ showed a
decrease in score 0 (DC: 0,3±0,15; PD: 1,2±0,32/n=60) in the group with of 6-9 years old, compared with other groups, although
a predominance of score 1 for DC (5.7 ± 0.74 / n = 110) is observed in the group with 3-6 years. The ‗C‘ and ‗PM‘ have the
dominance of the score 1 for DC already at age 0-3 years (5.0 ± 0.57 / n = 32, 5.1 ± 0.56 / n = 128, respectively), with ‗C‘
exhibiting the same trend for PD (6,75±0,25/n=32), but not the ‗PM‘, where the PD is expressed significantly at age 3-6 years
(5,75±0,33/n=176).
Conclusions:
According to the results, it appears that the dental groups most affected by DC and PD are the ‗C‘ and ‗PM‘. These changes begin
early in life of dogs (0-3 years), and considering the consequences on the health care of them, justify the adoption of preventive
measures to maintain health and welfare of these animals.
Keywords: periodontal disease, dental calculus, dogs
Resumo:12-029
THE INCREASED LEVELS OF PHOSPHO-EIF2-ALPHA INDUCES PHOTORECEPTOR DEGENERATION IN
MURINE MODELS OF RETINITIS PIGMENTOSA(RD1)
Gonçalves, B. S. ; Munk, N. ; Vieira-barroso, L. ; Chiarini, L. B.
Universidade Federal do Rio de Janeiro, UFRJ
Objectives:
The retinitis pigmentosa is characterized by death of photoreceptors in the retina what causes blindness. Therefore, the analysis of
the mechanisms responsible for the death of retinal photoreceptors is critical for the development of therapeutic strategies.
Recently, endoplasmic reticulum stress, activation of PERK and an increase of phosphorylation of eIF2alpha were described in
rd1 mice, a mutant model of retinitis pigmentosa. However, the role of PERK/eIF2alpha pathway, in this context, have not been
evaluated. Our purpose was to test the role of phosphorylation of eukaryotic translation initiation factor 2 alpha subunit
(eIF2alpha) in the degeneration of photoreceptors in retinal tissue from mice RD1 C3H-HeJ, a murine model of retinitis
pigmentosa. This is an animal homozygous for mutation in the gene encoding the β subunit of cGMP phosphodiesterase. This is
one of the most common mutations associated with autosomal recessive retinitis pigmentosa in humans.
Methods and Results:
The degeneration of photoreceptors was assessed by measuring the thickness of the Outer Nuclear layer (ONL, formed by the
photoreceptors) and by detection of DNA fragmentation in situ (TUNEL assay). C57-BL6 wild-type mice were used as control
group. Retinal explants were maintained in vitro for 24 hours in the presence of salubrinal, an inhibitor of the dephosphorylation
of eIF2alpha. In our experimental conditions, the degeneration of the photoreceptor layer of RD1 C3H-HeJ begins on the
fifteenth day post-natal (P15) and is time dependent. We found by western blot an increase of phosphorylated eIF2alpha content
that coincides with the period of the degeneration of the photoreceptor cells (P15). The treatment of retinal explants from
rd1C3H/HeJ mice with salubrinal, the inhibitor of the dephosphorylation of eIF2-alpha, increases the incidence of TUNEL
positive photoreceptors and decreases the thickness of ONL.
Conclusions:
These results indicate that the accumulation of phosphorylated eIF2alpha induces the death of photoreceptors in this model of
retinitis pigmentosa.
Keywords: Retina, Apoptosis, UPR, eIF2, Retinitis Pigmentosa
Financial Support: CNPq, FAPEJ, UFRJ-PIBIC.
Resumo:12-030
INVOLVEMENT OF THE P53 TUMOR SUPPRESSOR PROTEIN IN RESVERATROL-INDUCED APOPTOSIS IN
MCF-7 AND H1299 CELLS
Costa, D. C. F. 1; Malheiros, M. S. 1; Campos, N. P. C. 1; Casanova, F. A. 1; Sanches, D. 1; Fialho, E. 2;
Silva, J. L. D. 1
1
Instituto de Bioquímica Médica - UFRJ, IBqM - UFRJ
2
Instituto de Nutrição Josué de Castro - UFRJ, INJC - UFRJ
Objectives:
Resveratrol (RV) is a promising chemopreventive agent, being able to modulate many cellular targets involved in cancer
signaling pathways. Since p53 has been suggested to be important for anticancer properties of RV, we investigated the RVinduced cytotoxicity in H1299 non-small lung cancer cells, which have a partial deletion of p53 protein-encoding gene, and
compared with MCF-7 breast cancer cells, which constitutively express wild-type p53.
Methods and Results:
RV (0-500μM) reduced both H1299 and MCF-7 cells viability in a dose- and time-dependent manner. However, MCF-7 cells
were more sensitive than H1299, when exposed to RV at concentrations higher than 100µM for 24h. RV (0-200μM) also
increased p53 protein levels in MCF-7, measured by western blotting, without altering p53 mRNA, detected by RT-PCR, thus
suggesting a possible modulation of this protein by post-translational processes. Under these experimental conditions, RV
stimulated phospho-Chk2 and p21 in MCF-7. Moreover, in these cells, RV-induced cytotoxicity was partially mediated by p53
and involved caspases 9 and 7 activation, as well as PARP cleavage, which is indicative of apoptosis. In H1299, cytotoxicity of
RV was less pronounced, as determined by MTT and live/dead assays and, unlike MCF-7, cell death were not accompanied by
caspases activation. These results also corroborate the finding that MCF-7 cells were positively labeled by TUNEL after
exposition to 100µM RV, but not H1299 cells at the same conditions. However, transient transfection of wild-type p53-GFP
renders H1299 more sensitive to the proapoptotic effects of RV.
Conclusions:
Modulation of p53 by RV seems to be an important mechanism by which this bioactive compound exerts its chemopreventive
effects. Our results suggest that H1299 requires p53 expression to undergo apoptosis in response to RV, similar to MCF-7 cells.
Keywords: resveratrol, cancer, MCF-7, H1299, p53
Financial Support: INBEB, FAPERJ, FAF and CNPq.
Resumo:12-031
INVESTIGATION OF YELLOW FEVER VIRUS-INDUCED ENDOPLASMIC RETICULUM STRESS
Sanches, D. 1; Campos, S. P. C. 1; Rocha, C. M. 1; Gonçalves, B. S. 2; Chiarini, L. B. 2; Gaspar, L. P. 3;
Freire, M. S. 3; Silva, J. L. 1; Gomes, A. M. O. 1; Oliveira, A. C. 1
1
Instituto de Bioquímica Médica, IBqM-CCS-UFRJ
2
Instituto de Biofísica Carlos Chagas Filho, IBCCF-UFRJ
3
Instituto de Tecnologia em Imunobiológicos, ITI-FIOCRUZ-RJ
Objectives:
Flaviviruses are arboviruses and cause diseases like Dengue and Yellow fever. These viruses have a particular importance for
public health mainly in South America, Central America and Asiatic southeast. Virus-induced apoptosis is related to a
cytopathological consequence of an infection in vivo or in vitro. During apoptosis, some cellular mechanisms occur, DNA
fragmentation and release of messengers of the apoptotic pathways. The endoplasmic reticulum stress (ERS) can be triggered by
an accumulation of unfolded protein leading to stress response (UPR) with BiP-PERK complex dissociation. Once PERK is
dissociated from BiP it can lead to eIF2á phosphorilation, inducing CHOP overexpression. CHOP is a nuclear factor that leads to
expression and translocation of pro-apoptotic Bcl-2 proteins from the cytosol to the mitochondria. Yellow Fever Virus (YFV) and
other members of the Flaviviridae family are endoplasmic reticulum (ER)-tropic viruses that are dependent on the host ER to
translate, replicate and package their genome. Here, we investigate the endoplasmic reticulum stress induced by YFV.
Methods and Results:
We infected Vero cells with YFV using a MOI = 1 and analyzed the cell viability using LIVE/DEAD kit assay and lactate
dehidrogenase (LDH) activity assay. The expression of BiP, PhosphoeIF2á and CHOP was followed by Western-blotting in a
time-dependent manner after 24, 48 and 72 hours post infection. The YFV-induced ERS was confirmed by PhosphoeIF2á
overexpression 24 hours post infection. We also observed a reduction of cytosolic BiP levels after 24 hours of infection,
suggesting ERS activation. CHOP overexpression was observed after 48 hours post infection. We analyzed the role of PERK
pathway activation during YFV-induced apoptosis by using PERK pathway inhibitor 4 phenylbutyric acid (4-PBA). We observed
that even using 4-PBA inhibitor, YFV still induces apoptosis after 96 hours post infection.
Conclusions:
Our results suggest that ERS could lead to apoptosis pathway activation through CHOP overexpression, but PERK activation
does not seem to be necessary to YFV-induced apoptosis.
Keywords: Yellow Fever Virus, Apoptosis, Endoplasmic Reticulum Stress, PERK
Financial Support: CNPq, CAPES, FAPERJ, FINEP/CT-INFRA, INBEB, PRONEX
Resumo:12-032
STUDY OF LOPAP (LONOMIA OBLIQUA PROTHROMBIN ACTIVATOR PROTEASE) AND DERIVED PEPTIDE
ACTIVITIES IN THE MODULATION OF HUVEC SURVIVAL.
Roedel, K. Q. ; Carrijo-carvalho, L. C. ; Flores, M. P. A. ; Chudzinski-tavassi, A. M.
Laboratório de Bioquímica e Biofísica/Instituto Butantan, IB
Objectives:
Obtention of recombinant Lopap (rLopap), the most abundant protein found in the bristles of Lonomia obliqua caterpillars, and
study of cytoprotective effects of this protein and derived peptide (P4) in cultures of HUVECs (Human Umbilical Vein
Endothelial Cells). Lopap was characterized as a prothrombin activator serine protease which belongs to lipocalin family. The
members of this family present a wide functional diversity, among then, the modulation of cell growth, proliferation and survival,
being Lopap considered the only member of this family to possess proteolytic activity.
Methods and Results:
The recombinant protein was expressed with a histidine tag using the expression vector pAE, which was transformed on E. coli
BL21-DE3 by heat shock. The expression was done in 2xYT medium and induced with IPTG (0.5mM) for 3h at 37°C. After that,
the recombinant protein was purified in Ni-Sepharose column and eluted with imidazole buffer (150mM), followed by desalting
in G-25 column to remove the imidazole salt. Steps of expression and purification were monitored by SDS-PAGE and the
concentration of rLopap was quantified by Bradford assay. The synthetic peptide (P4) was obtained by chemical synthesis in
solid phase according to a conserved domain of lipocalin family found in the Lopap sequence. The HUVECs were cultivated
following the method described by Jaffe et al. (1973), kept on deprived fetal bovine serum medium (RPMI + 1% FBS) and
treated with rLopap (10μg⁄ml, 5μg⁄ml and 2.5μg⁄ml) and P4 (0.9μg⁄ml, 0.3μg⁄ml, 0.1μg⁄ml, 0.03μg⁄ml) (n=4) for 48 and 72h.
Cultures kept with 10% and 1% of serum and no treatment were used as control, being the cell viability measured by MTT assay.
The Lopap was obtained in the recombinant form with yield of 2mg⁄L. When tested on HUVECs for 48h and compared with the
control 1% FBS, rLopap promoted an increase of 16% (p=0.0001) and 22% (p=0.002) in cell viability, at concentrations of
5μg⁄ml and 10μg⁄ml, respectively. On the other hand, P4 (0.1μg⁄ml) increased cell viability by 11% (p=0.006). After 72h,
compared with the control 1% FBS, rLopap induced a viability increase of 11% at 2.5μg⁄ml (p=0.001), 15% (p=0.0005) at 5μg⁄ml
and 42% (p=0.0005) at 10μg⁄ml. Cells treated with 0.1μg⁄ml of P4 showed an increase in viability of 16% (p=0.01).
Conclusions:
Our results indicate that rLopap, as well as P4, have potential properties on modulation of cell survival, which is independent of
the proteolytic activity such as predicted for this molecule.
Keywords: Lopap, Peptide, HUVEC, Cytoprotection
Financial Support: FAPESP (2010⁄03516-0, 2010⁄00600-0)
Resumo:12-033
PROTECTIVE EFFECT OF GLUTAMINE AND ALANIL-GLUTAMINE ON CLOSTRIDIUM DIFFICILE TOXIN AINDUCED INJURY IN HUMAN AND RAT INTESTINAL EPITHELIAL CELLS IN VITRO
Santos, A. A. Q. A. 1; Neto, M. B. B. 1; Freire, R. 1; Santiago, T. M. 2; Ribeiro, R. 1; Brito, G. A. C. 3
1
Farmacologia, UFC
2
física , UFC
3
morfologia, UFC
Objectives:
Aim.: Clostridium difficille is one of the most frequent causes of diarrhea associated to the use of antibiotics. The increase of CDI
incidence and severity has been related to the upbringing of a high virulent strain NAP1/B1/027 which produces wider quantities
of toxin A (TcdA) and Toxin B (TcdB), as well as Binary Toxin (CDT). TcdA causes the Rho proteins family
monoglycosylation, which results in an actin cytoskeleton disorganization, cell rounding and loss of cellular adhesion. Glutamine
(Gln) is a critical requirement for the dynamic proliferating intestinal cell population but Gln has tendency to hydrolyze to
potentially toxic glutamate. Differently of the Alanyl-glutamine (AG) that is stable, well tolerated and repair intestinal injury in
vitro. We evaluated the effect of Gln or AG on the epithelial cell lines:HTC- 8 and IEC-6 following exposure to TcdA of
Clostridium difficile.
Methods and Results:
Four well slide chambers were seeded with HTC-8 cells in RPMI medium. After 48h, cells were incubated in media without Gln,
standard media or for 24 h with TcdA (10 ng / mL), TcdA + Gln (10 mM) and TcdA + AG (10 mM). After 24h of exposure, the
cells were fixed in 4.0% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Cells were stained with FITC-phalloidin
and DAPI to performed confocal microscopy. Cell proliferation was evaluated using the tetrazolium salt WST-1 (4-[3-(4iodophenyl)-2H-5-tetrazolio]-1-3-benzene disulfonate). HCT8-cells were seeded and allowed to attach for 48 hours. Afterwards,
wells were incubated for 48h with 10ng/mL TcdA, TcdA + Gln (10mM) and TcdA + AG (10mM). Wells were incubated for 4
hours with tetrazolium salt and the absorbance was measured using an ELISA (450nm). In another set of experiment, cell culture
plates with 13 mm diameter glass coverslips. It was seeded with IEC-6 cells and grown for 24 h in DMEM media. Afterwards,
the wells were incubated for 24h with TcdA (10ng/mL) in DMEM without Gln or DMEM with Gln or AG. Cells were than fixed
in 4% formaldehyde for 14h. Slides were covered with a 15 mm gold film for conductivity by sputter and examined in a field
emission SEM. TcdA caused cell disruption, changes in F-actin cortex distribution, cell orientation and fragmentation of
cytoskeleton. Supplementation with both Gln and AG partially reverted the changes. Treatment with TcdA at 10ng/mL caused a
reduction of 8.4% on cell proliferation after 48h (p=0.01). Supplementation with 10mM of Gln and with of AG significantly
increased cell proliferation (12.7% and 13,2%, respectively) after 48h, compared to TcdA group; P<0.05.
Conclusions:
Gln and AG partially reverted these changes. Through SEM was observed that cells treated with TcdA shows a cellular
elongation and cytoskeleton disorganization. Those morphological alterations might be caused by disaggregation of actin
cytoskeleton as shown by confocal microscope. These results reinforce the Gln and AG potential as adjuvant therapeutic
measures in C.difficile enteritis.
Keywords: clostridium difficile, glutamine, Toxin A, alanil-glutamine
Financial Support: CNPq/PIBIC
Resumo:12-034
P2X7 RECEPTOR ACTIVATION INHIBITS THE PROLIFERATION OF CHICK RETINAL GLIAL PROGENITORS
IN CULTURE.
Silva, T. M. ; Ventura, A. L. M.
Neurobiology Department / Federal Fluminense University, UFF
Objectives:
ATP induces the proliferation of retinal glial progenitors at early periods of development through the activation of P2Y1
receptors (Intl. J. Devl. Neurosci., 20:21-27, 2002). However, in cultured astrocytes, ATP is also capable of inhibiting cell
proliferation via activation of P2X7 receptors (J. Neuroci. Res. 86:3096, 2008). In this study, we decided to evaluate whether
activation of P2X7 receptors is also able to inhibit ATP-induced proliferation of retinal progenitors in culture.
Methods and Results:
Cultures obtained from retinas of 7-day-old chick embryos were used in immunocytochemistry experiments and incorporation of
[3H]-thymidine assays. When retinal cells cultured for 2 days (E7C2) were treated for 24 h with 500 μM ADP, an increase of
162% above the basal incorporation of [3H]-thymidine was observed. Addition of increasing concentrations of Bz-ATP to the
cultures, a selective agonist for the P2X7 receptor, was able to completely inhibit the proliferative effect of ADP at this stage of
development (in cpm/culture: basal = 905.1 ± 124.8; ADP = 2373 ± 159; ADP + 10 μM Bz-ATP = 1456 ± 154.7; ADP + 30 µM
Bz-ATP = 1118 ± 190.9; ADP + 100 μM Bz-ATP = 692 ± 89.6 p
Conclusions:
Although the two antagonists did not block the effect ob Bz-ATP, our data suggest that activation of P2X7 receptors by ATP
inhibits the proliferation of glial progenitors at an early stage of culture development.
Keywords: ATP, Proliferation, P2X7 Receptors , Glial Progenitors, Retina
Financial Support: FAPERJ, CNPq, Proopi-UFF, MCT-Pronex
Resumo:12-035
FERROMAGNETIC NANOPARTICLES: A TOOL TO LABELING AND TRACKING NEURAL STEM CELLS "IN
VIVO"
Rangel, B. 1,2,4,3; Santos, G. M. 1,2,4,3; Carvalho, A. B. 1,2,4,5; Rachid, R. 1,2,4,3; Attias, M. 1,2,5; Azevedopereira, R. L. 1,2,4,3; Mendez-otero, R. 1,2,4,3
1
Instituto de Biofísica Carlos Chagas Filho, IBCCF
2
Inst Nac de Ciência e Tecnol de Bio Estrut e Bioimagem, INBEB
4
Universidade Federal do Rio de Janeiro, UFRJ
3
Programa de Terapia Celular e Bioengenharia, PTCB
Objectives:
Neural stem cells (NSCs) are a source of new neural cells with capacity of proliferation and differentiation into neurons and glia.
This capacity opens a possibility for replacement cell therapy in neurological disorders. Preclinical and clinical research will
benefit from reliable in vivo tracking of transplanted cells. Here, we investigated the potency of superparamagnetic iron oxide
particles (SPION) to label NSCs, the effect of SPION on: 1) survival; 2) proliferation; 3) cell migration and 4) differentiation into
neural cells
Methods and Results:
NSCs were isolated from E12.5 Swiss mice and cultured as neurospheres in DMEM/F12 supplemented with B27 and epithelial
growth factor (EGF; 20ng/mL) and bFGF (10ng/mL). To label NSC, neurospheres were incubated overnight with 50ug/mL of
SPION (Endorem) and 0.1UI/mL of protamine sulfate. After 24 hours, SPION were observed manly in periphery of the
neurospheres. The areas of neuropheres with and without SPION were analyzed after 48 hours. We did not observe difference
between the areas of neurospheres indicating that SPION did not impair the growth of neurospheres. After 24 hours of labeling,
the neurospheres were dissociated and analyzed by FACS. Two different cell populations were found in the neurospheres by
setting forward and side scatters to linear amplification: 26-30% of the cells were high in forward and side scatter, while 10.412.5% of the cells were low in forward and side scatter. Twenty six percent of the high in forward and side scatter cells were
labeled with SPION, while 20.4% of the low in forward and side scatter cells were labeled. We observed that 44% of the cells
were stained by propidium iodide indicating high mortality after neurospheres dissociation. To access if the neurospheres labeled
with SPION could differentiate in neural cells, we cultured them onto coverslips previously coated with poly-L-lysine (10ug/mL)
and laminin (20ug/mL). To differentiate preferentially into neurons, the cells were cultivated in Neurobasal medium with B27
and N2 supplement. SPION are detected at 3 and 7 in neurons, astrocytes and oligodendrocytes from NSCs. In addition,
transmission electron microscopy (TEM) was performed in neurospheres 24 hours after incubation and SPION were detected
scattered in cellular cytoplasm.
Conclusions:
Our data indicate that NSCs can be labeled with SPION without impairment of neurospheres growth, differentiation and cellular
migration.
Keywords: Nanopaticles, Neural Stem cells, Neuron, Stem Cell
Financial Support: Edital CT-Biotecnologia/MCT/CNPq/MS/SCTIE/DECIT, CAPES, FAPERJ e INCT.
Resumo:12-036
EFFECT OF TREATMENT WITH PURIFIED BIOACTIVE FRACTION OF UNCARIA TOMENTOSA (WILLD) DC.
ON CELL GROWTH OF T24 HUMAN BLADDER CANCER CELL LINE.
Dietrich, F. ; Rockenbach, L. ; Cunha, F. M. D. ; Cappellari, A. R. ; Battastini, A. M. O.
Dep. Bioquímica- Universidade Federal do Rio Grande do Sul, UFRGS
Objectives:
Bladder cancer is the most common malignancy of the genitourinary tract and the effectiveness of treatments, most often, is
inadequate. Extensive studies in recent years, has demonstrated strong evidence for the involvement of purinergic signaling in the
physiology and in the tumor progression of the urinary bladder. Plant species of Uncaria tomentosa, widely used in traditional
medicine because of the many pharmacological properties attributed to it, already showed the presence of metabolites with
potential antitumor effect on different cell lines. Considering these informations, the objective of this study is to investigate the
antitumor effect of purified bioactive fractions of U. tomentosa on the ecto-nucleotidases activity and in cell proliferation on the
T24 bladder cancer cell line.
Methods and Results:
The alkaloidal fraction (AF) and triterpenoid fraction (TF) were obtained by maceration process from the barks of Uncaria
tomentosa. The T24 cell lines were cultured in RPMI culture medium supplemented with 10% fetal bovine serum in standard
conditions. For the evaluation of the antitumoral activity and its effect on cell proliferation, the cell number was determined after
treatments, where the cells were released, dissociated and counted by hemocytometer. The cell viability was evaluated by MTT
assay. The ecto-5'-nucleotidase/CD73 activity was determined by the inorganic phosphate released measured by Malachite Green
method, after different treatments of the bladder cells. Cell adhesion was also assessed, where adherent cells were fixed and
stained, and staining intensity was determined by optical density measurement. The anti-proliferative effect was found to TF after
treatment for 24, 48 e 72h at concentrations of 100 and 150µg/mL, where the inhibition was 29,7% (±9,49 SD) and 39,1% (±3,98
SD); 58,5% (±8,68 SD) and 68,5% (± 6,24 SD); 69,8% (±4,33 SD) and 85,3% (±5,59 SD), respectively. Still, in 72h significant
effect was observed at concentration of 50µg/mL with inhibition of 37,2% (±7,96 SD). The alkaloidal fraction did not affect cell
proliferation in any treatment. The treatment with TF significantly decreased cell viability, following the same profile observed
for cell proliferation. No change in the ecto-5'-nucleotidase/CD73 activity was observed when T24 cell lines were treated with
both fractions (TF and AF) for 10 min (direct effect) or when the cells were treated for 48h. In cell adhesion assay, there was an
increase of 40.8% (± 0.063 SD) in adhesion of T24 cells exposed to TF at concentration of 25µg/mL, unlike the AF which was
not significantly changed on cell proliferation.
Conclusions:
Considering the proposed objectives, it becomes evident that this investigation showed promising biological properties for the TF
of Uncaria tomentosa, which a significant inhibition of T24 human bladder cancer cells proliferation when compared to AF.
However, the involvement of the enzyme ecto-5'-nucleotidase/CD73 was not observed after treatments with both fractions,
excluding its involvement in the death of T24 tumor cells. Furthermore, the increased adherence evidenced after treatment with
the TF, brings new perspectives for research, since the search for treatments that can improve adherence and, consequently,
inhibit the migration are important for decreasing the process of tumor metastasis.
Keywords: bladder cancer, Uncaria tomentosa, cell proliferation
Financial Support: CNPQ
Resumo:12-037
CREB SIGNALING IN NEURONAL SURVIVAL AND DIFFERENTIATION.
Santana, T. T. S. ; Araújo, J. A. D. M. ; Costa, M. R.
Departamento de Neurobiologia Celular, Instituto do Cérebro, UFRN
Objectives:
In the last two decades, several approaches have been proposed to replace neurons lost in the course of central nervous system
diseases (Nature 441, 1094:1096, 2006). However, neuronal integration, differentiation and survival following transplantation are
still a bottle neck for such approaches. Our main goal is to establish cell culture models to investigate genetic and
pharmacological manipulations leading to increased neuronal differentiation and survival in cell-based therapies. Here, we study
the role of CREB (cAMP-response element binding) signaling.
Methods and Results:
Embryonic day 18 cortical cells from C57/Bl6 mice were digested in 0,05% Trypsin, dissociated and grown as primary
neurospheres in DMEM/F-12 supplemented with 2% B27, (1% Penicillin/Streptomycin, 4.500 mg/L glucose, 1% glutaMAX) 10
ng/mL basic fibroblast growth factor (FGF), and 10 ng/mL epidermal growth factor (EGF). After 7 days, primary neurospheres
were plated onto PDL-coated glass cover slips in DMEM F-12/(1% Penicillin/Streptomycin, 4.500 mg/L glucose, 1%
glutaMAX)/2% B27/1% fetal bovine serum for differentiation. Two hours after plating, cells were transfected with GFP, ACREB
(negative dominant of CREB) or CREB s142l (constitutively active) either by retrovirus or Ca2+-phosphate. To manipulate
signaling cascades involved in the activation of CREB-dependent-transcription, cultures were treated with H-89 (PKA-inhibitor),
KN-62 (CAMK-inhibitor), U0126 (MAP/ERK-inhibitor) or Wortmannin (PI3K- inhibitor) 24h after plating. Cultures were then
maintained under differentiation condition for 7-10 d before fixation with 4% PFA and immunocytochemistry for GFP and
neuronal markers (β-III tubulin or MAP2). To analyze the morphology of the neurons, images were taken with an epifluorescence microscope and analyzed in Image J v1.44 (National Institute of Health). Total dendritic length, number of dendritic
branches and longer dendrites were measured. Our preliminary results indicate that Ca2+-phosphate methods is more efficient
than retroviruses to transfect neurons derived from cortical neurospheres. Dendritic morphology of cortical neurons is
significantly affected by treatment with H-89 (PKA-inhibitor), KN-62 (CAMK-inhibitor), U0126 (MAP/ERK-inhibitor) and
Wortmannin (PI3K- inhibitor), similar to what has been described following forced expression of CREB.dominant negatives
(Neuron 34, 999:1010, 2002).
Conclusions:
Our results suggest that CREB mediated transcription can be activated through different signaling pathways and is important for
dendritic differentiation of newly generated neurons.
Keywords: CREB, signaling cascades , Neurospheres, neuronal differentiation and survival, dendrites
Financial Support: FAPERN, CAPES and CNPq.
Resumo:12-038
INVESTIGATION OF PURINERGIC SYSTEM IN NON-DIFFERENTIATED AND DIFFERENTIATED STEM
CELLS
Iser, I. C. 1; Bracco, P. A. 1; Zaninn, R. F. 5; Lenz, G. 4; Nardi, N. B. 2; Battastini, A. M. O. 3; Wink, M. R. 1
1
Departamento de Ciências Básicas da Saúde, UFCSPA
2
Departamento de Genética, UFRGS
3
Departamento de Bioquímica, UFRGS
4
Departamento de Biofisica, UFRGS
5
Faculdade de Farmácia, PUCRS
Objectives:
Extracellular nucleotides are signaling molecules, involved in important processes in the body. Recently, it has been suggested
that the purinergic system may be involved in the biology of mesenchymal stem cells (MSCs). A particular interest has been
focused on the role of this system in the proliferation and differentiation of this cells. In view of this, our goal was to analyze the
degradation rate of adenine nucleotides as well as the expression of NTPDases and ecto-5'-nucleotidase (CD73) in nondifferentiated and differentiated MSCs.
Methods and Results:
The cultures of MSCs from BALB/c mice non-differentiated and differentiated into adipocytes and osteoblasts, were submitted to
the determination of enzymatic activity for hydrolysis of ATP, ADP and AMP, by measuring the release of inorganic phosphate
by the Chan method. Protein content was performed by the Comassie blue method. The real-time PCR technique was performed
to verify the expression of NTPDases, using primers for the six types of NTPDases and CD73. Our data suggest that ATP, ADP
and AMP hydrolysis increase when the cells are differentiated in osteoblasts, however, in the adipogenic differentiation, there is
increase only in the AMP hydrolysis. Moreover, non-differentiated and differentiated MSCs express NTPDase 1,2, 3, 5 and 6 and
CD73 in similar levels.
Conclusions:
ATP may be required during the process of differentiation in osteoblasts but not necessary when differentiation is complete,
explaining its high hydrolysis after differentiation. Moreover, we believe that adenosine generated might be involved in the
processes of MSCs differentiation, since the presence of receptors for this molecule has been reported in the process of
differentiation of hematopoietic precursor cells. Finally, the presence of these ectoenzymes and the differential ectoncleotidases
activities in undifferentiated and differentiated cells suggest that these enzymes can modulate the concentration of extracellular
nucleotides and nucleosides, generating a signal that can be important in physiological regulation and maintenance of
undifferentiated and differentiated MSCs.
Keywords: stem cells, purinergic, differentiation
Financial Support: CAPES and CNPq.
Resumo:13-068
α-MSH EFFECTS ON RHODOPSIN, TYROSINASE AND MC1R GENES IN B16 MUS MUSCULUS
MELANOCYTES.
Glória, T. H. R. ; Castrucci, A. M. D. L.
Departamento de Fisiologia, Instituto Biociências, USP, IB
Objectives:
In vertebrates, skin color is given by pigments, synthesized and/or stored in cutaneous pigment cells. The vertebrate color change
is mainly regulated by α-MSH and a family of melanosome enzymes, which includes tyrosinase and tyrosinase-related proteins 1
and 2 (TRP-1 and TRP-2, respectively). α-MSH action is associated with melanosome dispersion and/or melanin synthesis,
processes which lead to body darkening, whereas melanin aggregation or synthesis inhibition results in skin lightening. Opsins,
such as melanopsin and rhodopsin, may be expressed in skin pigment cells, besides being present in the retina, and may mediate
non-visual photo-responses such as cell proliferation and melanosome dispersion. The aim of this study was to investigate the
temporal expression of rhodopsin, tyrosinase and of α-MSH receptor MC1R, as well as the effects of 10-7 M and 10-8 M α-MSH
for 24 hours in Mus musculus B16 melanocytes, kept in constant darkness.
Methods and Results:
In this work we used quantitative PCR to investigate the expression of rhodopsin, tyrosinase and the MC1R receptor genes in
Mus musculus melanocytes submitted to constant darkness regimen, in the presence or absence of α-MSH. Cultured melanocytes
were seeded (1x106 cells) in 25 cm2 culture flasks, and kept at 37°C in constant darkness for 2 days. Total RNA extraction was
performed every four hours along 20 hours starting at 0 hour of the third day. The samples were submitted to RT-PCR followed
by quantitative PCR for mRNA quantification of MC1R receptor, tyrosinase and rhodopsin. Using real time PCR (quantitative)
we demonstrated that 10-7 M α-MSH does not modulate MC1R mRNA levels, as compared to the control group, although a
tendency to reduction was evident. On the other hand, at the concentration of 10-8 M, we observed a statistically significant
increase of the transcript level at hour 20, as compared to the control group. For rhodopsin, we demonstrated that 10-7 M α-MSH
modulates mRNA levels, as compared to the control group, demonstrating a statistically significant decrease at hour 12. At 10-8
M, α-MSH significantly increased the transcript levels at hour 4 and 20, as compared to the control group. The same pattern was
observed for tyrosinase, demonstrating a statistically significant decrease in the concentration of 10-7 M at hour 0 and a
significant increase in the concentration of 10-8 M at the hour 8.
Conclusions:
Using real time PCR (quantitative) we demonstrated that α-MSH promotes a dose-dependent response of MC1R receptor,
tyrosinase and rhodopsin gene expression, but was not able to synchronize the expression of these genes.
Keywords: MC1R, Melanin, α-MSH, Rhodopsin, Tyrosinase
Financial Support: FAPESP and CNPq grants. THRG is a fellow of FAPESP.
Resumo:13-069
AN ASSESSMENT OF THE DIABETES KNOWLEDGE AMONG HIGH-SCHOOL STUDENTS IN THE CITY OF
ITABAIANA, SERGIPE, BRAZIL
Lima, D. B. 1; Santos, D. M. 1; Camporez, J. P. G. 2; Aires, M. B. 1; Rodrigues, T. M. A. 1; Aragão, J. A. 1;
Carvalho, C. R. O. 2; Marçal, A. C. 1
1
Departamento de Morfologia, Universidade Federal de Sergipe, UFS
2
Deptº de Fisiologia e Biofísica, Universidade de São Paulo, USP
Objectives:
Evaluate the knowledge of high school students in the city of Itabaiana-SE about diabetes, and also estimate the number of
students with the disease.
Methods and Results:
This was an exploratory descriptive study involving 712 high school students (14 to 44 years old) of the urban area in Itabaiana
City. It was applied an structured questionnaire with subjective questions covering the general knowledge about diabetes mellitus
(DM), the etiologic classification and symptom identification related to Diabetes type 1 and 2.The body mass index was
calculated based on weight and height self-reported. The results were expressed as average, confidence intervals (CI) or
percentages (p
Conclusions:
The diabetic knowledge results showed a lack of information about the disease symptoms and an ineffective comprehension of
the diabetes types and the specific symptoms.
Keywords: Diabetes, Knowledge, Students
Resumo:13-070
USE OF MARKERS OF OXIDATIVE STRESS AS INDICATORS OF INJURY OF THE LOWER EXTREMITIES IN
PATIENTS WITH TYPE 2 DIABETES MELLITUS.
Teixeira, C. J. 1; Oliveira, A. C. P. 1; Stefanello, T. F. 1; Carrara, M. A. 5; Sá-nakanishi, A. B. D. 2; Comar,
J. F. 2; Santos, C. A. D. 3; Mareze-costa, C. 4; Bazotte, R. B. 5; Batista, M. R. 1
1
Clinical Analysis Depart. / State University of Maringá, UEM
2
Biochemistry Departament / State University of Maringá, UEM
3
Statistical Departament / State University of Maringá, UEM
4
Morphological Sciences Depart. / State University of Maringá, UEM
5
Pharmacology Departament / State University of Maringá, UEM
Objectives:
Oxidative stress, caused by an imbalance between production of reactive oxygen species and endogenous antioxidant capacity is
a major determinant of diabetic complications. We investigated the role of oxidative stress markers as indicators of risk of injury
of the lower extremities in patients with type 2 diabetes mellitus (PPDMT2).
Methods and Results:
In a group of 29 patients (the Ethics Committee of the State University of Maringá - Protocol no. 381/2010) were assessed the
following parameters: 1. Anthropometric measures (weight and height) for calculating the Body Mass Index (BMI); 2. Glycated
hemoglobin (HbA1c); 3. Three blood markers of oxidative status (total antioxidant capacity - TAC, reduced proteins thiol groups
and reduced levels of protein carbonyls), and 4. Test of sensation in the feet, as determined by to the National Hansen's Disease
Program (HRSA – U.S.). Patients were divided into two groups according to the index of sensation in the feet: group 1 (low risk
of injury – 18 PPDMT2) and group 2 (high risk of injury – 11 PPDMT2). It is noteworthy that the metabolic evaluations were
performed at two moments in each group, baseline (time 0) and later (110 days). The results were: 1. The group 1 had a mean age
56.9±8.7 years and the median duration of disease of 120 [0-240] months, while group 2 had a mean age of 63.4±7.6 years and
the median of disease duration of 180 [60-480] months (P
Conclusions:
The results showed an increase in HbA1c levels reflected in the increase of the marker pro-oxidant and reduction in markers of
antioxidant. Thus, we conclude that these markers of oxidative stress may be a good parameter to assess the degree of impairment
or worsening of vascular lesions in the lower extremities in type 2 diabetes.
Keywords: type 2 diabetes mellitus, diabetic foot, oxidative stress
Financial Support: Fundação Araucária.
Resumo:13-071
MATERNAL NICOTINE EXPOSURE DURING LACTATION ALTERS HYPOTHALAMUS- PITUITARYADRENAL FUNCTION IN MALE ADULT OFFSPRING.
Younes-rapozo, V. ; Bernardino, D. N. ; Peixoto-silva, N. ; Rodrigues, C. P. ; Santos-silva, A. P. ; de
Moura, E. G. ; Manhães, A. C. ; de Oliveira, E. ; Lisboa, P. C.
Departamento de Ciências Fisiológicas/IBRAG, UERJ
Objectives:
We have previously showed that maternal exposure to nicotine during lactation - from postnatal day 2 (P2) to P15, causes some
metabolic disturbance in pups, such as higher serum leptin, higher catecholamine content, lower tyrosine hydroxilase expression
and higher serum corticosterone at the end of treatment (J Endocrinol 205 159-170, 2010), evidencing an acute effect of nicotine.
Our objective here was to evaluate the long-term effects of maternal nicotine exposure exclusively during lactation, specifically
in the hypothalamic-pituitary-adrenal axis, by the immunostaing of corticotropin-releasing factor (CRF) and adrenocorticotropic
hormone (ACTH) and the analyses of serum corticosterone.
Methods and Results:
At postnatal P2, dams were subcutaneously implanted with osmotic minipumps releasing nicotine (NIC - 6mg/Kg/day) or saline
for 14 days (control group). P180 male offspring (n=4/group, one animal from each litter) were intracardially perfused with 0.9%
saline solution followed by 4% paraformaldehyde and 4% paraformaldehyde plus 10% sucrose for cryoprotection. Brains and
pituitaries were sectioned at 20micrometers on a cryotome at - 20ºC. Hypothalamus sections were incubated with anti-CRF
(1:100 from Abcam) and pituitaries with anti-ACTH (1:100 from Santa Cruz). Immunoreaction was visualized by incubation
with secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 555 (1:400 from Invitrogen, Molecular Probes) and
sections were also counterstained with DAPI (1:5000 from Sigma). Serum corticosterone was measured using a specific
commercial RIA kit (ICN Biomedicals Inc) (n=6/group, one animal from each litter). NIC animals showed a higher CRF
immunostaining, and CRF-positive fibers were apparently thicker but smaller, in comparison to control group. In pituitaries, NIC
male offspring showed ACTH-positive cells with increased immunoreactivity in comparison to control animals. Concerning
serum corticosterone, NIC group presented higher levels (+77%, p
Conclusions:
Our results suggest that the adrenal dysfunction of male offspring in early life by maternal nicotine exposure can alter
permanently the hypothalamic-pituitary-adrenal axis at adulthood, stimulating all the axis, reinforcing the idea that maternal
smoking during lactation can be a risk factor for development of later hormonal disorders.
Keywords: nicotine exposure, lactation, CRH, ACTH, corticosterone
Financial Support: CAPES, FAPERJ, CNPq.
Resumo:13-072
GHRELIN SIGNALING IN ADIPOSE TISSUE OF OBESE MICE IS ALTERED DURING THE DEVELOPMENT.
Soares, V. . M. ; Garcia-souza, E. P. ; Miranda, G. L. ; Neves, F. A. ; Bernado, A. F. ; Gomes, I. A. ; Lessa,
J. G. ; Moura, A. S.
Instituto de Biologia Roberto Alcantra Gomes - UERJ, IBRAG
Objectives:
Human overnutrition has caused a rise in the prevalence of obesity in recent years. In addition to the deleterious effects of obesity
during childhood, the long-term effects in adulthood have also been described. Intensive efforts are underway to clarify nutrienthormone interactions contributing to weight gain. The gastric peptide hormone ghrelin, an endogenous ligand for GHS-R, has
generated considerable interest as an important stimulus for weight gain and control body fat mass by modulating the storage and
oxidation of fatty acids. This study aims to evaluate the association of ghrelin signaling in adipose tissue in mice overfed during
lactation after weaning and during adulthood.
Methods and Results:
To induce early postnatal overnutrition, Swiss mice were overfed through litter size reduction. In this model, the litter was
adjusted to only three male pups per litter three days after birth (overfed group; OG). For the control group, the litter size was
adjusted to nine newborns (CG).The CG and OG were studied at 21 and 180 days. We evaluated anthropometric parameters as
body mass and visceral adipose tissue. Fasting glucose was measured using glucometer and test strips. Histological sections of
adipose tissue were paraffin embedded and stained with hematoxylin and eosin to determine the area and diameter of adipocytes,
using Olympus BX40 microscope and analyzed with Image-Pro Plus software version 4.5.1. Analysis of the protein content of
GHS-R 1a, CPT1, PPAR, UCP2, AKT, pAKT, PI3K and actin were detected by Western blotting method. Statistical analysis was
performed using Student's t-test and values expressed as mean and standard error. Results: In these animals, overfeeding during
lactation was able to induce a significant increase in body weight starting at the 21th day of life, and this increased weight
persisted until 180 days of age (21 days- CG: 13.43 ± 0.76, OG: 19.57 ± 0.29 g, N=8, 180 days - CG: 48.15 ± 0.85, OG: 67.8 ±
1.9 g, n = 17). We observed a significant increase in fasting glucose between groups at 180 days (C: 114.0 ± 9205, O: 160.2 ± 5.9
mg / dl), and of visceral adipose tissue weight at 21 days (CG: 0.03 ± 0.005 g, N=6, OG: 0.25 ± 0.05g N=6) and 180 days(CG:
2.3 ± 0.67 , OG: 5.31 ± 0.37 g, n = 8) compared to CG animals. OG Adipocytes showed a larger area (CG: 3095.7 ± 484, OG:
6292.9 ± 902 ìm2, n = 5) and diameter (CG: 75.7 ± 1.85, OG: 104.7 ± 4 , 0 mM, n = 5) compared with CG. At 21 days of age OG
presented a expressive increase in the GHSR1a (CG: 18,330 ± 4433, OG: 44,460 ± 1778 AU, n = 5) and PI3K content (CG:
14,560 ± 1355, OG: 39,620 ± 5920 AU n = 6) in adipose tissue. However, this profile has changed in adulthood, since GHSR1a,
PI3K, AKT, pAKT and PPAR gamma content were reduced in adipose tissue of OG compared to CG at 180 days old.
Conclusions:
Overfeeding during lactation induces obesity, increased visceral fat mass, adipocyte hypertrophy. Ghrelin receptor expression and
key signaling proteins of the hormone action were found altered. The data suggested an association of the hormone with storage
and oxidation of fatty acids during mice developme
Keywords: Ghrelin, overnutrition, adipose tissue
Financial Support: CNPq, Capes, FAPERJ, Vital Brasil
Resumo:13-073
LONG-TERM EFFECTS OF NEONATAL HORMONAL MANIPULATION ON BONE MINERAL AND
BIOMECHANICAL PROPERTIES OF FEMUR RATS
Mello, W. G. D. 1,4; Morais, S. R. L. D. 1,4; Biffe, B. G. 1,4; Louzada, M. J. Q. 2; Dornelles, R. C. M. 4;
Nakamune, A. C. D. M. S. 4; Antunes-rodrigues, J. 3; Bedran de Castro, J. C. 4
1
Multicentric Graduate Studies Prog. in Physiological Science, SBFis
2
Department of Support to Production and Health Animal , FMVA-UNESP
3
Department of Physiology, FMRP-USP
4
Department of Basic Sciences , UNESP - Araçatuba-SP
Objectives:
Several functions wich are controlled by the brain are expressed differently in male and female mammals, including the central
regulation of bone mass. Therefore, the aim this study was analyze the long-term changes in bone mineral and biomechanical
properties induced by sexual dimorphism in the hypothalamic control of bone mass
Methods and Results:
Wistar rats, newborns, were divided into four groups (n = 08 per group). Male pups were cryoanesthetized and castrated or
sham/castrated by 24 hours after birth; female pups from separate litters were injected SC with testosterone propionate 100 ìg in
50µL corn oil (androgenized female) or oil vehicle 50µL (control female) and were observed for 20, 40 and 120 postnatal day,
both femurs were collected and preserved in physiological saline at -20 °C by the day of experiments. The length and thickness of
the left femurs were performed with aid of a caliper; after measuring, they were dried overnight at 90 °C and ashed at 900 °C for
24 h for determined the mineral content. The bone mineral density (BMD) was determinate in the right femurs by dual-energy Xray absorptiometry (DXA) and, the mechanical properties were assessed by Three-Point Bending Testing; these data were used
for the acquisition and calculation of the structural properties: ultimate strength (N), toughness (N.mm), and stiffness (N/mm).
Data were analyzed using ANOVA and Tukey and expressed as the means±SE. The results showed that bone mineral content and
biomechanical properties in all groups increased rapidly with aging. However, the neonatal exposed to androgen induced changes
in growth, BMD and bone mass quality in androgenized females, leading to a masculinization pattern in development. On the
other hand, neonatally castrated males had the bone development and quality of the mechanical properties similar to those control
females.
Conclusions:
In conclusion, our work indicated that the long-term organizational influence of the neonatal castration and androgenization,
which is known inducing irreversible effects on CNS and hypothalamus, also induced a long-lasting modification in bone
structure and strength. These results provide new insight into the dynamic complexity of bone mass homeostasis.
Keywords: ANDROGEN, BONE PROPERTIES, SEXUAL DIMORPHISM
Financial Support: CAPES
Resumo:13-074
DPP-IV ACTIVITY AND INFLAMMATORY BIOMARKERS IN TYPE 2 DIABETES MELLITUS
Bellé, L. P. 1; Bitencourt, P. E. R. 2; Bona, K. S. 2; Moretto, M. B. 2
1
Departamento de Análises Clínicas e Toxicológicas , USP
2
Departamento de Análises Clínicas e Toxicológicas, UFSM
Objectives:
Type 2 diabetes mellitus (type 2 DM) is one of the world‘s most important diseases and is associated with a general activation of
the innate immune system, in which there is a chronic state of low-grade inflammation (Trends Endocrinol. Metab. 19; 10, 2008).
Dipeptidyl peptidase IV (DPP-IV, CD26) is a glycoprotein widely expressed in lymphocytes surface, where it is binding with
adenosine deaminase (ADA) (J. Immunol, 156; 1349, 1996). Moreover, DPP-IV has been proposed as a target for
pharmacological intervention in patients with type 2 DM (Curr. Diabetes Rev. 5; 92, 2009). Thus, the aim of this study was to
investigate CD26 expression and activity as well as ADA and n-acetyl-β-d-glucosaminidase (NAG) activities in lymphocytes
from type 2 DM and control subjects.
Methods and Results:
We obtained lymphocytes from 32 fasting type 2 DM patients (HbA1c= 7.36 ± 0.22%) and 23 fasting control subjects (HbA1c=
5.00 ± 0.17%). In lymphocytes we determinated DPP-IV, ADA and NAG activity and CD26 expression. The results were
described in mean ± S.E. We observed an increase in ADA (C=7.75±0.49; type 2 DM=9.37±0.51U/L) and NAG (C=23.63±2.5;
type 2 DM=29.86±1.4mmol/L) activities in lymphocytes from type 2 DM when compared with control subjects. DPP-IV activity
(C=83.39±6.5; type 2 DM=89.12±4.5U/L) did not alter between the groups and the CD26 expression decrease in type 2 DM
patients (C=14.68±1.7, type 2 DM=8.96±1.2%). Moreover, we observed a positive correlation between ADA and NAG activity
(R= 0.4272; P
Conclusions:
We conclude that in type 2 DM there is no coestimulatory activity (low CD26 expression), possible because the no alteration in
DPP-IV activity. The increase in ADA and NAG activity indicate an inflammatory state in these patients. This result is reinforced
by the positive correlation between NAG and ADA activity and the negative correlation between NAG and CD26 once it is
known that there is an inverse relationship between CD26 and inflammation.
Keywords: type 2 diabetes mellitus, lymphocytes, dipeptidyl peptidase IV, adenosine deaminase, n-acetyl-b-d-glucosaminidase
Financial Support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Resumo:13-075
GENOTYPIC DISTRIBUTION OF THE FAT MASS–AND OBESITY-ASSOCIATED (FTO) GENE
POLYMORPHISMS AND ASSOCIATION WITH C-REACTIVE PROTEIN AND METABOLIC VARIABLES IN
RECENT POSTMENOPAUSAL WOMEN RECEIVING HORMONE THERAPY
Ramos, R. B. ; Casanova, G. K. ; Spritzer, P. M.
Gynecological Endocrinology Unit, UFRGS/HCPA, GEU, UFRGS/HCPA
Objectives:
Postmenopausal women are at a higher risk of hypertension, unfavorable changes in the lipoprotein profile, diabetes, and
cardiovascular disease as compared with their premenopausal counterparts. Hormone therapy (HT) seems to have different
impact on cardiovascular (CV) risk, according to the age and time after menopause and ultra sensitive C reactive protein (usCRP)
has been regarded as a good marker in predicting this CV risk. The fat mass–and obesity-associated gene (FTO), located in the
chromosome 16, is associated with fat mass and body mass index (BMI), risk of obesity, metabolic syndrome and high CV risk.
The present study aimed to assess the genotypic distribution of the single-nucleotide polymorphisms (SNPs) rs9939609 T>A and
rs8050136 A>C located in the intron 1 of the FTO gene and verify whether these gene variants are associated with usCRP and
metabolic variables in recent postmenopausal women receiving HT.
Methods and Results:
Sixty-six participants (51± 3 years, 22± 10.2 months since menopause) consulting for symptoms of estrogen deficiency were
clinically evaluated before and after 6 months of HT. DNA was extracted from peripheral blood by a standard salting out
procedure and genotyped by allelic discrimination assay with Real Time PCR. The genotypic distribution of the SNPs rs9939609
(TT: 47%, TA: 42.4%, AA:10.6%) and rs8050136 (AA: 13.6%, AC:34.9%, CC: 51.5%) were similar to previous studies. During
HT, waist circumference and systolic blood pressure were reduced. Women with the homozygous polymorphic AA genotype of
the SNP s9939609 showed a decrease in usCRP [2.1 (0.9 -4.8) to 1.2 (0.5 -1.9) mg/L, p= 0.02] and triglycerides [158.7±16.9 to
129.1±22.3 mg/dL, p=0.02] after HT, while no changes were observed in women with the TT+TA genotypes. In contrast,
considering the SNP rs8050136, only women with the wild AA genotype presented a decrease in the usCRP [1.84 (0.77 -4.49) to
1.23 (0.61-2.23) mg/L, p0.008] after HT, but not those with the polymorphic AC+CC genotypes. Anthropometric and other
metabolic variables remained unchanged before and after HT in both polymorphisms.
Conclusions:
Our results indicate that these FTO gene variants may exert dissimilar influence on the effects of HT: the wild genotype of the
SNP rs8050136 and the homozygous polymorphic genotype of SNP rs9939609 presenting a beneficial impact on usCRP. These
data suggest a possible linkage desequilibrium in the 1 and 2 intronic regions of the FTO gene.
Keywords: FTO gene, Hormone Therapy, Menopausal Women, Polymorphisms
Financial Support: CAPES, INCT Hormones and Women‘s Health- CNPq
Resumo:13-076
ENDOCRINE AND METABOLIC FUNCTIONS ARE ALTEREDED IN A MODEL OF GENERALIZED ANXIETY
DISORDER
Mousovich-neto, F. 1; Lonrenço, A. L. 1; Landeira-fernandez, J. 2; Corrêa-da-costa, V. M. 1
1
Instituto de Biofísica Carlos Chagas Filho/UFRJ, IBCCF/UFRJ
2
Departamento de Psicologia/Pontifícia Universidade Católica, PUC
Objectives:
Freezing in response to threat is used by many species as an adaptative-defensive strategy. Carioca High-Freezing rats (CHF) are
animals selected by their high freezing response in contextual fear conditioning and represent a great model of generalized
anxiety disorder. Here we evaluated some aspects of endocrine and metabolic functions in those animals, by evaluating
corticosterone and testosterone serum levels, thyroidal function, serum colesterol and triglycerides, fasted glucose serum levels,
glucose tolerance, oxigen consumption and epididymal and retroperitoneal fat depots weight.
Methods and Results:
Control male Wistar rats (C) and CHF animals were sacrificed by decaptation and blood was collected and centrifuged at 1200x
g, to obtain serum. Corticosterone, testosterone and thyroid hormones were measured by specific radioimunoassays, and
colesterol and triglycerides through an enzymatic-colorimetric assay. Oxygen consumption was measured using an air
composition analyzer. Epididymal and retroperitoneal fat depots were removed and weighted. Glucose tolerance test were
performed in fasted animals by administration of 1.83 x 10-3 mol/glucose/100g of body weight and glycemia measurements were
done 30, 60, 120 and 180 minutes after the glicemic orogastric infusion. Fast glycemia was considered the first measure of the
glucose tolerance test. Serum corticoesterone was higher in CHF animals (C: 118.9±27.97 vs CHF: 339.0±49.38 ng/mL), while
serum testosterone was decreased (C: 3.33±0.294 vs CHF: 2.03±0.29 ng/mL). Serum T3 is decreased (C: 58.40±2.401 vs CHF:
46.95±2.846 ng/dL) while T4 and thyrotropin serum levels were unaltered in CHF animals. Serum colesterol was higher if
compared with control group (C: 181.6±5.61 vs CHF: 226.4±13.04 mg/dL) as well as serum triglycerides (C: 41.4±6.03 vs CHF:
82.2±17.4 mg/dL). Fat depots were significantly higher in CHF animals; epididymal (C: 2.4±0.26 vs CHF: 4.3±0.38 g) and
retroperitoneal (C: 1.8±0.212 vs CHF: 3.8±0.58 g). Oxygen consumption was lower in CHF animals when compared to control
animals (C: 10.55 vs CHF: 7.95 VO2 ml/min/kg0.75). Fast glucose was higher in CHF animals (C: 68.7±3.04 vs 82.3±2.9
mg/dL) however there was no difference between groups in glucose tolerance test.
Conclusions:
In CHF animals, anxiety disorder induces important endocrine and metabolic disfunctios
Keywords: anxiety, endocrine disorders, metabolic disorders
Financial Support: CNPq, FAPERJ, CAPES and SBEM Thyroid Department.
Resumo:13-077
SODIUM APPETITE AFTER SODIUM RESTRICTION: AN INTEGRATIVE VIEWPOINT
Mecawi, A. S. 1,2; Fonseca, F. V. 2; Vilhena-franco, T. 1; Reis, L. C. 2; Elias, L. L. K. 1; Antunes-rodrigues,
J. 1
1
Department of Physiology, FMRP-USP
2
Department of Physiological Sciences , UFRRJ
Objectives:
Salt intake is a behavior implicated in the integrative regulation of extracellular volume, plasma tonicity and blood pressure and is
directly influenced by changes in sodium homeostasis. Sodium dietary restriction is an interesting noninvasive model to study the
sodium appetite. Our aim was to investigate the integrative control of salt and water intake after dietary sodium restriction.
Methods and Results:
To this purpose, Wistar male rats received normal- (1% NaCl) or low- (< 0.01) associated with a decrease in water intake (9.6 ±
0.8 mL vs. 13.1 ± 0.5 mL, p < 0.05) and a decrease in the extracellular volume as demonstrated by increase in hematocrit (42.8 ±
0.7 % vs. 38.8 ± 0.6 %, p < 0.01) and plasma protein (7.0 ± 0.1 mg/dL vs. 5.7 ± 0.1 mg/dL, p < 0.001). Additionally the low-Na+
diet decreased urinary volume (5.4 ± 0.6 mL vs. 7.6 ± 0.5 mL, p < 0.05) and sodium excretion (0.26 ± 0.01 mE/100g/day vs. 1.66
± 0.07 mE/100g/day, p < 0.01). We observed a discrete decreased arterial pressure (101 ± 1.5 mmHg vs. 105 ± 1.1 mmHg, p <
0.05) without effect in heart rate. There was no effect of low-Na+ diet on plasma atrial natriuretic peptide and vasopressin
concentrations. An increase in plasma angiotensin II (119 ± 12 pg/mL vs. 39 ± 11 pg/mL, p < 0.001) and decrease in plasma
oxytocin (0.60 ± 0.04 pg/mL vs. 0.99 ± 0.12 pg/mL, p < 0.05) concentrations after low-Na+ diet was observed. These animals
also presented an increase in 1.8% NaCl intake at 30 min (0.44 ± 0.05 mL vs. 0.06 ± 0.02 mL, p < 0.05) and 5 hours (1.65 ± 0.13
mL vs. 0.57 ± 0.05 mL, p < 0.001) after four days of low-Na+ diet. Associated to this, low-Na+ diet group also presented sodium
intake-dependent increase in water intake at 1 hour (0.99 ± 0.09 mL vs. 0.49 ± 0.12 mL,p < 0.05) and 5 hours (2.86 ± 0.13 mL vs.
1.64 ± 0.21 mL, p < 0.001). Regarding the sodium appetite, the low-Na+ diet induced an important increase in sodium preference
at 30 min (31.4 ± 3.5 % vs. 12.9 ± 4.8 %, p < 0.001) and 5 hours (34.1 ± 1.9 % vs. 22.1 ± 1.9 %, p < 0.01).
Conclusions:
Our results indicate that sodium appetite control and the strong increase in sodium preference after sodium restriction is
dependent on a variety of factors, that could activate sodium appetite signals (volume/ baroreceptors and peripheral Ang II) and
inhibition of sodium satiety signals, as oxytocin.
Keywords: renin-angiotensin system, oxytocin, low sodium-diet, salt intake
Financial Support: CNPq/FAPESP
Resumo:13-078
ENDOCANNABINOID SYSTEM AND INFLAMMATORY RESPONSE IN ADIPOSE TISSUE DURING OBESITY.
Gotardo, É. M. F. ; Santos, A. N. ; Ribeiro, M. L. ; Gambero, A.
Clinical Pharmacology and Gastroenterology Unit, USF
Objectives:
Obese animals and humans over activated the endocannabinoid system (CB1 and CB2 receptors, endogenous ligands and
enzymes for synthesis or degradation of ligands) in several tissues involved in metabolic process, including adipose tissue (AT).
The endocannabinoid system also modulates inflammatory response, mainly through CB2 activation in inflammatory cells. Thus,
we investigated if endocannabinoid system expression is altered in AT and in macrophages infiltrated in AT from high-fat diet
mice. We also analyzed the CB2 cannabinoid agonist action in macrophage/adipocyte co-culture system, an in vitro model of
inflammation in adipose tissue.
Methods and Results:
Swiss mice were feed with high-fat diet (HFD) or standard diet (N) during 12 or 24 weeks (n=12/group). Body weight and
glucose homeostasis (glucose basal and Insulin test tolerance) were evaluated. Epididymal adipose tissue was collected for gene
expression analysis by qRT-PCR (CB1, CB2, N-acyl-phosphatidylethanolamine (NAPE), fatty acid amide hydrolase (FAAH),
diacylglycerol lipase (DAGL)). 3T3-L1 adipocytes and RAW 264.7 macrophages were co-cultured in presence or absence of
CB2 agonist JWH-015 (1, 3 e 10 µM). TNF-&alpha was measured by ELISA in co-cultures stimulated or not by
lipopolysaccharide (LPS, 1 ng/ml). HFD mice presents high body weight (41.3±1.1 and 56.8±1.8 g for C and HFD 12 weeks;
45.5±1.2 and 62±2.2 g for C and HFD 24 weeks) and insulin resistance (3.38±0.75 and 1.17±0.09 for C and HDF 12 weeks;
1.79±0.6 and 0.502±0.09 for C and HFD 24 weeks), confirming the obesity status. CB2 receptor gene expression in AT was
higher in obese mice after 12 or 24 weeks (14.0±3.3 and 3.3±0.4 arbitrary unit (AU) for HFD and C 12 weeks group,
respectively; 19.8±2.1 and 11.3±2.0 AU for HFD and C 24 weeks group, respectively). NAPE expression was reduced only after
24 weeks in AT (15.5±4.2 and 38.8±5.8 AU for HFD and C 24 weeks, respectively). When isolated macrophages were analyzed
no differences were observed in CB2 or NAPE expression. Basal or LPS-stimulated TNF-&alpha release in the co-culture system
were reduced by 10µM JWH 015 (1243,2±60,4 e 982,0±141,9 pg/ml in the absence and presence of JWH 015 after LPS
stimulus).
Conclusions:
Increased CB2 expression observed in AT from obese mice could be due an increased macrophage infiltration, because in
isolated macrophage these alterations were not detected. Anyway, the stimulation of CB2 receptor reduces TNF-&alpha
production by adipocytes/macrophages suggesting an anti-inflammatory action that could be very important in the control of AT
inflammation during obesity.
Keywords: inflammation, macrophages, adipocytes, CB2 receptors, adipokines
Financial Support: FAPESP and CNPq
Resumo:13-079
MATERNAL HIGH-FAT DIET DURING PREGNANCY AND LACTATION PROGRAMS THE OFFSPRING FOR
OBESITY AND LEPTIN RESISTANCE IN MICE.
Volpato, A. M. ; Magalhães-da-costa, E. ; Monterlei, R. K. S. ; Aguila, M. B. ; Mandarim-de-lacerda, C. A.
; Correia, M. L.
Departamento de Anatomia , UERJ
Objectives:
Fatty diet during pregnancy in rat dams programs for metabolic syndrome (MS) in the offspring. We tested the hypothesis that
the offspring of dams fed high fat diet during pregnancy and lactation develops MS and leptin resistance.
Methods and Results:
Pregnant C57BL6 mice (n=20) were fed either standard chow (SC; 19% fat) or high fat diet (HF; 49% fat). After weaning, male
offspring was divided in four groups according to diet of dams and offspring: SC(dams)/SC(offspring), SC/HF, HF/SC and
HF/HF (n=12/gp). MS was characterized by weight gain curve measured weekly (g); tail-cuff systolic pressure (mmHg), areas
under the curve after oral glucose tolerance test (OGTT) and fat mass depots performed at 12 weeks of age (g). To analyse leptin
sensitivity, each group was divided into two groups (vehicle or leptin-5µg) to verify the feeding response (Kcal) after acute
intracerebroventricular (ICV) treatment (n=6/gp). Offspring born from HF dams does not present higher body mass at birth and at
weaning (data not shown). However, at 6 weeks of age, HF/HF animals were heavier than SC/SC (+10%, p
Conclusions:
In conclusion, HF diet during pregnancy and lactation programs the offspring to the development of obesity. Patterns of blood
pressure and glucose metabolism can be affected only if HF diet persists after weaning. Importantly, HF diet during gestation and
lactation; only after weaning or during whole life alter the feeding response to leptin, which could mechanistically contribute to
the development of obesity.
Keywords: obesity, programming, leptin resistance
Financial Support: Cnpq, FAPERJ
Resumo:13-080
TYPE 2 IODOTHYRONINE DEIODINASE ACTIVITY IS INHIBITED BY THIMEROSAL
Pantaleão, T. U. ; Padrón, A. S. ; Ferreira, A. C. F. ; Carvalho, D. P. D. ; Rosenthal, D. ; Guimaraes, J. R. D.
; Costa, V. M. C. D.
INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO/UFRJ, IBCCF/UFRJ
Objectives:
Sodium ethyl mercury thiosalicylate (thimerosal) is an ethylmercury (Et-Hg)-containing preservative that has been used for over
80 years as an antimicrobial agent in vaccines to prevent contamination. Until the removal of thimerosal from most pediatric
vaccines in 2001, the largest human exposure in the US was in children under 18 months of age. Prior to 2001, a child may have
received a cumulative dose of over 200 mg/kg in the first 18 months of life. In the body, ethylmercury can be converted to
inorganic mercury, which then accumulates in the kidney and brain. Thyroid hormones, triiodothyronine (T3) and thyroxine (T4),
play an essential role in the central nervous system development. In the brain, the T3, the metabolic active hormone is locally
produced by T4 deiodination mediated by type 2 iodothyronine deiodinase (D2). So, D2 plays an important role in adequately
maintaining local T3 levels in the brain and consequently normal brain development. We aimed to examine the effect of
thimerosal on D2 activity.
Methods and Results:
We obtained brown adipose tissue, hypothalamus and hippocampus of adult female Wistar rats. D2 activities was determined by
quantification of the radioiodine released by 125I-T4 under standardized conditions, and expressed as fmol T4 min-1.mg.ptn-1.
D2 inhibition was determined, in the presence of different concentrations of thimerosal (6mM; 1,2mM; 0,6mM; 0,2mM; 100µM).
In vivo effect of thimerosal was also evaluated in female rats treated with two different doses of thimerosal (0,25 µg/100g BW or
250µg/100g BW, i.m.), administered twice a week for one month. Animals were sacrificed and hypothalamus, pituitary,
cerebellum and hyppocampus were obtained and processed for determination of D2 activity. Thimerosal was able to completely
inhibit the enzyme in brown adipose tissue, hypothalamus and hippocampus. The activity was reduced by 50% (IC50) in the
thimerosal concentration of 0.47 mM in BAT, 0,19mM in hypothalamus and 0,25mM in hippocampus. Despite of the clear
inhibition shown in vitro, in vivo treatment inhibited D2 activity in pituitary (C: 3.06±0.178 vs 2.62±0.140 or 2.358±0.082 fmol
T4 min-1.mg.ptn-1 in a dose of 25 µg/100g BW and in a dose of 250µg/100g BW, respectively), but no statisticaly differeces
were detected in hippocampus, cerebellum, or hypothalamus tissues.
Conclusions:
Our data show that thimerosal is able to inhibit D2 activity both in vitro and in vivo, although the inhibition was not observed in
all tissues studied. Since this enzyme is important for local T3 generation, our results indicate a possible deleterious effect of
thimerosal, especially in the higher dose. On the other hand, there must be defense mechanisms against the effect of thimerosal
on D2 activity, since important targets of T3, such as cerebellum, hypothalamus and hyppocampus, showed no inhibition of D2
by this drug.
Keywords: DEIODINASE ACTIVITY, THIMEROSAL, THYROIDAL HORMONE METABOLISM
Financial Support: PRONEX/FAPERJ, FAPERJ, CNPq, MCT
Resumo:13-081
HIGH CONCENTRATIONS OF RESISTIN AND TNF-Á MAY RESULT IN REDUCED INSULIN SIGNAL IN RATS
CHRONICALLY TREATED WITH NAF
Silva, V. C. ; Chiba, F. Y. ; Shirakashi, D. J. ; Colombo, N. H. ; Garbin, C. A. S. ; Sumida, D. H.
Department of Basic Sciences, FOA - UNESP
Objectives:
The excessive (F) fluoride intake can lead to acute or chronic poisoning, such as dental fluorosis and disturbances in glucose
homeostasis. Further studies from our laboratory have demonstrated that chronic treatment with NaF causes insulin resistance
(IR) and decreased insulin signal (IS). In the literature there are several studies that correlate increased concentration of resistin
and TNF-á with IR and decreased IS. Knowing that chronic treatment with F can interfere with IR and IS, the aim of this study
was to determine whether alterations in concentrations of resistin and TNF-á could be involved in the reduction of IS in rats
treated with F. In order to do that, the experiment was carried out to evaluate: 1) the IRS-1 serine phosphorylation status in
epididymal white adipose tissue (WAT), 2) the plasma concentrations of resistin and TNF-á in rats.
Methods and Results:
Four-week-old male Wistar rats (32 animals) were castrated. After 30 days this castration, the animals were divided into two
groups: 1) control group (CN); 2) fluoride group (FN), which was submmited for treatment with NaF (3.1 mg F / kg b.w.) in
drinking water for 42 days. After 6 weeks, the animals were subjected to 14-hour fast and then they were anesthetized to: 1)
quantify the IRS-1 serine phosphorylation status after insulin stimulation in WAT by the method of "western blotting", 2) collect
the blood samples for evaluation of plasma concentrations of resistin and TNF-á by ELISA method. The results showed that the
FN group, compared to the CN group, showed a significant increase (p
Conclusions:
The increase in plasma concentrations of resistin and TNF-á may have caused the decrease in IS, because an increase in the IRS-1
serine phosphorylation status in WAT was observed. Based on this, it is recommended to reduce the fluoride concentration (1100
ug F / g) in toothpaste used mainly by children with diabetes, as the ingestion of toothpaste containing fluoride can lead to
worsening in the health of these children. Research "in vitro" (Caries Res 41: 263, 2007) using a more acidic toothpaste (pH =
4.5) with 412 and 550 ug F / g has observed that these acidic toothpastes had the same effectiveness of neutral toothpaste (pH =
7.0) with 1100 ug F / g. These toothpastes (acidified with low fluoride concentration) would be a great option for young children
who often take in large amounts of toothpaste, with a major impact on the health of diabetic children.
Keywords: Diabetes Mellitus, Fluorine, Fluoride Poisoning , Insulin, Insulin resistance
Resumo:13-082
ANALYSIS OF THE OXYTOCIN EFFECTS ON BONE REMODELING IN SENILE RATS
1
Colli, V. C. 1; Spritzer, P. M. 2; Dornelles, R. C. M. 1
Multicentric Studies Graduate Prog in Physiological Sciences, UNESP
2
Department of Physiology,, UFRGS
3
Department of Basic Sciences, UNESP
Objectives:
Treatments for osteoporosis aim increase the bone density, acting in the decreased of reabsorption or increased the bone
formation. Studies have been developed to find new therapy to bone formation and oxytocin has been suggested as anabolic bone
mass regulator. The aim of this study was to verify the oxytocin action on bone metabolism in senile rats by analysis of alveolar
bone repair as well as the concentrations of markers of cellular activity.
Methods and Results:
Female Wistar rats (24-mo-old) in the diestrus cycle phase were divided into two groups (8 rat in each group). Two i.p. injections
of saline (NaCl 0.9%-control group) or Oxytocin (45 µg/rat- treated group) was given 12 h apart. Seven days after, the right
maxillary incisor was extracted. At 35 days after oxytocin treatment the blood was collected and the right maxilla was removed.
The plasma concentrations of calcium, phosphorus and alkaline phosphatase were determined for spectrophotometry (Labtest
kits, São Paulo, Brazil). Serum osteocalcin (OC) and tartrate resistant acid phosphatase (TRAP) levels were determined (ELISA USCN Life Science Inc.). Tests were performed in duplicate samples. Middle thirds stained sections of the of the alveolus were
examined by light microscopy under 10X objective lenses, and images were obtained with a digital camera (JVC TK-1270 Color
Video Camera) mounted on the microscope, and analyzed with Leica Qwin Color/RGB software. Differences between groups
were tested using a Student t-test as the criteria of normal and equal variances between groups was met with a significance level
of 0.05 using GraphPad Prism 3.02 (GraphPad Software, Inc.; La Jolla, CA, USA). All data are reported as means with their
standard errors of the mean (SEM). The plasma concentration of calcium (control = 10.70 ± 0.6580; treated = 10.41 ± 0.6663
mg/dL) and phosphorus (control =4.880 ± 0.3216; treated = 5.629 ± 0.3932 mg/dL) was not significantly changed by treatment
with oxytocin, however, the ALP (control = 73.20 ± 4.510; treated =130.8 ± 11.37 U/L) and OC (control = 0.7280 ± 0.02956;
treated = 1.117 ± 0.04839 ng/mL) serum level was significantly higher in the treated group. The TRAP serum level (control =
2.404 ± 0.3724; treated = 0.5867 ± 0.07200 U/L) was significantly decreased after oxytocin treatment. Histomorphometric
analysis (control = 29.95 ± 1.807; treated = 44.36 ± 2.697 %) showed higher areas of bone formation in treated group.
Conclusions:
This results evidence the peripheral action of oxytocin in bone metabolism of the senile females and suggests involvement of this
hormone in decreased bone resorption.
Keywords: bone remodeling, oxytocin, senile rats
Financial Support: CAPES
Resumo:13-083
ARGININE INDUCES GH GENE EXPRESSION BY ACTIVATING NOS/NO SIGNALLING IN RAT ISOLATED
HEMI-PITUITARIES
Olinto, S. C. F. 1; Gomes, M. A. 2; Barbosa, T. D. C. 3; Silva, F. G. D. 3; Nunes, M. T. 3
1
Faculdade De Ciências Integradas do Pontal, UFU
2
Depart.of Animal Morphology and Physiology , UFRPE
3
Department of Physiology and Biophysics, ICB-USP
Objectives:
Aim: The amino acid arginine (Arg) is a recognised secretagogue of growth hormone (GH), and has been shown to induce GH
gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many effects of Arg, such as GH
secretion. Arg was also shown to increase calcium influx in pituitaries cells, which might contribute to its effects on GH
secretion. Although the mechanisms involved in Arg effects on GH secretion are well established, little is known about them on
the control of GH gene expression. This study aimed to investigate whether the NO pathway and/or calcium are involved with the
Arg effects on GH gene expression in rat isolated pituitaries.
Methods and Results:
Methods and Results: Pituitaries of Wistar rats weighing approximately 250 g were removed, divided in two halves, pooled and
incubated or not with Arg as well as with different pharmacological agents, to attain this purpose. It was observed that Arg 15
mg/ml; NO donor SNP - sodium nitroprusside: 10-3 and 10-4 M; and cGMP analogue 8-Br-GMPc 10-3M increased GH mRNA
expression 60 min afterwards: Control (C) 1,00 ± 0,17 vs Arg 15 mg 1,90 ± 0,30 , p
Conclusions:
Conclusion: The present study increases the body of evidence showing that Arg induces GH gene expression in hemi-pituitaries
and further demonstrates that NOS/ NO signalling pathway and calcium are essential for this effect.
Keywords: Arginine, Calcium , cGMP, GH mRNA , Nitric Oxide/NO sintase
Financial Support: Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP.
Resumo:13-084
VENTRICULAR T3-INDUCED HYPERTROPHY IS ACCOMPANIED BY SIRTUIN1 AND PPAR ALPHA PROTEIN
EXPRESSION DOWNREGULATION
Oliveira, L. S. ; Cordeiro, A. ; Souza, L. L. ; Pazos-moura, C. C.
INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO, UFRJ
Objectives:
Sirtuin1 (SIRT1) is a NAD+-dependent deacetylase that acts on transcription factors, modifying their activity. SIRT1 have
similar roles as the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) on the metabolism regulation. In
the liver, increased SIRT1 and PPARα protein contents are normally observed in fasting, an important response for adaptation to
deprivation conditions. PPARα activation increases SIRT1 expression, whereas SIRT1 knockout mice have disturbed PPARα
transcriptional activity. It was described that SIRT1 and PPARα are downregulated in cardiac hypertrophy models by
isoproterenol administration or pressure overload, and these reports characterized them as part of protection response to
hypertrophy. Since thyroid hormone status variation promotes structural cardiac changes, we aimed to investigate SIRT1 and
PPARα expression in ventricle of hypo- and hyperthyroid mice, and its possible correlation with thyroid hormone-induced
hypertrophy.
Methods and Results:
Male mice (2 months of age) were rendered hypothyroid (HYPO) by addeding propyl-thyouracil 0.15% in the chow during 4
weeks and, hyperthyroidism (HYPER) was induced by T3 subcutaneous daily injections for 14 days (50 ug/100g body weight).
These mice were compared to euthyroid littermates (EU). Another experimental protocol was developed to compare hormone and
food restriction effects. A group of HYPER mice received the same food amount as EU mice consumed, as well as, a group of
EU animals were submitted to equal percentage of restriction as HYPER restricted, in mean 40% restriction during 13 days.
Ventricular SIRT1 and PPARα protein content were analyzed by Western blotting. One-way ANOVA followed by the StudentNewman-Keuls test was used for comparisons between different groups and, differences were considered significant at p
Conclusions:
We demonstrated that SIRT1 and PPARα protein levels in the cardiac ventricle are downregulated in hyperthyroidism and
upregulated in hypothyroidism. These results suggest that SIRT1 and PPARα could be involved in thyroid hormone effects on
ventricular mass.
Keywords: THYROID HORMONE, HYPERTROPHY, SIRTUIN1, PPAR ALPHA
Financial Support: CNPq, CAPES and FAPERJ
Resumo:13-085
EVALUATION OF GADD45γ AND MEG3 EXPRESSION BY QUANTITATIVE REAL TIME PCR IN SPORADIC
PITUITARY ADENOMAS.
Pesce, F. G. ; Mezzomo, L. C. ; Gonzales, P. H. R. ; Kretzmann, N. A. ; Oliveira, M. C. ; Kohek, M. B. F.
Universidade Federal de Ciências da Saúde de Porto Alegre, UFCSPA
Objectives:
This study evaluated MEG3 and GADD45γ gene expression by qRT-PCR in sporadic functioning and clinically non-functioning
human pituitary adenomas morphologically characterized by immunoistochemical analysis.
Methods and Results:
Fourteen clinically nonfunctioning, 10 GH-secreting, 9 PRL-secreting, and 5 ACTH-secreting pituitary adenomas from patients
who had undergone hyphophysectomy at São José Hospital of Irmandade Santa Casa de Misericórdia in Porto Alegre, were
included in this study. RNA was obtained by Trizol method and cDNA was synthesized using Superscript 1st strand (Invitrogen).
We evaluated the tumor-type specific MEG3 and GADD45γ gene expression by quantitative RT-PCR and correlated with clinical
features. The qRT-PCR was performed using 10μL working mix containing 2μL of the cDNA template in 2x TaqMan™
universal Master Mix (Applied Biosystems) and 100nM final concentration of the primers and the probe. A comercial pool of
RNA from normal pituitary gland was used as a calibrator. The quality control was done by the simultaneous amplification of the
constitutive gene GAPDH. Statistical analysis was done using Kruskal-Wallis followed by post-hoc evaluation. To compare
median between groups, (clinically non-functioning adenomas vs. functioning adenomas) Mann-Whitney test was performed.
Significance was taken at the 5% level. The presence of the GAPDH amplicon by duplicate qRT-PCR confirmed individual
cDNA integrity in all of the samples examined. MEG3 expression was lost in 20 of 38 (53%) pituitary adenomas. Thus far,
clinically non-functioning pituitary adenomas had a significant decrease in MEG3 relative expression compared with functioning
adenomas (p=0,001). In contrast, qRT-PCR showed detectable and variable relative expression of this gene in all functioning
pituitary adenomas (p=0,001), while GH-secreting adenomas (p=0,01) also had a significant variability when compared with
clinically nonfunctioning adenomas. In the same background, GADD45γ transcript was not detected in 24 of 38 adenomas
(63,2%). Interestingly, clinically nonfunctioning adenomas had a significant decrease (pGADD45γ relative expression when
compared with functioning adenomas. However, some functioning pituitary adenomas demonstrated detectable and significant
variable GADD45γ relative expression (pp=0,001) presented increased and variable levels of GADD45γ expression when
compared with other tumors, followed by ACTH-secreting adenomas (p=0,05).
Conclusions:
MEG3 and GADD45γ gene expressions are significantly lost in almost all clinically nonfunctioning adenomas studied, which are
of gonadotroph origin. Other pituitary tumor phenotypes examined expressed both genes in significantly different levels, and
even with overexpression in some cases. These data suggest that those genes probably were in association with uncontrolled cell
growth and tumor development in the human pituitary gland.
Keywords: Cell Cicle, GADD45γ, MEG3, Pituitary Adenomas
Financial Support: Programa de Apoio à Pesquisa (PROAP) - UFCSPA
Resumo:13-086
VITAMIN D RECEPTOR GENE POLYMORPHISMS AND SEX STEROID SECRETION IN GIRLS WITH
PRECOCIOUS PUBARCHE IN SOUTHERN BRAZIL.
Santos, B. R. 1,2; Mascarenhas, L. P. G. 3; Satler, F. 2; Boguszewski, M. C. S. 3; Spritzer, P. M. 1,2
1
Department of Physiology, UFRGS
2
Division of Endocrinology/HCPA, UFRGS
3
Department of Pediatrics, UFPR
Objectives:
Evidence suggests that precocious pubarche [PP] girls may have higher risk of developing polycystic ovary syndrome [PCOS] at
later ages. Vitamin D receptor [VDR] gene polymorphisms have been implicated in the risk of diabetes and PCOS, but little is
known about the role of VDR in PP. The aims this work were to assess the frequencies of VDR gene ApaI, TaqI, BsmI and FokI
polymorphisms and to determine whether these variants are associated with sex hormone concentrations in patients with PP and
controls from southern Brazil.
Methods and Results:
Blood was collected from 36 girls with PP (age 11.27 ± 3.79 years at the time of the study) and 197 controls (12.92 ± 2.09 years
at the time of the study) for genotyping of BsmI and FokI polymorphisms using real time polymerase chain reaction [PCR] and of
ApaI e TaqI polymorphisms using restriction fragment length polymorphism [PCR-RFLP], all in vitamin D receptor gene.
Hormone levels were also determined. Genotype GG of the ApaI single nucleotide polymorphism [SNP] was more frequent in PP
(30.6%) than in controls (16.2%) (odds ratio [OR]: 2.269; confidence interval 95%[95%CI]:1.015-5.076; p = 0.042). This
genotype was also associated with lower estradiol (35.30 [14.80-50.48] pg/mL vs. 12.22 [6.49-23.69] pg/mL; p = 0.030) and total
testosterone levels (0.52 [0.39-0.84] ng/mL vs. 0.20 [0.11-0.47] ng/mL; p = 0.009), but not DHEAS (148.20 [79.25 - 202.50]
μg/dL vs. 83.03 [62.00 - 174.00] μg/dL; p = 0.186), as compared with the TT + TG genotypes in girls with PP. The distribution
of TaqI, BsmI and Fokl SNPs was similar in PP and controls, and no association was found between these polymorphisms and
sex steroid levels.
Conclusions:
The present findings suggest that the ApaI polymorphism of the VDR gene is associated with PP and seems to modulate ovarian
steroid secretion in affected girls. Further studies are needed to better clarify the biological mechanisms by which the ApaI
polymorphism influences PP risk and sex steroid secretion.
Keywords: vitamin D receptor, precocious pubarche, steroid hormones, single nucleotide polymorphism
Financial Support: National Institute of Hormones and Women‘s Health
Resumo:13-087
MODULATION OF PI3K/AKT PATHWAY BY RENIN-ANGIOTENSIN SYSTEM INHIBITION IN OBESITY
EXPERIMENTAL MODEL.
Bulla, M. L. ; Bertholdo, M. C. D. M. ; Haidar, A. A. ; Hirata, A. E.
Fisiologia/Universidade Federal de São Paulo , UNIFESP
Objectives:
The objective was to evaluate IRSs/PI3-K pathway in insulin resistant obese animals treated with AT1 blocker.
Methods and Results:
Insulin resitant animals were obtained by neonatally treatment with glutamate monosodium (MSG). MSG animals had higher
body weight than the control animals at seven months old (7-mo). Furthermore, 7-mo MSG animals showed no change in
pressure blood compared to controls and treatment with losartan did not alter it. Evaluating the association of IRS1 with PI3K in
muscle, we noted that the acute stimulation with insulin can promote the association in the control group that received vehicle.
Muscle of MSG animals presented decreased IRS1/PI3K interaction after acute insulin stimulation however no statistical
significance. The treatment with losartan did not change this response. However, IRS2/PI3-K association increased significantly
after losartan treatement. When evaluating the adipose tissue of these animals, we observed an improvement not statistically in
the IRS1/PI3K interaction comparable to the controls suggesting higher sensitivity of this tissue to the hormone But with that
increase group experiemntal. On the other hand, when assessing IRS2/PI3K interaction we observed a strong tendency decrease
in adipose tissue of MSG suggesting locally resistance to insulin action.
Conclusions:
Neonatal treatment with monosodium glutamate (MSG) induced metabolic syndrome. Treatment with losartan did not alter
parameters such as body weight and blood pressure of animals MSG. Treatment with losartan for one week showed a tissue
specific modulation of IRSs/PI3K interaction. Possibly the increased insulin resistance reflected by lower IRS2/PI3K association
in adipose tissue after losartan treatment may contribute to decreased glucose uptake in this tissue and thus would tend to
decrease fat mass and production of various adipokines that are known to work for the installation and worsening of metabolic
syndrome. Our results are not conclusive, being necessary to perform experiments complemetares increasing n order to elucidate
results.
Keywords: Diabetes, Hypertension, Insulin
Financial Support: Scientific activity performed from January 2010 to 2011 grant from FAPESP.
Resumo:13-088
IDENTIFICATION OF NOVEL VARIANTS OF RET ONCOGENE POTENTIALLY LINKED TO THE
PATHOGENESIS OF PHEOCHROMOCYTOMA AND MEDULLARY THYROID CARCINOMA
Bim, L. V. ; Cerutti, J. M.
Universidade Federal de São Paulo, UNIFESP
Objectives:
The RET gene encodes a transmembrane receptor with tyrosine kinase activity linked to signaling pathways involving growth,
migration and cell development. Activating mutations of RET in cell germlines are associated with an autosomal dominant
hereditary syndrome called multiple endocrine neoplasia type 2 (MEN 2), which can be classified into three subtypes: familial
medullary thyroid carcinoma (FMTC), MEN 2A and MEN 2B. MEN 2A is characterized by the presence of medullary thyroid
carcinoma (MTC), pheochromocytoma (PHEO) and hyperparathyroidism. FMTC is characterized by the presence of MTC as the
only clinical feature. Our group recently described a novel heterozygous mutation in exon 8 of RET gene associated with FMTC,
which leads to the substitution of a glycine by a cysteine at codon 533 (p.G533C). Later a member of the family with FMTC was
diagnosed with PHEO, suggesting that p.G533C mutation leads to the MEN 2A phenotype. Interestingly, in addition to the RET
mutation p.G533C, two novel variants within RET oncogene were found in DNA isolated from PHEO, which were not detected
in the DNA extracted from the blood of the patient. This finding suggests that additional genetic events in somatic tissues may be
associated with tumorigenesis of MEN 2A-associated tumors. Whether this event is associated with pathogenesis of sporadic
PHEO or MTC, is still unclear. We here aim to investigate the prevalence of new variants in the RET gene exon 8 (G548V) and
exon 9 (S556T) in sporadic and familial PHEO and MTC. We additionally will investigate whether these variants activate the
MAPK pathway, through in vitro phosphorylation of ERK.
Methods and Results:
A set of 25 PHEO and 43 MTC, sporadic and MEN 2A-associated tumors were selected from the files of the Department of
Pathology of UNIFESP. Detection of RET variants in paraffin-embedded pheochromocytoma specimens will be performed by
PCR sequencing. Specific primers were designed for amplification of exons 8 and 9 of the RET gene. Of the 12 samples, 6 have
already had the genetic material extracted and primers are in the process of standardization. For functional analysis, cDNA RET
Wt and new mutant‘s cDNA will be cloned in expression vector (pBUD4.1), there will be a site-directed mutation to obtain the
G533C mutation, the plasmid will be then transfected into HEK 293 cells for protein expression and RET phosphorylated ERK
analysis by Western blotting. Specific primers for subcloning and site directed mutation of RET were designed and are being
standardized.
Conclusions:
Recent data on the exon 8 mutations and its genotype-phenotype suggest that they are more aggressive and less rare than
originally imagined. The two new mutations never described before, found in the patient, suggest that a second hit in the RET
gene can be a potential mechanism for the formation of pheochromocytoma in patients with MEN 2A. Thus, the results of this
project can and aims to help clarify such issues.
Keywords: MTC, Mutations, Pheochromocytoma, RET oncogene
Financial Support: FAPESP
Resumo:13-089
INTERMITTENT APNEA INDUCES INSULIN RESISTANCE IN CONSCIOUS RATS.
Quadros, C. D. M. 1; Haidar, A. A. 1; Nejm, M. B. 3; Rocha, M. S. 2; Carpinelli, A. R. 2; Cravo, S. L. D. 1;
Schoorlemmer, G. H. M. 1; Hirata, A. E. 1
1
Depto. de Fisiologia/ Universidade Federal de São Paulo, UNIFESP
2
Depto. de Fisiologia e Biofísica, Universidade de São Paulo, USP
3
Depto. de Neurociências/ Universidade Federal de São Paulo, UNIFESP
Objectives:
Diabetes mellitus is a very common finding in patients with sleep apnea, but whether apnea causes diabetes, diabetes causes
apnea, or both are consequences of obesity is not clear. We used a new method to induce intermittent apnea in rats and measured
the consequences of apnea on glucose tolerance and insulin sensitivity.
Methods and Results:
We used male Wistar rats weighing 300 – 500 g. Rats were anesthetized with ketamine and xylazine, and a balloon-tipped
polyurethane catheter was implanted in the trachea. The balloon was contained in a rigid Teflon tube so that inflation of the
balloon blocked the airway, without inducing tracheal pain. A silicone rubber cannula was inserted in the femoral vein and
advanced to the thoracic vena cava. Both balloons were exteriorized on the rat‘s back. These implants allow induction of apnea
and removal of blood samples in conscious, unrestrained rats. Rats were allowed at least a week to recover pre-operative body
weight. To measure insulin sensitivity, insulin (0.75 U/kg body weight) was injected through the venous cannula. Blood glucose
concentration was measured just before, and 4, 8, 12, and 16 min after insulin injection. To measure glucose tolerance, glucose (1
g/kg body weight) was injected through the venous cannula, and blood glucose concentration was measured just before, and 4, 8,
12, 16, 20, 24, and 28 min after glucose injection. Glucose tolerance and insulin sensitivity were measured a few days before and
30 min after 8 h of intermittent apnea (14 s every 2 min). Apnea did not significantly change glucose tolerance (AUC in controls
1110 ± 52 mg/dl*min, after apnea 1181 ± 55 mg/dl*min, n = 8/5), but it significantly reduced insulin sensitivity: the glucose
decay constant fell from 5.3 ± 0.4 to 3.9 ± 0.5 %/min (n=8/7).
Conclusions:
Our data suggest that apnea contributes to development of insulin resistance. However, we are not yet sure that the mechanism of
insulin resistance is the same in human patients with sleep apneas and in rats with a tracheal balloon. We are planning to increase
the delay between the end of the apneas and measurement of insulin sensitivity to reduce acute effects of stress.
Keywords: Glucose tolerance, Insulin resistance, Intermittent apnea
Financial Support: CAPES e FAPESP
Resumo:13-090
THYROID STATUS MODULATES THE EXPRESSION OF THYROID HORMONES TRANSPORTERS MCT8 AND
MCT10 IN THE LIVER
Pereira, G. F. ; Ramos, R. G. ; Império, G. E. D. ; Santiago, L. A. ; Faustino, L. C. ; Ortiga-carvalho, T. M.
Instituto de Biofísica Carlos Chagas Filho, UFRJ
Objectives:
The idea that thyroid hormones (TH) entered cells passively through the plasma membrane was abolished after the discovery of
transporters that mediate the cellular influx and efflux of TH. Two members of the monocarboxylate transporters (Mct) family
can transport TH, Mct8 and Mct10, which are expressed in many tissues. However, it is unknown whether TH can change its
transport into liver cells via regulation of its transporters. Here, we aim to evaluate the mRNA expression of Mct8 and Mct10 in
the mice liver after TH deprivation, chronic treatments with both TH biologically active forms (T3 and T4) and acute treatment
with T3 only.
Methods and Results:
In 12-week-old male mice, hypothyroidism (Hypo group) was induced by feeding them a 0.15 % PTU diet for 3 weeks. Daily sc
injections of T3 (50 �g /100g BW) were given for 2 weeks to induce hyperthyroidism in the group called HyperT3, while sc
injections of T4 at the same concentration were given during the same period to induce hyperthyroidism in the group called
HyperT4. Four animals were used in each group, except the control group, composed by five animals, totalizing 17. Acute
treatment was done by one single sc injection of T3 (50 �g/100g BW). Mice were sacrificed 15, 30, 60, 180 and 360 minutes
after injection. Control groups were injected with saline. Data were reported as mean ± SEM. One-way ANOVA followed by
Student-Newman-Keuls multiple comparisons test and Two-way Anova were employed. Five animals were used in each group,
resulting in 50 treated animals. Liver mRNA levels of Mct8 and Mct10 were analyzed by real time PCR, samples were
normalized to euthyroid controls. Unexpectedly, in chronically treated mice, both hypo- and T3 treated hyperthyroidism led to the
reduction of mRNA expression of Mct8 (Hypo: 0.5± 0.01; HyperT3:0.4±0.1,p0.05). However, there is a tendency to increase the
mRNA expression in HyperT4 compared to HyperT3, although not significantly different. Conversely, acute T3 injection
increased mRNA expression of Mct8 (60:2.3±0.2,180:1.6±0.14,360:1.9±0.37) p
Conclusions:
Our preliminary data showed that, chronically, hypothyroidism and T3 treatment decreased Mct8 and Mct10 mRNA expression
in mice liver. Acutely, T3 increased Mct8 and Mct10 mRNA expression, suggesting a non genomic action of T3.
Keywords: thyroid hormones transporters, thyroid status, liver
Financial Support: CNPq, CAPES, FAPERJ, Dept. Tireóide SBEM.
Resumo:13-091
ADHERENCE OF PATIENTS IN AN EDUCATION AND CONTROL PROGRAM OF DIABETES IN THE CITY OF
JATAÍ - GO
Andrade, D. O 1; Ferreira, N. S. 1,1; de Lira, C. A. B. 1,1,3; Ferri, L. P. 4; Moraes, L. C. 4; Cintra, C. E. 4;
Benite-ribeiro, S. A1
1
Universidade Federal de Goiás, UFG
2
Universidade Federal de Goiás, UFG
3
Universidade Federal de Goiás, UFG
4
Secretaria Municipal de Saúde, SMS
5
Secretaria Municipal de Saúde, SMS
6
Universidade Federal de Goiás, UFG
Objectives:
Diabetes is an important metabolic disease and it is a foremost public health problem, affecting more than five million Brazilian
individuals. The patient with diabetes can be benefit from intervention programs that focus on drug treatment and nonpharmacologic procedures, such as regular physical exercise (PE), diet and body mass (BM) control. So, adherence (AD) to
therapy is essential to success of treatment. AD can be defined as the extent to which a person performs recommendations from a
health care provider. In Jataí – GO, there is a Program of Education and Control of Diabetes (PECD), conducted by a
multiprofessional health care team (MHC). In this program, patients are given drug therapy and are advised to practice PE and
hypocaloric calorie diet (HCD). Thus, this study aimed to assess patients AD to the PECD and the effects of counseling done by
the MHC.
Methods and Results:
The study included patients with diabetes treated by the PECD. We evaluated the number of patients who after being registered in
the program (1st visit, n=102) attended all the queries until the 4th and 8th visits. We analyzed the prevalence of patients with
postprandial plasma glucose (PG) decompensated (PG ≥ 180 mg/dL), patients with overweight (BMI≥ 25m.cm2) and the patients
reports about the practice of PE and HCD. The prevalence were tested by Chi square test and the statistical significance was
p≤0.05. Data are presented as mean±SD. It was observed that of 102 patients registered at the 1st consultation only 44 (43.1%)
remained until 4th visit, and 20 (19.6%) patients continued until 8th visit, showing a low frequency of patients to the queries. At
the 1st visit, there was prevalence of overweight (75.8%), and the percentage of patients with decompensated PG was 59.3%,
non-practicing HCD was 43.1%, and non-practicing PE was 46%. At 4th visit there was prevalence of overweight (71.9%) and
practicing PE (70.4%), where as 48.3% have not yet followed HCD. Although there are 20 patients in the 8th consultation, the
records were incomplete, and were not possible to do statistical analysis. Despite of weak AD, patients who did both the PE and
HCD had compensated PG at 4th consultation (in mg/dL): PE practicing= 159±32.71, PE non-practicing= 182.9±88.78; HCD
practicing= 149.9±58.78, and HCD non-practicing= 202.3±102.0.
Conclusions:
In this study was observed low AD of the patients to the PECD, both by reducing the number of patients who attended all visits,
as by no patients AD to advice from the MHC. Problems with AD to therapeutic recommendations are common in almost all
diseases, which impact negatively the effectiveness of the treatment. Probably the factors related to the AD are due to the
complexity of the therapeutic schedule and the adaptability of the recommendations to the usual habits of the person. Indeed, the
knowledge of the patients about disease, the relationship with the health care time and the patients perception of health and the
benefits of treatment are factors associated with adherence. So, we suggest that the interventions need to be easier to apply and
that the MHC combined cognitive and behavioral strategies, planning activities of community meetings to increase patients
knowledge about the disease and the benefits of AD to the program. Nevertheless, patients who adhered to the PECD had better
control of PG, demonstrating the importance of MHC work in the control of diabetes.
Keywords: diabetes, adherence, physical exercise, hypocaloric diet, multiprofessional health care
Resumo:13-092
SERUM AMYLOID A (SAA) EXPRESSION IS MODULATED BY HYPOXIA IN ADIPOCYTES
Oliveira, E. M. ; Monteiro, F. B. F. ; Sandri, S. ; Knebel, F. H. ; Contesini, C. G. I. ; Campa, A.
Department of Clinical Chemistry / FCF, USP
Objectives:
In obesity, adipocyte hypertrophy and hyperplasia might generate hypoxic areas within the tissue. In a close relationship with
hypoxia, the adipose tissue produces inflammatory molecules involved in obesity-related complications, such as cardiovascular
disease and metabolic disorders. Besides the hypoxia-inducible factor 1α (HIF-1α), several proteins have their stability or
expression modulated by hypoxia. It was also recognized that adipocyte size was correlated with the expression and production of
the serum amyloid A (SAA). This protein acts as a potent stimulus for the production of cytokines in immune cells. Thus, the aim
of the present study was to assess the influence of hypoxia on the expression of adipocyte-derived protein SAA isoforms, and
investigate, in normoxia, the effect of SAA on the production of proinflammatory cytokines by 3T3-L1 cells and human
adipocytes.
Methods and Results:
The cell culture was performed according to a standard protocol using a murine fibroblast cells (lineage 3T3-L1) from ATCC and
human adipocytes from abdominal subcutaneous adipose tissue, derived from plastic surgery and obtained from five healthy
donors (BMI between 25 and 35). The ambient hypoxia was generated by filling in a sealed acrylic chamber (self-designed) with
low-oxygen air that contained 1% oxygen, 5% carbon dioxide, and 94% nitrogen. Western blotting was used to measure HIF-1α
and SAA; for the last, Real Time PCR analyses were also performed. The production of the cytokines TNF-α, IL-6, IL-1β and IL8 in cell culture supernatants was determined by ELISA assays. Ambient hypoxia (1% O2) caused a two (2,02 ± 0,08 *p
Conclusions:
Our data show that the SAA gene is responsive to hypoxia, and that SAA possibly favors the improvement of the inflammatory
profile of the adipose tissue that may contribute to the overall health complications related to obesity, including insulin resistence.
The study was approved by the Faculty of Pharmaceutical Sciences Human Ethics Committee.
Keywords: 3T3-L1, CYTOKINES, HYPOXIA, HUMAN ADIPOCYTE, SERUM AMYLOID A
Financial Support: FAPESP; CAPES; CNPq.
Resumo:13-093
THIRST AND SODIUM APPETITE: EFFECT OF ADRENALECTOMY AND ESTRADIOL TREATMENT IN
OVARIECTOMIZED RATS
Almeida-pereira, G. ; Elias, L. L. K. ; Antunes-rodrigues, J
Physiology Dept/ FMRP-USP, USP
Objectives:
The regulation of body fluids in many species is under the influence of ovarian hormones, especially estrogen. In addition, there
are several experimental evidences demonstrating the involvement of hypothalamic pituitary adrenal axis (HPA) in the control of
hydromineral homeostasis, as well as the influence of estrogen in the modulation of this axis. In this context, our objective was to
study the influence of estrogen replacement in ovariectomized rats on the activity of the HPA axis using the adrenalectomized
animal, with or without hormone replacement, for the evaluation of behavioral responses involved with control of body fluids
homeostasis.
Methods and Results:
Wistar rats (220-240g, N = 11-14) were bilaterally ovariectomized and treated with estradiol (20 ìg/animal/day, sc, OVX-E2) or
vehicle (corn oil, 0.2 ml daily, sc, OVX) started 24 after surgery and maintained for 14 days. On the seventh day of replacement
the animals were submitted to bilateral adrenalectomy (ADX-OVX, OVX-E2-ADX) or sham surgery (SHAM-OVX, OVX-E2SHAM) and they were treated with corticosterone (10 mg/kg/day, sc, OVX-SHAM-B, OVX-E2-SHAM-B, OVX-ADX-B, OVXADX-B-E2) or vehicle (corn oil containing 5% ethanol, sc, 0.2 ml daily) for 7 days. Animals pre-habituated to the metabolic cage
had the daily water and hypertonic saline (1.8%) intake evaluated (18:00h) during the six days after ADX or sham surgery. The
results are expressed as mean ± SEM of cumulative intake. Data analysis were performed using three-way ANOVA, followed by
post-test Newman-Keuls and the level of significance was set at p<0.001).
Conclusions:
Our results demonstrate that E2 attenuates sodium intake induced by ADX, can be postulated that E2 might reduce ANGII
secretion, and thereby reducing sodium intake after ADX. However, the effect of E2 reducing sodium appetite could involve
regulation of other factors than ANGII, since this effect was also observed in animals with intact adrenals treated with B. The E2
also modulates the mechanisms involved in water intake in ADX animals, but only in the presence of B, since in group OVX-E2ADX-B was observed reversal of the effect induced by ADX.
Keywords: Adrenalectomy, Corticosterone, Estrogen, Sodium intake
Financial Support: CAPES, CNPq and FAPESP
Resumo:13-094
MODULATION OF GLUCOSE TRANSPORTERS GLUT2 AND SGLT2 IN KIDNEY: PARTICIPATION OF AKT,
INSULIN AND HNF-3 BETA TRANSCRICIONAL FACTOR.
Freitas, H. S. ; Okamoto, M. M. ; Beloto - Silva, O. ; Sabino-silva, R. ; David-silva ; Furuya, D. T. ; Souza,
M. O. ; Machado, U. F.
Dep. Fisiologia e Biofísica - Inst. de Ciências Biomédicas , ICB / USP
Objectives:
The Akt is one downstream target of phosphatidylinositol 3-kinase and plays an important role in mediating effects of insulin on
hepatic glucose production, glycogen, and protein synthesis. Upon activation, Akt is translocated to the nucleus where it exerts
effects on gene activity by phosphorylation of target proteins. Genetic studies have shown that the activation of PI3-kinase-Akt
pathway by insulin induces HNF-3 phosphorylation; this transcricional factor physically interacts with Akt and is phosphorylated
at a single site (T156). Renal glucose reabsortion is a coordinated process, which takes place in the epithelial cells of the proximal
tubule, involving two classes of glucose transporters, the Na - glucose transporters (SGLTs) and the facilitative diffusion
transporters (GLUTs). Increases in the renal glucose transporter gene expression are involved in renal tubule - glomerular
diseases. We showed that GLUT2 and SGLT2 are increased in diabetes and treatment with insulin (4 hours to 2 days) in kidney
of rats. That overexpression of GLUT2 and SGLT2 is related to the increased expression and activity of HNF-3 beta transcription
factor. Thus, the aim of this study is to establish involvement of the insulin, kinase Akt and HNF-3 beta in GLUT2 and SGLT2
modulation in renal territory.
Methods and Results:
The GLUT2, SGLT2, HNF-3â and p-Akt (Ser473) expressions as well the activity of HNF-3 transcription factor were analyzed in
IRPTC (immortalized renal proximal tubular cells) cells cultivated under such conditions: low (L) or high glucose (H), high
glucose plus insulin (I), high glucose, insulin and Akt inhibitor (Ii), and high glucose plus Akt inhibitor (Hi). In comparison to L,
p-Akt expression was increased in H (498.0±15, P<0.001).
Conclusions:
Our results clearly demonstrated that insulin regulates GLUT2 and SGLT2 expressions by increasing p-Akt protein, HNF-3 beta
expression and its binding activity. Moreover, this study suggests that Akt could play a key role in renal glucose reabsorption, by
modulating glucose transporters expressions.
Keywords: Rim, GLUT2, SGLT2, Insulina, Akt
Financial Support: FAPESP 05/60588-5 and 07/50554-1
Resumo:13-095
PROGRAMMING OF ADRENAL MEDULLARY FUNCTION BY NEONATAL OVERFEEDING IN RATS
Santos, A. S. ; Conceição, E. P. S. ; Oliveira, E. ; Pinheiro, C. R. ; Trevenzoli, I. H. ; Cardoso, F. S. ;
Moura, E. G. ; Lisboa, P. C.
DCF/UERJ, IBRAG
Objectives:
Several studies reveal that nutritional, hormonal and environmental factors at prenatal and neonatal period may influence organs
and tissues development, and this could be related with late diseases, like diabetes and cardiovascular disturbances (1, 2).
Postnatal overfeeding increases development risk to obesity and cardiovascular diseases. It has been shown that overfed neonate
rats show higher visceral adiposity and hyperleptinemia at weaning and they were programmed for obesity, higher food intake
and hypertension at adulthood (3). Previously, we evidenced that neonatal hyperleptinemia induces adrenal medullary
hyperfunction in early and late life (4). In the present study, we evaluated the adrenal function of obese adult rats that were
overfed during lactation. We also evaluated indirectly the visceral adipocyte sensitivity to serum catecholamine, through β3adrenergic receptor (ADRB3).
Methods and Results:
To induce early obesity by overfeeding, the litter size was reduced from ten to three male pups at third day of lactation until
weaning (small litter group, SL) while the control group stayed with ten males by litter over the lactation (normal litter group,
NL). After weaning, one pup from each litter (8/group) had free access to standard diet and water until the 180 days old when
they were killed and were collected samples of visceral adipose tissue (VAT), liver and the adrenal glands. Significant differences
had p
Conclusions:
The higher adrenergic catecholamine profile can help to explain the reported hypertension in this model. Paradoxically, those
animals had higher liver glycogen content and VAT, which suggest a participation of other factors than adrenaline, such as a
preferential use of lipids as energy source, saving glycogen.
Keywords: adrenal medullar function , catecholamine, postnatal overfeeding
Financial Support: CNPq, FAPERJ and CAPES
Resumo:13-096
AMP-ACTIVATED PROTEIN KINASE MEDIATES GLUCOSE UPTAKE IN RAT THYROID CELLS THROUGH A
TSH-INDEPENDENT MECHANISM
Cazarin, J. M. 1; Andrade, B. M. . 1; Rodrigues, D. C. 1; Zancan, P. 2; Ceddia, R. B. 3; Carvalho, D. P. 1
1
Instituto de Biofísica Carlos Chagas Filho - UFRJ, IBCCF/UFRJ
2
Faculdade de Farmácia-UFRJ, FF/UFRJ
3
School of Kinesiology and Health Science - Faculty of Health, York University
Objectives:
Glucose is an essential substrate for several cell functions. Glucose uptake is mediated by a family of glucose transport proteins
(GLUTs) expressed in a tissue-specific manner. Several glucose transporters are expressed in rat thyroid lineage cells and rat
thyroid gland with predominant expression of GLUT-1. However, the mechanisms that regulate GLUT1 expression and
translocation in thyroid cells are not completely understood. Several studies described that some thyroid carcinomas exhibit a
phenotype of low iodide uptake and increased glucose uptake. Recently our group showed for the first time that AMP-activated
protein kinase (AMPK) is expressed in the rat thyroid gland and decreased iodide uptake by the thyroid follicular cell, modulating
a major step involved in thyroid function (Am J. Physiol, in press, March 2011). Although many studies demonstrated that
AMPK regulates glucose uptake in different tissues, none have looked to this potential role of AMPK in glucose metabolism in
thyroid cells. Therefore, the study of AMPK signaling effects in thyroid cell can help to understand some acpects of the thyroid
carcinogenesis. The purpose of this study was to investigate the role of AMPK in the regulation of glucose uptake in rat thyroid
cells.
Methods and Results:
PCCL3 cells, a thyroid cell lineage, were cultivated in Ham's F12 medium supplemented with 5% fetal bovine serum and
containing six components: 1mU/ml bovine TSH, 10µg/ml insulin, 5 µg/ml transferring, 10nM hydrocortisone, 6nM
Somatostatin, and 2.5µM Gly-His-Lys. The cells were then treated with an AMPK activator, AICAR (A-1mM), or an AMPK
inhibitor Compound C (CC-20µM), or both. Besides, some cells were submitted to TSH deprivation for 24h. The incubation with
AICAR for 24h induced a significant (C 1,01&plusmn0.05; A0.5mM 0.87&plusmn0.02; A1mM 1.55&plusmn0.13; A2mM
2.00&plusmn0.05; n=6 P<0.05).
Conclusions:
Our results suggest that AMPK regulates glucose uptake in rat thyroid cells independent of the TSH, and might be involved in the
increased glucose uptake that is characteristic of tumoral cells.
Keywords: AMPK, Thyroid, Glucose uptake, GLUT-1, PCCL3
Financial Support: FAPERJ, CNPq and CAPES
Resumo:13-097
IN VIVO CROSS-TALK BETWEEN THYROID HORMONES AND ESTRADIOL IN RENAL GLUTATHIONE STRANSFERASE ALPHA REGULATION
Faustino, L. C. 1; Almeida, N. A. 2,1; Pereira, G. F. 1; Ramos, R. G. 1; Santiago, L. A. 1; Cordeiro, A. 1;
Ortiga-carvalho, T. M. 1
1
IBCCF, UFRJ
2
Depto de Ciencias Fisiológicas, UFRRJ
Objectives:
Aim: Glutathione S-transferase alpha (Gsta) represents an essential component of cellular antioxidant defence mechanism in
liver, and also in kidney. Mice liver Gsta mRNA was described to be repressed by thyroid hormones (TH) only in hypothyroid
state. Our previous data have shown an essential role of TRbeta1 in mediating liver Gsta suppression in response to T3, and in the
absence of a functional TRbeta, there is a compensatory action of TRalpha1. Kidney is one of the most important tissues
responsible for detofication processes. Until today, there is no published data about Gsta regulation by TH in the kidney.
Recently, we observed gender-dependent kidney Gsta regulation. In males, we saw an increase of Gsta levels in hypothyroidism
whereas in females, surprisingly, there was an unexpected reduction of renal Gsta expression suggesting a possible cross talk
between TH and estradiol/progesterone regulation pathways. Therefore, this work aims to evaluate, in vivo, the interaction among
TH and sex hormones in kidney Gsta expression.
Methods and Results:
All animals used in this work were 8-week-old females subjected to ovariectomy (OVX). After OVX, mice were induced to
hypothyroidism (HYPO) and hyperthyroidism (HYPER). HYPO was induced by PTU diet for 4 weeks. Mice were rendered
hyperthyroid by daily sc injections of T3 (50 g/100 g BW) for 15 days. Aiming to investigate which ovarian sex hormone could
be responsible for gender differences in renal Gsta expression regulation, we performed OVX followed by treatment with
estradiol benzoate (Es). Mice received Es injections at 0.1 mg/kg BW for 15 days. One control group was sham operated females
and another control group was male mice. In another experiment, after OVX and TH modulation, mice were also treated with Es
for 15 days. Gsta mRNA and protein levels were evaluated using quantitative RT-PCR and Western Blot, respectively. After
ovariectomy (OVX), Gsta mRNA was increased in HYPO (2.2±0.2; p
Conclusions:
Our data show a synergic interaction of TH and estradiol regulating renal Gsta expression, in which Gsta appears to be
downregulated by T3 in the absence of estradiol, and upregulated by T3 when estradiol is present.
Keywords: thyroid hormones, estradiol, glutatione S transferase, kidney, cross-talk
Financial Support: CNPq, CAPES, FAPERJ, Dept. Tireóide SBEM.
Resumo:13-098
MORPHOLOGICAL ANALYSIS OF FIBER-SKELETAL MUSCLE OF ADULT RATS SUBMITTED TO
PERINATAL PROTEIN MALNUTRITION
Tófolo, L. P. ; Mendes, F. C. V. ; Ribeiro, T. A. D. S. ; Fabricio, G. S. ; Oliveira, J. C. D. ; Torrezan, R. ;
Agostinho, A. R. ; Silva, P. M. D. S. ; Lopes, D. A. ; Rinaldi, W.
Depart of cell biology and genetic, UEM
Objectives:
It has been showed that early undernutrition is associated with being overweight in adult life and leads serious pathophysiologys
such as cardiovascular diseases, hypertension and type 2 diabetes. The low protein diet during early life might lead to irreversible
changes on glucose homeostasis, insulin secretion and hypoinsulinemia in rats. Insulin is an important hormone for growth and
development of the muscle. Thus, the objective this work was to study the effects of metabolic programming by maternal protein
restriction in the morphology of skeletal muscle.
Methods and Results:
Dams received a poor protein diet (4%) during initial 2/3 of lactation (RP), while, control-dams (NP) received normal protein diet
(23%), after that both offspring ate normal diet until 90-day-old. Adult rats were sacrificed and biometric parameters were
analyzed. The retroperitoneal and periepididymal fat pads were removed and weighed. The soleus muscle was removed, weighed
and frozen in liquid nitrogen. The cross section‘s area soleus muscle (120 fibers/animal, 10 fibers/field, 3 fields/cut and 4
cut/animal of each experimental group) were staining by hematoxylin and eosin (HE), after that the images were taking through
the stereomicroscope to subsequent quantification. Results were evaluated through the Student‘s t-test (p
Conclusions:
Maternal protein restriction during lactation does not impair the morphology of the soleus muscle.
Keywords: Protein restriction , insulin secretion, soleus muscle, glucose homeostasis
Financial Support: CNPq, CAPES and Fundação Araucária
Resumo:13-099
INTERFERON-GAMMA DECREASES RAT PINEAL NFKB NUCLEAR TRANSLOCATION AND POTETIANTES
MELATONIN PRODUCTION
Barbosa Lima, L. E. ; Fernandes, P. A. C. M. ; Markus, R. P.
Dep. de Fisiologia/ Instituto de Biociências-USP, IB-USP
Objectives:
The cross-talk between the pineal gland and the immune-system characterizes an immune pineal axis (Neuroimmunomodulation
14, 126, 2007). We have previously shown that the activation of nuclear factor kappa B (NFKB) by TNF and LPS pathway
inhibits melatonin production, while inhibition of NFKB by glucocorticoids has the contrary effect. In order to understand how
the pineal gland activity is altered during the development of an inflammatory response, we aim to evaluating the effect of other
cytokines that peaks lately than TNF. In the present work, we analyzed the effect of interferon (IFN)-gamma, by determining
NFKB nuclear accumulation and noradrenaline-induced melatonin synthesis.
Methods and Results:
Male Wistar rats aging 2-3 months were used in all the experiments. All animal procedures were performed according to
approved institutional protocols (048/2007). Pineal glands cultivated in BGJB medium for 48 h were incubated with IFN-gamma
(30 ng/mL) for 1, 5, 10, 15 or 30 min. In order to determine the effect of IFN-gamma on noradrenaline-induced melatonin
production, the glands were pre-incubated for 30 min with IFN-gamma (10 or 30 ng/mL) and then noradrenaline (10 nM) was
added for 5 h. NFKB nuclear content was determined by electromobility shift assay (EMSA), and melatonin content in the
medium was measured by HPLC. Maximal inhibition of NFKB nuclear translocation by IFN-gamma (30 ng/mL) was observed
after 10 min (28.1 ± 11.4%, n=3, compared to vehicle). This effect is probably transient, as after 15 min there was an increase of
the percentage of nuclear NFKB (54.0 ± 15.1%, n=3). The same profile of IFN-gamma effect was observed in the glands
incubated IFN-gamma plus noradrenaline. In addition, IFN-gamma (10 and 30 ng/mL) potentiates noradrenaline-induced
melatonin basal production (24.3 ± 4.2 ng/mL, n=8) in 43.6 ± 6.9% (n=4) and 60.4 ± 11.8% (n=4), respectively. Therefore, our
data show that INF-gamma potentiates melatonin, simultaneously with inhibition of nuclear translocation of NFKB.
Conclusions:
Taking into account that the potentiation of melatonin production by glucocorticoids was due to NFKB inhibition, our data
strongly suggest that the effect of IFN-gamma is also mediated by this transcription factor. In addition, we may propose that
during the development of the inflammatory response melatonin pineal production is restored by the interference of IFN-gamma.
Keywords: EIXO IMUNE-PINEAL, INTERFERON-GAMMA, MELATONINA, NFKB, PINEAL
Financial Support: FAPESP and CNPq
Resumo:13-100
CHALCONE ANALOGUE STUDIES ON INSULIN SECRETION AND IN VITRO DISACCHARIDASES ACTIVITY
IN RATS
Pereira, D. F. 1; Tavares, L. C. 2; Kappel, V. D. 1; Cazarolli, L. H. 3; Guesser, S. 1; Pizzolatti, M. G. 2; Silva,
F. R. M. B. 1
1
Departamento de Bioquímica/Univ. Fed. de Santa Catarina, UFSC
2
Departamento de Química/ Univ. Fed. de Santa Catarina, USFC
3
Universidade Federal de Fronteira Sl, UFFS
Objectives:
It is describe that some chalcones show anti-hyperglycemic activity when administered in hyperglycemic normal rats. In
adipocytes, chalcone analogues stimulate the glucose uptake and potentiate insulin-induced glucose uptake. The aim of this study
was to investigate the mechanism of chalcone QC10 on oral glucose tolerance curve improvement, on insulin secretion and in
vitro disaccharidase activity (maltase).
Methods and Results:
METHODS: Male Wistar rats (180-250 g) were deprived of food for at least 16 h but allowed free access to water. Fasted normal
rats were loaded with glucose (4 g/ kg p.o.) plus different doses of the chalcone (5, 10 and 20 mg/ kg). The glycemia (mg/ dL)
was measured at zero, 15, 30, 60 and 180 min after treatment, by glucose oxidase method. Serum samples from rats untreated
(control) or treated with QC10 (10 mg/ kg), by oral gavage, were used to determine insulin secretion. The insulin levels were
measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer‘s instructions. The range of values
detected by this assay was: 0.2–10 ng/ml. The intra- and inter-assay coefficients of variation (CV) for insulin were 3.22 and 6.95,
respectively, with a sensitivity of 0.2 ng/ml. To disaccharidase activity, duodenum was removed, homogenized in saline and kept
on ice until the moment of the experiment. Aliquots of the homogenate were pre-incubated for 5 min in the absence (controls) or
in the presence of chalcone. After, duodenum homogenates were incubated for 5 min with the substrate maltose. Chalcone was
used in several dosages, such as 25; 50; 100; 200 and 400 mM. The glucose produced was measured by glucose oxidase method
and its concentration was used to determine the maltase activity. Previous data demonstrated the higher activity of maltase in the
duodenum portion (basal activity). RESULTS: Chalcone caused significant acute anti-hyperglycemic effect when compared to
respective hyperglycemic control group (0 min: 114 ± 2; 15 min: 170 ± 3; 30 min: 191 ± 4; 60 min: 170 ± 3 and 180 min: 128 ± 6
mg/ dL). Serum glucose-lowering was detected at 15, 30 and 60 min after oral treatment to any dose tested. The serum glucoselowering was around 12% ate 15 min and 20% at 30 and 60 min for 5 mg/ kg QC10. The anti-hyperglycemic activity was higher
to 10 and 20 mg/ kg QC10 at 15 min: 24%; 30 min: 30% and 60 min: 20%, respectively. The insulin secretion increased at 60
min (1.4 ± 0.2) and can represent the classical second phase of insulin secretion when compared with basal insulin (0.54 ± 0.04).
Chalcone was able to inhibit the specific activity of maltase with 100, 200 and 400 mM when compared with the basal enzyme
activity.
Conclusions:
CONCLUSION: From these results we can conclude that this class of chalcone (QC10) improves glucose tolerance curve,
probably, by acting as insulin secretagogue. Also, an alternative intestinal target to this chalcone (maltase) is associated to
ameliorate glycemia balance.
Keywords: Chalcone, insulin secretion, disaccharidases
Financial Support: CNPq; CAPES, FAPESC, UFSC
Resumo:13-101
CPG ISLANDS SELECTION FOR STUDYNG METHYLATION PATTERNS OF RET (MAPK), HES1 (NOTCH) AND
WNT5A (WNT/ß-CATENINA) REGULATORY REGIONS IN MEDULLARY THYROID CARCINOMA
Cardoso, M. G. ; Maciel, R. M. D. B. ; Jasiulionis, M. G. ; Ierardi, D. F. ; Aguiar, G. ; Harada, M. Y. ;
Kizys, M. M. L. ; Silva, M. R. D. D.
Universidade Federal de São Paulo, UNIFESP
Objectives:
Differential patterns in DNA 5-methyldeoxycytidine influence gene expression for many genes in development, differentiation,
aging and carcinogenesis. It has been known for decades that Familial Medullary Thyroid Carcinoma (FMTC) is caused by
activating mutation in the oncogene RET, but the role of DNA methylation at its promoter region is scarcely investigated.
Intriguingly, family members bearing the same RET mutation present contrary prognosis, follow-up and tumor progression. This
observation has raised the hypothesis of epigenetics modifying events on oncogenes and tumor suppressor genes as RET
(MAPK), HES1 (NOTCH) and WNT5A (WNT/ß-CATENINA), genes and cell signaling implicated in carcinogenesis. In this
initial phase of the project we aim at selecting in silico the best predictive RET, HES1 and WNT5A regulatory regions enriched for
CpG islands, thereby, following the study of bisulfite methylcytosine sequence mapping.
Methods and Results:
We performed a detailed analysis of potential CpG island using the bioinformatics programs: Methprimer and MethylPrimer
Express. We opted to select two regulatory regions for each gene, termed as alternative A and B, hence, to proceed with BSPdesigned primers. We standardized paraffin-tumor DNA extraction, bisulfite treatment and DNA purification for the following
BSP-PCR amplification and then bisulfite genomic sequencing. Bisulfite-treated PCR amplified fragments were also inserted into
pCR4.1 sequencing vector to ensure 16 sequenced colonies for comparison and confirmation for methylated cytosines with direct
PCR product sequence. PCR condition consisted of forty-five cycles at 95°C for 1min, 53°C for 1min and 72°C for 1min using
both primers and final extension at 72°C for 10 min. We were able to successfully design BSP primers and bisulfite-sequence
large CpG islands of RET, HES1 and WNT5A regulatory regions, overcoming the challenge of obtaining sufficient amount of
DNA from FMTC paraffin tumor samples, and improve the readability of GC-rich sequences usually seen at the promoter
regions.
Conclusions:
we optimized the conditions for studying methylation pattern changes to understand the discrepancy in inherited tumor phenotype
in patients with same RET mutation. In the second ongoing phase, we will evaluate whether the hypermethylation of HES1 and
WNT5A, and hypomethylation of RET are linked with transcription repression and activation, respectively, therefore, imposing a
worse FMTC prognosis.
Keywords: epigenetics, Thyroid Carcinoma, Methylation, RET, HES1 and WNT5A
Financial Support: FAPESP
Resumo:15-032
IDENTIFICATION OF NOR-β-LAPACHONE DERIVATIVES AS POTENTIAL ANTIBACTERIAL COMPOUNDS
AGAINST ENTEROCOCCUS FAECALIS CLINICAL STRAIN
Lourenço, A. L. 1; Abreu, P. A. 1,2; Rodrigues, C. R. 2; Castro, H. C. 1; Milazzotto, T. L. 1
1
Instituto de Biologia / Universidade Federal Fluminense, UFF
2
Faculdade de Farmácia, UFRJ
Objectives:
A broad-spectrum antibiotic therapy has led to medical complications and emergence of multiresistant bacteria including
Enterococcus faecalis. Naphthoquinones have been focused in a variety of studies because of their various biological
activities. In this study, we designed, synthesized, and evaluated the antibacterial activity of 13 nor-β-lapachone
derivatives against a drug resistant E. faecalis strain using a molecular modeling approach.
Methods and Results:
All the substances were obtained from lapachol, extracted and purified from the heartwood of Tabebuia sp. (Tecoma) and
further submitted to Antibacterial susceptibility and Minimal Inhibitory Concentration tests against clinical E. faecalis
strains obtained from Ant�nio Pedro University Hospital from Fluminense Federal University, which were originally
isolated from patients and donated to our laboratiories under research purposes. All molecular computations were
performed using SPARTAN 08� software. The structures were optimized to a local minimum and the equilibrium
geometry obtained in vacuum using AM1 semi-empirical methods. Subsequently, molecules were submitted to a singlepoint energy ab initio calculation, at the 6-31G* level, to calculate some stereoelectronic properties and perform the SAR
studies. Promising MIC values were identified for two triazole substituted compounds (1e = 8 lg/ml and 1c = 16 lg/ml) and
also for the non-substituted derivative (1a = 8 lg/m). In addition, molecular modeling studies pointed the low HOMO
energy values, HOMO density concentrated on the nor-b-lapachone ring, lipophilicity, solubility, and number HBA as
important stereoelectronic features for the antibacterial profile. The triazole compounds presented low theoretical toxicity
profile and drug-score higher than commercial antibiotics also fulfilling the Lipinski ��Rule of Five�.
Conclusions:
Overall this work pointed the presence of the triazole groups as important but not essential for the activity of nor-βlapachone derivatives. Our SAR analysis suggests the low HOMO energy values, HOMO density concentrated on the norb-lapachone ring, lipophilicity, solubility, and number HBA as important stereoelectronic features for the antibacterial
profile. In addition triazole compounds presented low theoretical toxicity profile and drug-score higher than commercial
antibiotics also fulfilling the Lipinski ��Rule of Five�, which pointed them as promising candidates for further
studies in infections caused by multiresistant E. faecalis hospital strains.
Keywords: Enterococcus faecalis, Nor-β-Lapachone Derivatives, Potential Antibacterial Compounds
Financial Support: FAPERJ, CNPq, CAPES, UFF-PROPPi
Resumo:15-033
SOCIOECONOMICS PROFILE STUDY AND ASSOCIATIONS WITH THE TUBERCULOSIS CLINICAL FORM,
MULTIDRUG RESISTANCE AND TERAPEUTIC APPROACHES USED IN PATIENTS OF A PUBLIC CENTER TO
RESPIRATORY DISEASES.
Nascimento, A. A. 1; Pinheiro, C. G. 1; Pereira, L. S. R. 1; Santos, C. B. S. 1; Carvalho, J. S. M. 2; Carvalho,
F. L. Q. 1
1
Departamento de Ciências da Vida, UNEB
2
Departamento de Saúde, Unijorge
Objectives:
Tuberculosis has important levels of morbidity and mortality among the contagious diseases in world, and is the main cause of
deaths by curable infectious diseases in adults. The low socioeconomic level inherent to most of these patients in treatment
reinforces the status of serious public health problem. In this work we aimed to study the socioeconomic profile of the patients
submitted to treatment and the possible associations with the clinical form of the tuberculosis, multidrug-resistance and
therapeutic approaches adopted in a public center to respiratory diseases in Salvador-BA.
Methods and Results:
All the data had been obtained from the medical records of 78 patients with 18 years old minimum hospitalized in the Octávio
Mangabeira Reference Hospital (OMRH), the public reference center to the respiratory diseases in this city, in 2010 with
tuberculosis diagnostic. This study was approved by the Ethics Committee of the Bahia State University (UNEB), protocol n.
0603100102672, as well as by the OMRH Center for Research Pneumology. From these data, it was possible the observe that
most of these patients were men, with age between 18 and 29 years old, low scholarity degree, and the maximum of one
minimum salary fee. More than 50% of them had no history of incomplete previous treatment, as well as were alcohol and
tobacco users. We also didn‘t detect any illicit drug users nor HIV carriers. 64% of those who still are in treatment use the
scheme 1 (izoniazide + riphampicin + pyrazinamide).Pulmonary tuberculosis was the predominant clinical form in the patients
studied. 4 % of the total 78 patients developed multidrug-resistance.
Conclusions:
From all the data obtained, we conclude that tuberculosis onset is strongly related to the socioeconomic conditions of life. The
low rate of multidrug resistance is related probably to the low level of abandoning the treatment, and to the unique scheme used
during the treatment. These are critical to the success of treatment.
Keywords: Pharmacological treatment, Socioecomics conditions, Tuberculosis
Financial Support: Financial Agency for the Support of Research of the State of Bahia (FAPESB).
Resumo:15-034
EVALUATION OF THE EFFECTIVENESS OF COSMETIC MESOTHERAPY FOR BODY CONTOURING
Soares, A. K. A. ; Maia, C. O. ; Azevedo, D. F. M. D. ; Lima, H. L. ; Raulino, G. P. ; Deocleciano, O. B.
UNIVERSIDADE DE FORTALEZA, UNIFOR
Objectives:
Mesotherapy is an invasive medical procedure that involves the application of intradermal injections and/or subcutaneous
injections of low concentrations of medicines directly into the area to be treated (Plast Reconstr Surg. 115:1425, 2005). This
method is indicated in the treatment of rheumatic diseases, sports medicine, dermatology and esthetics (Indian J Dermatol
Venereol Leprol. 73:60, 2007). In this context, this study is intended to evaluate the effectiveness of mesotherapy when used in
beauty treatments for body contouring.
Methods and Results:
This is a retrospective study of quantitative and descriptive character, from analysis of medical records of customers of a beauty
clinic located in Fortaleza-Ceará. Data were collected between november to march this year, with a sample of 20 participants
from both sex. The evaluation of the effectiveness of mesotherapy was determined by reports of improvement of the doctor and
the patients described on the medical records. These parameters were based on the appearance of the treated area like, reduction
of measures, cutaneous sagging and of the fatty tissue. Also was used as criteria of evaluation, the comparison of waist measure
on the umbilical region (C1), 5cm above (C2), 5 cm below the same (C3), performed at the beginning and the end of treatment
using ANOVA as statistical test. The study was approved by the Research Ethics Committee of the University of FortalezaUNIFOR and is protected by resolution 196/96 of the National Commission of Research Ethics.The sample used in this study was
20 patients, 90% (n=18) of female and 10% (n=2) male. The mean age was 32.79 years (SD+10) and all patients were in
treatment for reduction of located fat. The treatment was performed completely by 60% of patients and the amount of
applications not mades per patient who left the treatment was 3.7 per session. The abandonment of 40% of cases were associated
with the discomfort of applications, mainly hematoma, pruritus and in some cases the availability of time of patients for the
weekly returns. The average loss measured after the treatment was -6.7cm in C1, C2 -5cm, and -2.5 in C3. Although not present
statistically significant differences the variations were considered satisfactory in most of the cases. One patient reduced 21 cm in
measure of C3. During treatment, there were reports of improvement, reported by the doctor in 90% and 70% for patients. About
19.04% (n= 4) reported improvement in sagging, 47.61% (n=10) in the measures, 9.52% (n=2) others and 19.04% (n=4) didn‘t
have information in the medical records. Mesotherapy in some patients was associated with other treatments such as physical
activity lymphatic drainage, endermologie, manthus, and ultrasound as mechanisms to enhance results.
Conclusions:
Positive results were found for mesotherapy to be on visual aspect of the treated areas such as in the required measurements of
treated areas. However, the success of treatment depends of individual variables, and their collaboration that goes from his
assiduity in applications to dieting, physical activity to enhance your results.
Keywords: INJECTIONS, INTRADERMAL, EFFICACY, MESOTHERAPY
Financial Support: PAVIC/UNIFOR
Resumo:15-035
ADVERSE EFFECTS CHARACTERIZATION OBSERVED IN TUBERCULOSIS PATIENTS.
Pinheiro, C. G. 1; Nascimento, A. A. 1; Pereira, L. S. R. 1; Santos, C. B. S. 1; Carvalho, J. S. M. 2; Carvalho,
F. L. Q. 1
1
Departamento de Ciências da Vida, UNEB
2
Departamento de Saúde, Unijorge
Objectives:
The efficacy to the tuberculosis (TB) treatment is observed when there is at least six months of pharmacological therapy,
although the development of important adverse effects (AE) frequently observed. Such effects can alter many physiological
processes and had been related as important factors to patients give up the treatment. In this work we aimed to characterize the
adverse effects caused by the anti-TB treatment, and its relationship between the protocols used with the abandon to treatment
rate by patients with or without multidrug-resistance in a public center to respiratory diseases in Salvador-BA.
Methods and Results:
All the data had been obtained from the medical records of 78 patients with 18 years old minimum who were hospitalized in the
Otávio Mangabeira Reference Hospital (OMRH), the public reference center to the respiratory diseases in this city, in 2010 with
tuberculosis diagnostic, with or without multidrug resistance. This study was approved by the Ethics Committee of the Bahia
State University (UNEB), protocol n. 0603100102672, as well as by the OMRH Center for Research Pneumology. We observed
that most of the patients submitted to TB treatment developed any kind of AE (61,5%). Minor effects were observed in 37
patients (77,08%), in special attention to gastric manifestations (48,6%), headache and humor alterations (24,3%). 20 patients
(41,66%) had major effects, specially hepatotoxicity and other metabolic alterations (32,4%). We consider here that some patients
had either minor or major effects. When we analyzed all the protocols used (scheme I, scheme IR, personalized scheme and
multidrug resistant scheme) we observed that the number of patients showing any AE (48) was higher than those who didn‘t
relate anyone in all the situations (30). This high frequency of AE is related to the inherent toxicity of each drug used in the
treatment, as well as to the high impact caused when the patient abandon the therapy in any moment. When we studied the
relationship between the AEs with the treatment abandon, we observed that such events were more frequent in those who didn‘t
give up, since they were more exposed to the chemotherapics, and had higher probability to develop the AEs. Importantly, all the
patients who abandoned the treatment had developed any kind of AE. This means that the total number of patients suffering any
AE would be higher.
Conclusions:
Our data show that most of the TB patients studied developed minor adverse effects; also, in all the protocols used, more people
developed AEs than who did not, and that the onset of adverse effects was strongly related to therapy abandon. This work helps
us to the know about tuberculosis and the therapeutic management, better identify the patients that have critical profile, which
facilitate the development of politics of prevention to complications, improving the therapeutic and public control of the disease.
Keywords: Adverse effects, Pharmacological treatment, Tuberculosis
Financial Support: Financial Agency for the Support of Research of the State of Bahia (FAPESB).
Resumo:15-036
INTERACTION OF RISPERIDONE AND SERUM ALBUMIN. A SPECTROFLUORIMETRIC STUDY.
Fragoso, V. M. S. 1; Silva, D. 2; Cruz, F. A. O. 3; Cortez, C. M. 2
1
Faculdade de Ciências Médicas, IBRAG - UERJ
2
Instituto da Matemática e Estatística, IME - UERJ
3
Departamento de Física, ICE - UFRRJ
Objectives:
Interaction mechanisms of risperidone to human (HSA) and bovine (BSA) serum albumins have been studied by using the
fluorescence quenching technique. Risperidone is an atypical antipsychotic drug used in many psychiatric disorders. The aim of
this study was to analyze the interaction of risperidone to HSA and BSA, characterizing the probable binding region, comparing
the results obtained for two albumins. The quenching curves were plotted, Stern-Volmer constants were estimated, and the nature
of the highest affinity binding sites of risperidone to the albumin were discussed.
Methods and Results:
Fluorescence measurements of risperidone with serum albumins were performed on a Hitachi - F3010 fluorescence equipment.
Quenching measurements were taken in 2ml of HSA and BSA 2x10−6M in 10 mM phosphate buffer, pH 7.4, when titrated with
risperidone (0.01 mM - 1.68 mM) at 25 °C and 37 °C. Using data from all experiments, graphs were plotted according to the
Stern–Volmer equation (Princ. of Fluores. Spectr. 3. ed. London: Plenum Press,2001). The graphs generated by the analysis of
the suppression of fluorescence were plotted from the arithmetic mean of three experiments for each temperature (standard
deviations lower than 10%), considering the average of two points around the maximum emission (λmax), λmax = 340 nm, for
BSA, and 338 nm, for HSA. In Stern-Volmer analysis, the cases for which the regression coefficient r2>0.9980 and p
Conclusions:
The decrease of Stern-Volmer constant caused by temperature elevation suggests the occurrence of static quenching for HSA
titrated by risperidone, interacting with this protein by forming complex risperidone - HSA. As the quenching intensity of BSA
was higher than of HSA, we are suggesting that the primary binding site for risperidone in albumin can be located close to
position 134 of the peptide chain in sub domain IB. At position 134 is a tryptophan residue in BSA.
Keywords: risperidone, albumin, antipsychotic drug, spectrofluorimetric
Financial Support: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro
Resumo:15-037
PRESYNAPTIC ADENOSINE AND MUSCARINIC RECEPTORS ARE INVOLVED IN HEXAMETHONIUM-, DTUBOCURARINE-, VECURONIUM-, AND ROCURONIUM-INDUCED TOFFADE.
1
1
1
2
1
Pereira, M. W. ; Bornia, E. C. S. ; Ambiel, C. R. ; Correia-de-sá, P. ; Alves-do-prado, W.
1
Department of Pharmacology and Therapeutic., UEM
2
UMIB / Universidade do Porto, ICBAS
Objectives:
The quotient between muscular tension produced by the fourth stimulus (T4) and first stimulus (T1) is the train-of-four (TOF) ratio (Rtof=T4/T1) when the neuromuscular preparation is indirectly
stimulated at 2.0 Hz for 2 s. Reductions in Rtof values are referred to as TOFfade. TOFfade is determined by drug-induced blockade of facilitatory nicotinic receptors on motor nerve terminals. Here,
we investigated the participation of presynaptic inhibitory (M2 and A1) and facilitatory (M1 and A2A) receptors in TOFfade induced by hexamethonium (HEX), D-tubocurarine (D-TC), vecuronium
(VEC), and rocuronium (ROC).
Methods and Results:
The Ethics Committee for Experimental Studies of the State University of Maringá approved the procedures (043-2007). The neuromuscular preparations of male wistar rats (250g) were assembled
according Bülbring (Br J Pharmacol. 1:38, 1946). The preparations were indirectly stimulated at 0.2 Hz for 15 min, and TOF stimulation was applied at 15s intervals for 15min. Muscarinic antagonists
(pirenzepine, methoctramine) or adenosine receptor antagonists (DPCPX, ZM241385) were administered 1 min before the second sequence of TOF stimulation. The antinicotinic agents were
administered 1 min before the third sequence of stimulation. The diaphragm muscle was coupled to force displacement transducer (Grass FT 03, connected to Chart Software Powerlab Instruments).
The lowest concentration of HEX, D-TC, VEC or ROC able to produce 25% TOFfade 3 min after its addition in the bath were determined and administered after 10nM Pirenzepine (PZP), 1µM
Methoctramine (MTC), 2.5 nM DPCPX or 10nM ZM241385 (ZM). Data were compared with ANOVA followed by the Bonferroni test (P1 antagonist) attenuated TOFfade produced by HEX
(5.0±0.4% n=6), D-TC (4.0±1.4% n=6), VEC (13.0±4.3% n=6), and ROC (7.0±1.8% n=6). MTC (M2 antagonist) fully prevented TOFfade caused by HEX (7.0±2.1% n=6). Surprisingly TOFfade
induced by D-TC (16.0±1.9% n=6), VEC (16.0±2.4% n=6) or ROC (15±2.4% n=6), was only partially attenuated by methoctramine. Antagonism of adenosine A1 receptors with DPCPX reduced
TOFfade caused by HEX (14±0.8% n=6), D-TC (18.0±2.8% n=6), VEC (11.0±1.7% n=6), and ROC (8.0 ± 1.8% n=6). Blockade of the A2A receptor with ZM partially reversed TOFfade induced by
D-TC (12.0±3.3% n=6), VEC (11.0±1.7% n=6), and ROC (5.0±0.2% n=6), but not that caused by a ―pure‖ neuronal nicotinic receptor antagonist, HEX, unless one increases the concentration to 50
nM (6.0±3.0% n=6).
Conclusions:
The results presented in this study add rationale for proposing an adequate use of subtype specific antagonists of muscarinic and adenosine receptors (like atropine- and metylxanthine-derivatives) in
patient‘s safe recovery from neuromuscular transmission block caused by antinicotinic agents. We provided new information regarding the need for a distinctive pharmacological management of
patients receiving antinicotinic drugs acting preferentially at pre- versus post-synaptic sites, or both.
Keywords: TOF fade, Hexametonium, D-tubocurarine, Vecuronium, Rocuronium
Financial Support: Araucaria Foundation and FADEC-UEM.
Resumo:15-038
CHRONIC CAPTOPRIL TREATMENT PREVENTS ERECTILE DYSFUNCTION IN STREPTOZOTOCIN
INDUCED DIABETIC RATS
Neves, N. C. V. 1; Damasceno, E. C. 1; Batista, K. F. 1; Assis, L. V. M. 1; Guimar�es, H. N. 2; Grabeguimarães, A. 1; Leite, R. 1
2
Escola de Engenharia, UFMG
1
Cipharma / Escola de Farmácia, UFOP
Objectives:
Diabetes mellitus when non-treated is commonly associated with many cardiovascular and neurological complications that could
lead to mild to severe male erectile dysfunction. The purpose of this study was to evaluate the beneficial effects of increasing
levels of circulating angiotensin-(1-7) [Ang-(1-7)] observed in chronic treatment with angiotensin-converting enzyme (ACE)
inhibitors such as captopril, on the erectile dysfunction (ED) that has been described in streptozotocin (STZ)-induced diabetic
male rats (Ethics Committee Protocol n. 2010/53).
Methods and Results:
Diabetic stage was induced by 3 consecutive injections of 40 mg/Kg of STZ in male Wistar rats (190-220g). Where considered
diabetic the rats presenting glucose level superior to 350 mg/dL 5 days after the last injection. Penile erection was evaluated in
control (normoglycemic), diabetic, and diabetic rats treated with captopril (25 mg/Kg) for 60 days after the injection of vehicle or
STZ. For that, the animals were anesthetized by ketamine/xylazine (100/14 mg/100g), had the major pelvic ganglion isolated and
electrically stimulated. Intracavernosal pressure (ICP) and mean arterial pressure (MAP) were measured and presented as an
index of erection (ICP/MAP). Frequency-response curves (1-12 Hz, 4V, 5ms pulse and 30 seconds for each frequency) were
obtained from rats of all groups. Erectile function was severely reduced in hyperglycemic compared to control normoglycemic
rats. Captopril treatment did not affect the ganglionic induced erectile response in normoglycemic rats, but significantly
improved, almost to a normal level, the impaired responses observed in diabetic hyperglycemic rats.
Conclusions:
Our data indicate that chronic treatment with ACE inhibitors such as captopril, that has been known to increase the circulating
levels of Ang-(1-7) and improve erectile function in spontaneously hypertensive rats, can also reverse the erectile dysfunction
observed in STZ-induced diabetic rats. We speculate that the high levels of Ang-(1-7) could be in part responsible for the
improvement of the erectile response in diabetic rats. These results can be very useful if applied to diabetic man who presented
ED.
Keywords: Angiotensin-(1-7), captopril, diabetes, erectile dysfunction
Financial Support: FAPEMIG, CNPq, UFOP.
Resumo:15-039
COMPARATIVE BIOAVAILABILITY OF BETAHISTINE TABLET FORMULATIONS ADMINISTERED IN
HEALTHY SUBJECTS
Arruda, A. M. M. ; Junior, P. P. D. ; Chen, L. S. ; Nucci, G. D.
Departamento de Farmacologia - Faculdade de Ciências Médicas, UNICAMP
Objectives:
To asses the comparative bioavailability of two formulations (16 mg tablet) of betahistine (CAS 5579-84-0) in healthy volunteers
of both sexes.
Methods and Results:
The study was conducted using an open, randomized, two-period crossover design with a 1-week washout interval. Plasma
samples were obtained for up to 36 h psot dose. Plasma 2-pyridylacetic acid concentrations were analyzed by LC-MS/MS with
positive ion electrospray ionization using multiple reaction monitoring (MRM). From the 2-pyridylacetic acid plasma
concentration vs. time curves, the pharmacokinetic parameters were obtained for AUClast and Cmax.
Conclusions:
The limit of quantification was 4 ng/ml for plasma 2-pyridylacetic acid analysis. The geometric mean and 90% confidence
interval (CI) of test/reference percent ratios were: 98.94% (92.21% - 106.16%) for Cmax and 95.42% (91.74% - 99.25%) for
AUClast. Since the 90% CI for Cmax and AUCs ratios were all within the 80-125% interval proposed by the US Food and Drug
Administration Agency, it was concluded that the test formulation is bioequivalent to the reference for both the rate and the
extend of absorption.
Keywords: bioavailability, betahistine, pharmacokinetics, bioequivalence, LC-MS/MS
Financial Support: FAPESP, CnPQ, Apsen Farmaceutica S.A.
Resumo:15-040
QT INTERVAL AND ITS DRUG-INDUCED PROLONGATION
Souza, A. C. M. 1; Guimarães, H. N. 3; Leite, R. 1; Sales, M. L. 2; Grabe-guimarães, A. 1
3
DEE, Escola de Engenharia, UFMG
1
DEFAR, Escola de Farmácia, UFOP
2
DECME, Escola de Farmácia, UFOP
Objectives:
Describe the QT interval and its prolongation induced by drugs.
Methods and Results:
Literature research on Medline database using the keywords ―QT interval prolongation‖ and ―cardiotoxic drugs‖. Theoretical
literature information was obtained after selection of articles that describe ECG alterations induced by drugs with different
therapeutic goals. According to ―FDA Guidance for Industry S7B Nonclinical Evaluation of the Potential for Delayed Ventricular
Repolarization (QT Interval Prolongation) by Human Pharmaceuticals‖ when QT interval is prolonged, there is an increased risk
of ventricular tachyarrhythmia, particularly when combined with other risk factors (e.g., hypokalemia, structural heart disease,
bradycardia). Thus, much emphasis has been placed on the potential proarrhythmic effects of pharmaceuticals that are associated
with QT interval prolongation. The rapidly and slowly activating components of the delayed rectifier potassium current, IKr and
IKs, have the most influential role in determining the duration of the action potential, and so, the QT interval. The human ether-ago-go-related gene (hERG) and KvLQT1 gene encode pore-forming proteins KCNH2 and KCNQ1 that are thought to represent
the α-subunits of the human potassium channels responsible for IKr and IKs, respectively. ECG abnormalities similar to those
seen in patients carrying mutations in the hERG gene can also be induced by direct blockade of hERG/IKr channels by a large
group of structurally diverse therapeutic compounds including many antiarrhythmics, antihistamines, antipsychotics and
antibiotics. The most common mechanism of drug induced QT interval prolongation is inhibition of the delayed rectifier
potassium channel that is responsible for IKr. At least three therapeutic compounds reduce hERG/IKr currents not by direct
blockade but by inhibition of hERG/IKr trafficking to the cell surface: (i) arsenic trioxide and and antimonial compounds, which
are used to treat acute promyelocytic leukaemia, (ii) the antiprotozoical agent pentamidine, and (iii) cardiac glycosides, which are
still used in heart failure. The antidepressive fluoxetine, represents the first member of a potentially large group of compounds
that directly block hERG and inhibit its traffic at the same time.(Biochemical Society Transactions 35:5, 2007).
Conclusions:
Therefore, it's important to investigate and understand the safety of therapeutic drugs that may induce arrhythmias due the QT
prolongation.
Keywords: cardiotoxic, drugs, prolongation, QT interval
Financial Support: FAPEMIG; UFOP; UFMG
Resumo:15-041
THERAPEUTIC EFFICACY STUDY OF MELAGRIÃO® SYRUP IN PATIENTS WITH CLINICAL DIAGNOSIS OF
ACUTE BRONCHITIS.
Nascimento, D. F. ; Leite, I. O. ; Andrade, W. S. ; Leite, A. L. A. S. ; Fechine, F. V. ; Oliveira, J. C. ; Frota
Bezerra, F. A. ; Moraes, R. A. ; Moraes, M. E. A.
Depto. de Fisiologia e Farmacologia, UFC
Objectives:
Aim: Melagrião® is a phytotherapic medicine composed of six medicinal plants with known action in the respiratory tract:
Mikania glomerata, Cephaelis ipecacuanha, Aconitum napellus, Polygala senega, Myroxylon balsamum and Nasturtium
officinale. The aim of this study was to evaluate the therapeutic efficacy of Melagrião® in patients with clinical diagnosis of
acute bronchitis.
Methods and Results:
Methods and Results: A double-blind, randomized, parallel study, with 86 patients of both genders. The patients were randomly
divided in two groups: Melagrião® (n = 43) or bromhexine (n = 43). They were all treated for 7 consecutive days using the
recommended dosage according to the treatment group and age. Clinical evaluations were performed on the pre-treatment and on
the seventh day of administration (post-treatment). To quantify the clinical response, we used a instrument composed of 12 items
related to signs and symptoms of respiratory infections (cough frequency, cough intensity, asthenia, nasal congestion, sore throat,
mucus elimination, anorexia, quality of sleep, headache , myalgia, fever and general condition), which were evaluated by using a
visual analog scale. In addition, we defined the general clinical evaluation as the arithmetic average of the 12 components of the
used instrument. In the analysis within the same group in all evaluated items, there was a significant improvement (P
Conclusions:
The Melagrião® syrup showed that the improvement of the bronchitis symptoms was equivalent to the group treated with
bromhexine, thus proving the efficacy of this phytotherapic therapy.
Keywords: Clinical Trial, Therapeutic Efficacy, Bronchitis, Phytomedicine
Financial Support: CNPq, CAPES, FUNCAP, FINEP, MS-RNPC-UNIFAC-HM, and Instituto Claude
Bernard.
Resumo:15-042
BIOLOGICAL POTENCY EVALUATION AND CHARACTERIZATION OF RHEPO IN PHARMACEUTICAL
FORMULATIONS
Schutkoski, R. ; Camponogara, R. L. ; Freitas, G. W. ; Scheeren, L. E. ; Vecchia, F. D. ; Dalmora, S. L.
Departamento de Farmácia Industrial, UFSM
Objectives:
Erythropoietin (EPO) is the primary regulator of the rate of erythropoiesis. The recombinant human erythropoietin (rhEPO)
consists of a 165 amino acids polypeptide chain heavily glycosilated at three N-linked and one O-linked sites yielding a
molecular mass of 30-34 kDa. About 40% of the fully glycosilated EPO molecule consists of carbohydrate. Therapeutically,
rhEPO is used to stimulate red cell production by promoting maturation and growth of erythroid precursor cells. The aim of this
study was perform the characterization and the potency evaluation of erythropoietin in pharmaceutical formulations, by the in
vivo biological assay and physico-chemical methods.
Methods and Results:
Samples of pharmaceutical products from different manufacturers were used and the purity and molecular weight were assessed
using SDS/polyacrylamide gel electrophoresis and the heterogeneity by isoelectric focusing gels and immunoblotting of IEF gels
which showed a broad band in the 30.4 kDa range. The normocythaemic mice bioassay was performed using eight-week old
female BALB/c mice, which received a multiple daily injections of standard and sample solutions (3+3), during four days. The
blood sampling was performed 24 hours after the last injection and the reticulocytes counted by automated flow cytometry.
Parameters such as age, sex, strain, different injection schedules, and animals per group were evaluated using the multiple daily
injections protocol and the single injection protocol. The results were calculated against the Ph. Eur. BRP for rhEPO, giving
potencies within 91.70 and 115.40%. The pharmaceutical samples were also analyzed by the reversed-phase chromatographic
method which showed mean difference between the estimated potencies of 5.20% lower compared to the bioassay, with
significant correlation (P>0.05).
Conclusions:
The normocythaemic mice bioassay was validated and applied for the potency evaluation of rhEPO pharmaceutical formulations,
trying to develop alternatives in the context of the 3Rs. The results were also correlated to the chromatographic method
evaluating correlations which could contribute to improve the quality control and to assure their therapeutic efficacy of the
biological medicine.
Keywords: Characterization and potency evaluation, Erythropoietin, In vivo biological assay, Isoelectric focusing,
SDS/polyacrylamide gel electrophoresis
Financial Support: CNPq.
Resumo:15-043
PREVALENCE OF SELF MEDICATION IN ADULT POPULATION FROM THE URBAN AREA OF THE CITY OF
FLORIANO – PI
Duarte, A. B. 1; Silva, D. J. S. D. 1,1; Santos, D. B. 1,1,1; Filho, M. D. S. 2; Martins, M. D. C. D. C. E. 2
1
Faculdade de Ensino Superior de Floriano, FAESF
2
Universidade Federal do Piauí, UFPI
Objectives:
To assess the prevalence of self medication in the adult population of the urban area of the city of Floriano - PI the categories of
medicines most used and the motivations to use.
Methods and Results:
The survey consisted of cross-sectional study of population with sample of 552 people aged between 20 and 59 years. The adults
were selected through random sampling proportional to population in the neighborhoods. Data were collected through
questionnaires with closed and open questions. The significance level was set at 5% and statistical evaluation of variables was
accomplished using Chi-square test. The prevalence of self medication was 96.9%. The main reason for self-medication was pain
(78.6%). Drug group most widely used was represented by the analgesics drugs (48.5%). Previous experience with the
medication was the justification for self-medication pointed to by 64.6% of the participants. The protocols used were approved by
the ethics committee under number 2010/19.
Conclusions:
This study showed high prevalence of self-medication in the adult population of the urban area of Floriano-PI. The pain was the
main reason for self-medication, which explains the consumption of analgesics by a majority of respondents. Previous experience
with the medication was the main justification for self-medication. This demonstrates the need for preventive strategies in health
that clarify this population about risks f self-medication and contributes to the rational use of medicines.
Keywords: self medication, adults, prevalence
Resumo:16-041
EFFECT OF ANGIOTENSIN II ON SMOOTH MUSCLE FUNCTIONS IS MODULATED THROUGH
ALPHA1BETA1 INTEGRIN SIGNALING ACTIVATION
Moraes, J. A. 1; Dias, A. M. 1; Rodrigues, G. 1; Marcinkiewicz, C. 2; Assreuy, J. 3; Arruda, M. A. 4; Barjafidalgo, C. 1
1
Departamento de Farmacologia / Instituto de Biologia, UERJ
2
Neurovirology and Cancer Biology, Temple University
3
Departamento de Farmacologia, UFSC
4
Divisão de Ensino, FIOCRUZ
Objectives:
The accumulation of vascular smooth muscle cell (SMC) in the atheromatous plaque is a key event of atherosclerosis. Under the
effect of different stimuli SMC migrate and proliferate, leading to formation of fibrous cap (atherosclerosis) or neointima
(restenosis). Angiotensin II (AngII), acting via its G-protein coupled receptor, AT1, is able to induce those effects through the
activation of PKC α and ROS production, also involving the activation of integrin-mediated signaling pathways. In an attempt to
elucidate the molecular mechanisms involved in signaling pathways that modulate the effects of AngII on SMC functions.
Methods and Results:
The migration of SMC (A7r5 cell linage) towards AngII (100nM) was analyzed in Boyden chambers after 4h incubation. The
chemotactic effect of AngII was abolished when the cells were pretreated with the α1β1 integrin selective ligand, the disintegrin
Obtustatin (Obt 100nM). Stimulation of SMC with AngII induces a PKCα-dependent FAK phosphorylation, uncoupling of ILK
to phosphorylated FAK and the association of ILK to actin, promoting cytoskeleton rearrangement. The treatment of cells with
Obt or with PKCα inhibitor abrogated AngII effect on FAK phosphorylation, maintained the association between FAKILK(analyzed by western blot), inhibiting ILK association to F-actin, as observed by fluorescence microscopy in AngII-treated
SMC. The data indicate that, in SMC, the activation of α1β1 integrin-signaling pathway is downstream to PKCα activation,
triggered by the binding of Ang-II to AT1-GPCR. Interestingly, the ROS production induced by Ang-II is more transient in cells
treated with Obt (DCF probe analysis). Additionally, it was demonstrated that the pretreatment with Obt induced G1 phase arrest
(cell cycle analysis) and diminishment of SMC proliferation (thymidine incorporation assay) of SMC treated with AngII.
Moreover, AngII-induced AKT phosphorylation and p21 expression reducement, is inhibited in cells treated with α1β1 integrin
inhibitor.
Conclusions:
In this work we suggest that α1β1 integrin may be an important target molecule to the development of more effective therapeutic
interventions in restenosis/atherosclerosis.
Keywords: ILK, Obtustatin, Angiotensin II, alpha1beta1 integrin, Smooth Muscle Cell
Financial Support: FAPERJ, CAPES, CNPq
Resumo:16-042
EFFECTS OF TREATMENT WITH RESVERATROL ON THE CONTRACTIONS STIMULATED BY KCL ON
ISOLATED AORTAS FROM HYPERTENSIVE RATS.
Antonieto, C. R. K. ; Oliveira, J. C. S. D. ; Mariinho, T. S. ; Scalabrini, A. C. ; Restini, C. B. A.
Medicine School - Universidade de Ribeirão Preto, UNAERP
Objectives:
Hypertension is a multifactorial clinical condition characterized by high and sustained levels of blood pressure. The vascular
endothelium develops important roles in physiologic and pathophysiologic hypertension. In experimental models to study the
hypertension the endothelium-dependent vasodilatation is severely reduced. The membrane of aorta rings isolated from rats
submitted to renovascular hypertension is more depolarized as compared to normotensive rats. The depolarization leads to the
activation of voltage-operated calcium channels (VOCCa2+) increasing the contractions in aorta from hypertensive rats, which in
general, are more sensitive to changes in Ca2+concentrations than in normotensive rats. Resveratrol, a polyphenolic compound
present in red wines has the ability to stimulate the vasodilatation due to the acute release of nitric oxide (NO) from vascular
endothelial cells. Aim: This study was designed to evaluate the participation of endothelial NO on the contractions stimulated by
depolarization induced with KCl in isolated aortic rings isolated from 2K-1C rats treated with resveratrol
Methods and Results:
Renovascular hypertension was induced in male Wistar rats (180g) by the implantation of a silver a Clip in a Kidney artery (2K1C). Control rats were submitted to abdominal laparatomy (2K). Resveratrol treatment (20 mg/kg, gavage) began one day after
the surgery and was performed tree times a week, during six weeks. After that, rats were killed by cervical dislocation and had
thoracic aortic isolated. The aortic rings with (E+) or without endothelium (E-) were set at a resting tension of 1.5 g in an organ
chamber containing Krebs solution, at 37¨¬C, pH 7.4. After testing the presence or absence of the E, concentration-response
curves (4.7-120 mM) for KCl were done before and 20 min after incubation 0.1¥ìM L-NAME (NOSynthase inhibitor). All
procedures and protocols were in accordance with the Guidelines for Ethical Care of the Experimental Animals; approved by
Institutional Animals Care and Use Committee (007/2010). Results were analyzed through One-way ANOVA; Newman-Keuls
post-hoc. Results: Maximum contractions were not altered in none of the conditions/groups of the reactivity studies. On the other
hand, the potency, evaluated by the negative log of concentration producing half the maximal effect values (pD2) was altered.
Presence of L-NAME increased (p
Conclusions:
Treatment with resveratrol does not alter the contractile response induced by KCl in aorta isolated from hypertensive rats. The
attenuation of the contractile response induced by KCl in aorta isolated from 2K-1C rats is dependent of the presence of
endothelium and of the NO production, but not to resveratrol treatment.
Keywords: hypertension, resveratrol, nitric oxide, endothelium, Potassium depolarization
Financial Support: CNPq, UNAERP
Resumo:16-043
ROLE OF ANG(1-7) IN THE CEREBRAL CIRCULATION OF 2K-1C HYPERTENSIVE RATS
Olivon, V. C. 1; Santiago, L. B. 1; Bonaventura, D. 2; Ramalho, L. N. Z. 3; Cortes, S. F. 2; Lemos, V. S. 1
1
ICB - Dep.Fisiologia e Biofisica, UFMG
2
ICB - Dep. Farmacologia, UFMG
3
FMRP - Dep. Patologia, USP
Objectives:
The renin-angiotensin system is fundamentally important for the development of renal hypertension. Ang (1-7) by activating the
receptor Mas produces relaxation, mainly via NO-cGMP. Hypertension can lead to a reduction in endothelium-dependent
vasodilation, caused in part by a reduction in the bioavailability of NO. The role of Mas receptors in the cerebral vascular
function is not yet well elucidated. The aim of this study was to investigate the role of Ang(1-7) in the carotid artery from 2K-1C
rats.
Methods and Results:
METHODS: We studied vascular reactivity in carotid artery rings from sham and 2K-1C Wistar rats. Ang (1-7) concentrationresponse curves were constructed in endothelium-intact or endothelium-denuded arteries pre-contracted with phenylephrine (0.10.03 µmol/L) from sham and 2K-1C Wistar rats in the following conditions: 1) in the presence of A-779 (Mas selective
antagonist, 1µmol/L); 2) in presence of PD 123,319 (AT2 selective antagonist, 1µmol/L)+losartan (AT1 selective antagonist,
1µmol/L)+HOE-140 (B2 selective antagonist, 1µmol/L) or 3) in presence of L-NAME (non-selective NOS inhibitor, 0.1mol/L).
Proteins expression and localization were evaluated by Western blot, immunohistochemistry and immunofluorescence. All
procedures are in accordance with the standards and policies of the Animal Care Committee of UFMG (164/10). Data analyses:
ONE-WAY ANOVA. RESULTS: In endothelium-intact vessels Ang (1-7) elicited a concentration-dependent relaxation, which
was more pronounced in 2K-1C (Emax=70.8±5.2%**) animals as compared with sham (Emax= 45.66±5.12%). L-NAME
reduced but not abolished, Ang (1-7) relaxation in sham (Emax= 12.0±0.9%) and in 2K-1C (Emax= 30.3± 0.18 %*) was still
increased in 2K-1C; A-779 also decreased Ang (1-7)-induced relaxation in 2K-1C (Emax= 40.2 ±3.8%*) and sham (Emax=
30.2±3.8%) but in the presence of A-779 the relaxant effect of Ang-(1-7) was still increased in 2K-1C. The blockade of AT1,
AT2 and B2 receptors evoked similar results as blockade of Mas; relaxation in response to Ang (1-7) was reduced in the presence
of the antagonists but remained larger in 2K-1C animals (Emax= 38.9±2.3%*) than sham (Emax= 22.4±1.2%). Endothelium
removal decreased Ang-(1-7)-dependent relaxation in sham (Emax= 29.0±3.1%) and 2K-1C (Emax= 50.6±3.7%*). However,
vasodilation remained bigger in 2K-1C vessels. In endothelium-denuded vessels, A-779 leveled Ang-(1-7)-induced relaxation
(sham=18.2±1.2%; 2K-1C=15.3±0.8%). We found similar results after simultaneous blockade of AT1, AT2 and B2 receptors
(sham=16.4±1.2%; 2K-1C=19.8±1.2%); L-NAME reduced Ang-(1-7)-induced relaxation in both groups (sham=10.4±1.0%; 2K1C=20.3±0.15%*). Mas receptor and endothelial NOS expressions were increased in 2K-1C group when to compare to sham
group. *p
Conclusions:
Together, our results show that in carotid artery of hypertensive animals, the vasodilator response to Ang-(1-7) is more
pronounced when compared to sham animals and mediated via Mas receptor. This increased vasodilator response is part mediated
by the endothelium with an important participation of NO. Another part of the increased vasodilator response is endotheliumindependent. Hence, it is possible that a compensatory mechanism mediated by Ang-(1-7) may exist to control the arterial
hypertension.
Keywords: Ang (1-7), carotid artery, hypertensive rats, Mas receptor
Financial Support: FAPEMIG and CNPq
Resumo:16-044
ROLE OF TLR4 IN BLOOD PRESSURE AND CARDIAC HYPERTROPHY OF SPONTANEOUSLY
HYPERTENSIVE RATS (SHR)
Echem, C. ; Bomfim, G. F. ; Dossantos, R. A. ; Fortes, Z. B. ; Carvalho, M. H. C.
Farmacologia / USP (ICB1), ICB/USP
Objectives:
AIM: Arterial hypertension is considered as a main risk factor for the development of cardiovascular disease (CVDs). As a
consequence, hypertension, leads to cardiac hypertrophy and a low degree chronic inflammation, with an increase in circulating
pro-inflammatory cytokines. Toll-Like Receptors (TLR) family was identified as one of the major factors involved in innate
immune response and its activation also induces pro-inflammatory cytokines production. Recently, TLR4 has been shown in
experimental models of atherosclerosis, heart failure, ischemic injury and septic cardiomyopathy indicating at with receptor could
be an important link between CVDs, inflammation and immune system activation. TLR4 expression was demonstrated to be
increased in heart with had undergone in a myocardial infarction, besides that TLR4-knockout mouse develop less severe cardiac
hypertrophy when compared to the wild type. Based on the evidences mentioned above, the purpose of this study was to
investigate the role of TLR4 in hypertension.
Methods and Results:
METHODS: SHR 5 and 15 weeks old (n=5) tail blood pressure (TBP) was measured by pletismography and cardiac TLR4
mRNA expression was evaluated by real time-PCR. Both parameters were also determined in SHR 15 weeks old (n=5), treated
with amlodipine (15mg/kgBW/day) and losartan (10mg/kgBW/day) for 15 days by gavage. Collagen I and III mRNA expressions
were also determined by real time-PCR in heart of SHR (n=6) untreated and treated with anti-TLR4 antibody (1ug/day, ip.) for 15
days. The wet and dry weight (mg) measurement was performed in the heart of SHR 15 weeks old (n=5) untreated and treated
with anti-TLR4 antibody. RESULTS: TBP (mmHg) of SHR 5 weeks old was 108±2, and of SHR 15 weeks old was 178±3. TBP
of SHR 15 weeks old treated with losartan (138±2) and amlodipine (142±5) was found lower than the untreated SHR. Cardiac
TLR4 mRNA expression of SHR 15 weeks old (1.0±0.13) was found higher than the SHR 5 weeks old (0.67±0.1).
Antihypertensive treatment decreased TLR4 mRNA expression when compared to the untreated SHR (untreated SHR: 1.0±0.13;
losartan: 0.48±0.17 and amlodipine: 0.53±0.12). The anti-TLR4 treatment decreased the mRNA expression of collagen I
(1.096±0.04 vs. 0.613±0.005) and collagen III (1.02±0.08 vs. 0.60±0.08) of SHR heart. This treatment also decreased the wet
weight (0.36±0.01 vs. 0.34±0.01) and dry weight (0.088±0.001 vs. 0.081±0.003) of SHR heart.
Conclusions:
CONCLUSION: We might suggest that TLR4 mRNA expression in the heart can be modulated by SHR blood pressure and
TLR4 seems to be involved in SHR cardiac hypertrophy.
Keywords: CARDIAC HYPERTROPHY, HYPERTENSION, TLR4
Financial Support: FAPESP and CAPES
Resumo:16-045
PHARMACOLOGICAL INHIBITION OF DIPEPTIDYL PEPTIDASE IV INCREASES GLUT4 PLASMA
MEMBRANE AND PROTEIN EXPRESSION IN SKELETAL AND CARDIAC MUSCLES OF SPONTANEOUSLY
HYPERTENSIVE RATS (SHR).
Salles, T. A. 1; Giannocco, G. 2; Oliveira, K. C. 2; Pacheco, B. P. M. 1; Girardi, A. C. C. 1
1
Instituto do Coração, HC-FMUSP
2
Faculdade de Medicina do ABC, FMABC
Objectives:
SHRs display changes on GLUT4 function, trafficking, and/or expression in insulin-sensitive tissues, suggesting that altered
regulation of this glucose transporter may account at least in part for the abnormalities of glucose metabolism in hypertension.
Inhibitors of the enzyme dipeptidyl peptidase IV (DPPIV) represent a novel class of antihyperglicemic agents that improve
glycemic control in type 2 diabetic patients. Furthermore, therapy with DPPIV inhibitors is associated with blood pressure
reduction. The present study was designed to test the hypothesis that pharmacological inhibition of DPPIV with sitagliptin may
improve glucose homeostasis in hypertensive animals by increasing skeletal and cardiac muscle GLUT4 expression and/or
plasma membrane (PM) translocation.
Methods and Results:
Studies were conducted in male SHR at 5 (Y) and 20 weeks (A) of age that were randomly divided into two groups and treated
twice a day, by oral gavage, with either sitagliptin (40 mg/day) (IDPPIV) or vehicle (water) for the period of ten days. Agematched Wistar Kyoto (WKY) rats served as normotensive controls. After sitagliptin treatment, Y-SHR + IDPPIV displayed
lower systolic blood pressure (SBP) than Y-SHR (119 ± 3 vs. 136 ± 4 mmHg; P < 0.05). In the A-SHR, sitagliptin treatment had
no significant effect on arterial blood pressure. Skeletal (soleus, EDL and gastrocnemius) and ventricular muscles were excised
from these experimental groups for analyses of GLUT4 expression and subcellular localization by immunoblotting. Y-SHR
exhibited less GLUT4 in the PM of the heart (-11.0 ± 0.3%), soleus (-25.0 ± 1.0%), EDL (-12.0 ± 0.24%), and gastrocnemius (18.0 ± 0.9%), as compared to Y-WKY. The reduction in PM GLUT4 expression in the heart, soleus and gastrocnemius were
accompanied by a slight, but significant decrease of total GLUT4 protein expression. The total protein abundance of GLUT4 in
these three types of muscles cells was normalized by the ten-day-treatment with sitagliptin. Interestingly, DPPIV inhibition
produced a significant increase (+20.0 ± 0.48%) on PM GLUT4 expression in the ventricular myocytes from Y-SHRs + IDPPV
vs. Y-WKY. Reduction of GLUT4 PM in the skeletal and cardiac muscle cells of A-SHR vs. A-WKY was even more
pronounced: heart (-22.0 ± 1.0%), soleus (-35.0 ± 3.4%), EDL (-15.0 ± 0.6%), and gastrocnemius (-30.0 ± 1.8%). In the adult
SHR, lower PM GLUT4 expression was associated with lower GLUT4 total abundance in all muscle cell types analyzed.
Sitagliptin increased GLUT4 PM and total protein expression in the heart, soleus, EDL and gastrocnemius of A-SHR + IDPPIV
to levels higher than those found in the normotensive adult animals.
Conclusions:
Taken together, our data demonstrate that DPPIV inhibition increases GLUT4 expression and PM translocation in both skeletal
and cardiac muscles of SHR. Changes on GLUT4 expression by sitagliptin do not seem to correlate with blood pressure reduction
in this rat lineage. Moreover, our study has shed light upon a new mechanism by which DPPIV inhibitors improve glycemic
control.
Keywords: Dipeptidyl peptidase IV, Glut4, Heart, Hypertension, Skeletal Muscle
Financial Support: FAPESP and CNPq
Resumo:16-046
THE INCREMENT IN NITRIC OXIDE MODULATION IN RAT CORONARY ARTERIES POST-MYOCARDIAL
INFARCTION IS A POSSIBLE MECHANISM INVOLVED IN PREVENTING THE ONSET OF HEART FAILURE
Couto, G. K. 1; Britto, L. R. G. 1; Mill, J. G. 2; Rossoni, L. V. 1
1
Department of Physiology and Biophysics/ ICB, USP
2
Department of Physiological Sciences/ CCS, UFES
Objectives:
The coronary circulation supplies the myocardium with oxygen and nutrients to maintain the cardiac function and then provides
the homeostasis. The endothelium, mainly by nitric oxide (NO) release, is able to adjust the coronary flow and it function has
been modified in some cardiovascular diseases, such as myocardium infarction (MI) and heart failure (HF). It is well known that
MI is followed by adaptive processes triggered in myocardium to maintain cardiovascular homeostasis which can retard the onset
of HF. We hypothesized that the coronary artery (CA) can be differently adjusted after MI in the presence and absence of HF.
Thus, we assessed the endothelium function in CA from infarcted rats with and without HF, focused in the nitrergic modulation.
Methods and Results:
Male Wistar rats, 10 weeks old, were anaesthetized (ketamine 50mg/kg plus xylasine 10mg/kg; i.p.) and submitted to left CA
ligation to produce MI or sham operation (SHAM; n=42). After 4 weeks, hemodynamic parameters were assessed in
anaesthetized rats and after the infarcted rats were subdivided in rats without (INF; n=32) or with HF (n=5). The results are
expressed as mean±SEM. Statistical analyses: ANOVA (pvs. SHAM; #vs. INF). Afterwards, we evaluated the infarction area by
% of left ventricle (LV) area and the hypertrophy index by ratio ventricular weight/ tibia length. The infarction size was similar
between groups. The LV end diastolic pressure was increased after MI, however this increment was biggest in HF as compared to
INF (SHAM: 8.2±0.2 vs. INF: 10.5±0.3* vs. HF: 26.7±1.6 mmHg*#). The contractile index (dP/dt+) was reduced after MI, but
HF rats showed a greater decrease as compared to INF (SHAM: 7122±160 vs. INF: 5960±131* vs. HF: 4570±125 mmHg/s*#).
On the other hand, MI reduced the relaxation index (dP/dt-) only in HF. The MI-induced right ventricular hypertrophy, but it was
greater in HF (SHAM: 5.1±0.1 vs. INF: 7.6±0.4* vs. HF: 11.0±0.7 g/cm*#). Afterwards, the septal CA was mounted on a wire
myograph and the endothelium-dependent (acetylcholine-ACh, 100ρM-100µM) and -independent (sodium nitroprusside-SNP,
100ρM-10µM) relaxation were evaluated. ACh-induced relaxation was decreased in CA of HF, while it was enhanced in CA of
INF as compared to SHAM (Emax: SHAM: 83±1 vs. INF: 91±1* vs. HF: 54±6 % relaxation*#). SNP-induced relaxation was
similar among groups. Further, some CA from SHAM and INF were incubated with NO synthase (NOS) inhibitor L-NAME (LN;
100µM) to evaluate the nitrergic modulation on ACh relaxation. LN incubation reduced the ACh-induced relaxation in CA of
SHAM and INF rats, in addition it abolished the differences observed between groups in the absence of this inhibitor (Emax:
SHAM/LN: 37±8 vs. INF/LN: 36±6 % relaxation). Also, endothelial (e) and neuronal (n) NOS isoforms, Akt and AMPK protein
expression were evaluated by Western Blot in CA from INF and SHAM. The eNOS (79%), nNOS (52%) and AKt (45%) protein
expression were greater in CA from INF as compared to SHAM, whereas the AMPK protein expression did not change between
groups.
Conclusions:
After MI the CA showed different adjustments. The CA from HF rats presented endothelial dysfunction, while the endothelial
function in CA from INF rats was increased due to enhanced nitrergic modulation probably by activation of nNOS and AKteNOS pathways. This adjustment could act as a compensatory mechanism that improve the perfusion to non-infarcted
myocardium and could be involved on the prevention of HF installation.
Keywords: Coronary artery, Myocardial infarction, Heart failure, Endothelium, Nitric Oxide
Financial Support: FAPESP and CNPq
Resumo:16-047
EFFECT OF PYRIDOSTIGMINE ON CARDIOVASCULAR AUTONOMIC CONTROL SIX TO SEVEN WEEKS
AFTER CORONARY ARTERY LIGATION
Sabino, J. P. J. ; da Silva, C. A. A. ; Fazan Jr. R. ; Salgado, H. C.
Depto. Fisiologia Universidade de São Paulo, FMRP-RP USP
Objectives:
Heart failure (HF) is characterized by remarkable changes in baro and chemoreflex control consisting of increased sympathetic
and reduced parasympathetic activity. While the sympathetic hyperactivity and its management in heart failure has been
thoroughly investigated, therapeutic options for reducing cardiac parasympathetic tone have not received the same attention.
Previous studies from our laboratory have demonstrated a protective effect of pyridostigmine (an acetylcholinesterase inhibitor)
on the autonomic control of arterial pressure and heart rate, due to the increase in parasympathetic function, 4 weeks after the
onset of heart failure in rats. However, the effect of pyridostigmine was not investigated during longer period of heart failure.
Thus, the present study evaluated the effect of pyridostigmine on the autonomic control of arterial pressure and heart rate, by
means of spectral analysis, six to seven weeks after coronary artery ligation.
Methods and Results:
HF was induced by coronary artery ligation of Wistar rats (250-300g) under anesthesia of ketamine/xilazyne (250 mg/kg, i.p.)
The procedure was similar for sham operated rats (Control), except that they did not undergo the ligation. After surgery the rats
received water or a solution of pyridostigmine (26 mg/kg/day, p.o.) during 6-7 weeks when the experiments were performed.
Beat-by-beat time series of systolic arterial pressure (SAP) were subjected to spectral analysis by Fast Fourier Transform. The
spectra of SAP were integrated into low (0.2 to 0.75 Hz) and high (0.75 to 0.75 Hz) frequency bands. The parasympathetic tonus
was evaluated by the tachycardic response caused by atropine (2 mg/kg, i.v.). HF rats exhibited reduced mean arterial pressure
(MAP) and no change in heart rate (HR) (96 ± 2 mmHg, 341 ± 9 bpm, n= 9) as compared to control rats (112 ± 2 mmHg, 325 ± 9
bpm, n= 12). Pyridostigmine impaired the decreased of MAP (105 ± 4 mmHg, n= 4) of HF rats. In addition, HF rats showed
smaller LF power (1.3 ± 0 mmHg2; n= 7) of arterial pressure as compared to control rats (3.1 ± 0 mmHg2; n= 10).
Pyridostigmine prevented the decrease of LF arterial pressure power (4.4 ± 1 mmHg2, n= 4) in HF rats. Parasympathetic tonus
was reduced in HF rats (30 ± 6 bpm, n= 6) as compared to control rats (90 ± 15 bpm, n= 7) while pyridostigmine protected the
parasympathetic tonus in HF rats (80 ± 11 bpm, n= 3).
Conclusions:
These results demonstrate that pyridostigmine prevented the decrease in MAP, LF arterial pressure power and parasympathetic
tonus. Accordingly, pyridostigmine was efficient to protect the autonomic imbalance observed in HF rats.
Keywords: coronary artery ligation , parasympathetic tonus, pyridostigmine, spectral analysis
Financial Support: Capes, CNPQ and FAPESP
Resumo:16-048
EVALUATION OF NON-ENZYMATIC ANTIOXIDANT DEFENSE IN PATIENTS WITH METABOLIC SYNDROME
Martins, C. C. 1; Baldissarelli, J. 1; Ferreira, R. D. 1; Bagatini, M. D. 2,1; Cardoso, A. M. 1; Fiorin, F. S. 1;
Rodrigues, M. V. 1; Pereira, L. B. 1; Morsch, V. M. 1; Shetinger, M. R. C. 1
1
Universidade Federal de Santa Maria, UFSM
2
Universidade Federal da Fronteira Sul, UFFS
Objectives:
Metabolic Syndrome (MS) is a multifaceted condition, which is associated with a collection of disorders which contributes to
cardiovascular risk. This syndrome is often accompanied by oxidative stress, which occurs when reactive oxygen species
production is higher than the antioxidant capacity of the organism. Then, we evaluated the non-enzymatic antioxidant defense
that includes ascorbic acid (vitamin C) and Non-protein thiols (NPSH) in patients with MS to better characterize the presence of
reactive species in this pathology.
Methods and Results:
Thirty volunteer MetS patients were selected for this study and thirty control patients. They were aged between 35 and 65 years,
both sexes and all subjects gave written informed consent to participate in the study. The protocol was approved by the Human
Ethics Committee of the Health Science Center from the Federal University of Santa Maria, protocol number 98/10, Brazil. The
Non-protein thiols were assayed in plasma by the method of Ellman (Arch Biochem Biophys, v. 82, p. 70:77, 1959) and Vitamin
C analysis was made in serum by the method described by Jacques-Silva (J Biol Chem, v. 147, p. 399:407, 1943). Data were
analyzed statistically using test-t. Differences were considered significant when the probability was P < 0.05. We observed that
Non-protein thiols was significantly decreased in the MetS patients (0,60 ± 0,15), when compared with the control group (1,15 ±
0,33); the same was observed with Vitamin C (27,65 ± 9,7 / 45,48 ± 9,8).
Conclusions:
The vitamin C counters free radicals and regulates vitamin E metabolism by recycling oxidized tocopherols and its decrease
could be due to the increasing utilization of vitamin C as an antioxidant defense against reactive oxygen species. Similarly, nonprotein thiols, as GSH, play an important role in the antioxidant defense system by scavenging reactive species and regenerating
other antioxidants, as vitamin C. Its decrease may serve as a marker of increased oxidation and an indirect sign of higher
oxidative stress. Then, the elevated oxidative damage in MS patients can be evidenced by decreased the non-enzymatic
antioxidant defense.
Keywords: Metabolic Syndrome, antioxidant capacity , ascorbic acid , Non-protein thiols , cardiovascular risk
Financial Support: CNPq, CAPES, FAPERGS.
Resumo:16-049
ACUTE RENAL FAILURE EFFECTS IN THE GLOBAL PATTERN GENE EXPRESSION OF HEART TISSUE
Nakama, K. ; Abrahão, M. ; Carneiro-ramos, M. S.
Universidade Federal do ABC, UFABC
Objectives:
Patients suffering from renal failure are more likely to develop cardiovascular diseases, which aggravate patient health and can
lead to death. The heart is a target organ in many regulatory systems, such as hormones, neuronal and immunological systems,
modulating heart trophism directly or indirectly. This study aimed to determine changes in gene expression of hypertrophied
cardiac tissue in a renal ischemia-induced systemic inflammation model.
Methods and Results:
C57BL/6 young (20g to 26g), male mice were submitted to ischemic protocol, 60 min left renal pedicle unilateral occlusion
followed by reperfusion 5, 8, 15 or 20 days. Hearts were removed and total RNA was extracted and purified for the synthesis of
cDNA and cRNA. Analyses were performed using GeneChip Mouse Genome chip 430 2.0 Array (Affimetrix) containing 45,000
mice genes. The software dCHIP (DNA Chip Analyzer) was used to normalize and model expression. Gene modeled expression
cutoff was set to 20, comparable to modeled expression for TRbeta. The same software was used to separate genes that showed a
20% increase or decrease of the probes fluorescence intensity in comparison to the control group. The analysis results indicated
17,413 up-regulated genes and 18,297 down-regulated genes in at least one of the test groups with different days of reperfusion,
according to established criteria of 20% change of expression. Subsequently, we used the GenMAPP software to group the
selected genes in signaling pathways, being the analysis done with the same criterion of 20% genes up or down regulated.
Preliminary results indicate, in at least one of the groups tested, 405 up-regulated and 300 down-regulated pathways.
Conclusions:
The analysis of modulated genes indicate a higher number of down-regulated genes in test groups 5 and 8 days, as the largest
number of up-regulated genes were found in test groups 15 and 20 days. Concerning the regulated pathways, our data shows that
there is a progressive enhancement of the up-regulated pathways in test groups 5, 8 and 15 days, followed by a decrease in the
group 20 days. Thus, regarding the down-regulated pathways, the number of pathways negatively modulated in the group 8 days
is lower in comparison to the group 5 days. However, there was a subsequent increase in the groups 15 and 20 days.
Keywords: cardiac hyperthrophy, microarray, renal failure
Financial Support: FAPESP, CNPq e UFABC
Resumo:16-050
PRELIMINARY CARDIAC CHARACTERIZATION OF P2X7 PURINERGIC RECEPTOR KNOCKOUTS MICE
IMMUNIZED WITH PLASMIDS ENCODING THE CARDIAC MUSCARINIC RECEPTOR SUBTYPE M2
Martinez, C. G. ; Ribeiro, J. M. ; Bellio, M. ; Persechini, P. M. ; Kurtenbach, E.
Universidade Federal do Rio de Janeiro, UFRJ
Objectives:
Dilated cardiomyopathy (DCM) can be associated with the presence of auto-antibodies against the muscarinic receptor subtype
M2 (m2-AChR) as showed for Chagas‘ disease patients and animals models immunized with M2-peptides or cDNA encoding for
M2-AChR. In these cases, the observed morphological and functional cardiac derangements was attributed to an increase in the
agonist action. Additionally an inflammatory response with an increase of fibrosis has been described, which in damage cells is
associated with the release of the pro-inflammatory mediator ATP, a ligand for purinergic P2X7 receptor in T cells. Thus, the
main purpose of this work is to determine if the absence of these receptors could interfere in the progression of DCM established
by the presence of anti-m2AChRs antibodies using C57Bl/6J knockout for P2X7 purinergic receptors.
Methods and Results:
For the development of DCM, C57Bl/6J mice, wild and P2X7 knockout, were immunized by biolistic with the plasmid
enconding for M2-AChR (pcDNA3-hM2). Other groups of animals, named controls, were immunized with the empty plasmid
pcDNA3. Twenty-three weeks after vaccination, cardio-respiratory function assessed by ergospirometry showed a significant
decrease in the time and speed of exercise in both groups of animals deleted for P2X7. All groups of animals showed an increase
in cardiac output that was dependent on an increase in heart rate. Analysis of regulatory T cells in the spleen through labeling for
Foxp3 showed no significant difference between those groups. However, the labeling for CD39, an ectonucleotidase
characteristic of immune cells, was increased in P2X7 knockouts vaccinated with plasmid pcDNA3 compared to the wild-type
mice group vaccinated with the same plasmid. Moreover the P2X7 knockout immunized with pcDNA3 group presents higher
levels of CD39 expression than P2X7 knockout immunized with pcDNA3-hM2. The last one present level comparable to wildtype mice vaccinated with pcDNA3 and pcDNA3-hM2. These dates could demonstrate a cross-talk beteween purinergic ang
muscarinic receptores. A few in vitro models demonstrated that might exist a compensatory action, which the absence of P2X7
can lead to an increase in function of CD39.
Conclusions:
We conclude that at this point, it was not possible to observe huge changes in the parameters analyzed, though the vaccination
promoted subtle immunological and functional changes, mainly in the cardiac output, frequency and expression of CD39.
Keywords: Cardiac muscarinic receptor subtype M2 , Dilated cardiomyopathy , P2X7 purinergic receptors
Financial Support: CAPES, CNPq and FAPERJ
Resumo:16-051
ECG ALTERATIONS INDUCED BY ANGIOTENSIN I AND ANGIOTENSIN II IN RATS
Silva, M. D. A. 1,1; de Paula, D. C. C. 1; Aguiar, L. D. 1; Grabe-guimarães, A. 1; Serra, C. P. 1; Guimarães,
H. N. 2,1
1
Universidade Federal de Ouro Preto, UFOP
2
Universidade Federal de Minas Gerais, UFMG
Objectives:
Aim: Many experimental models were developed for the hypertension study and screening of potential therapeutic agents that
may contribute to the prevention and treatment of human hypertension. Induce hypertension via activation of RAS (Renin
Angiotensin System) is a classic model but the electrocardiogram (ECG) induced alterations are not described. The evaluation of
drug effects on QT interval of ECG is a fundamental component required for safety database, because its prolongations is
associated with arrythmias and sudden cardiac death. The aim of this study was to evaluate the pressor effect and its ECG
associated alterations at different doses of angiotensin I (ANG I) and angiotensin II (ANG II).
Methods and Results:
Methods: All the procedures were approved by the UFOP Ethical Committee (number 2010/83). Male Wistar rats (260 ± 10 g)
were divided into 4 groups (n=6) that received: 1) Vehicle + ANG I; 2) Captopril + ANG I; 3) Vehicle + ANG II; 4) Losartan +
ANG II. The captopril or losartan (30 mg/kg) were orally administered 20 minutes before the animals were anesthetized with
quetamin/xilasin (100/14 mg/100g). Catheters were inserted into a femoral artery and vein, in order to obtain arterial pressure
(AP) signal and for drugs administration, respectively. Electrodes were inserted subcutaneously to obtain ECG signal (DII). After
surgical procedures, 3 doses of ANG I (1, 10 and 100 pmol) or ANG II (0,5; 5 and 50 pmol) were i.v. administered. The data
were analyzed with one-way ANOVA. Results: It was obtained significant and dose-dependent increase in blood pressure in
response to ANG I and ANG II. The increase of MAP was from 6 to 42 mmHg for ANG I, from 7 to 40 mmHg for ANG II. The
QT interval prolongation was significant for ANG I at 1 pmol (73,50 ± 3,10 ms), 10 pmol (76,65 ± 2,26 ms) and 100 pmol (78,73
± 2,80 ms) versus control (69,92 ± 2,36 ms); and for ANG II at 5 pmol (74,54 ± 2,96 ms) and 50 pmol (78,31 ± 3,44 ms) versus
control (69,37 ± 2,78 ms). The PR interval of ECG also presented significant increase after 1 pmol (57,17 ± 1,04 ms), 10 pmol
(58,64 ± 1,53 ms) and 100 pmol (60,73 ± 1,05 ms) for ANG I versus control (54,96 ± 1,12 ms); and after 5 pmol (58,67 ± 3,00
ms) and 50 pmol (60,46 ± 2,86 ms) for ANG II versus control (54,15 ± 2,17 ms). The treatment with captopril reduced about 50%
and Losartan reduced about 80%. No changes were observed in the ECG, expected result for the acute treatment.
Conclusions:
Conclusion: The models of hypertension induced by ANG I and ANG II are also able to induce ECG alterations, mainly QT
interval prolongation. So they could be used for screening drugs with potential cardioprotective action.
Keywords: Angiotensin I, Angiotensin II, Cardioprotection, Eletrocardiogram, Hipertension
Financial Support: FAPEMIG, CNPq, UFOP
Resumo:17-135
TRPA1 RECEPTOR ACTIVITY EXPRESSED IN BLADDER DORSAL ROOT GANGLION NEURONS IS
REGULATED BY NEUTROPHIC FACTORS FOLLOWING SPINAL CORD INJURY
Andrade, E. L. 1; Ferreira, J. 2; de Castro, C. J. 3; Bento, A. F. 1; Souza, A. H. 3; Gomez, M. V. 3; Calixto, J.
B. 1
1
UNIVERSIDADE FEDERAL DE SANTA CATARINA, UFSC
2
UNIVERSIDADE FEDERAL DE SANTA MARIA, UFSM
3
UNIVERSIDADE FEDERAL DE MINAS GERAIS, UFMG
Objectives:
We previously observed that alterations in TRPA1 (Transient Receptor Potential Ankyrin 1) expression and activity in bladder
after spinal cord injury (SCI) contribute to the development of overactive bladder in rats (Am. J. Physiol. Renal Physiol., 2011).
Studies suggest that neurotrophic factors are involved in the regulation of TRPA1 sensitivity and expression (J. Clin. Invest., 115;
2393-2401, 2005; Dent. Res., 86; 550-555, 2007; Neurosci. Lett., 438; 221-227, 2008; Brain Res., 1284; 54-67, 2009). Therefore,
the aim of this study was to evaluate the influence of neurotrophic factors on the responsiveness and functionality of TRPA1 in
DRG neurons innervating bladder following SCI.
Methods and Results:
Spinal cords of anesthetized male Wistar rats (270-300g) were injured at T10 level by inserting a Fogarty 2F embolectomy
catheter (ethical committee process 000156). Dorsal root ganglia neurons (L6-S1 segment) of naive, sham-operated or SCI rats
were isolated 14 days after surgery and cultured in absence or presence of neurotrophic factors. Twenty-four hours later, the
calcium mobilization assay was performed. In other experiments, the neurotrophic factors levels in DRG neurons were evaluated
2, 7, 14 and 28 days after surgery. SCI induced increase in the proportion of DRG neurons responsive to TRPA1 agonist
cinnamaldehyde (300 ìM) (87%, 65/75 responsive cells/total cells) in comparison to non-operated group (naive) (68%, 30/44).
Moreover, SCI increased the calcium mobilization to cinnamaldehyde stimulus (72.7 ± 21.9%; n = 5 experiments, 30-80 cells) in
comparison to sham-operated group. The exposure of DRG neurons of naive animals to neurotrophin BDNF (brain-derived
neurotrophic factor; 100 ng/mL) increased the proportion of DRG neurons responsive to cinnamaldehyde (89%, 80/90) in
comparison to control group (BDNF absence) (68%, 30/44). In addition, the exposure of DRG neurons of naive animals to NGF
(nerve growth factor; 100 ng/mL) or BDNF increased the calcium mobilization induced by cinammaldehyde (NGF: 93.3 ±
25.3%; BDNF: 97.8 ± 17.9%; n = 5 experiments, 30-80 cells) in comparison to control group. The NGF and BDNF levels were
significantly increased in DRG neurons on day 28 (2.5-fold) and on days 2 and 28 (3-fold) after SCI, respectively.
Conclusions:
Taken together, the results suggest that NGF and BDNF may be directly involved in the changes of TRPA1 responsiveness and
functionality induced by SCI. These events could contribute to the pathogenesis of overactive bladder following SCI.
Keywords: SPINAL CORD INJURY, BLADDER, NEUROTROPHIC FACTORS, OVERACTIVE BLADDER, RATS
Financial Support: CNPq, CAPES, FINEP, FAPESC, PRONEX
Resumo:17-136
BOTHROPS JARARACA HIGH MOLECULAR MASS KININOGEN (BJHK) INHIBITS CHANGES IN
LEUKOCYTE-ENDOTHELIAL INTERACTIONS INDUCED BY B. JARARACA SNAKE VENOM
METALLOPROTEINASES
Sanson, A. L. ; Zychar, B. C. ; Clissa, P. B. , ; Portaro, F. C. V. , ; Gonçalves, L. R. C. ,
Instituto Butantan, IBU
Objectives:
Aim: The Bothrops jararaca (Bj) snake plasma is rich in protease inhibitors, some of which have inhibitory activity on toxins
from its own venom. One of these, which have a molecular mass of 110 kDa, is a potent inhibitor of cysteine proteases and
releases a peptide that induces contraction of homologous smooth musculature. For these characteristics this protein, named
BjHK (Bothrops jararaca High Molecular Weight Kininogen), was correlated to mammalian high molecular weight kininogens.
Moreover, it was found that this protein inhibits metalloproteases present in the Bj venom. Severe local inflammatory symptoms
are common in envenoming induced by B. jararaca snakebites, and It is known that metalloproteases is the main class of toxins
involved in the inflammatory activity of this venom. Our goal in this study was to test the effect of the BjHK on changes in the
leukocyte-endothelial interactions induced by Bj venom or by an isolated Bj metalloproteinase, in vivo, using intravital
microscopy.
Methods and Results:
Methods and Results: Bj venom (1 µg) or Jararhagin (JAR) (0.5µg), a hemorrhagic P3 metalloproteinase isolated from Bj venom,
incubated (30 min, 37 ºC) or not with BjHK (2 µg) were injected (100µL) into the scrotal bag of Swiss mice (25 to 28g body
weight, n= 5/ group). The microcirculation of cremaster muscle was analyzed 2 or 24h after the injections. We analyzed a
segment (100 µm) of a post-capillary venule during 5 min and counted the number of adhered and emigrated leukocytes,
statistically comparing with the observed in control group injected with sterile buffered saline (significant when p< 0.05). The
results were the following: Adhesion 2h: control= 1.4±0.6; BjHk= 1.8±0.6; VBj= 12.2±0.6; JAR= 15.2±1.0; VBj+BjHK=
4.0±0.6; JAR+BjHK= 4.0±0.9; Adhesion 24h: control= 1.3±0.7; BjHK= 1.7±0.8; VBj= 4.2±0.4; JAR= 4.6±0.5; VBj+BjHK=
2.2±0.8; JAR+BjHK= 2.75±0.8; Emigration 2h: control= 1.0±0.3; BjHK= 0.6±0.4; VBj= 3.8±0.4; JAR= 6.4±0.5; VBj+BjHK=
2.6±0.6; JAR+BjHK= 1.7±0.5; Emigration 24h: control= 1.2±0.4; BjHK= 0.6±0.4; VBj= 14.4±0.6; JAR= 16.8±0.6; VBj+BjHK=
2.3±0.5; JAR+BjHK= 3.25±0.5. Results showed that BjHK did not induce changes in leukocyte-endothelial interactions, when
compared to the control group. In groups injected with Bj venom or with JAR, adhered and emigrated leukocytes were hugely
increased in all times studied. Adhered cells were significantly diminished after 24h of the venom or toxin injections when
compared to 2h, but emigrated cells were significantly increased. When incubated with BjHK, neither the Bj venom nor the JAR
induced significant changes in leukocyte-endothelial interactions in microcirculation when compared to the control group. This
effect was similar to the observed when Bj venom and JAR were treated with ο-phenanthroline, despite of the mechanism of
action of BjHK on the catalytic activity of JAR not be like a chelating agent.
Conclusions:
Conclusion: We conclude that BjHK can inhibit this inflammatory activity of the Bj venom probably by inhibiting the activity of
their metalloproteinases.
Keywords: kininogen, Bothrops jararaca, leukocyte, venom, metalloproteinases
Financial Support: FAPESP, CAPES, CNPq, and INCTTOX.
Resumo:17-137
CONSTITUTIVE NITRIC OXIDE SYNTHASES REGULATE NF-κB NUCLEAR TRANSLOCATION AND SMOOTH
MUSCLE CELL ACTIVATION.
Scheschowitsch, K. ; Assreuy, J.
Dept of Pharmacology , UFSC
Objectives:
Cell stimulation with pro-inflammatory agents, such as lipopolyssacharide (LPS) and interferon-γ (IFN), causes cell activation
and tissue inflammation in vivo by increasing a variety of pro-inflammatory proteins through nuclear factor transcription kappa-B
(NF-kB) activation. During sepsis, a systemic inflammatory disease, a large amount of nitric oxide (NO) is produced in response
to cell activation and contributes to persistent hypotension. Results of our laboratory show that hypotension and mortality during
sepsis is prevented by early administration of NO synthase (NOS) inhibitors, suggesting that constitutive NOS play an important
role in sepsis. Vascular smooth muscle cells are the major component of vascular tonus maintenance and thus we decided to
investigate the importance of constitutive NOS in smooth muscle cell activation in vitro.
Methods and Results:
To evaluate cell activation a cell line of rat aorta smooth muscle cells (A7r5) were plated on a 96-well culture plate and
stimulated with LPS 1 µg/mL and IFN 200 U/ml (LPS/IFN) for 24, 48 and 72 h. At each time period, 100 μL of culture
supernatant was collected and nitrite concentration (μM) determined by Griess reaction (n=3). Constitutive NOS activity was
investigated by plating A7r5 cells on a 96-well black culture plate, DAF-FM DA loading and stimulation with LPS/IFN in the
presence or the absence of NOS inhibitors (7-NI and L-NA, 200 μM) or NO scavenger (PTIO, 100 μM). Fluorescence intensity
was measured 30 minutes after stimulation and compared to control group and expressed as the percentage of fluorescence (n=3).
The effects of constitutive NOS inhibition on NF-kB nuclear translocation was evaluated by immunofluorescence. Cells were
plated in appropriate conditions and stimulated with LPS/IFN for 30 minutes, 2 and 4 hours. Images were obtained with confocal
microscopy (63x immersion) and quantified by LAS® software. Results were expressed as the mean of fluorescence (in arbitrary
units, AU) in the nucleus (n=20 nucleus). All results are representative of 3 independent experiments ± SEM and statistical
comparisons performed by two-way ANOVA followed by Tuckey test. Nitrite concentration in stimulated cells was statistically
greater (3.7 ± 0.3; 8.4 ± 0.9; 13.9 ± 0.4 μM) compared to control group (0 μM), respectively, in all the evaluated times. Cell
stimulation with LPS/IFN causes a NO pulse (3.7 ± 0.5 versus control group 1.0 ± 0.1), that was diminished in the presence of 7NI, L-NA and PTIO (2.5 ± 0.3; 2.0 ± 0.3; 1.4 ± 0.3, respectively). Also, nuclear translocation of NF-kB in stimulated cells (56.3
± 0.7; 43.2 ± 1.9; 14.9 ± 0.5, respectively, to 30 minutes, 2 and 4 h) was diminished in the presence of these inhibitors compared
to stimulated cells in all the evaluated times (46.9 ± 1.8; 37.1 ± 1.5; 39.8 ± 1.3, respectively, in the presence of 7-NI, L-NA and
PTIO, 30 minutes after stimulation). Reduced NO pulse and NF-kB translocation leads to diminished cell activation, as assessed
by nitrite concentration 48 h after cell stimulation.
Conclusions:
Smooth muscle cell stimulation with pro-inflammatory agents induces constitutive NOS activity and a NO pulse generation that
plays an important role in NF-kB nuclear translocation and cell activation.
Keywords: Nitric oxide synthases, NF-kB, smooth muscle cell
Financial Support: CAPES, CNPQ and FAPESC.
Resumo:17-138
EFFECTS OF RESVERATROL IN ANIMAL MODELS OF ACUTE NOCICEPTION INDUCED BY GLUTAMATE
AND CAPSAICIN IN MICE.
Bazzo, K. O. 1; Gomez, M. V. 3; Souza, A. H. D. 3; Campos, M. M. 1,2
2
Pontífica Universidade Católica do Rio Grande do Sul, PUCRS
1
Mestrado em Medicina e Ciências da Saúde, PPGMCS
3
Universidade Federal de Minas Gerais, UFMG
Objectives:
Resveratrol (RSV) is a naturally-occurring polyphenol with well-characterized anti-inflammatory and antioxidant actions.
Moreover, significant antinociceptive effects have been demonstrated for RSV in the inflammatory hyperalgesia induced by
carrageenan in rats (Life Sci. 1317:21,2001). In the present study we have evaluated the effects of RSV in two acute models of
spontaneous nociception, induced by capsaicin and glutamate in mice.
Methods and Results:
Male Swiss mice (30 to 35 g, N=8 per group) were used. All the experimental protocols were approved by the local Animal
Ethics Committee (protocol number: 10/00175). The animals were pre-treated with RSV by oral route (50, 100 or 200 mg/kg), 30
min before the induction of nociception. In a separate series of experiments, RSV was administered intraplantarly (200 µg/paw),
5 min beforehand. The spontaneous nociception was induced by the intraplantar injection of capsaicin (1.6 µg/paw) or glutamate
(20 µg/paw) into the right hindpaw. The time spent licking or biting the injected paw was registered during 5 and 15 minutes,
respectively. The results demonstrate that oral administration of RSV was able to significantly reduce capsaicin-induced
spontaneous nociception, at the doses of 50 and 100 mg/kg (25±11% and 36±10%, respectively). Furthermore, the oral treatment
with resveratrol (100 mg/kg) also produced a significant reduction of nociceptive behavior elicited by glutamate (39±9%).
Nevertheless, the local administration of RSV failed to significantly affect either capsaicin-induced nociception, whereas it
partially reduced the nociception induced by glutamate (33 ± 9 %).
Conclusions:
The present findings suggest that RSV has antinociceptive effects in the acute nociception induced by both capsaicin and
glutamate, which appear to be related to an interference with central pathways of pain transmission. Additional experiments are
currently being performed to further characterize the mechanisms underlying the antinociceptive effects of RSV.
Keywords: Capsaicin, Glutamate, Nociception, Resveratrol
Financial Support: CAPES, CNpq and PUCRS
Resumo:17-139
LIPID BODY FORMATION INDUCED BY BOTHROPS JARARACA (BOTHROPOIDES), BOTHROPS INSULARIS
(BOTHROPOIDES), BOTHROPS ATROX AND BOTHROPS MOOJENI SNAKE VENOMS IN PERITONEAL
LEUKOCYTES OF MICE.
Nascimento, N. G. 1; Leiguez, E. J. 1,2; Giannotti, K. C. 1; Viana, M. D. N. 1; Teixeira, C. 1
1
Laboratório de Farmacologia, Instituto Butantan - SP. , IBU
2
Universidade de São Paulo, USP
Objectives:
Lipid bodies (LBs) are cytosolic inclusions present in most eukaryotic cells, containing neutral lipids surrounded by a single
phospholipid membrane and specific proteins, such as the protein related to differentiation of adipocytes (ADRP). LBs are
involved in lipid metabolism and generation of inflammatory mediators. Moreover, increased numbers of LBs has been
demonstrated in inflammatory leukocytes. Venom from Bothrops genus snakes induce a marked inflammatory local reaction
characterized by leukocyte influx and release of a variety of inflammatory mediators. However, formation of LBs in leukocytes
present at the site of Bothrops venom injection is still unknown. In this study the ability of snake venoms from distinct species of
Bothrops genus, such as B. jararaca (BjV), B. insularis (BiV), B. moojeni (BmV) and B.atrox (BatxV) to induce LBs formation
and upregulation of ADRP protein expression was comparatively evaluated in mice peritoneal leukocytes.
Methods and Results:
Male Swiss mice were used (Butantan Institute Ethical Committee ref. 729/10). These animals received intraperitoneal (i.p)
injection of inflammatory doses of BjV (0.250 mg/g) or BmV (0.250 mg/g) or BiV (0.050 mg/g) or BatxV (0,025 mg/g) or
apyrogenic saline (control). At selected periods of time (1, 3, 6, 12, 24, 48 or 72 h) after each venom injection, the inflammatory
exudates were harvested to determine total number of leukocytes in Neubauer chamber and subtypes of leukocytes using Hema3
stained cell smears. LB formation was determined in macrophages stained with osmium tetroxide (1%) followed by counting
under phase contrast microscopy. ADRP protein expression was evaluated by Western blotting. Results showed that i.p injection
of BjV or BiV or BmV or BatxV caused a significant increase in the numbers of LBs from 1 up to 24 h (200%) when compared
with control cells (2.0 ± 0.5 LB/cell, n=4), with no statistical difference among venoms from studied snake species. The highest
LB numbers were detected at the 12 h (300%) after venom injection in comparison with controls (2.4 ± 0.7 LB/cell, n=4).
Moreover, all studied venoms caused a significant increase in levels of ADRP protein expression in leukocytes collected 12 h
after their injection.
Conclusions:
In conclusion, these data indicate the ability of Bothrops venoms either from the mainland or island to induce lipid body
formation and upregulate ADRP expression in leukocytes. Moreover, these data suggest that LBs have a role in the inflammatory
reaction induced by snake venoms from Bothrops genus.
Keywords: Snake Venom, Bothrops, Leukocytes, Lipid Body
Financial Support: FAPESP; CAPES.
Resumo:17-140
URB602N, AN INHIBITOR OF MONOACYLGLYCEROL LIPASE, DECREASES INFLAMMATION IN A MURINE
MODEL OF ACUTE LUNG INJURY
Costola-de-souza, C. ; Ribeiro, A. ; Ferraz-de-paula, V. ; Matazo, M. P. ; Palermo-neto, J.
Patologia Experimental e Comparada Universidade de São Paulo, VPT- FMVZ/USP
Objectives:
Recent studies indicate the importance of the endocannabinoid system, into different pathophysiological processes (Guindon,
Hohmann, 8:403,2009), where the endocannabinoids, such as 2-Arachidonoylglycerol (2-AG), modulate the immune response
(Cabral, Griffin-Thomas, 11:1, 2009). Based on these facts, the use of [1,1'-biphenyl]-3-yl-carbamic acid, cyclohexyl ester
(URB602) might be a nice opportunity to investigate the roles of 2-AG, since it blocks 2-AG degradation, making possible to
amplify 2-AG intrinsic actions. Thus, we analyzed the effects of URB602 under a neuroimmune perspective. Specifically, our
objective was to establish a dose-response curve for the anti-inflammatory effects of URB602 in a murine model of acute lung
injury.
Methods and Results:
Thirty-two C57BL/6 male mice were used; mice were divided randomly into six groups (n=4-6/group). Group 1 (G1) received
1.0mg/kg; group 2 (G2) 5.0mg/kg; group 3 (G3) 10.0mg/kg and group 4 (G4) 20.0mg/kg URB602 i.p. 60min prior a nasal
instillation of LPS (100ug/mL). Two Control groups received vehicle i.p. 60min prior to nasal instillation of LPS (GC1) or sterile
PBS (GC2). Each mice received 1ul of LPS/g bw. Twenty-four hours followed LPS or PBS nasal instillations, mice were
submitted to euthanasia and bronchoalveolar lavage fluid (BAL) as harvested for cell counting. Data were analyzed using Oneway ANOVA followed by Tukey-Kramer multiple comparison test. We observed that LPS treatment increases the number of
cells in the BAL compared to animals treated with PBS. Further statistical analysis showed that only animals treated with
20mg/kg of URB602 (F(5,19)=12,90;p,0,0001) showed no increases in the number of inflammatory cells induced by LPS in
BAL.
Conclusions:
We showed that URB602 (20mg/kg) decreases some inflammatory parameters in a murine model of acute lung injury.
Keywords: URB602, Acute lung injury, murine
Financial Support: CAPES
Resumo:17-141
REACTIVITY OF LUNG FIBROBLASTS FROM SILICOTIC MICE: ANALYSIS IN 3D CELL CULTURE MODEL
Guimarães-silva, A. M; Trentin, P. G. ; Dalzy, D. V; Perez, S. A. C; Martins, M. A. ; Silva, P. M. R
Instituto Oswaldo Cruz, FIOCRUZ
Objectives:
Silicosis is a chronic lung disease caused by the inhalation of crystalline silica particles, characterized by a slow and progressive
evolution leading to an intense fibrogenic response. Fibroblasts are considered crucial target cells in chronic fibrotic diseases and
over the past years, the development various types of 3D culture systems have garnered much attention and gained increased
recognition as important research tools. In this study we evaluated the reactivity of lung fibroblasts from normal or silicotic mice
using a 3D cell culture (spheroid) assay in vitro.
Methods and Results:
Male Swiss-Webster mice were instilled with intranasal crystalline silica particles (10 mg/50 µL) or with the same volume of
saline. After 7 days, lungs were perfused with PBS and digested with enzyme (collagenase A) to obtain the fibroblasts. The cells
were cultured in DMEM + fetal bovine serum (10%), and after they entered in confluence they were replicated three times. Cells
were put in a 96 wellplate, previously covered with agarose, in a density of 1.25x104 cells/well. The analysis was performed in
the period of 1-4 days and included parameters such as size/diameter and cell proliferation, evaluated by image analyses (Image Pro Plus program) and thymidine [H3] uptake, respectively. All experimental procedures were performed in accordance with the
guidelines of the Committee on Use of Laboratory Animals of the Oswaldo Cruz Foundation (L-034/09).The growth kinetics
revealed that at early time-points (1-2 days), the spheroids obtained from normal mice fibroblasts presented lower density and
higher size/diameter as compared to those at later time points (3 - 4 days). Spheroids from cells obtained from silicotic mice
exhibited a similar profile, though showing themselves bigger than those from normal mice. The proliferation rate was also
increased in the case of fibroblasts from silicotic mice as compared to that of normal cells (192.3±15.6 vs. 113±17.4,
respectively) (mean ± SEM; n=6). When the two spheroid populations were submitted to stimulation with the pro-fibrotic
cytokine IL-13 (40 ng/mL), for 1-4 days, we noted that cells were activated as attested by a significant increase of size/diameter
and of proliferation rate. Notably, under condition of IL-13 stimulation, silicotic spheroids presented higher size/diameter and
proliferation rate when compared to normal spheroids (rate proliferation: 1373±147.3 vs. 630±117.8; respectively).
Conclusions:
Altogether, our results show that fibroblasts from adult mice lungs can be cultured in a 3D culture system and that the spheroids
generated from silicotic fibroblasts were more activated than the normal ones. The 3D model for lung fibroblasts seems to be a
quite reproducible and easy-handling culture, representing a promising assay for the search of anti-fibrotic drugs.
Keywords: silicosis, lung fibroblasts, 3D culture
Financial Support: FIOCRUZ/CNPq/FAPERJ
Resumo:17-142
REGULATORY ROLE OF THE PROTEIN ANNEXIN-1 ON THE LIPOPOLYSACCHARIDE INDUCED LUNG
INJURY IN MICE
Matheus-souza, D1; Arantes, A. C. S1; Ferreira, T. P. T1; Trentin, P. G. 1; Pires A. L. A1; Flower, R. 2;
Perretti, M. 2; Martins, M. A. 1; Silva, P. M. R 1
2
Dept.of Biochemical Pharmacology,The William Harvey Institut, WHI
1
Fundação Oswaldo Cruz, FIOCRUZ
Objectives:
Introduction: Escherichia coli lipopolysaccharide (LPS) induces an acute lung injury characterized by vascular permeability
increase and leukocyte infiltration in the air space and lung parenchyma, leading to impairment of the respiratory function.
Glucocorticoids are considered as critical agents based on their anti-inflammatory properties, which is shown to be at least
partially dependent on the induction of an intermediate protein called annexin-1. In this study we aimed to investigated the
potential regulatory role of annexin-1 on the acute lung injury caused by LPS in mice.
Methods and Results:
Methods: Anesthetized male Balb/c (ANX1+/+) mice and annexin-1 knockout mice (ANX1-/-) received a single intranasal
instillation of LPS (25µg/ 50 µL) or saline. The analyses were made 24h-post LPS provocation. Lung function (resistance and
elastance) and airways hyperreactivity to methacholine (3 - 27 mg/mL) were evaluated by whole body invasive plestimography
(Finepoint, Buxco System). Total and differential leukocytes in bronchoalveolar lavage (BAL) were counted in Neubauer
chamber and cytospin preparations stained with May-Grunwald-Giemsa dye, respectively. Collagen was measured by Sircol
technique and cytokine/chemokine generation by ELISA. All experimental procedures were performed in accordance with the
guidelines of the Committee on Use of Laboratory Animals of the Oswaldo Cruz Foundation (L-034/09). Results: ANX1+/+
mice when stimulated with LPS showed increased levels of basal lung resistance and elastance as well as airways hyperreactivity
to aerosolized methacholine. A marked inflammatory response was noted in LPS-provoked mice, including leukocyte infiltration
(mainly neutrophils) in the BAL fluid as well as generation of chemokines (KC and MIP-2) and cytokine (TNF) in the lung
tissue. In the case of ANX1-/- mice, an exacerbation of lung function compromise, leukocyte infiltration in the BAL fluid and
cytokine/chemokine generation in the lung tissue was evidenced. A marked increase in the tissue collagen production was also
detected in the ANX1-/- animals as compared to ANX1+/+ mice (from 47,43 ± 3,566 to 26,42 ± 2,848 μg/right lung,
respectively. Mean ± SEM, n=6)
Conclusions:
Conclusion: Altogether our data show that the annexin-1 seems to effectively regulate the inflammatory response caused by LPS
in mice, indicating that treatment with this protein or even with its derivatives could be an important pharmacological tool for the
treatment of inflammatory diseases such as acute lung injury.
Keywords: Annexin-1, Inflammation, Lipopolysaccharide, Lung, Mice
Financial Support: Financial Support : FIOCRUZ, CNPq, FAPERJ and CAPES.
Resumo:17-143
ANTI-INFLAMMATORY ACTIVITY OF COCOS NUCIFERA L. (ARECACEAE), GIANT VARIETY
Silva, R. R. 1; Silva, D. O. 1; Zardo, R. S. 2; Gonçalves, M. R. 2; Alviano, C. S. 1; Fernandes, P. D. 2;
Alviano, D. S. 1
1
Instituto de Microbiologia Paulo de Góes (UFRJ), IMPPG
2
Instituto de Ciências Biomédicas (UFRJ), ICB
Objectives:
The fruit of Cocos nucifera L. (Arecaceae) has a fiber husk rich in polyphenolic compounds that is popularly used to several
disorders. Recent results show that aqueous extracts from husk fiber of C. nucifera present different biological activities. In this
study our objectives were to evaluate the anti-inflammatory activity of a variety of C. nucifera that has not been investigated yet.
Methods and Results:
The Cocos nucifera L. (Arecaceae), giant variety, was collected in Aracaju, Brazil (in May, 2007). The husk fiber (414 g) was
dried in the sun, finely ground and the powder soaked for 3 h in 6 L of boiling distilled water. Then, the extract was filtered,
lyophilized and stored at −200C, yielding around 10% of the dry weight of starting material. The aqueous extract was
resuspended in distilled water in all the experiments. The anti-inflammatory activity was evaluated in models of abdominal
writhing induced by acetic acid (2%, ip), formalin-induced licking response (2%, intraplantar) and subcutaneous air pouch (SAP)
induced by carrageenan (1%) injection. Swiss mice (20-25 g, n = 5-6) were orally pre-treated with the aqueous extracts from C.
nucifera (doses 50, 100, and 150 mg/kg). The use of animals was approved by the ethical committee of animal experimentation
from Centro de Ciências da Saúde (UFRJ) and received the number ICBDFBC015. The results are presented as the mean ± S.D.
and statistical analysis was ANOVA followed by Bonferroni (*p
Conclusions:
Our results suggest that the aqueous extracts from husk fiber of Cocos nucifera L. (Arecaceae), giant variety, have significant
anti-inflammatory activity. Since the husk fiber is an industrial reject, the results obtained in the present study suggest that it
might be a very inexpensive source of anti-inflammatory drug that warrants further investigation.
Keywords: anti-inflammatory activity, Arecaceae, Cocos nucifera, extract, giant
Financial Support: CAPES, CNPq, and FAPERJ.
Resumo:17-144
AGLUTININ ISOLATED FROM THE INVERTEBRATE MARINE HOLOTHURIA GRISEA (HGA) REDUCES
INFLAMMATORY HIPERNOCICEPTION
Luz, P. B. 1; Aragão, K. S. 1; Osório, C. B. H. 2; Moura, R. D. M. 1; Melo, A. A. D. 1; Carneiro, R. F. 1;
Cavada, B. S. 1; Alencar, N. M. N. D. 1
1
Departamento de Fisiologia e Farmacologia/ Faculdade de Medi, UFCe
2
Departamento de Bioquímica e Biologia Molecular/ Centro de C, UFCe
Objectives:
The present work aimed to evaluate the effect of lectin's Holothuria grisea (HGA) in the model of mechanical
hipernociception induced by carrageenan (Cg).
Methods and Results:
Animal handling and experimental protocols were approved by the Institutional Ethics Committee under number 60/09.
Male Swiss mice (n=6, 25-30g) were placed in acrylic cages (12×20×17cm) with wire grid floors, 15–30 min before the
beginning of the test. The test consisted of evoking a hindpaw flexion reflex with a hand-held force transducer adapted
with a 0.5mm² polypropylene tip. The investigator was trained to apply the tip perpendicularly to the central area of the
hindpaw with a gradual increase in pressure. The end point was characterized by the removal of the paw followed by
clear flinching movements. After the paw withdrawal the intensity of the pressure was automatically recorded, and the
value for the response was obtained by averaging four measurements. The animals were tested before (T=0) and after
treatment (1, 2, 3 and 4 hours). HGA (1 e 10mg/kg) or saline were administred i.v. 30 min before injection of Cg in right
hind paw (300µg/paw). For measurement of myeloperoxidade activity (MPO) the mice were treated with HGA 10mg/kg
i.v. or saline, 30 min later they received 300µg/paw of Cg, 4h later they were sacrificed and a sample of tissue (50mg) has
been removed. The results were presented as number of neutrophils×106/mg tissue (MPO activity). Nitrite (NO2 −)
concentrations were determined in serum obtained from the animals 4h after intraplantar injection of Cg. HGA (10
mg/kg) or saline was administered i.v. 30 min before Cg. The results were expressed as micromoles of nitrite. Regarding
statistics we used ANOVA/Bonferroni’s test. P
Conclusions:
The results indicate that HGA shows a potent antinociceptive effect on mechanical inflammatory hypernociception
evoked by Cg may be associated with the inhibition of neutrophil migration and NO production.
Keywords: AGLUTININ, HIPERNOCICEPTION, INFLAMATION, INVERTEBRATE
Financial Support: CAPES and CNPq
Resumo:17-145
ROLE OF TRPA1 RECEPTOR IN NOCICEPTION INDUCED BY MONOSODIUM URATE (MSU) CRYSTALS IN
RATS.
Trevisan, G. ; Hoffmeister, C. ; Rossato, M. F. ; Ferreira, J.
Departamento de Química/Universidade Federal de Santa Maria, UFSM
Objectives:
Gout is characterized by the deposition of monosodium urate (MSU) crystals in joints. Despite being one of the most painful
forms of arthritis, the mechanisms responsible for acute painful gout attacks are poorly understood. Recently, our research group
has shown the involvement of TRPV1 receptors in the acute nociception and edema evoked by MSU in rats. As TRPA1 receptor
are co-expressed with TRPV1 in capsaicin-sensitive fibers, the present study aimed to investigate if TRPA1 receptors also
contribute to the nociceptive and edematogenic response induced by MSU crystals in rats.
Methods and Results:
Adult male Wistar rats (200-300 g) were used. We observed the signs of spontaneous nociception, cold alodynia and edema in the
animals produced by MSU (0.25 mg/paw, 100 ìl) administered subcutaneously (s.c). Separate groups of animals received instead
the s.c. injection of the vehicle (PBS). Animals were placed individually in chambers and adapted for 20 min before injection.
The numbers of flinch responses were observed during 10 minutes after the injection and it was recorded and considered as
indicative of spontaneous nociception. Cold allodynia was observed as a score (0= non response, 1= flinching, 2= shaking,
4=licking of the paw when 50 uL of acetone was delivery). The edema formation was assessed as an increase in paw thickness
measured by a digital calipter, 15 min after s.c. injection and the results were expressed as the difference between the basal value
and the test value. HC0300313 (100 ug/paw) and camphor (150 nmol/paw), a selective and a non-selective TRPA1 antagonist,
respectively, were also subcutaneously injected with MSU. Allyl isothiocynate (AITC, 1 nmol/paw) a TRPA1 agonist, was used
as a positive control in all experiments. To further explore the role of capsaicin-sensitive fibers in the nociceptive and
edematogenic effect induced by MSU, animals were submitted to a perineural capsaicin desensitization, were the sciatic nerve
received a 10 µL injection of 2% capsaicin or vehicle (10% ethanol, 10% Tween 80 and 80% PBS). After 7 days, animals were
submitted to a subcutaneous injection of MSU, AITC or PBS. Treatment with the selective TRPA1 receptor antagonist
HC030031 inhibited spontaneous nociceptive responses to MSU and AITC (Imax of 87±6 and 617±3 %, respectively). Camphor
also reduced MSU-induced nociceptive response induced by MSU and AITC (Imax of 74±9 and 75±5 %, respectively). The cold
allodynia induced by MSU or AITC (94±6 and 92±8, respectively) was also effective reduced by the treatment with TRPA1
antagonists HC 030031 and Camphor (I max 92±8 and 92±8, respectively).The edema formation induced by MSU or AITC was
also reduced by camphor (Imax of 65±17 and 83±3 %, respectively) or HC030031 (I max of 79±12 and 67±16 %, respectively).
The nociception and edema induced by AITC or MSU were largely inhibited in 76±5 and 88±4 or 92±4 and 68±12%,
respectively, when animals were submitted to a perineural capsaicin desensitization protocol.
Conclusions:
Collectively, the present findings demonstrate that MSU produces nociceptive and edematogenic responses through, at least in
part, TRPA1 receptor activation.
Keywords: Gout, Nociception, TRPA1
Financial Support: CNPQ, CAPES
Resumo:17-146
CANNABIDIOL PRODUCES A LONG-TERM ANTI-INFLAMMATORY EFFECT IN A MURINE MODEL OF
ACUTE LUNG INJURY: ROLE FOR A2A ADENOSINE RECEPTOR
Ribeiro, A. 1; Ferraz-de-paula, V. 1; Pinheiro, M. L. 1; Almeida, V. I. 1; Quinteiro-filho, W. M. 1; Akamine,
A. T. 1; Hallak, J. E. C. 2; Zuardi, A. W. 2; Crippa, J. A. 2; Palermo-neto, J. 1
1
Departamento de Patologia / FMVZ-USP, FMVZ-USP
2
Departamento de Neurociencias e Comportamento FMRP-USP, FMRP-USP
Objectives:
Endocannabinoid system has become a topic of great interest in pharmacology due to its remarkable distribution in mammal
organisms and capacity to play a modulatory role on diverse physiological functions, including immune system modulation.
Acute lung injury (ALI) is a murine model of Acute Respiratory Distress Syndrome (ARDS), a condition developed by many
patients with sepsis, which affects thousands of people every year. We have previously shown that cannabidiol (CBD) decreases
leukocyte migration into the lungs and TNF concentration in the bronchoalveolar lavage fluid on ALI. Here our goal was to
analyze the effects of CBD in the progression of ALI and the mechanism of action involved.
Methods and Results:
C57BL/6 male mice were used and divided randomly in 3 groups: vehicle+PBS, vehicle+LPS, and CBD+LPS (n=5-7/group).
Experimental groups received CBD (20 mg/kg) i.p. 60 min prior to nasal instillation of LPS (100 ug/mL). Control groups
received vehicle i.p. 60 min prior nasal instillation of LPS or sterile PBS. Each mouse received 1 uL of LPS or PBS per g of
mouse weight. One, two, four, and seven days after LPS or PBS nasal instillation, mice were submitted to euthanasia and
bronchoalveolar lavage fluid (BAL) was harvested for cell counting and cytokines/chemokines measurement, and a lung fraction
was collected for myeloperoxidase (MPO) analysis. Moreover, an antagonist of A2A adenosine receptor (ZM241385) was used
(30 min prior to CBD treatment) to investigate the involvement of adenosine signaling in the effects observed. Data was analyzed
using One-way ANOVA followed by Tukey-Kramer multiple comparison test or Kruskal-Wallis followed by Dunn‘s multiple
comparison test. We show that vehicle+LPS group presented a remarkable increasing on BAL cell counting following 1 (F (2,13)
= 23.58; p < 0.0001), 2 (F (2,16) = 36.83; p < 0.0001), and 4 (F (2,17) = 17.27; p < 0.0001) days of the induction of lung
inflammation. We observe that CBD treatment decreased BAL cell counting following 1, 2, and 4 days of the induction of
inflammation. We also show that CBD treatment decreased cytokine/chemokine concentration in the BAL, such as TNF (day 1)
(F (2,12) = 11.38; p < 0.001), IL-6 and MIP-2 (day 2) (F (2,14) = 6.33; p < 0.01 and KW = 10.56; p < 0.01, respectively).
Moreover, we show that CBD treatment decreased MPO activity following 1 (F (2,12) = 16.39; p < 0.001), 2 (F (2,18) = 17.21; p
< 0.0001), and 4 (F (2,17) = 49.55, p < 0.0001) days of the induction of lung inflammation. Notably, when mice received
ZM241385, CBD was not able to decrease BAL cell count as previously observed on day 1, 2, and 4 after the induction of
inflammation.
Conclusions:
We show that a single CBD treatment is able to produce a long-term decrease on several inflammatory parameters in a model of
ALI. Partially, we also show that A2A adenosine receptor is involved on those effects. Therefore, we believe that CBD might be
a useful therapeutic tool in the treatment of inflammatory lung diseases.
Keywords: Cannabidiol, Acute lung inflammation, Adenosine receptor
Financial Support: FAPESP (2009/51886-3) and CNPq.
Resumo:17-147
NEURAL MOBILIZATION REVERSES PAIN BEHAVIORAL AND INCREASE PROTEIN ZERO (PO)
EXPRESSION IN SCIATIC NERVE AFTER NEUROPATHIC PAIN IN RATS
Santos, F. M. ; Giardini, A. C. ; Silva, J. T. ; Chacur, M.
Departamento de Anatomia, Universidade de São Paulo, USP
Objectives:
The neural mobilization (MOB) technique is a noninvasive method that has proved clinically effective in reducing pain
sensitivity and consequently in improving quality of life after neuropathic pain. The present study examined the effects of neural
mobilization on pain sensitivity induced by chronic constriction injury (CCI) in rats. In addition, we also evaluated the protein
zero (PO) expression in sciatic nerve after neuropathic pain and MOB treatment.
Methods and Results:
The CCI was performed on adult male rats, submitted thereafter to 10 sessions of MOB, each other day, starting 14 days after the
CCI injury. Over the treatment period, animals were evaluated for nociception using behavioral tests, such as tests for allodynia
and thermal and mechanical hyperalgesia. At the end of the sessions, the sciatic nerve was analyzed using Western blot assays for
neural protein zero (PO). The results showed that MOB treatment induced an early reduction (in the second session) of the
hyperalgesia and allodynia in CCI-injured rats, which persisted until the end of the treatment. On the other hand, only after the
4th session we observed a blockade of thermal sensitivity. Regarding cellular changes, we observed a increase of PO expression
after MOB in the ipsilateral nerve when compared to CCI animals.
Conclusions:
In conclusion, we suggest the involvement of PO in rats after the treatment with MOB. Our findings contribute to the
understanding of the mechanisms involved in the therapeutic potential of neural mobilization as a treatment for neuropathic pain.
Keywords: Neural Mobilization, Western blot , chronic constriction injury, hyperalgesia, sciatic
Financial Support: Fapesp, CAPES
Resumo:17-148
EVALUATION OF THE THERAPEUTIC POTENTIAL OF CANNABIDIOL IN A MURINE MODEL OF LPSINDUCED ACUTE LUNG INFLAMMATION.
Almeida, V. I. ; Ribeiro, A. ; Ferraz-de-paula, V. ; Pinheiro, M. L. ; Akamine, A. T. ; Quinteiro-filho, W. ;
Palermo-neto, J. ; Gomes, P. F.
Patologia/Faculdade de Medicina Veterinária e Zootecnia, USP
Objectives:
Endocannabinoid system has become a topic of great interest in pharmacology due to its remarkable distribution in mammal
organisms and capacity to play a modulatory role on diverse physiological functions, including immune system modulation.
Acute lung injury is a murine model of Acute Respiratory Distress Syndrome (ARDS), a condition developed by many patients
with sepsis, which affects thousands of people every year. Experimental data from our laboratory show that CBD has antiinflammatory activity if given previous to the induction of acute lung inflammation. Therefore, the goal of our work was to
evaluate possible therapeutic effect of CBD on ARDS, i.e., given after (6 h) the induction of inflammation.
Methods and Results:
Twenty-one C57BL/6 mice were used and divided randomly into 3 groups: PBS+vehicle, LPS+vehicle, and LPS+CBD (n = 45/group). Each group received CBD (20mg/kg) or vehicle i.p. 6 hours after nasal instillation of LPS or sterile PBS (1 uL / mouse
g). Twenty-four hours following nasal instillation of LPS or PBS, mice were euthanized and bronchoalveolar lavage (BAL), bone
marrow, spleen and blood were harvested to the analysis of total leukocyte count. It was also harvested a fraction of the lung to
measure myeloperoxidase (MPO) activity. Data were analyzed using ANOVA followed by Tukey-Kramer multiple comparison
test. We showed that LPS+vehicle group increased the total cell count in the BAL (F(2,14)=33.9,p
Conclusions:
We have previously shown that CBD given in a prophylactic manner was able to decrease several inflammatory parameters. Here
we showed that CBD (20mg/kg) given following a therapeutic protocol, i.e., after the induction of lung inflammation, reduce the
acute lung inflammation. Literature data point forwards some possible mechanisms involved in the anti-inflammatory action of
CBD, such as inhibition of NF-kB. In addition, there are reports that anti-inflammatory properties of CBD are triggered by A2A
adenosine receptor. These factor are new being analyzer.
Keywords: Acute lung inflammation, Cannabidiol, Cannabinoids, Lipopolysaccharides, Neuroimunomodulation
Financial Support: FAPESP
Resumo:17-149
ATTENUATION OF ACTIVITY IN AN ENDOGENOUS ANALGESIA CIRCUIT DURING THE CRONIFICATION
PERIOD OF INFLAMMATORY PAIN IN RATS.
Miranda, J. 1; Tambeli, C. H. 2; Lamana, S. M. 1
1
Faculdade de Odontologia de Piracicaba, FOP - UNICAMP
2
Universidade Estadual de Campinas, UNICAMP
Objectives:
The aim of this study was to assess whether persistent mechanical inflammatory hyperalgesia, during its induction period, affects
the analgesia induced by activation of an endogenous pain modulation circuit that can be activated by peripheral nociceptive
stimulation. Once activated, this neural circuit named ascending nociceptive control (ANC) induces analgesia mediated by
endogenous opioids in Nucleus Accumbens. ANC was activated by capsaicin injection in the forepaw, and the nociceptive test
was applied in a distant region, i.e. the hind paw.
Methods and Results:
Methods: Male Wistar rats (180-220g) were used. Persistent mechanical hyperalgesia was induced by the subcutaneous injection
of PGE2 (100ng/50µl) into the hind paw of rats for a period of 14 consecutive days. To verify the progression of mechanical
hyperalgesia, the basal nociceptive threshold was assessed on days 1 and 7 of the induction period of persistent hyperalgesia and
on days 1,7,14, and 21 after discontinuation of the treatment with PGE2. The nociceptive mechanical threshold was evaluated by
the method of compression of the paw of the animal, first described by Randall & Selitto (1957). To verify whether peripheral
noxious stimulation attenuates the hyperalgesic response, the nociceptive threshold was assessed on day 1 of the induction period
of persistent hyperalgesia, 3 hours after the injection of PGE2 and on day 7of the induction period of persistent hyperalgesia, 3
hours after the injection of PGE2. Saline or Cys2,Tyr3,Orn5,Pen7 amide (CTOP), was administered intra-nucleus accumbens 10
min before the intraplantar injection of capsaicin (125µg/50µl) or vehicle into the forepaw of rats, and the nociceptive threshold
of the animals was assessed 0, 15, 30, 45 and 60 minutes later, in the hind paw. The intra-nucleus accumbens administration was
performed by a cannula stereotaxically positioned. The locomotor activity was evaluated by Rotarod after nociceptive testing. A
two-way repeated-measure ANOVA with one between subjects factor (i.e., treatment) and one within subjects factor (i.e, time)
followed by Tukey test, was used to determine if there were significant (p ≤ 0.05) differences in nociceptive mechanical threshold
among the groups. Results: Daily injections of PGE2 induced persistent inflammatory hyperalgesia from day 7 of induction, since
the nociceptive baseline threshold significantly decreased on day 7 (42,5%) of induction and on days 1 (49,5%), 7 (27,8%), 14
(29,5%) and 21 (20,1%) of the period of maintenance of hyperalgesia compared to the basal nociceptive threshold of the 1st day
of the induction period (p
Conclusions:
These findings suggest that inflammatory pain attenuates the activity in endogenous pain control circuits during the period of its
chronification.
Keywords: antinociception, mechanical hyperalgesia, nociceptive stimulation
Financial Support: FAPESP
Resumo:17-150
P-CYMENE ATTENUATES MECHANICAL HYPERNOCICEPTION IN MICE.
Santana, M. F. ; Chaves, D. O. ; Santana, M. T. ; Oliveira, M. G. B. ; Melo, M. S. ; Siqueira, R. S. ;
Cavalcanti, S. C. H. ; Araújo, A. A. S. ; Bonjardim, L. R. ; Júnior, L. J. Q.
Universidade Federal de Sergipe, UFS
Objectives:
This study investigated the effect of p-cymene (PC), a biological monoterpene precursor of carvacrol, on inflammatory
hypernociception induced in mice.
Methods and Results:
Male Swiss mice were divided into five groups (n = 6, per group), pre-treated with vehicle (saline + tween 80 0.2%), PC (25, 50
or 100 mg/kg, i.p.), 0.5 h after the subplantar injection of 20 μl of carrageenan (CG; 300 µg/paw), tumor necrosis factor-α (TNFα; 100 pg/paw), prostaglandine E2 (PGE2; 100 ng/paw) or dopamine (DA; 30 μg/paw). Hypernociceptive behavior was measured
with a digital von Frey apparatus at period 0.5, 1, 2, and 3h after the hyperalgesic stimuli. Experimental protocols were approved
by the Animal Care and Use Committee at the UFS (Nº 26/09). The data were evaluated by one-way analysis of variance
(ANOVA) followed by Tukey‘s test (p < 0.05). Acute pretreatment with PC, in all doses, inhibited the development of
mechanical hypernociception induced by CG and TNF-α (0.5, 1.0, 2.0 and 3.0h; p < 0.001)when compared with the vehicle
group. Additionally, treatment inhibited the hypernociception induced by final mediators of the cascade hypernociceptive, PGE2
(0.5h, in the doses of 25 mg/kg p < 0.05, 50 mg/kg p < 0.001 and 100 mg/kg p < 0.001; 1.0, 2.0 and 3.0h; p < 0.001 in all doses)
and DA (0.5, 1.0, 2.0 and 3.0h; p < 0.001 in all doses)when compared with the vehicle.
Conclusions:
Our results suggest that acute treatment with PC produces anti-hypernociceptive effect in mice probably by a mechanism
dependent on inhibition of cytokines production and/or the final hypernociceptive mediators. Therefore, it´s tempting to speculate
that PC may represent a new compound for the treatment of painful conditions.
Keywords: HYPERNOCICEPTION, MONOTERPENE, PAIN, p-CYMENE
Financial Support: FAPITEC-SE, CNPq.
Resumo:17-151
HEXANIC AND N-BUTANOLIC FRACTION OF COMBRETUM DUARTEANUM CAMBESS REDUCES
OROFACIAL NOCICEPTIVE BEHAVIOR IN MICE
Melo, M. S. 1; Oliveira, G. F. 1; Souza, T. T. 1; Oliveira, M. G. B. 1; Santana, M. F. 1; Santana, M. T. 1;
Xavier, M. A. 1; Agra, M. F. 2; Tavares, J. F. 2; Quintans-junior, L. J. 1
1
Universidade Federal de Sergipe, UFSE
2
Universidade Federal da Paraiba , UFPB
Objectives:
The purpose of the present study was to evaluate the antinociceptive effect of the hexanic (FH) and n-butanolic fractions (FB) of
Combretum duarteanum Cambess in formalin- and glutamate-induced orofacial nociception on mice.
Methods and Results:
Male Swiss mice (25-30 g), with 2-3 months of age, were used throughout this study. Mice (n = 8/ per group) were pretreated
with FH or FB at doses of 100, 200 and 400 mg/kg (i.p.), 30 min before of the tests. In all models, nociception was quantified at
those periods by measuring the time (s) that the animals spent face-rubbing in the injected area with its fore- or hindpaws. The
experimental protocols were approved by the Ethics Committee on Animal Research at the Federal University of Sergipe (CEPA:
17/10). The results were analyzed using ANOVA followed by post-Tukey test, were considered significant at p < 0.05. In the
formalin test, was found that the FH reduced orofacial nociception in first phase of the test (48.9%, 60.4%, 47.6%) at doses of
100, 200 and 400 mg/kg and the FB (41.5%, 65.3%) at doses of 100 and 400 mg/kg, p < 0.001. In second phase, FH and FB were
also effective to reduce orofacial nociception (40.0%, 50.4%, 55.3%) and (65.0%, 77.8% and 96.0%, p < 0.001), respectively, at
doses of 100, 200 and 400 mg/kg. In glutamate-induced orofacial nociception, FH and FB produced a significant reduction in
orofacial nociceptive behavior (60.9%, 66.4%, 52.5%) and (63.4%, 66.9%, 90.3%) with p < 0.001, respectively
Conclusions:
Our results suggest that Combretum duarteanum might represent an important tool for treatment of orofacial pain. Further studies
currently in progress will enable us to understand the precise action mechanisms
Keywords: Combretum duarteanum, orofacial pain, formalin, glutamate
Financial Support: CNPq and FAPITEC
Resumo:17-152
ANTINOCICEPTIVE ACTIVITY OF A NEW ALKALOID ISOPROPYL N-METHYLANTHRANILATE FROM THE
ESSENTIAL OIL OF CHOISYA TERNATE KUNTH AND THE ANALOGS METHYL AND PROPYL NMETHYLANTHRANILATE
Pinheiro, M. M. G. 1; Radulovic, N. S. 2; Miltojevic, A. B. 2; Mcdermott, M. 3; Waldren, S. 4; Parnell, J. A.
4
; Fernandes, P. D. 1; Menezes, F. D. S. 3
1
Laboratorio Farmacologia da Inflamaçao e do NO/ICB, UFRJ
2
Department of Chemistry/ University of Niš, University Nis
3
Panoz Institute/Trinity College Dublin, TCD
4
Department of Botany/Trinity College Dublin, TCD
Objectives:
Aim:The infusion of leaves of Choisya ternate Kunth (Rutaceae) is popularly employed for their antispasmodic property. The
study the detailed GC-MS analyses of the essential oil of C. ternate revealed the presence of isopropyl N-methylanthranilate
(ternanthranin) (ISOAN) and other volatiles compounds. The essential oil of leaves (EO), ternanthranin and the analogs methyl(MAN) and propyl-N-methylanthranilate (PAN) were assayed for nociception.
Methods and Results:
Material and methods:EO of C. ternateleaves, collected at Dublin, was obtained by hydrodistillation. The oil and compounds
were analyzed by CG-MS and spectrally characterized (UV-Vis, IR, NMR and MS). Male Swiss mice (20–25g; n=6-8) were
orally pre-treated 60 minutes before with EO (at doses 10-100 mg/kg), ISOAN, MAN, or PAN (at doses 0.3-3 mg/kg) and
evaluated in the acetic acid (2%, ip) induced writhing (AA) and hot plate (HP) models. Animal care and research protocols were
in accordance with the principles and guidelines adopted by the Brazilian College of Animal Experimentation (COBEA),
approved by the Ethical Committee for Animal Research/UFRJ (Protocol number, DFBCICB-015). The results are presented as
mean ± S.D. Statistical significance between groups was determined by ANOVA followed by Bonferroni´s test (*p
Conclusions:
EO and its minor alkaloid constituents from the leaves of C. ternate produce dose-related antinociceptive action. Furthermore, the
antinociceptive action demonstrated in the present study supports, at least partly, the ethnomedical uses of this plant.
Keywords: anthranilate, nociception, choysia ternate
Financial Support: CAPES, CNPq, FAPERJ.
Resumo:17-153
HEME OXYGENASE 1 INDUCTION REDUCES LUNG OXIDATIVE STRESS AND INFLAMMATION AFTER
INTESTINAL ISCHEMIA AND REPERFUSION INJURY
Bertoni, J. A. 1; Correa-costa, M. 2; Breithaupt-faloppa, A. C. 1; Lino dos Santos Franco, A. 1; Vitoretti, L.
B. 1; Saraiva Câmara, N. O. 2; Tavares-de-lima, W. 1
1
Farmacologia / Universidade de São Paulo, ICB - USP
2
Imunologia/Universidade de São Paulo, ICB - USP
Objectives:
Intestinal ischemia and reperfusion (intestinal I/R) frequently occurs in human pathological conditions, and it has been considered
as a significant cause of Acute Lung Injury (ALI). In your most severe form Acute Respiratory Distress Syndrome (ARDS) is
responsible for significant morbidity and mortality. It is thought that polymorphonuclear neutrophils and mediators generated in
the setting of oxidative stress, such as reactive oxygen species (ROS), have important roles in its pathophysiology. In this sense,
we hypothesize that heme oxygenase-1 (HO-1) up regulation, an enzyme with antiinflamatory and antioxidant activities, could
modulate the expression of antioxidant enzymes and neutrophils recruitment in the lung after an intestinal I/R, therefore inducing
cytoprotection.
Methods and Results:
Upon anesthesia male Wistar rats (60 days) weighing 200 ~ 300 g were subjected to 45 min occlusion of the superior mesenteric
artery (SMA) and followed by 2 hr of reperfusion. Before ischemia, one group (n=5) of rats had the lymphatic thoracic duct
sectioned and another group of rats (n=5) was treated 48 h and 24 h before induction of SMA occlusion with an HO-1 inductor,
Hemin (10 mg/kg, i.p.). Neutrophils recruitment to the tissues was indirectly measured by myeloperoxidase (MPO) activity. HO1, superoxide dismutase (SOD) 1 and 2, catalase (CAT) mRNA were determined in the lung tissue. Intestinal I/R determined a
significant increase of MPO activity in lung that was reduced in rats upon lymphatic duct sectioned. Hemin treatment of rats
increased the expression of HO-1, SOD-1 and SOD-2, but did not modify the expression the CAT in lung tissue. In addition, the
MPO activity in lung tissue was significantly reduced in hemin-treated group as compared with non treated rats upon intestinal
I/R.
Conclusions:
Our data indicate that HO-1 decreases neutrophils recruitment and upregulates the expression of antioxidant enzymes, reducing
inflammation of lung tissue after intestinal I/R. Moreover lymph factors seem not be involved with HO-1 effects.
Keywords: Acute respiratory distress syndrome, Inflammation, Intestinal ischemia and reperfusion , Heme Oxygenase 1, Lung
Financial Support: FAPESP (2007/07139-3 and 2009/54823-2) / CAPES and CNPq
Resumo:17-154
LOCAL ANTINOCICEPTIVE EFFECT OF CANNABINOIDS IN A RAT MODEL OF PAINFUL DIABETIC
NEUROPAHTY
Schreiber, A. K. 1; Neufeld, M. 1; Enzo, J. 1; Pinto, R. B. 1; Cunha, T. M. 2; Cunha, F. D. Q. 2; Cunha, J. M.
D. 1
1
Farmacologia, UFPR
2
Farmacologia, FMRP - USP
Objectives:
Painful neuropathy is one of the most common secondary complications associated with diabetes. This kind of pain often
responds poorly to standard pain treatments and often a better management is required to treat this condition. Based on reported
antinociceptive properties of cannabinoids, this study was designed to investigate the intraplantar (i.pl.) effect of AM404 (N-(4hydroxyphenyl) arachidonoylethanolamide; an endogenous cannabinoid reuptake inhibitor, or ACEA (arachidonyl-2chloroethylamide), a potent CB1 agonist in model of neuropathic pain induced by streptozotocin (STZ) in rats.
Methods and Results:
After a 12h-fast, male Wistar rats received one intraperitoneal (i.p.) injection of STZ (50 mg/kg freshly dissolved in citrate buffer,
10 mM, pH 4.5). Hyperglycemia was confirmed 3 days later and at the finish of each study using blood taken by tail prick and a
strip-operated reflectance meter. Only rats with a blood glucose concentration higher than 250 mg/dL at the start and end of the
study were included. The formalin test was performed 4 weeks after the onset of hyperglycemia. After 20 min of vehicle or
cannabinoid i.pl. treatments, normoglycemic (Ngl) and diabetic (Dbt) rats (n=6-10 each group) received an ipsilateral injection of
formalin (0.5%, 50 µL) in the hind paw, and flinches were counted in 5 min blocks for 1 hour. All procedures were approved by
the Ethics Animal Experiment Committee of Federal University of Paraná. Formalin induced a biphasic pattern of flinching
responses, from 0 to 10 min (first phase; 1P) and from 15 to 60 min (second phase, 2P). The two phases were separated by a
quiescent period (QP; 10-15 min post injection). As previous described (Eur J Pharmacol 285:189, 1995), at any given
concentration of formalin, Dbt rats show a raised rate of paw flinching. In this study, vehicle-treated Dbt rats displayed a nonsignificant trend to increase flinching during 1P but exhibited increased response during QP and 2P. Three different doses of
ACEA (10, 30 or 100 ug, in 50 uL) were tested at both Ngl and Dbt rats. In both groups, only the two higher doses affected pain
scores during the 1P of formalin test. In Ngl rats, treatment with ACEA (30 or 100 ug) reduced the frequency of flinches by 45%
and 58%, respectively. In Dbt group, the pain scores were reduced by 58% and 82% after 30 or 100 ug ACEA-treatment,
respectively. Interestingly, the i.pl. treatment with AM 404 (75 ug) promoted significant reduction of all phases of formalin test in
Dbt rats, reaching the inhibition of 33%, 71% and 52%, during 1P, QP and 2P, respectively. Using the same dose of AM404 in
Ngl rats, the chemical hyperalgesia was reduced by 43% and 77% during 1P and 2P, respectively. Additional dose of 50 ug of
AM 404 was used in Dbt animals, leading a decrease of 41%, 51% and 60% in 1P, PQ and 2P, respectively. Neither ACEA nor
AM404 treatments altered plasma glucose levels when compared with control rats.
Conclusions:
The results of this study show that local administration of selective CB1 agonist or endogenous cannabinoid reuptake inhibitor
are effective in ameliorating chemical hyperalgesia in STZ-diabetic rats. Further studies aimed to investigate the involvement of
CB1 and/or CB2 receptors in these responses. Besides, the results suggested that endocannabinoid system could be an interesting
target for new drugs, as seen that an important antinociceptive effect was obtained after very low doses treatment.
Keywords: Diabetic, neuropathy, endocannabinoids, rat, local
Financial Support: CAPES; Fundação Araucária.
Resumo:17-155
POLYSACCHARIDES OF CAESALPINIA FERREA PODS INHIBITS CELL MIGRATION AND PROTEIN
LEAKAGE INDUCED BY CARRAGEENAN
Silva, R. O. 1; Pereira, L. D. P. 1; Marques-domingos, G. F. O. 1; Assreuy, A. M. S. 1; Pereira, M. G. 1,2
1
Instituto Superior de Ciências Biomédicas, ISBC-UECE
2
Faculdade de Educação, Ciências e Letras do Sertão Central, FECLESC-UECE
Objectives:
In vivo experimental studies testing total stem extracts of Caesalpinia ferrea showed analgesic and anti-inflammatory activities in
the model of paw edema induced by carrageenan. Further results obtained from our research group demonstrated antiinflammatory effect of a polysaccharide extract from C. ferrea pods, especially on the late phase of carrageenan-induced paw
edema in rats, an effect that was shown to involve prostaglandins and nitric oxide. In this study, it was evaluated the effect of a
polysaccharide isolated from C. ferrea pods in a cellular model of acute inflammation.
Methods and Results:
Wistar rats (150-250g) were handled following the principles of the Ethics Committee-UECE (Nº 09204632-0). The
polysaccharide fraction (FIII) was isolated by ion exchange chromatography (DEAE-cellulose). Animals received intravenous
(i.v.) injection of FIII (1mg/kg in 0.1 mL/100 g weight) or 0.9% saline, 30 min before intraperitoneal (i.p) injection of indirect
(carrageenan; 500 mg) or direct (N-formil-methionil-leucil-fenilalanina fMLP; 50 εg) chemoatractants. Animals were sacrificed
4 h later and peritoneal fluid collected for total and differential leukocyte counts and dosage protein dosage. Results were
expressed as mean ± S.E.M. (n=6) and analyzed by ANOVA and Bonferroni‘s test, considering values p
Conclusions:
The polysaccharide isolated from pods of C. ferrea showed anti-inflammatory effect by inhibiting cell migration, acting both
directly and indirectly on neutrophils. This finding supports the popular use of C. ferrea involving inflammatory desordes.
Keywords: Carrageenan, Caesalpinia ferrea, Cell migration, Polysaccharides, Protein leakage
Financial Support: CAPES, CNPq, FUNCAP; Marques- Domingos G.F.O.
Resumo:17-156
KETAMINE INDUCES PERIPHERAL ANTINOCICEPTION IN RAT BY OPIOID AND CANNABINOID SYSTEM
ACTIVATION.
Petrocchi, J. A. 1; Queiroz-junior, C. M. 2; Paiva-lima, P. 1; Caliari, M. V. 2; Di Marzo, V. 3; Duarte, I. D. G.
1
; Romero, T. R. L. 1
1
Laboratório de Dor e Analgesia, Departamento de Farmacologia, ICB-UFMG
2
Lab. de Patologia Odontológica, Faculdade de Odontologia, Fac. Odonto-UFMG
3
Inst. of Biomol. Chemistry, Consiglio Nazionale Ricerche, C.N.R.I
Objectives:
Although ketamine shows a peripheral analgesic component, the base of this mechanism is not completely elucidated. Thus the
aim of this study was obtain evidences for the involvement of endogenous opioids peptides and endocannabinoids in the
peripheral antinociceptive effect induced by ketamine.
Methods and Results:
The rat paw pressure test was used and hyperalgesia was induced by intraplantar injection of prostaglandin E2 (2 µg/paw). The
immunohistochemistry and the HPLC chromatography were performed to verify the participation of beta-endorphin and
endocannabinoids, respectively, in the ketamine-effect. All drugs were administered locally into the right hind paw of Wistar
male rats with n > 4 animals per group. The data were statistically analyzed by one-way analysis of variance (ANOVA) and the
post-hoc Bonferroni test for multiple comparisons. Probabilities of less than 5% (P
Conclusions:
The results provide evidences that ketamine probably induces peripheral antinociceptive effect by release of beta-endorphin and
anandamide to activate the selective µ, delta-opioid receptors and CB1 cannabinoid receptor.
Keywords: Peripheral antinociception, Ketamine, opioid system, Cannabinoid system
Financial Support: CNPq (473758/2007-5) fellowships by CAPES.
Resumo:17-157
OXIDATIVE STRESS IN CHRONIC POST-ISCHEMIA PAIN (CPIP), A MOUSE MODEL OF COMPLEX
REGIONAL PAIN SYNDROME-TYPE I
Bratti, T. 1; Bobinski, F. 1; Martins, D. F. 1; Mazzardo-martins, L. 1; Winkelmann-duarte, E. C. 2; Santos, A.
R. S. 1
1
Depto.C.Fisiológicas, Universidade Federal de Santa Catarina, UFSC
2
Depto.C.Morfológicas, Universidade Federal de Santa Catarina, UFSC
Objectives:
AIM: Rats presented microvascular injury after they were submitted to hindpaw ischemia and reperfusion (IR) and developed
chronic post-ischemia pain (CPIP) with mechanical hypersensitivity (Pain Med, 11: 1224, 2010). Here we investigate in mice
whether this hypersensitivity is associated with oxidative stress.
Methods and Results:
METHODS: 70 male Swiss mice (25–35 g, n=14) were divided in 5 groups: (1) CPIP + NAC: submitted to CPIP and treated with
N-acetylcysteine (NAC) (500 mgkg, i.p.); (2) CPIP + V: submitted to CPIP and treated with vehicle (10 ml/kg, i.p.); (3) CPIP:
submitted to CPIP; (4) S: Sham; (5) N: Naive. CPIP induction in mice was adapted from rats (Pain. 112: 94, 2004) and the
experiments were performed after approval of the protocol PP00509 by the Institutional Ethics Committee for Animal Research
(CEUA) of UFSC. Mice were anesthetized with chloral hydrate (7%, 0.6 ml/kg, i.p.) and an elastic O-ring for braces (Elástico
Ligadura 000-1237, Uniden) was placed around the mouse‘s right hindlimb just proximal to the ankle joint producing a tight-fit
that induced ischemia and it was left on the limb for 3 h. Sham mice received the same treatment, except that the O-ring was cut
so that it only loosely surrounded the ankle. To establish the potential antihypersensitivity effect of free radical scavengers,
groups 1 and 2 were pre-treated with NAC or vehicle. The mechanical hypersensitivity was measured according the modified up
and down method using von Frey filament both before and at different time-points after treatment (0.5, 1, 2, 4, 6 and 24 hours) at
2 and 7 days after ischemia and reperfusion (IR). Besides, seven days after IR, groups 3, 4 and 5 were killed by decapitation
without anesthesia and both the plantar surface of the hind paw and the lumbar portion of the spinal cord (L1 to L6) were
collected for thiol levels estimation. These tissue was homogenized and then centrifuged. The supernatant was divided into 2
aliquots and one was used to determine total thiol content while the other was used to determine the non-protein thiol content in
the sample. The color of the solution resulting from the reaction was measured using ELISA (R&D systems). Protein thiol
content was calculated by subtracting the values of non-protein thiols from total thiols. Statistic analysis was performed using
one-way ANOVA, with the Student-Newman-Keuls test as the post-hoc test or t test when appropriate. Results are presented as
the mean ± S.E.M. for each group. p < 0.05 was considered significant. RESULTS: The time course trials of both 2 and 7 days
following hind paw IR demonstrated von Frey thresholds that were significantly lower than baseline (p < 0.001). Conversely, 30
minutes following treatment with NAC, von Frey thresholds were significantly different from mice treated with vehicle (p <
0.001), suggesting that NAC reversed the mechanical hypersensitivity caused by IR for at least 2 hours. Seven days after IR, mice
had increased total thiol levels, protein thiol levels and nonprotein thiol levels (p < 0.001) in both hind paw and spinal cord when
compared to naive.
Conclusions:
CONCLUSION: The present study demonstrates that NAC suppressed pain behavior and hind paw tourniquet produced an
increase in thiols levels, suggesting a key role of free radicals that might be able to cause damage to the venules and capillaries.
Keywords: N-acetylcysteine , tiols, pain
Financial Support: UFSC and Capes
Resumo:17-158
EFFECTS OF LOW LEVEL LASER (LLL) AND LIGHT EMITTING DIODE (LED) TO THE LOCAL EFFECT
INDUCED BY BOTHROPS MOOJENI SNAKE VENOM (BMJV)
Nadur- Andrade, N. 1; Zamuner, S. R. 1; Barbosa, A. M. 2; Cogo, J. C. 2
1
Ciências da Reabilitacão/ Universidade Nove de Julho, UNINOVE
2
IP&D/ Universidade do Vale do Paraíba, UNIVAP
Objectives:
Bothrops snake venoms produce marked local effects including edema, hemorrhage, pain and necrosis. The currently treatment of
Bothrops accidents is the soroterapy. However, this treatment is inefficient in neutralizing the local effects. In the present study
the therapeutic effect of the LLL and LED therapy was evaluated on edema formation and hemorrhage, induced by BmjV.
Furthermore, the effectiveness of antivenom used alone or in combination with LLL or LED treatment was also evaluated.
Methods and Results:
Male Swiss mice (22-25 g) were used. Edema was measured by plethismography after subplantar injection of BmjV (1
&microg/paw), or saline solution (control) at 15, 30 min, 1, 3 and 6 h. The hemorrhagic activity was evaluated after injection of
20 &microg of BmjV, measuring the diameter of the hemorrhagic area on the inner skin of mice, 2 h after venom injection. The
LLL was used in 685 nm, 30 mW of power, 15s irradiation time and irradiated area of 0,2 cm2 which correspond to a laser dose
of 2,2 J/cm2, and the LEDs in the wave lengths of 945nm and 635 nm, 110 and 120 mW of power, density of energy 4 J/cm2,
irradiated area of 1,2 cm2, 41s and 38s irradiation time for the red LED (LEDr) and infra-red (LEDinf) respectively, with two
applications, at 30 min and 3 h after the venom injection or saline. The antivenom (0.1 mL/5mg; i.v.) was applied i.v 30 min after
venom injection. Ethics Committee: A017/CEP/2008. BmjV induced an edematogenic effect from 30 min up to 6 h with
maximum peak at 1 h, and also caused a significant hemorrhagic activity in the skin of the animals 2 h after venom injection. The
edema caused by the BmjV was reduced by 77, 84 and 70 % by LLL, LEDinf and LEDr, respectively. Antivenom treatment did
not neutralize the edematogenic effect caused by BmjV. The combined treatment with antivenom therapy and LEDs or LLL
significantly reduced the edema at the maximum peak by 55% for LLL, 64% for the LEDinf and 53% for LEDr. The hemorrhage
caused by the BmjV was reduced by 47, 39 and 47% with LLL, LEDinf e LEDr, respectively. The antivenom, administered
through i.v 30 min after venom injection, had a significant effect on the neutralization of hemorrhage halo induced by BmjV,
causing a reduction of 25% of the hemorrhagic halo. Likewise, treatment of antivenom therapy combined with laser and LEDs
was effective to reduce the hemorrhagic halo in the order of 25% for the LLL, 36% for the LEDinf and 27% for LEDr.
Conclusions:
These results highlight the benefit that the LLL and LED offers as an alternative therapy in the treatment of snake accidents
caused by bothropic venom.
Keywords: low level LASER, Light emitting diode, Bothrops moojeni, edema, hemorrhage
Financial Support: FAPESP nº 2008/02297-2X, FVE-UNIVAP, CNPq.
Resumo:17-159
ANTINOCICEPTIVE ASSESSMENT OF SNAKE VENOM CAUDISONA DURISSA COLLILINEATA AND
IDENTIFICATION OF THE ACTIVE FRACTION.
Oliveira, S. A. 1,2; Magalhães, M. R. 2; Costa, E. A. 1
1
Laboratório de Farmacologia de Produtos Naturais, ICB, UFG
2
Laboratório de Toxinologia,CEPB, PUC-GO
Objectives:
The venoms of snakes of the subfamily Crotalinae have a great diversity of biological activities. The venom of the snake
Caudisona durissa promotes a series of neurologic and myotoxic accidents in humans without causing pain. In contrast victims
feel analgesia at the injection site a few minutes after the accident. Studies show that the snake venom of Caudisona durissa
terrificus has analgesic activity and other activities. In addition to molecules that act on the circulatory system and coagulation,
several other studies are being developed aiming to isolate molecules that may have analgesic effects (Toxicon. 44:1, 2004, Drug
News Perspect.19: 381, 2006) antimicrobial (Biopolymers. 47: 415, 1998; Toxicon. 44:305, 2004; Toxicon. 45:817, 2005;
Toxicon. 45:807, 2005; Toxicon. 48:227, 2006, Biochem J. 402:93, 2007), or anti-inflammatory (Biochem Biophys Res
Commun.302: 193, 2003).Despite the venom in nature be considered unsuitable for therapeutic use there is the possibility of
isolated constituents to be useful for the treatment of different pathologies. The aim of this study was to identify the fraction with
antinociceptive activity of snake venom of Caudisona durissa collilineata.
Methods and Results:
The crude venom was subjected to fractionation by HPLC with a gradient 20−70% acetonitrile with 0.1% TFA at a flow rate of
1.0 mL/min. Fractions of 1.0 mL were collected. The antinociceptive activity was evaluated against a chemical stimulus (writhing
test induced by acetic acid 0.6%, 0.1 mL/10g,) 20 min after i.p. treatement and 45 min after p.o. treatement and the writings
number recorded by 20 min. In this test the pre-treatment with the crude venom of Caudisona durissa collilineata (Cdc-b) at
doses of 20, 40 and 60ìg/kg, by intraperitoneal route, significantly inhibited the writhing of 40.12 ± 6.24 control group (saline,
ip) to 14.12 ± 5.26∗, 7.75 ± 2.48 ∗∗, 13 ± 2.52∗∗∗, respectively. The fraction that exhibited antinociceptive activity, called FrS at
a dose of 40ìg/Kg i.p, reduced the number of writhing (40.12 ± 6.24) control group (saline, ip) to 11.43 ± 2.46 ∗∗. The oral
treatment with Cdc-b and 40ìg/Kg FrS 40ìg/Kg reduced the number of contortions of 41.44 ± 0.62 (control group) ± 2.33 to
19.00 ∗∗∗ and 2.77 ± 0.75 ∗∗∗, respectively.
Conclusions:
The present study revealed that the FrS fraction of the venom of South American rattlesnake, Caudisona durissa collilineata
contributes to the antinociceptive effect caused for crude venom by both oral and intraperitoneal treatment. We have also
demonstrated that the effects of FrS fraction, administered orally, should be mediated by activation of opioid receptors. The
antinociceptive properties of FrS, its special quality of being orally active in low doses, and its long-lasting effect emphasize its
therapeutic potential.
Keywords: Caudisona durissa collilineata, active fraction , writings, antinociception
Financial Support: CAPES, CNPq, FAPEG, PUC-GO.
Resumo:17-160
FEMALE SEX HORMONES MODULATE DIFFERENTIAL CELLULAR RECRUITMENT AND TRACHEAL
REACTIVITY IN A MICE MODEL OF LIPOPOLYSACCHARIDE-INDUCED LUNG INFLAMMATION
Gimenes-junior, J. A. 1; Lino-dos-santos-franco, A. 1; Vitoretti, L. B. 1; Ligeiro-de-oliveira, A. P. 2; Moriya,
H. T. 3; Palermo-neto, J. 4; Oliveira-filho, R. M. 1; Vargaftig, B. B. 1; Tavares-de-lima, W. 1
1
Depto. de Farmacologia / Instituto de Ciências Biomédicas I, ICB I / USP
2
Depto. de Imunologia / Instituto de Ciências Biomédias IV, ICB IV / USP
3
Laboratório de Engenharia Biomédica / Escola Politécnica, LEB / PTC / EPUSP
4
Depto. Patologia / Fac. de Medicina Veterinária e Zootecnia, VPT / FMVZ / USP
Objectives:
Though it is known that acute lung injury (ALI) induced by lipopolysaccharide (LPS) exposure is characterized by cell migration,
edema and altered airway responsiveness, the role of lung inflammation on the induction of altered airway reactivity after
endotoxin exposure remains undefinied. Female sex hormones (FSH) are implicated in innate and adaptative immune responses
and estradiol reportedly protects or deteriorates asthma symptoms. Previous data from this laboratory revealed that ovariectomy
(OVx) blunted LPS-induced lung inflammation in mice after 24 h. Here, we expanded the study and assessed the influence of
FSH on airway smooth muscle reactivity to methacholine (MCh).
Methods and Results:
7 day-OVx female mice (C57BL/6, n = 5-8/group, 7-10 wk old, mean body weight = 23 g) were subjected to intranasal
instillation of LPS (Escherichia coli, 100 μg/ml, 1 μl/g) or saline (0.9%, 1 μl/g). Mice were euthanized 24 h later and the cells
recovered in bronchoalveolar lavage (BAL). Total cells coutings were made in Neubauer chambers and differential coutings were
made upon optical microscopy. The thacheal reactivity to MCh were determined using a Powerlab™ system. Otherwise intact
animals subjected to false operation (Sham-OVx) served as controls. Data (mean ± SEM) were analysed by ANOVA followed by
the Student Newman-Keuls post test (cells BAL) and the Student´s tailed paired t-test (thacheal reactivity). The GraphPad
Prism™ version 5.03 was used for this purpose. P
Conclusions:
FSH control lung inflammation by downregulating accumulation of inflammatory cells induced by LPS exposure. Thus, it is
conceivable that FSH-depriving conditions could blunt endogenous control of inflammatory process. The OVx-induced increase
in cellular recruitment after LPS coexisted with a reduced tracheal reactivity, clearly showing that the lung inflammatory
response and the tracheal responsiveness to LPS exposure are under distinct pathways of control by FSH.
Keywords: Lung, Inflammation, Lipopolysaccharide, Female Sex Hormones, Mice
Financial Support: FAPESP (2009/52782-7 and 2009/51886-3).
Resumo:17-161
α-TERPINEOL REDUCES NOCICEPTIVE BEHAVIOR IN MICE
Oliveira, M. G. B. D. 1; Santana, M. F. D. 1; Santana, M. T. 1; Santos, A. B. D. 1; Guimaraes, A. G. 1;
Siqueira, J. S. 1; Sousa, D. P. D. 1; Almeida, R. N. D. 2; Bonjardim, L. R. 1; Quintans-júnior, L. J. 1
1
UNIVERSIDADE FEDERAL DE SERGIPE, UFSE
2
Universidade Federal da Paraíba, UFPB
Objectives:
α-Terpineol (TPN) is a monoterpenoid alcohol present in the essential oils of several species of the Eucalyptus genus
(Myrtaceae). The aim of the present study was investigate the antinociceptive of TPN in rodents.
Methods and Results:
The antinociceptive effect of TPN was examined using the acetic acid (0.85%) writhing reflex, formalin (20 μl of 1%), glutamate
(20 μl of 20 μmol), and capsaicin (20 μl of 1.6 μg)-induced nociception tests. Mice divided into five groups (eight per group),
were pretreated with vehicle (saline + tween 80 0.2%) or TPN (25, 50 or 100 mg/kg, i.p.) 0.5 h before experiments. Experimental
protocols were approved by the animal care and use Committee (CEPA/UFS: 26/09) at the Federal University of Sergipe. The
obtained data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's of test. These results were
presented with Mean ± SEM, in all cases, differences were considered significant if p < 0.05. TPN produced significant a
analgesic effect by reduction at the early and late phases of paw licking, 1st phase: 25 (46.0 ± 9.2), 50 (49.1 ± 4.9) and 100 mg/kg
(40.5 ± 6.2), 2nd phase: 25 (17.3 ± 8.9), 50 (0.4 ± 0.3) and 100mg/kg (14.1 ± 10.3), with p< 0.01 in the formalin test and reduced
the writhing reflex in mice 25 (4.5 ± 1.3), 50 (1.5 ± 0.7) and 100mg/kg (0.7 ± 0.4) in the writhing test with p< 0.01) nociceptive
protection, 25 (32.5 ± 5.8), 50 (32.1 ± 5.2) and 100mg/kg (33.0 ± 3.4). When the capsaicin-induced nociception test was
conducted, TPN produced dose-related inhibition of the nociceptive behavior, 25 (25.5 ± 4.4), 50 (13.2 ± 2.7) and 100 mg/kg (4.5
± 1.0) with p< 0.01 or p< 0.001). Such results were unlikely to be provoked by motor abnormality.
Conclusions:
Together, these results suggest that TPN, an important monoterpene of the Eucalyptus species, might represent an important tool
for treatment of pain conditions. Further studies currently in progress will enable us to understand the precise action mechanisms.
Keywords: α-terpineol, formalin, glutamate, monoterpene, pain
Financial Support: FAPITEC-SE, CNPq
Resumo:17-162
RELATIONSHIP BETWEEN NIGROSTRIATAL DOPAMINE AND NOCICEPTIVE MODULATION: THALAMIC
HYPERACTIVATION IN HEMIPARKINSONIAN RATS
Domenici, R. A. 1; Maciel, S. T. 1; Fonoff, E. T. 2; Pagano, R. D. L. 1
1
Hospital Sirio-Libanes, IEP- HSL
2
Dep. Neurology, University of Sao Paulo Medical School, USP
Objectives:
Parkinson Disease (PD) is a chronic progressive neurodegenerative disorder characterized by selective loss of nigrostriatal
dopaminergic neurons, modifying the extrapyramidal motor control. Chronic pain is a common complaint in PD, which may
precede the motor symptoms and is commonly neglected and poorly understood. Therefore, it was showed a reduction of
nociceptive threshold in hemiparkinsonian rats in different models of nociception. The aim of this study was to investigate the
mechanical and thermal nociceptive threshold of the rats submitted to a PD model and consequently to evaluate the neuronal
activation pattern of the neurocircuitry involved in this response.
Methods and Results:
Adult male Wistar rats (200-250 g), deeply anesthetized (tribromoethanol 2,5%), were submitted to a PD model induced by
unilateral striatal stereotaxic injection of the neurotoxin 6-hydroxydopamine (6-OHDA; 12 µg/2µl), on the left side. Rats injected
with saline or naïve were used as control (n=5 animals per group). After 14 days of the lesion, rats were evaluated in the open
field test and no significant change in the motor behavior or stereotyped movements were noted. After 7 and 14 days of the
neurotoxin injection, mechanical and thermal hyperalgesia were investigated by paw pressure and plantar tests, respectively. In
all times evaluated, it was observed a reduction (55%) in the mechanical nociceptive threshold of the hemiparkinsonian rats,
when compare with control group. However, no difference was observed in the thermal response. After 1 hour of the nociceptive
tests, the immunoreactivity (IR) for tyrosine-hydroxylase (TH; dopaminergic cell marker) in the substantia nigra (SN) was
determined. Nigrostriatal damage was characterized by the loss of 62% in TH-IR in cells of the SN, in the injection side, and 21%
in the contralateral side. We also evaluated the IR for Fos protein, a neuronal activation marker, in the periaqueductal gray
(PAG), the SN and the ventral posterior lateral and medial nuclei (VPL/VPM) of the thalamus. Fos-IR increased (30%) in the
VPL/VPM of the lesioned animals, on the injection side, when compared to the pattern observed in the control animals. No
significant change in Fos-IR in the SN or in the PAG was noted.
Conclusions:
These results show that unilateral dopaminergic inhibition induces bilateral mechanical hyperalgesia, suggesting that the
dopamine in the mesostriatal system has an important role in modulating nociceptive process. Thalamic hyperactivation suggests
a close interaction between nigrostriatal dopaminergic system and pain afferent pathway in PD. Further investigation of the
mechanisms involved in these nociceptive modulation may contribute to improve the clinic treatment of persistent pain observed
during Parkinson´s disease.
Keywords: 6OHDA, Fos protein, Mechanical hyperalgesia, Parkinson disease, Thalamic nuclei
Financial Support: FAPESP and Hospital Sírio-Libanês
Resumo:17-163
ANTINOCICEPTIVE EFFECTS OF AN ALKALOID EXTRACTED FROM FRUITS OF OCOTEA PUBERULA.
Montrucchio, D. P. 1; Szyminovicz, M. 2; Bobinski, F. 2; Borges, F. R. M. 2; Santos, A. R. S. 2
1
PPG Farmacologia, UFSM
2
Depto de Ciências Fisiológicas, UFSC
Objectives:
Ocotea puberula is a native brazilian tree found mainly in the south region, and its barks and leaves has been a target for several
phytochemical and some pharmacological studies. The aim of this study was to investigate the antinociceptive properties of
dicentrine, an alkaloid extracted from O. puberula fruits as the majoritary compound, in animal models of acute and chronic
inflammatory pain.
Methods and Results:
Ocotea puberula fruits collected in Curitiba, state of Paraná, were extracted with ethanol in a soxhlet apparatus for 24 hours,
filtered and concentrated under reduced pressure. The concentrated ethanolic extract was then fractioned with solvents of
increasing polarity: n-hexane, chloroform and ethyl acetate. The chloroform fraction, rich in alkaloids, was then submitted to a
column chromatography, from which it was isolated an alkaloid identified as dicentrine. The antinociceptive properties of
dicentrine were tested in male adult Swiss mice, weighing between 25-35g, in an acute nociception model (formalin) and in a
chronic inflammatory nociception model (CFA). In the formalin model, three groups (n=6) were pretreated with a solution of
dicentrine in doses of 10, 30 and 100 mg/kg by oral route 60 minutes prior to the injection of 20μL of a 2,5% formalin solution in
the right hindpaw of mice. The time spent licking the injected paw was counted during the first 5 minutes (neurogenic phase) and
between the 15th and 30th minute (inflammatory phase). Dicentrine was able to diminish the licking time in the inflammatory
phase in a dose-dependent manner, with the best result at the dose of 100mg/kg (MI=62±14%). In the chronic model, an
inflammation was inducted by the injection of 20μL of CFA 50% in the plantar surface of mice‘s right hindpaw. After 24h, the
mechanical hypersensitivity was evaluated as the withdrawal response to 10 applications of 0,4g von Frey hair, and the thermal
hypersensitivity was evaluated in the hot plate (50ºC) as the latency for paw withdrawal (cut off 20s). Dicentrine (100 mg/kg,
n=8) administered by oral route was able to diminish the mechanical, but not thermal hypersensitivity, up to 2 hours after
administration, during 14 days of daily treatment. After the last von Frey evaluation, the mice were submitted to the open field
test and showed no effect in the locomotor activity. The weight gain was monitored along the 14 days and there was no difference
between treated and control groups.
Conclusions:
The results suggest that dicentrine has an antinociceptive effect on inflammatory conditions, without compromising the
locomotor activity in animal models.
Keywords: Dicentrine, Nociception, Ocotea puberula
Resumo:17-164
SURVIVAL AND OXIDATIVE STRESS OF ZEBRAFISH LARVAE EXPOSED TO ESCHERICHIA COLI
LIPOPOLYSACCHARIDE
Concatto, S. C. 1,2; Leite, C. E. 1,4,5; Bonan, C. D. 3; Menezes, F. P. 3; Campos, M. M. 1; O. Battastini, A. M.
4,5
; Morrone, F. B. 1,2
1
INSTITUTO DE TOXICOLOGIA , INTOX, PUCRS
2
FACULDADE DE FARMÁCIA, FFARM, PUCRS
3
4
FACULDADE DE BIOCIÊNCIAS, FABIO, PUCRS
DEPARTAMENTO DE BIOQUÍMICA ICBS, DEPBIOQUIMICA, PUCRS
5
FACULDADE DE MEDICINA, FAMED, UFRGS
Objectives:
Zebrafish (Danio rerio), known in Brazil as Paulistinha, has been used in various stages of drug discovery process. It is a useful
alternative, of great scientific value and lower cost, in comparison to other animal models (eg, rodents, dogs and pigs). The
standardization of a model of inflammation in zebrafish larvae, as well as the characterization of the systems involved in this
process might contribute to the validity and safety of its use as a model for screening new drugs. The aim of this study was to
begin the characterization of inflammation in zebrafish larvae exposed to Lipopolysaccharide (LPS) from Escherichia coli, by
assessment of survival and oxidative stress.
Methods and Results:
Larvae obtained and selected during the experiments were kept in an incubator and later transferred to Petry plates until use. The
experiments were performed in 6-well plates with up to 13 larvae per well. Zebrafish larvae were exposed to Escherichia coli
LPS at concentrations of 0, 50, 100, 150, 200 and 300 µg/ml. The survival was assessed for 72 hours. The oxidative stress was
assessed, 4 hours after LPS exposure, by catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) assay. The
control group and those exposed to 50 and 100 µg/ml showed higher survival rates (higher than 95%). The groups receiving 150,
200 and 300 µg/ml showed survival rates of 88±5%, 57±5% and 0 %, respectively. The groups treated with 150 µg/ml of LPS
showed a significant decrease of CAT activity (24±9%), allied to SOD and MDA increase (18±7% and 29±9%, respectively).
Conclusions:
The survival curve shows that the concentration of 300 µg/ml induced 100% of fish death and, oxidative stress was induced by
150 µg/ml. Additional studies are being conducted to determine the mechanisms involved in the induction of oxidative stress and
death of animals, but probably these events occurred in consequence of over-activation of defense mechanisms of the zebrafish,
similar to sepsis in mammals.
Keywords: LIPOPOLYSACCHARIDE, OXIDATIVE STRESS, ZEBRAFISH LARVAE
Financial Support: FAPERGS/CNPq
Resumo:17-165
MODULATION OF ENDOTHELIN PRODUCTION BY LIPOXIN A4 SUPPRESSES ARTICULAR INFLAMMATION
Conte, F. P. 1; Junior, O. M. D. L. 1; Junior, W. A. V. 2,3; Cunha, F. Q. 3; Penido, C. 1; Henriques, M. G. M.
O. 1
3
Farmacologia / Universidade de São Paulo, USP
1
Farmacologia / Farmanguinhos-Fundação Oswaldo Cruz, FIOCRUZ
2
Ciências Patológicas / Universidade Estadual de Londrina, UEL
Objectives:
Endothelins (ETs) are pro-inflammatory peptides involved in pain, fever, edema and cell migration. Rheumatoid arthritis (RA) is
a chronic autoimmune inflammatory disease characterized by joint inflammation, as well as local and systemic increased ET-1
levels. The present study demonstrates the effect of ET antagonism in articular inflammation on zymosan- and collagen-induced
arthritis and also show the modulation of ET-1 levels by lipoxin (LX)A4, an endogenous anti-inflammatory mediator.
Methods and Results:
Two hours after induction of zymosan (500 g/cav, i.a)-induced arthritis in C57BL/6 mice, ET-1 mRNA expression levels were
increased in synovial extracts, which was followed, at 6 and 24 h, by massive edema formation and neutrophil influx into
inflamed joint. Pre-treatment with bosentan (10 mg/kg; i.v.), a dual pharmacological ET receptor antagonist, significantly
impaired 6 h zymosan-induced leukocyte accumulation and edema formation. ET receptor blockade also decreased zymosaninduced production of TNF- CXCL1 and leukotriene(LT) B4 within 6-24 h. Bosentan (10 mg/kg; i.p.) post-treatment effectively
attenuated clinical score of established collagen-induced arthritis in male DBA/1 mice and caused a significant decrease on paw
edema (30 and 35% of inhibition, respectively). In addition, bosentan post-treatment reduced lymph node weight and cellularity,
with concomitant suppression in the numbers of CD3+CD25+CD71+ cells. LXA4 in vivo pre-treatment (20 ng/cav, i.a.)
significantly impaired zymosan-induced preproET-1 mRNA expression (80 % of inhibition; n=5) in C57BL/6 mice with
concomitant suppression of zymosan-induced edema formation and neutrophil influx (74 and 61% of inhibition, respectively;
n=6). In addition, in vitro pre-treatment of neutrophils with LXA4 (1-100 nM) inhibited ET-1 (100 nM)-induced activation and
chemotaxis, evaluated by shape change and boyden chamber assay, respectively.
Conclusions:
Our results suggest that pharmacological blockade of ET receptors, or the modulation of ET-1 expression by LXA4, may serve as
a possible novel therapeutic tool for the treatment of inflammatory and autoimmune articular diseases.
Keywords: artrite, bosentan, endotelina, lipoxina A4, neutrófilo
Financial Support: CNPq
Resumo:17-166
NORADRENALINE INJECTION INDUCES PERIPHERAL ANTINOCICEPTION BY ADRENERGIC,
OPIOIDERGIC AND CANNABINOIDERGIC SYSTEM INTERACTION.
Souza, T. C. 1; Di Marzo, V. 2; Romero, T. R. L. 1; Duarte, I. D. G. 1
1
Laboratório de Dor e Analgesia, Departamento de Farmacologia, ICB-UFMG
2
Inst. of Biomol. Chemistry, Consiglio Nazionale Ricerche., C.N.R.I
Objectives:
Despite the classical peripheral pronociceptive effect of noradrenaline (NA), recently studies showed the involvement of this
endogenous adrenoceptor agonist in intrinsic control of peripheral pain under interaction with the immune system. Thus, the aim
of this study was to verify the opioid and the cannabinoid system participation in the peripheral antinociceptive effect of NA.
Methods and Results:
The rat paw pressure test was used and hyperalgesia was induced by intraplantar injection of prostaglandin E2 (2 µg/paw). All
drugs were administered locally into the right hind paw of Wistar male rats with n > 4 animals per group. The HPLC
chromatography was performed to verify the participation of endocannabinoids in this event. NA (05, 20 and 80 ng/paw) elicited
a local inhibition of hyperalgesia (35%, 55% and 85%, respectively). The non selective alpha2 adrenoceptor antagonist
yohimbine (05, 10 and 20 µg/paw), the non selective alpha1 adrenoceptor antagonist prazosin (0.5, 1 and 2 µg/paw) and the non
selective beta adrenoceptor antagonist propranolol (150, 300 and 600 ng/paw) blocked (20%, 60% and 100%; 22%, 65% and
100%; 20%, 50% and 100%, respectively) the antinociception effect induced by NA, highest dose. The local effect of NA was
antagonized by the non selective opioid receptor naloxone (25, 50 and 100 µg/paw) (35%, 50% and 100%, respectively), as well,
by the selective µ-opioid receptor antagonist clocinnamox (10, 20 and 40 µg/paw) (15%, 40% and 100%, respectively) and by the
selective delta-opioid receptor antagonist naltrindole (15, 30 and 60 µg/paw) (20%, 50% and 100%), but not by the selective
kappa-opioid receptor antagonist NOR BNI (100 µg/paw). In addition, the enkephalinase inhibitor bestatin potentiated the
antinociceptive effect of a low dose of NA from 60% to 90%. The CB1 cannabinoid selective antagonist AM251 (20, 40 and 80
µg/paw) (30%, 55% and 100%, respectively) but not the CB2 cannabinoid selective antagonist AM630 (100 µg/paw) blocked the
noradrenaline-effect. The anandamide amidase inhibitor MAFP (2 µg/paw) and the anandamide reuptake inhibitor VDM11 (20
µg/paw) increased from 50% to 90% and from 52% to 95%, respectively, the peripheral antinociceptive effect of the NA low
dose. In additional, the dosage of endocannabinoids (AEA, 2-AG, PEA and OEA) indicated that noradrenaline induces a selective
anandamide (AEA) release in the peripheral site with an increase twice in the lipid extract dosage.
Conclusions:
The results provide evidences that NA probably induces peripheral antinociceptive effect by activation of adrenoceptors in the
resident immune cells to release beta-endorphine or anandamide that activate selectively µ, delta-opioid receptors or CB1
cannabinoide receptor in peripheral site.
Keywords: Peripheral antinociception, Noradrenaline, Adrenergic system, Opioidergic system, Cannabinoidergic system
Financial Support: CNPq and fellowships from CAPES.
Resumo:17-167
DUAL EFFECT OF INTRAPLANTAR INJECTION OF PERTUSSIS TOXIN IN NOCICEPTION.
Poloni, R. ; Schivo, I. R. ; Cunha, T. M. ; Ferreira, S. H.
Farmacologia/Faculdade de Medicina de Ribeirão Preto, FMRP-USP
Objectives:
Pertussis toxin (PTX) is widely used as a pharmacological tool for inhibiting Gi and G0 proteins, e.g., in studies of the
mechanism of action of antinociceptive drugs. Intrathecal PTX induces hypernociceptive signals compared to neuropathic
syndromes in rats and mice. However, there is a study wherein intraplantar (i.pl.) PTX prevents prostaglandin E2 (PGE2)-evoked
mechanical hypernociception in rats. Due to this contradiction found in literature, the present study evaluated the i.pl. PTX effects
(0.01 - 1 µg.paw-1) in nociception and inflammation, as well as i.pl. PGE2-evoked hypernociception (100 ng.paw-1).
Methods and Results:
Nociceptive evaluation was made through constant pressure and increasing pressure apparatuses on the animal‘s paw (modified
Randall-Selitto and electronic von Frey, respectively). The variation in paw volume was measured by using an
hydroplethismometer. The number of neutrophils was indirectly checked by a colorimetric assay which evaluated the
myeloperoxidase activity. The cytokine concentration was measured by using ELISA. Low doses of i.pl. PTX (0.1 - 0.15 µg.paw1) blocks the PGE2-evoked hypernociception in rats, which was confirmed in mice (0.03 - 0.1 µg.paw-1). The i.pl. pretreatment
with selective inhibitors of nitric oxide (NO) synthase (l-NOARG), guanylyl cyclase (ODQ), protein kinase G (KT5823) or ATPsensitive K+ channel blocker (glybenclamide) prevented the antinociceptive effect caused by i.pl. PTX in PGE2-evoked
mechanical hypernociception, confirming the data previously revealed by our group. However, if the PTX dose is increased (0.3 -
1 μg.paw-1), the rats present mechanical hypernociception, confirmed in mice (0,6 - 1 µg.paw-1), and edema, which depends on
the neutrophil migration and increased pro-inflammatory cytokine‘s synthesis/release to the injection situ. Such effects were
partially prevented by the pretreatment with intravenous fucoidin or intraplantar indomethacin, atenolol or dexamethasone. Posttreatment with i.pl. morphine prevented the PTX-evoked mechanical hypernociception. Further, pretreatment with adenylate
cyclase, protein kinase (PK) A or PKC inhibitors also prevented PTX-caused mechanical hypernociception. Mice which are
mutant for toll-like 4 receptors (TLR4) and knockout for MyD88 adaptor molecule did not present either i.pl. PTX-evoked
mechanical hypernociception or neutrophil migration, when compared to the respective wild animals.
Conclusions:
The present study showed that i.pl. PTX at low doses, through the activation of the Arginine/NO/PKG/ATP-sensitive potassium
channel pathway, blocked the PGE2-induced mechanical hypernociception. However, in higher doses, i.pl. PTX is capable of
provoking an inflammatory response, with induction of the mechanical hypernociception, edema, neutrophil migration and
inflammatory mediators synthesis/release. The mechanical hypernociception seems to depend on the signalization pathway which
involves adenylil cyclase / protein kinase (PK) A and PKC. The mechanical hypernociception and the neutrophil migration
evoked by the i.pl. PTX depend on toll-like 4 receptors, as well as MyD88 molecules.
Keywords: Pertussis toxin, Hypernociception, Antinociception, Cytokines, TLR4
Financial Support: CNPq
Resumo:17-168
EHRLICH ASCITIC TUMOR (EAT): STANDARDIZATION OF THE MODEL 1MACHADO, L.S.,1ZARDO,
R.S.,1FERNANDES, P.D. 1LABORATÓRIO DE FARMACOLOGIA DA INFLAMAÇÃO E DO ÓXIDO NÍTRICO,
ICB, CCS, UFRJ, RIO DE JANEIRO, RJ.
Machado, L. D. S. ; Zardo, R. S. ; Fernandes, P. D.
FARMACOLOGIA/FARMACIA, UFRJ
Objectives:
Ehrlich tumor is a spontaneous murine mammary adenocarcinoma with rapidly growing and aggressive behavior. Following,
intraperitoneal inoculation of Ehrlich tumor cells, the ascitic volume and number of tumor cells increase progressively. Ascitis is
probably formed in consequence of tumor-induced inflammation, due to the increase in peritoneal vascular permeability. The aim
of this study was standardizes the Ehrlich ascitic tumor (EAT) model to be used as a tool for anti-cancer drug discovery.
Methods and Results:
0.5 x 106 EAT cells were intraperitonealy injected into Swiss mice (20-25g, n=5-6, ethical committee license #ICBDFBC-015).
Every two days a group of mice were sacrificed, samples of blood and bone marrow lavage were collected for the determination
of total leukocytes count. Ascitic fluid was collected for the determination of volume, total leukocytes count and dosages of
cytokines, nitric oxide (NO), and protein. Results are expressed as media ± DP. In days 1th, 2sd, 4th, 6th, 8th, 10th,12th,14th
days after tumor inoculation it could be observed an increase in ascitic volume (1.8±0.1; 1.8±0.2; 2,1±0.15; 1.9±0.4; 2.7±0.3;
6.3±1.4; 9.1±2.6; 15.2±2.3 mL, respectively), in tumor cells count (0.2±0.08; 0.3±0.06; 0.4±0.07; 1.5±0.04; 4.2±0.6; 5.2±1.1;
5.1±0.2; 5.2±0.8 x107 cell/mL, respectively), in animals weight (0.1±0.05; 0.3±0.2; 0.8±0.2; 1.6±0.9; 3.8±1.7; 6.1±1.9; 7.6±1.4;
10.6±1.3 g, respectively). It was also observed a significant increase in total leukocytes count in bone marrow (3.8±1.1; 3.9±1.2;
4.0±0.3; 4.6±1.0; 4.9±0.9; 3.2±0.6; 3.6±0.5; 4.4±0.6 x106 cells/mL) and blood (2.7±0.4; 2.7±0.2; 2,3±0.07; 2.8±0.5; 2.4±0.2;
2.4±0.2; 3.6±0.5; 4.4±0.6 x106 cell/mL). A significant increase in protein extravased (6.4±1.5; 5.8±2.6; 40.9±4.6; 72.8±2.7;
141.7±46.0; 281.1±33.4; 386.6±29.8; 429.4±27.1 µg/mL), in cytokines (TNF: 0.3±0.05; 0.3±0.08; 0.31±0.02; 0.36±0.06;
0.57±0.09; 1.0±0.2; 1.2±0.2; 1.3±0.2 pg/mL and IL-6: 0.6±0.09; 0.65±0.09; 0.77±0.1; 0.8±0.2; 1.3±0.4; 2.0±0.45; 2.0±0.42;
2.1±0.4 pg/mL) and NO (6.6±4.2; 8.3±3.3; 32.8±8.1; 69.6±13.3; 92.5±9.5; 150.4±11.7; 192.4±10.4; 241.5±36.4 µM) were also
observed. After the 10th day an intense hemorrhage was observed and animals died between 15th and 16th days.
Conclusions:
we could observe an increased of ascitic fluid together with tumor cells growth. This phenomenon was accompanied by an
inflammatory response with increase in number of leukocytes, protein extravased, pro-inflammatory cytokines, and nitric oxide.
In conclusion, the EAT model can be used for studies of new anti-inflammatory and anticancer drugs, with treatment during 10
consecutive days.
Keywords: EHRLICH TUMOR, STANDARDIZATION , CANCER
Financial Support: CNPq and FAPERJ
Resumo:17-169
PLATELET ACTIVITY PROFILE IN A MURINE MODEL OF ALLERGIC ASTHMA
Baldissera Jr, L. ; Calixto, M. C. ; Mendes, C. B. ; Antunes, E.
Departamento de Farmacologia/Faculdade de Ciências Médicas, UNICAMP
Objectives:
Introduction: Platelet activation has been reported in a variety of inflammatory diseases, including bronchial asthma (Clin Exp
Allergy. 36:399-401, 2006). In asthma, platelets have been found to actively participate in most of its main features, including
bronchoconstriction, airway inflammation and airway remodeling (Platelets. 18:319-28, 2007). Obesity is associated with
increased atherothrombotic morbi-mortality risk, and is associated with deterioration in asthma outcomes. However, little is
known about the mechanisms by which platelets are activated in allergic diseases, and whether obesity contributes to the platelet
activation in allergic conditions. Aim: In this study we have examined the in vitro platelet adhesion in lean and obese mice
previously sensitized and challenged with ovalbumin-(OVA).
Methods and Results:
Methods: Male C57BL/6 mice (15 g) were fed for 10 weeks with standard chow (lean group) or high-fat diet (obese group). On
the eighth week, animals were actively sensitized twice with ovalbumin (100 μg of OVA, s.c.) on days 0 and 7. At days 14 and
15, mice were intranasally challenge with OVA (10 mu;g) twice a day (6 h between challenges). Control group were challenged
with saline (50 μL). In separate groups, lean and obese mice were treated with anti-TNF-α antibody (2 mg/kg, i.p) given 30 min
before first challenges. Venous blood was collected in 3.8% sodium citrate (1:9 v/v) 48 h after OVA challenge, and centrifuged at
100 g, 20°C for 15 min. Platelet-rich plasma (PRP) was centrifuged at 800 g, 20°C for 13 min. Isolated platelets were then
resuspended in Krebs-Ringer solution (1 mM CaCl2). The adhesion assays (1.2x108 platelets/mL) were carried out in 96-well
plates coated with fibrinogen solution (50 μg/ml). Platelet adhesion were induced with ADP (10 μM) or thrombin (100 mU/mL).
Results: No significant differences were observed between spontaneous and stimulated-platelet adhesion between asthmatic and
non asthmatic groups. ADP-induced platelet adhesion was significantly lower in obese asthmatic (17.8±5.0%; p<0.05).
Conclusions:
Conclusion: Our data show that obesity decreases the stimulated platelet adhesion to immobilized fibrinogen at 48 h post OVAchallenge by mechanisms involving TNF-α inhibition.
Keywords: asthma, adhesion, obesity, platelet, thrombin
Financial Support: CNPq
Resumo:17-170
EFFECT OF MEDIUM CHAIN TRIGLYCERIDES, LINOLEIC ACID, SOY LECITHIN AND VITAMINS A ON
WOUND HEALING IN WISTAR RATS.
Magalhães, M. S. F. 1; Macedo, R. N. 2; Monteiro, D. L. S. 2; Oliveira, C. C. 2; Moraes, R. A. 1;
Nascimento, D. F. D. 1; Linhares, J. H. 2; Linhares, A. E. M. S. 2; Moraes, M. E. A. 1; Moraes, M. O. 1
1
Depto. Fisiologia e Farmacologia, UNIFAC-UFC
2
Faculdade de Medicina, UFC
3
Depto. de Cirurgia, UFC
Objectives:
The wound can be defined as any alteration in the anatomic integrity of the skin, resulting from any type of trauma, where it can
even be classified as intentional (surgical incisions) or accidental. The aim of this study was to determine the effects of a
combination of medium chain triglycerides, linoleic acid, soy lecithin and vitamins A and E, in a cutaneous wound model,
considering the cellular events involved in the cascade of wound healing.
Methods and Results:
A total of 45 young Wistar rats were used, in which a 4 cm2 full thickness skin segment was removed. The rats were distributed
randomly into three groups of 15 animals, Control, Reference and Triglyceride groups, which were treated topically with 0.9%
NaCl, clostebol + neomycin sulfate and the test formulation, respectively, during 12 days. Histological sections were stained with
hematoxylin and eosin, toluidine blue and Masson‘s trichrome. The healing process was assessed using the criteria of Meyers and
special software which quantified mastocytes, collagen fibers and neovessels. The collagen density measured in the Triglyceride
group was significantly higher (P<0.01).
Conclusions:
Therefore, these findings suggest that the test formulation enhances the process of tissue repair.
Keywords: Wound Healing, Medium Chain Triglycerides, Linoleic Acid, SOY LECITHIN, VITAMINS
Financial Support: CNPq, CAPES, FUNCAP, FINEP, MS-RNPC-UNIFAC-HM, and Instituto Claude
Bernard.
Resumo:17-171
SPLENECTOMY INCREASES NEUTROPHIL MIGRATION BUT DOES NOT AFFECT MORTALITY IN LETHAL
SEPSIS
Kanashiro, A. 1; Ferreira, A. E. 1,2; Figueiredo, J. G. 1; Souto, F. O. 1; Alves-filho, J. C. 1; Cunha, T. M. 1;
Cunha, F. Q. 1
1
Farmacologia / Faculdade de Medicina de Ribeirão Preto, FMRP
2
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, FCFRP
Objectives:
Sepsis has been considered the leading cause of death in intensive care units. During severe sepsis, a marked failure of neutrophil
migration leading to infectious focus has been observed and is associated with the spreading of infection, resulting in a high rate
of mortality1. This is due to the fact that neutrophils are responsible for controlling the spread of microorganisms. Several
strategies have been investigated to improve this high sepsis mortality associated with the failure of neutrophil migration.
Currently, the role of the spleen in sepsis is contradictory. Recently, it has been demonstrated that splenectomy changes
neutrophil recruitment during a localized inflammatory process2. So, the present study aimed to investigate the role of
splenectomy in polymicrobial sepsis induced by cecal ligation and puncture (CLP) focusing on a study of functionalneutrophil
alterations.
Methods and Results:
This study was approved by the Ethics Committee of the School of Medicine of Ribeirão Preto - USP, (protocol n°. 041/2011).
Spleens were removed from anesthetized mice (Balb/c, male, 22-26 g) and sham animals underwent laparotomy without
splenectomy. Ten days after splenectomy, mice were subjected to CLP surgery under anesthesia. After 6 hours into the neutrophil
migration experiment, the peritoneal cavity was washed with 3 mL of normal saline containing EDTA. Total leukocyte counts
were performed in a Neubauer Chamber while differential leukocyte counts were performed in cytospin preparations. For the
survival study, the animals were monitored daily for 1 wk. Finally, chemotaxis (using Boynder Chamber counts) and ―killing‖
(evaluated by bacteria killing function) properties as well as CXCR2 and CD11b receptors expression (evaluated by flow
cytometry analysis) were studied in peripheral neutrophils isolated from splenectomized and sham mice. In the present study,
although an improvement in the failure of neutrophil migration was observed after the development of intra-abdominal sepsis, no
survival benefit was shown for splenectomized animals. Neutrophil properties as well as receptor expression of neutrophils
isolated from splenectomized animals were not altered when compared with the sham operated animals.
Conclusions:
Under our experimental conditions, it was demonstrated that the migration neutrophil improvement after splenectomy surgery did
improve neither sepsis survival nor altered neutrophil functions. Other humoral or cellular immune component(s) present in the
spleen, which may be related to infection control during polimicrobial sepsis, are under investigation.
Keywords: Neutrophil migration, Sepsis, Spleen, Splenectomy, Survival
Financial Support: Santander, CNPq, FAPESP.
Resumo:17-172
A RAPID AND RELIABLE MODEL OF ORAL OEDEMA TO SCREEN FOR NEW ANTI-INFLAMMATORY
DRUGS.
Ortolani, P. L. 1; Reis, W. G. P. D. 1; Bakhle, Y. S. 2; Francischi, J. N. D. 1
1
Depto. Farmacologia, Inst. de Ciências Biológicas, UFMG
2
Faculty of Medicine, National Health and Lung Institute, Imperial College
Objectives:
The present study aimed to develop a model to screen for anti-inflammatory effects in the oral cavity.
Methods and Results:
A range of doses (25 - 1000 mcg in 0.1 ml) of carrageenan (CG) were injected into the upper lips of anesthetized animals
(mixture of ketamine/xylazine,15/90 mg/kg, subcutaneous), or intraplantarly (ipl) in Holtzman rats (150-180 g, male) at time
zero. Control rats (C) received the same volume locally (0.1 ml), either in the lip or the hind paw of physiological saline at time
zero. Increased thickness of the lip or hind paw was measured with a digital caliper (Mytutoyo, Japan) for up to 6 h following
injections. CG induced a significant and dose-dependent increase in lip thickness in comparison with controls (Delta CG at 500
mcg/site= 4.12 ± 0.05 mm; Delta C= 0.023 ± 0.005 mm with a maximal effect occurring at 1 h after injection, in contrast to 3 h
(Delta CG at 500 mcg/site =1.68 ± 0.04mm; Delta C= 0.08 ± 0.004 mm) in rat paws. At these times, non-selective (2 mg/kg
indomethacin; 20 mg/kg ibuprofen), selective (12 or 30 mg/kg celecoxib) non-steroidal anti-inflammatory drugs (NSAIDs) or
dexamethasone (1 mg/kg), given s.c. 30 min before CG, reduced oral and paw oedema to a similar extent.
Conclusions:
Although hind paw oedema is highly predictive of the clinical efficacy of NSAIDs (Inflamm. Res. 45; 531-540, 1996), our results
would suggest that oral oedema in rats could be as useful a model and would take less time in screening for new antiinflammatory drugs.
Keywords: anti-inflammatory, carrageenan, inflammation, oedema, oral cavity
Financial Support: CNPq, FAPEMIG.
Resumo:17-173
HEME MODULATES GASTRIC AND INTESTINAL EPITHELIAL CELLS ACTIVATION
Barcellos-de-souza, P. 1; Moraes, J. A. 1; de Freitas Junior, J. C. 2; Morgado-díaz, J. A. 2; Nasciutti, L. E. 3;
Barja-fidalgo, C. 1; Arruda, M. A. 1,4
1
Departamento de Farmacologia e Psicobiologia/IBRAG, UERJ
2
Divisão de Biologia Celular, INCA
3
Instituto de Ciências Biomédicas, UFRJ
4
Farmanguinhos, FIOCRUZ
Objectives:
Gastrointestinal epithelium (GE) works as an intrinsic barrier against microbial invaders. However, the role of GE in immunity is
beyond physical intervention, since it is recognized that GE is able to regulate cellular mechanisms that can distinguish
potentially pathogenic microorganisms from endogenous bacterial flora, as well as coordinate the proper biological response
against them. In many pathological situations arising from chronic inflammatory conditions such as gastritis and inflammatory
bowel disease, GE epithelial cells are deprived of the protection of the mucus secreted by GE-specialized cells. In these
circumstances, notably in gastric and duodenal ulcers, disruption of blood vessels and subsequent lysis of erythrocytes are
common. This may lead to the release of high amounts of heme, which interacts with GE. Previous works from our group have
shown that heme itself is a proinflammatory molecule, activating a number of phlogistic signaling events in a nicotinamide
adenine dinucleotide phosphate oxidase (NADPHox)-dependent manner. In this study we aim to evaluate the effects of heme
upon GE epithelial cells.
Methods and Results:
A well established non-transformed rat small intestine epithelial cell lineage (IEC6) and a human gastric epithelial cell lineage
(HGE3) were used in this study. Intracellular reactive oxygen species (ROS) generation was measured using a cell-permeable,
oxidation-sensitive dye CM-H2DCFDA (10 μM). Cell proliferation was evaluated by tritiated thymidine (10 μM) incorporation
to cell DNA. Wound healing assay was performed by scratching confluent cultures and then the number of cells that migrated to
the injured area was counted. Total cell extracts were obtained for immunoblotting. Our results show that free heme, in
concentrations easily found at hemorrhagic sites (about 20 μM), evokes intracellular ROS production by IEC6 and HGE3 cells,
which is inhibited when cells are pretreated with diphenyleneiodonium (DPI, 10 μM), a NADPHox inhibitor. FAK
phosphorylation, which is related to several cellular responses, is increased by heme (for up to 2 hours) in a NADPHox activity
dependent manner. Heme, in NADPHox-activating concentrations, is involved with IEC6 and HGE3 proliferation and wound
healing. The expression of Heme Oxygenase-1, an enzyme which is involved in heme degradation and is induced during
inflammatory processes, is up-regulated by heme in a NADPHox-dependent manner. Heme effects on transepithelial electrical
resistance (TEER) and on modulating other proinflammatory molecules expression are under investigation.
Conclusions:
These data indicate a prominent role for heme-derived signaling in the pathophysiology of gastrointestinal mucosa dysfunction.
Keywords: Epithelial cells, Heme, NADPH oxidase, Reactive oxygen species
Financial Support: CNPq, FAPERJ, SR-2/UERJ, ABC/UNESCO/L‘Oreal.
Resumo:17-174
JUVENILE IMMUNE CHALLENGE HYPERNOCICEPTION ARE NOT PREVENTED BY ANTINFLAMMATORY
TREATEMANT.
Ferreira, M. S. ; Giusti-paiva, A. ; Nascimento, C. G. O.
INSTITUTO DE CIENCIAS BIOLÓGICAS/UNIFAL, ICB
Objectives:
Aim: Adult hyperalgesia has some involvement with inflammatory and expressive nociceptive neonatal or juvenile experiences.
In a similar way, lipopolysaccharide (LPS) induced sickness syndrome in neonatal and juvenile subjects can also be responsable
for nociceptive and inflammatory adult response, leading to hypernociception in thouse adult animals. Thus, the aim of this study
was to evaluate the possible adult antihyperalgesic effect of nonsteroidal anti-inflammatory drugs therapy in juvenile rats.
Methods and Results:
Methods and results: Twenty one days male Wistar rats received one intraperitonial LPS injection (100 µg/Kg). Some of those
LPS treated animal received three days of anti-inflammatory analgesic therapy with dipyrone, nimesulide or indometacin. After
become adult (75 days after birth), the mechanical sensibility was measured by electronic von Frey apparatus, where alodinia
could be observed. The results show that IP LPS juvenile treatment induced hypernociception in adult rats (39,6±3,7 to 82,8±6,7).
Unlike the presumptions, all the three nonsteroidal anti-inflammatory therapy could not prevent the adult hypernociception. In
fact those animals demonstrated more mechanical sensitivity and alodinia then the only LPS treated group (77,25±6,25 to
34,4±4,5) relative to dipyrone. The results were presented as mean and standard error. Analyzed by ANOA and Tukey test.
Conclusions:
Conclusion: The systemic LPS treatment in young rats is able to lead to hypernociception by decreasing the nociceptive threshold
to mechanical stimulus in adult animals. It is important to emphasize that all therapies with nonsteroidal anti-inflammatory drugs
led to a bigger sensitivity to mechanical nociception, and there is probably no influence of those treatments on adult inflammatory
induced hypernociception.
Keywords: ANTINFLAMMATORY, ALODINIA, HYPERNOCICEPTION
Financial Support: FAPEMIG
Resumo:17-175
ANTI-INFLAMMATORY EFFECT OF NEW TYROSINE KINASES INHIBITORS PLANNED FROM IMATINIB.
Zardo, R. S. 1; Sampaio, T. S. 2; Gonçalves, M. R. 1; Lima, L. M. 2; Barreiro, E. J. 2; Fernandes, P. D. 1
1
Instituto de Ciências Biomédicas, ICB-UFRJ
2
Faculdade de Farmácia, LASSBio-UFRJ
Objectives:
The aim of this study was to evaluate the anti-inflammatory effect of new drugs planned from imatinib structure, first tyrosine
kinase inhibitor (TKI) approved for the treatment of cancer. These analogues were designed for a dual action: anti-tumor and
anti-inflammatory, from the implementation of strategies for molecular modification characteristics of medicinal chemistry, as
regioisomers, retroisosterism, simplification, and molecular bioisosterism in classic rings to obtain the target compounds.
Methods and Results:
The use of animals in this work was approved by the ethical committee of animal experimentation from Centro de Ciências da
Saúde (UFRJ), and received the number ICBDFBC-015. Male Swiss mice (20-25g, n=5-7) were used in the formalin-induced
licking response (2.5%, intraplantar) and in the carrageenan (1%)-induced inflammation in the subcutaneous air pouch (SAP)
model. Animals received oral administration of LASSBio1597, LASSBio1598, or LASSBio1599 (30 mg/kg), 1h before formalin
injection. In SAP these drugs were administrated at 1 and 10 mg/kg (LASSBio1597, LASSBio1598 and LASSBio1599), 1h
before and 23h after carrageenan injection. Statistical analyses was performed by ANOVA followed Bonferroni‘s post test (*p
Conclusions:
These results indicate that LASSBio1587, LASSBio1598, and LASSBio1599 have significantly anti-inflammatory action.
Keywords: anti-inflammatory, formalin, air-pouch, tyrosine kinases inhibitors, imatinib
Financial Support: INCT-INOFAR, CNPq, and FAPERJ.
Resumo:17-176
ANTI-INFLAMMATORY PROPERTIES OF THYMOL
Marinho, R. R. ; Santos, J. S. ; Camargo, E. A. ; Thomazzi, S. M.
Departamento de fisiologia/ Universidade Federal de Sergipe, UFS
Objectives:
Thymol is a monoterpene phenol derivative of cymene, found in oil of plants such as Thymus vulgaris, Lippia gracilis, Lippia
sidoides, and Origanum vulgare. It exhibits multiple biological activities, such as antibacterial, antifungal, anti-inflammatory, and
also possesses antioxidant, free radical scavenging, and antilipid peroxidative properties. Previous study shown that thymol
possesses significant inhibitory activity against at least one COX form at concentrations comparable to the active one of
indomethacin and suggest that this agent should be further studied for possible use as non-steroidal anti-inflammatory drugs
(Planta Med. 71:739, 2005). In order to evaluate the actions of this compound, studies were performed on anti-inflammatory
activity.
Methods and Results:
Methods: Male Swiss mice (20-30 g) and Wistar rats (120-180 g) were obtained from the Central Biotery of the Federal
University of Sergipe and complied with the guidelines on animal care of the Ethics Committee for Animal Use in Research
(CEPA/UFS 55/10). The animals were pre-treated with thymol (10, 30, or 100 mg/kg), dexamethasone (2 mg/kg) or vehicle (n =
6/group). The anti-inflammatory activity was studied using the paw edema model induced by 1% carrageenan (100 µL/paw) and
the volume of the paw was measuread at the time 0 and the intervals of 1, 2, 3, and 4 h. The leukocyte migration was induced by
injection of carrageenan (1%, 250 µL, i.p.) into the peritoneal cavity of mice and 4 h after carrageenan injection the total cells
were counted. Results: Oral treatment with the thymol (60 min before of stimulation) did not show any significant alteration on
paw edema model, but was capable of reducing the carrageenan-induced MPO activity at 100 mg/kg (5.4 ± 1.87 and 14.26 ± 1.05
UMPO/mg tissue for thymol and vehicle, respectively, p
Conclusions:
Thymol shows anti-inflammatory activity in rodents.
Keywords: anti-inflammatory , Inflammation, paw edema model , leukocyte migration , Thymol
Financial Support: Capes
Resumo:17-177
EXPRESSION OF HO-1 AND COX-2 IN NYLON THREAD LIGATURE-INDUCED PERIODONTITIS IN RATS.
Oliveira, J. M. 1; Souza, R. B. 1; Chaves, H. V. 1; Vieira, A. M. 1; Ribeiro, K. A. 2; Cunha, R. M. S. 2; Silva,
A. A. R. 1; Pereira, K. M. A. 1; Pinto, V. P. T. 1; Bezerra, M. M. 1
1
Universidade Federal do Ceará, UFC
2
Universidade Estadual Vale do Acaraú, UVA
Objectives:
Periodontitis is an inflammatory disease characterized by alveolar bone resorption. During inflammatory response heme
oxygenase-1/ biliverdin/carbon monoxide (HO-1/BVD/CO) and cyclooxygenase-2 (COX-2) pathways are activated. The aim of
this study was to analyse both COX-2 and HO-1 mRNA expression during periodontitis in rats.
Methods and Results:
Wistar rats received a nylon thread ligature around the molars and sacrificed after 3, 7, 11 or 14 days. Alveolar bone loss (ABL)
was measured using the ImageJ® software or histopathologic analysis. Quantitative Real Time PCR (qRT-PCR) for COX-2 and
HO-1 mRNA expression in gingival tissues was perfomed using Mastercycler® ep realplex4 (Eppendorf) and Power SYBR
Green Master Mix® (Applied Biosystems). Primers were designed by PrimerBlast using a GenBank (NCBI) Reference Sequence
of COX-2 (NM_017232.3) and HO-1 (NM_012580.2) to Rattus norvegicus. The efficiency amplification for all genes was
verified. The ΔΔCt method was used to calculate COX-2 and HO-1 mRNA relative expression levels. Expression level of the
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was using as the endogenous control (housekeeping). Data are
shown as mean±SEM. Significant (P
Conclusions:
The present data suggest that both HO-1/BVD/CO and cyclooxygenase-2 (COX-2) pathways could be involved in the
progression of periodontitis.
Keywords: Alveolar bone, Cyclooxygenase-2 , Heme oxygenase-1, mRNA, Periodontitis
Financial Support: Funcap; CNPq
Resumo:17-178
ALLERGIC INFLAMMATION MODIFIES THE PHENOTYPE AND FUNCTIONALITY OF LUNG FIBROBLASTS
IN 3D-CULTURE
Dalzy, D. V. ; Guimarães-silva, A. M. ; Garzoni, L. R. ; Perez, S. A. C. ; Silva, P. M. R. ; Martins, M. A.
FUNDAÇÃO OSWALDO CRUZ, FIOCRUZ
Objectives:
Subepithelial fibrosis is a prominent feature of lung remodeling in asthma of all severities. The phenomenon strongly correlates
with decline in lung function, but its mechanistic basis remains poorly understood. Since lung fibroblasts are major structural
cells orchestrating fibrotic remodeling in asthma, we have here studied and compared the morphological and functional changes
of lung fibroblasts, from normal and ―asthmatic‖ mice, cultured in a three-dimensional organized cellular arrangement
(spheroids). The central idea was to have lung fibroblast spheroids as an alternative system to study lung tissue remodeling in
vitro.
Methods and Results:
Sensitized Balb/C mice were challenged with ovalbumin or saline three times a week for two consecutive weeks, starting 30 days
post-sensitization. 24 h after last provocation, digested lung cells were cultured, and homogenous lung fibroblasts (obtained in
three passages) were eventually plated on agarose coated 96-well plastic dishes, without/with IL-13 (10-80 ng/ml). Subepithelium
fibrosis, leukocyte infiltration, cytokine release, collagen and fibronectin production were assessed by specific staining, ELISA,
Sircol and immunostaining, respectively. All procedures involving care and use of laboratory animals were approved by the
Animal Ethics Committee of Fiocruz (CEUA-FIOCRUZ, Prot. 0213-4). We found that allergen provocation led to marked lung
inflammatory response - predominantly eosinophilic- and expressive signs of airway remodeling and matrix protein deposit.
These changes were accompanied by elevation in IL-13 (~150%), eotaxin-1 (~400%), IL-4 (~500%) and IL-5 (~86%) levels,
measured in lung tissue samples. We also noted that lung fibroblasts obtained from healthy mice (control lung) or actively
sensitized and challenged mice (remodeled lung) consistently evolved to spheroid clusters within 24 h of culture. Spheroids from
fibroblasts from control lungs were found to be smaller (217.9±5.0 µm compared to 270.2±6.9 µm (mean±SEM; n=6; P
Conclusions:
In conclusion, these findings show that cultured spheroid formed by lung fibroblasts coming from allergen-challenged mice
exhibit phenotype and functional differences, related to repair and airway remodeling, reinforcing the view that changes acquired
by fibroblast experiencing remodeling events may persist in future generations of these cells, what could contribute to the
severity of asthma.
Keywords: ASTHMA, LUNG FIBROBLASTS, 3D-CULTURE
Financial Support: CAPES, FAPERJ, CNPq, PAPES and PDTIS
Resumo:17-179
CANAVALIA LECTINS: STRUCTURE VERSUS ANTINOCICEPTIVE ACTIVITY
Pinto, N. V. 1; Brito, L. F. 1; Pires, A. F. 1; Cavada, B. S. 2; Assreuy, A. M. S. 1
1
Instituto Superior de Ciências Biomédicas, ISCB-UECE
2
Departamento de Bioquímica e Biologia molecular, UFC
Objectives:
Diocleinae lectins, despite of the highly structural homology, express diverse biological activities, differing also in potency and
efficacy for the same activity. In this study it was investigated the different profiles in nociception models of lectins isolated from
Canavalia genus that posse high degree of similarity in their sequence of amino acids and three-dimensional structures.
Methods and Results:
Lectins from seeds of C. gladiata - CGL; C. maritima - ConM, C. brasiliensis - ConBr were isolated and purified by affinity
chromatography. Evaluation of antinociceptive activity was conducted in Swiss mice (25-35g), manipulated according to the
principles established by the Ethics Committee of UECE (No. 10130208-8/40). Mice received intraplantar (i.pl) injection of
formalin 20μl (2.5% v / v) in the right hind paws and the time (seconds) that animal spent licking its paws was recorded at
neurogenic (F1: 0-5 min) and inflammatory (F2: 15-30 min) phases. In the Hot Plate test, the reaction latency (time displayed
before appearance of licking and shaking hind paws or jumping) of mice to thermal stimuli (55°C) was recorded before and after
(30, 60, 90, 120, 150 and 180 min) lectins administration. Mechanical hyperalgesia was induced by i.p. injection of carrageenan
(Cg, 300 mg) and assessed by the use of Von Frey filaments (0.8 g). Finally, to eliminate the interference effects in areas
responsible for motor function was performed rota-rod test and the number of falls and the time that the animals remained in the
apparatus was recorded. Animals were treated with lectin (1 mg/kg) intravenously (i.v.) 30 min. before protocols. Results are
presented as mean ± SEM analyzed by ANOVA followed by Bonferroni test, considering p
Conclusions:
Canavalias lectins, although present structural similarity, inhibited the nociception inflammatory pain models by peripheral
mechanisms apparently by different pathways. These differences can be associated to pH-dependent oligomerization and to
substitutions of key aminoacids involved in the structure of the carbohydrate-binding sites and to quaternary structures.
Keywords: Antinociceptive activity, Canavalias, Lectin, Structure
Financial Support: CNPq, CAPES, Marques-Domingos, G.F.O.
Resumo:17-180
ANTINOCICEPTIVE EFFECT OF TEPHROSIA TOXICARIA PERS IN TEMPOROMANDIBULAR JOINT
ARTHRITIS INDUCED BY ZYMOSAN: INVOLVEMENT OF NITRIC OXIDE, ATP-SENSITIVE POTASSIUM
CHANNELS, HISTAMINE AND HEMEOXYGENASE-1.
Val, D. R. ; Filgueira, A. A. ; Gondim, C. B. ; Rios, L. C. ; Arriaga, N. C. ; Chaves, H. V. ; Silva, A. A. R. ;
Filho, G. C. ; Oliveira, J. M. ; Bezerra, M. M.
Faculdade de Medicina, UFC
Objectives:
Temporomandibular joint (TMJ) disorders are a group of important clinical entities that result in TMJ inflammatory pain. In the
context of inflammatory responses nitric oxide, ATP-sensitive potassium channels, histamine and hemeoxygenase-1 are
important regulators. Plants of the genus Tephrosia are distributed in tropical and subtropical regions and they are used by
community in allergic and inflammatory conditions, such as asthma and rheumatism. The present study investigated the
antinociceptive effect of Tephrosia toxicaria (Tt) pers in TMJ arthritis induced by zymosan and also verified the possible role of
nitric oxide, ATP-sensitive potassium channels, histamine and hemeoxygenase-1 in this action.
Methods and Results:
42 Male Wistar rats (160-220 g) were pretreated with Tephrosia toxicaria (0,2, 2,0 or 20 mg/kg; per os) 60 min before the
injection (intra-articular-i.a) of 40 µL zymosan (2 mg) into the left TMJ. Zymosan group (Zy) received saline (s.c) 60 min before
TMJ arthritis. Sham group (SH) received saline into left TMJ (i.a). Indometacin (5 mg/kg) was used as a positive control group.
Von Frey test was used to assess mechanical hipernociception (4th hour) and the animals were sacrificed 6h after Zy. In other
series of experiments, before Tt (20 mg/kg) injection, animals were pretreated with L-NAME (30 mg/kg; i.p.), a non-specific
inhibitor of the activity of nitric oxide synthase isoforms; glibenclamide (10mg/kg, i.p.), an ATP-sensitive potassium channel
antagonist; meclizine (40 mg/kg; s.c.), an inhibitor of histamine H1 receptors; or ZnPP-IX (3mg/kg, s.c.), a specific HO-1
inhibitor, followed by Zy injection (i.a). Histopathological analysis of TMJ were performed to evaluate cell influx. Results are
expressed as mean ±S.E.M. Histological data are expressed as medians. P
Conclusions:
These results suggest that the impairment of hypersensitivity elicited by Tt depends on the integrity of both NO and HO-1
pathway, as well as, ATP-sensitive potassium channels, and H-1 receptors.
Keywords: Antinociceptive , Arthritis, ATM, Tephrosia toxicaria, Zymosan
Financial Support: Funcap and CNPq
Resumo:17-181
CARVACROL ATTENUATES MECHANICAL HYPERNOCICEPTION IN MICE
Guimarães, A. G. ; Xavier, A. M. ; Santana, M. T. ; Cavalcanti, S. C. H. ; Bonjardim, L. R. ; Camargo, E.
A. ; Antoniolli, A. R. ; Santos, M. R. V. ; Quintans-júnior, L. J.
Fisiologia/Universidade Federal de Sergipe, UFS
Objectives:
Carvacrol (5-isopropyl-2-methylphenol) is a phenolic monoterpene present in the essential oil of the family Lamiaceae, which
includes the genera Origanum and Thymus. The purpose of the present study was to evaluate the anti-hypernociceptive activity of
carvacrol (CARV) in mice.
Methods and Results:
The anti-hypernociceptive activity of CARV (25, 50 and 100 mg/Kg; i.p.) was tested in male Swiss mice (n=8/ per group)
through models of mechanical hypernociception induced by carrageenan (CG; 300 μg/paw) and the involvement of important
mediators of its signaling cascade, as TNF-α (100 pg/paw), PGE2 (100 ng/paw) and dopamine (30 μg/paw). Mechanical
hypernociception was evaluated using digital Von Frey apparatus (Insight®, Brazil), before and at 0.5, 1, 2 and 3 h after the
administration of hypernociceptive agents. The experimental protocols were approved for the UFS Ethic Committee (CEPA:
43/08). Data were evaluated by ANOVA followed Tukey‘s test (p
Conclusions:
Our results show that CARV reduces inflammatory hypernociception and supporting the idea of this monoterpene acts inhibiting
the sensitization of the nociceptor by anti-inflammatory pathways.
Keywords: Monoterpene, Carvacrol, Hypernociception
Financial Support: CAPES, CNPq and FAPITEC-SE
Resumo:17-182
STEM CELL FACTOR STIMULATES SMOOTH MUSCLE CELLS FROM TRACHEA TO EXPRESS TGF-ALFA.
Oliveira, Lcf ; Oliveira, Shp
Depto Ciências Básicas/Faculdade de Odontologia de Araçatuba, UNESP
Objectives:
The airways smooth muscle cells plays a central role in asthma. These structures being recognized a great potential for active
participation in airway allergic processes for the synthesis of cytokines, chemokines and adhesion molecules with capacity to
promote and perpetuate the inflammatory mechanisms in the pathology present. The Stem cell factor(SCF) is an important
component of this synthetic process, working in maintenance and survival of mast cells, inducing chemotaxis and taking crucial
role in its adhesion to the extracellular matrix. The aim of this study was to evaluate the role of SCF on the Transforming Growth
Factor-beta(TGF-beta)expression on smooth muscle cells of the trachea of mice.
Methods and Results:
We used Balb/c young male mice (six animals per group) weighing 18-20g with free access to food and water before being
subjected to testing. The animals were sacrificed for collection of the trachea that was treated, washed and perforated to obtain
the smooth muscle cells that were stimulated by SCF. The expression of TGF-beta was analysis by RT-PCR. The smooth muscle
cells were stimulated with SCF (1, 10 e 100 ng/mL) at 1, 6 e 24h. After 6h hours, SCF in the concentration of 100 ng/mL was
able to stimulate smooth muscle cells from trachea to express TGF-beta. However, 1 and 24 hours we are not able to observe any
mRNA TGF-beta expression.
Conclusions:
TGF-beta is known cytokine with a role in regulating inflammation and airway remodeling, with isoforms related to the
epithelium and smooth muscle tissues of the lung. The elucidation of the mechanisms involved in the production of TGF-beta by
smooth muscle cells of airways, as well as evaluation of possible related signaling pathways are important tools for better
understanding of the etiology of asthma and to identify potential cellular targets of more selective drugs that can be used in its
pharmacotherapy.
Keywords: Airways smooth muscle cells, SCF, TGF-beta
Financial Support: FAPESP, CAPES.
Resumo:17-183
ROLE OF FRACTALKINE EXPRESSED IN DORSAL ROOT GANGLION IN INFLAMMATORY
HYPERNOCICEPTION
Souza, G. R ; Cunha, T. M. ; Lotufo, C. M. C. ; Talbot, J. ; Bozzo, T. A. ; Cunha, F. Q. ; Ferreira, S. H.
Departamento de Farmacologia - USP-RP, FMRP-USP
Objectives:
Glial activation in the central nervous system has been implicated in the development of neuropathic and inflammatory pain.
More recently, it was found that activation of satellite cells present in dorsal root ganglion (DRG) seems also to play a role in
nociception. The chemokine, Fractalkine (CX3CL1) is expressed by primary sensory afferent, whereas its receptor (CX3CR1) is
predominantly found in the glial cells, indicating that fracktalkine could participate in the process of glial activation. This
chemokine is tethered to the extracellular surface of neurons and when released constitute a diffusible signal that contributes to
neuron-glial interaction. The aim of this study was to test whether activation of satellite cells by fractalkine is involved in the
cascade of events responsible for the genesis of the inflammatory pain.
Methods and Results:
Mechanical nociceptive threshold was evaluated with an electronic version of von-Frey test (electronic pressure-meter paw test)
in Wistar rats weighing 150-200 g. Firstly, it was observed that the decrease in mechanical nociceptive threshold
(hypernociception) observed during peripheral inflammation of rat paw by intraplantar injection of carrageenan was inhibited by
the anti-CX3CR1 administered into the dorsal root ganglion (DRG-L5). Furthermore, intraplantar injection of carrageenan
induced significant increase in GFAP mRNA expression. This increase in GFAP mRNA expression was blocked by the treatment
with a specific antibody against CX3CL1 (10µg/i.gl). In agreement, direct administration of fractalkine into the DRG (L5) of rats
produced mechanical hypernociception of hind paws in a dose- and time- dependent manner. Regarding the mechanism by which
glanglionar fractalkine mediates inflammatory hypernociception, it was observed that fractalkine hypernociceptive effect was
blocked by the treatment with a specific antibody against CX3CR1 (10µg/i.gl) and CX3CL1 (10µg/i.gl), infliximab (50ng/i.gl.),
IL1-ra (300ng/i.gl.), indomethacin (50µg/i.gl) as well as dexamethasone (20ng/i.gl). In vitro, incubation of isolated satellite cells
in culture with fractalkine induced the release of TNF-alpha and IL-1beta.
Conclusions:
Overall, these results suggest that during peripheral inflammation fractalkine is released in DRG and contributes to the genesis of
inflammatory hypernociception by a mechanism dependent on stimulation of satellite cells to produced cytokines (TNF-alpha and
IL-1beta). Finally, these cytokines might activate COX enzyme triggering the release of prostanoids which are responsible for the
nociceptor sensitization and possibly maintaining inflammatory pain.
Keywords: dorsal root ganglion, fractalkine, glial cell, hyperalgesia, satellite cell
Financial Support: FAPESP, CNPq e FAEPA
Resumo:17-184
ANTINOCICEPTIVE AND ANTI-INFLAMMATORY EVALUATION OF NEW SUBSTANCES DERIVED FROM
ISATIN
Gonçalves, M. R. 1; Zardo, R. S. 1; Silva, B. V. 2; Pinto, A. C. 2; Fernandes, P. D. 1
1
Laboratório de Farmacologia da Inflamação e do Óxido Nítrico, ICB - UFRJ
2
Instituto de Química, IQ-UFRJ
Objectives:
Isatin (1H-indole-2,3-dione) is a synthetically versatile substance employed for the synthesis of a large variety of compounds and
with several biological effects. The objective of this work was to evaluate the antinociceptive and anti-inflammatory properties of
new substances derived from isatin.
Methods and Results:
Four substances derived from isatin (named Isaox 1, 2, 3, and 4) were tested in the licking response induced by formalin (2.5%,
intraplantar), acetic acid induced abdominal writhing (2%) and in the subcutaneous air pouch (SAP) model. Male swiss mice (2025g, n=4-5) received oral administration of the isatin derivatives (0.1 to 30 mg/kg), 1h before formalin or acetic acid injection. In
SAP, animals received the substances (30 mg/kg) 1h before and 23h after carrageenan injection (1% in SAP). The use of animals
was approved by the ethical committee of animal experimentation from Centro de Ciências da Saúde (UFRJ), and received the
number ICBDFBC015. Statistical analyses was performed by ANOVA and Bonferroni‘s test (*p
Conclusions:
These results suggest that the news substances present anti-inflammatory and antinociceptive effects. Thus, pharmacological
studies are continuing in order to characterize the mechanism(s) responsible for the antinociceptive and anti-inflammatory action.
Keywords: anti-inflamatory, antinociceptive, evaluation, isatin, new derivatives
Financial Support: CAPES, CNPq and FAPERJ
Resumo:17-185
PROTEINASE ACTIVATED RECEPTOR-4 (PAR-4) EXPRESSED IN SKIN MAST CELLS AND PRURICEPTIVE
NEURONS MEDIATES ITCH-LIKE BEHAVIOUR IN MICE.
Patricio, E. S. ; Costa, R. ; Figueiredo, C. P. ; Calixto, J. B.
Farmacologia/Universidade Federal de Santa Catarina, UFSC
Objectives:
Itch is a common symptom present in several cutaneous and systemic diseases. Recently, it has been suggested a role for PAR-4
in itch transmission (J Neurosci, 28: 4331, 2008), and PAR-4 agonists induced itch-like behaviour in mice (J Pharmacol Sci., 108:
385, 2008). The present work aims to investigate cellular and pharmacological mechanisms underlying the pruriceptive actions of
the selective PAR-4 agonist AYPGKF-NH2 (AYP) in mice.
Methods and Results:
Methods: Female adult CD-1 mice (25-30 g, n=6) received a dorsal intradermal (i.d.) injection of AYP (30-500 nmol/site), and
the frequency of scratching bouts with the hind paws toward the injected site was quantified by 30 min (Ethics Committee
protocol number: PP00497). Results: I.d. administration of AYP (30-500 nmol/site) elicited a marked and dose-related scratching
behaviour. The effective dose ranged from 200 to 500 nmol/site [p<0.05]).
Conclusions:
These findings provide new evidence that PAR-4 activation on mast cells and pruriceptive neurons induces scratching behaviour
in mice. In spite of the mast cell involvement, the itching elicited by PAR-4 activation seems to be independent of histamine,
serotonin or mast cell protease release.
Keywords: gastrin-releasing peptide, mast cells, PAR-4, scratching behaviour
Financial Support: CAPES/CNPq/FAPESC.
Resumo:17-186
COMPARATIVE STUDY OF ENZYMATIC, IMMUNOCHEMICAL AND BIOLOGICAL PROPERTIES OF
BOTHRIECHIS SCHLEGELII SNAKE VENOM FROM COLOMBIA AND COSTA RICA
Prezotto-neto, J. P. ; Kimura, L. F. ; Gutiérrez, J. M. ; Otero, R. ; Santoro, M. L. ; Barbaro, K. C.
Instituto Butantan, IBu
Objectives:
Bothriechis schlegelii is a relatively small snake found in Mexico, Central America and northwest of South America. Bites in
humans are characterized by pain, edema and ecchymosis at the site of bite, and slight defibrinating effect. The aim of this work
was to carry out a comparative study of enzymatic and immunochemical properties and toxic activities of B. schlegelii snake
venom from Colombia (BsCo) and Costa Rica (BsCR). Furthermore, this work also investigated the efficacy of polyspecific antiBothrops serum (ABS), produced by Butantan Institute, to recognize and neutralize some toxic activities of BsCo and BsCR
venoms.
Methods and Results:
Silver stained SDS-PAGE (12 %) was used to compare the protein profile of BsCo and BsCR venoms (5 µg). Many components
with similar molecular masses between 150 – 22 kDa were observed in both venoms, but some protein bands of 62, 33, 25 and 22
kDa were observed exclusively in BsCo venom. Zymography was employed to detect proteolytic and hyaluronidase activities of
venoms, using fibrinogen (0.5 mg/mL), gelatin (2 mg/mL), casein (2 mg/mL) or hyaluronic acid (170 µg/mL). Fibrinogenolytic
activity was observed using 40 µg of BsCo venom (bands of approximately 46, 42 and 30 kDa) and BsCR venom (band around
43 kDa). Different profiles of gelatinolytic activity were also observed using 20 µg of BsCR (bands in 43 and 40 kDa region) and
BsCo (bands between 54 - 25 kDa). BsCo and BsCR venoms exhibited caseinolytic spots between 50 – 23 kDa. Hyaluronidases
band of 60 kDa was observed only in BsCR venom (40 µg). Intense cross-reactivity between BsCo and BsCR was detected by
ELISA using ABS. Many different components located around 150 and 15 kDa in BsCo and BsCR venoms were recognized
using ABS by Western blotting. BsCo and BsCR venoms (0.5, 1, 2, 4 and 8 µg) were injected in mice footpad (Swiss, n= 6) to
evaluate edema evolution in 0.5, 1, 2, 4, 24, 48 and 72 h. The highest edematogenic activity of venoms was noticed at 30 min.
However, BsCR venom was more edematogenic when compared to BsCo venom, since the edema induced by 8 µg dose persisted
up to 48 h. The edema induced by BsCo and BsCR venoms was partiality neutralized after pre-incubation with ABS in the first 4
h. The ABS also neutralized around 87 % of the edema induced by BsCR venom (8 µg) in 48 h. Both BsCo and BSCR venoms
induced hemorrhage 2 h after i.d. injection in mice dorsum (n= 6). However, BsCR venom was almost 4 times more hemorrhagic
when compared with BsCo venom. ABS (80, 40 and 20 µL) totally neutralized the hemorrhagic activity induced by BsCo and
BsCR venoms.
Conclusions:
Geographic distribution can influence the composition and activity of B. schlegelii venoms. High antigenic cross-reactivity was
observed in both venoms (BsCo, BSCR) using ABS. Moreover ABS was effective to neutralize some toxic activities from BsCo
and BsCR venoms.
Keywords: Bothriechis schlegelii, veonom, antivenom, edema
Financial Support: FAPESP, CAPES
Resumo:17-187
STUDY OF PARAOXONASE 1 AND 2 IN PATIENTS WITH SICKLE CELL DISEASE.
Macedo, C. G. D. 1; Maselli, L. M. F. 1,3; Gualandro, S. F. M. 2; Fonseca, G. H. H. 2; Chamone, D. D. A. F.
2
; Bydlowski, S. P. 1
1
Laboratório de Genética Molecular e Hematologia/Fac Medicina, LIM-31 FMUSP
2
Serviço de Hematologia do Hospital das Clínicas da FMUSP, HCFMUSP
3
Fundação Pró-Sangue, Hemocentro São Paulo
Objectives:
The prevalence of sickle cell trait in Brazil is estimated as ranging from 2-8%. Anemia due to this trait presents extremely
heterogeneous clinic. Among other events, there is inflammation and changes related to the redox phenomena. PON1 is an
enzyme present in serum, strongly associated with apoA-I/HDL. It‘s primary physiological role is protect LDL against oxidative
modifications.The association between inflammatory diseases and the decreases of apo A1 and apo A2, whose are related to the
concentration and activity of PON1, are known. However, a significant reduction of apo-A1 during sickling crisis was already
reported. Polymorphisms in candidate genes that affect vascular and inflammatory components involved in the mechanisms of
disease may have prognostic utility. Among them, the PON family of enzymes has been widely studied concerning to protection
against oxidation and its behavior in the inflammatory processes. The present study aims to evaluate, in patients with sickle cell
disease, arylesterase activity of PON1, as good as PON1 and PON2 polymorphisms.
Methods and Results:
Genomic DNA was obtained from 5mL of whole blood from 16 patients with sickle cell disease and 20 from healthy people. The
polymorphisms were assayed by RFLP-PCR using Hinf I. The restriction product was analyzed in 2% agarose gel stained with
ethidium bromide. Arylesterase activity of PON1 was performed by specthrophotometry using phenylacetate in kinetic enzyme
method. The lipid profile (total cholesterol, HDL, LDL and VLDL cholesterol, triglycerides and apo A1) was determined as
described worldwide. Results showed an allelic frequency of 62,5% and 37,5% (controls), 43,75% and 56,25% (patients) for the
alleles PON1-192Q and PON1-192R; 60% and 40% (controls), 84,38% and 15,62% (patients) for the alleles PON1-55L and
PON1-55M; and 85% and 15% (controls), 84,38% and 15,62% (patients) for PON2-148A and PON2-148G. Arylesterase activity
of PON1 in patients showed mean of 70(±29) U/mL and the control showed mean 93,9(±26,52). Means observed for total
cholesterol were 128,7(±26,39)mg/dL; 34,35(±11,22)mg/dL for HDL-cholesterol; 69,7(±22,1)mg/dL for LDL;
24,64(±8,53)mg/dL for VLDL; 123,23(±43,16)mg/dL for triglycerides; and 107,44(±21,88)mg/dL for apo A1. This study was
approved by the ethics committee CAPPesq (0285/10).
Conclusions:
The distribution of alleles PON1-192Q and PON1-192R and PON1-55L and PON1-55M differed between groups, while PON2148A and PON2-148G showed similar frequencies. The values of apo A1 and HDL, in patients, were reduced, a finding that
could contribute to increased inflammatory activity in these patients.Hemolytic stress could be associated with a significant
reduction in plasma lipids and lipoproteins. It appears that patients with sickle cell anemia are at a lower risk for coronary artery
disease.
Keywords: Inflammation, PON1, PON2, Sickle cell
Financial Support: CAPES
Resumo:17-188
ANALYSIS OF THE ANTINOCICEPTIVE ACTIVITY OF &BETA-IONONE
Freitas, L. B. N. 1; Luz, P. B. 1; Osório, C. B. D. H. 1; Olinda, T. M. D. 1; Sousa, T. D. F. G. D. 1; Carmo, L.
D. D. 1; Alencar, N. M. N. D. 1; Sousa, D. P. 2
1
2
Faculdade de Medicina, UFC
Departamento de Fisiologia, UFS
Objectives:
The &beta-ionone (4 - [2,6,6-cyclohexene-1-trimetill]-3-butene-2-one) is a sesquiterpene (degraded terpenoid - C13) present in
the molecular structure of retinol, &beta-carotene and acid retinoic, formed from the mevalonate pathway in different types of
plants. The research on the biological activities of &beta-ionone are still incipient and limited results with this compound are
found in the literature. The objective of this study was to investigate the effect of &beta-ionone in classic models of nociception.
Methods and Results:
Animal handling and experimental protocols were approved by the Ethical Committee for Animal Research/UFC under number
0286. To this end, we used female Swiss mice (n = 10, 23 ± 2g). The animals were treated 30 minutes before each experiment
with &beta-ionone (12.5, 50 and 200mg/kg i.p.), Tween 1% (vehicle 10ml/kg i.p.), morphine 5mg/kg s.c. or diazepam 2mg/kg
s.c. Writhing Abdominal were induced by acetic acid 0.85% (10ml/kg i.p.) and after 10 min, the number of writhing were
counted for 20 min. In the formalin test, the time at which the animal is still licking the injected paw with formalin (1,2%, 20 mL
/ paw, s.c.) was recorded in the first 5 min (1st phase) and 20-30 minutes (2nd phase). In the hot plate test, reaction time of
animals was monitored at 55 º C at 0, 30, 60, 90, 120 and 150 min. Rota Rod test was performed in order to study a possible
activity of &beta-ionone on the locomotor system, mice were placed in the swivel bar (4 rpm) and the residence time was clocked
in 2 minutes. The &beta-ionone (50 e 200mg/kg) significantly reduced the writhing induced by acetic acid, compared with
vehicle (8.33 ± 2.46, 0.75 ± 0.41 and 39.6 ± 3, 8, respectively). In the formalin test, the &beta-ionone (200mg/kg) significantly
reduced response compared with the vehicle, in the 1st phase (3.14s ± 1.03, 63s ± 6.58, respectively), whereas in 2nd phase of the
test, all doses of &beta-ionone (12.5, 50 and 200mg/kg) were able to reduce the time to lick when compared to vehicle (45.8s ±
7.66, 38.8s ± 8.75, 0s and 121s ± 14.5 respectively). In the hot plate test, the &beta- ionone (200 mg/kg) significantly increased
the reaction time of animals at 30 and 60 min. (27.1s ± 3.8, 22.4s ± 3.1, respectively) compared with vehicle (11.7s ± 1.1, 12.1s ±
1.8, respectively). In Rota Rod, the &beta-ionone did not alter the time spent in the swivel bar. Statistical analysis was performed
by ANOVA followed by Student Neuman-Keuls.
Conclusions:
The results show the &beta-ionone has antinociceptive activity, by peripheral and central mechanism of action to be clarified.
Further researches are being developed to explain the exact mechanism of these effects.
Keywords: &beta-ionone, nociception, sesquiterpene
Financial Support: CAPES and CNPq
Resumo:17-189
PARADOXICAL EFFECTS OF BRAIN DEATH AND ASSOCIATED TRAUMA ON RAT MESENTERIC
MICROCIRCULATION: AN INTRAVITAL MICROSCOPIC STUDY.
Simas, R. ; Sannomiya, P. ; Cruz, J. W. M. C. ; Correia, C. J. ; Zanoni, F. L. ; Menegat, L. ; Moreira, L. F.
P.
Depto Cardiopneumologia FMUSP, InCor HC-FMUSP
Objectives:
Aim:To evaluate the role of brain death (BD) compared with BD-associated trauma on the development of the inflammatory
response at the mesenteric microcirculation and its systemic effects.
Methods and Results:
Methods and Results: Male Wistar rats (weighting 250-300 g) were anesthetized with isoflurane 5-2 %, intubated and
mechanically ventilated with a tidal volume of 10 mL/kg. Carotid artery was accessed to measure mean arterial pressure (MAP),
heart rate (HR) and blood sampling. Jugular vein was used to administration of saline 2 mL/h. To induce BD, a balloon catheter
was placed intracranially and rapidly inflated with 500 µL of water. BD was confirmed by sharp rise of MAP, maximal pupil
dilatation, absence of reflex and apnea. Sham operated animals were trepanned only. Animals (7 per group) were evaluated 30
and 180 minutes thereafter. The mesenteric microcirculation was analyzed by intravital microscopy in situ. Mesentery was
exposed and continually perfused by Krebs-Henseleit solution (pH 7.2-7.4), and three fields were obtained to quantify percentage
of perfusion and number of roller, adhered and migrated leukocytes. Expression of adhesion molecules, P-Selectin and ICAM-1,
was performed by immunohistochemistry of mesentery. Serum concentration of TNF-α, IL-1β, IL-6, IL-10, CINC-1 and CINC-2
was determined by ELISA. Number of total and differential white blood cells was determined. The methodology was approved
by the ethics committee (CAPPesq 0226/09). After induction of BD, it was observed an immediate increase in MAP values,
followed by hypotension. MAP values did not change over time in Sham rats. There were no differences in HR values among
groups. Proportion of perfused small vessels (≤30 μm diameter) was reduced in BD-rats compared to Sham rats either at 30
minutes (BD 30.2±0.06 % vs Sham 77.9±0.07 %, p
Conclusions:
Conclusion: BD-associated trauma is responsible for most of the inflammatory events observed. Otherwise perfusion of
mesenteric microvessels was promptly interrupted by BD itself. This was accompanied by a pronounced leucopoenia, and
increased ICAM-1 expression and migrated leukocytes.
Keywords: Brain Death, Cytokines, Microcirculation
Financial Support: Financial support: FAPESP.
Resumo:17-190
CENTRAL INTERACTIONS BETWEEN SUBSTANCE P AND PROSTAGLANDINS DURING FEVER.
Brito, H. O. ; Reis, R. C. ; Barbosa, F. L. ; Fraga, D. ; Zampronio, A. R.
Dept Pharmacology, UFPR
Objectives:
We showed previously that the neuropeptide substance P (SP), acting on NK1 receptors is involved in the febrile response
induced by lipopolysaccharide, tumor necrosis factor-α (TNF-α) and interleukin(IL)-6 but does not participate in the fever
induced by IL-1β and macrophage-inflammatory protein-1α. Since TNF-α and IL-6 induce a prostaglandin-dependent febrile
responses we decided to investigate the involvement of prostaglandins (PG) in the febrile response induced by SP.
Methods and Results:
Male Wistar rats (200 g) were implanted with a guide cannula in lateral ventricle and with data loggers (for measurement of body
temperature in the peritoneal cavity) one week before the experiments under the same anesthesia. Body temperature (•C) was
registered every 15 min. The room temperature was kept at 28•C. All the methods were previously approved by Institution´s
Ethics Committee on Animal Use under protocol # 384. Central administration of SP (250, 500 e 750 ng/2μl) induced a dosedependent increase on body temperature in captopril-treated rats (5 µg/2µl, 30 min before). The febrile response induced by
500ng SP was completely blocked (100%) by the non-peptide NK1 receptor antagonist SR140333 (3.0 µg/2µl, icv). To evaluate
the involvement of PG in this response rats were treated with PG-synthesis inhibitor indomethacin (IND, 2 mg/kg, i.p.) or the
same volume of vehicle (Tris buffer, pH 8.2). After 30 min, animals received captopril (5 µg/2µl) followed by an i.c.v. injection
of SP (500 ng) or vehicle (2 µl) 30 min later. As a positive control, another group of animals were treated with the same dose of
IND or vehicle and after 30 min they received IL-1β (3.1ng, i.c.v.). SP and IL-1β produced an increase in body temperature of
about 1 and 1.5•C, respectively, which peaked at 2h. IND reduced the febrile response induced by both SP (53%) and IL1β(86%). In a final set of experiments, animals were treated with SR140333 (3.0 µg/2µl, icv) and after 30 min they received an
i.c.v. injection of PGE2 (250 ng/2µl) or vehicle (2 µl). PGE2 induced an increase of 1.5•C in the body temperature in the first 30
min after injection. Surprisingly, SR140333 abolished (100%) the febrile response induced by PGE2.
Conclusions:
Our results show that SP,administered in the central nervous system, induces fever in rats when its metabolism is blocked by
captopril. The febrile response induced by SP seems to be dependent on PG synthesis, similarly to the response induced by TNFαand IL-1β. However, the release of SP may also be important after PG synthesis, although further studies are necessary for the
full understanding of those interactions.
Keywords: febrile response, prostaglandin, substance P, NK1 receptor, SR140333
Financial Support: CNPq, Fundação Araucária, CAPES and Sanofi-Aventis.
Resumo:17-191
STUDY OF MESENTERIC MICROCIRCULATORY ALTERATIONS ON BRAIN DEAD RATS.
Kase, M. ; Sannomiya, P. ; Simas, R. ; Cruz, J. W. M. C. ; Moreira, L. F. P.
Depto. Cardiopneumologia, FMUSP
Objectives:
Aim: To evaluate the role of brain death (BD) on mesenteric microcirculatory perfusion, by real-time observation.
Methods and Results:
Methods and Results: Male Wistar rats (250-350g, 2 months of age) were anesthetized with isoflurane (2-5%) and intubated. A
balloon catheter was placed into intracranial cavity and inflated to induce BD. Mesenteric microcirculation was analyzed by
intravital microscopy (5 BD-induced rats) 30 minutes before, during BD, and 30 minutes thereafter. BD was confirmed by sharp
rise of mean arterial pressure, maximal pupil dilatation, absence of reflex and apnea. The mesenteric microcirculation was
analyzed by intravital microscopy in situ. Mesentery was exposed and continually perfused by Krebs-Henseleit solution (pH 7.27.4). Five fields (1 mm2/field) of mesenteric microcirculation were randomly selected for analysis. Vessels (¡Ü30 ¦Ìm and >30
¦Ìm diameter) were evaluated in each field, and classified as perfused or non-perfused microvessels (0226/09 InCor-HC-FMUSP).
Before BD, perfusion was presented in 85¡À6 % of total vessels (¡Ü30 ¦Ìm). After induction of BD, there was a reduction to
42¡À5 % of total vessels (¡Ü30 ¦Ìm; p<0.001).
Conclusions:
Conclusion: The perfusion of mesenteric microvessels was promptly interrupted by BD itself, and was restricted to ¡Ü30 ¦Ìm
diameter vessels compromising, therefore, the viability of the mesentery.
Keywords: Brain Death, Intravital Microscopy, Microcirculation
Financial Support: Financial support: FAPESP.
Resumo:17-192
EFFECTS OF HYDROGEN SULFIDE (H2S) ON LUNG ALLERGIC RESPONSE IN MICE
Benetti, L. R. 2; Campos, D. 2; Almeida, M. F. 2; Guedes, C. E. V. 2; Barillas, S. G. 1; Antunes, E. 1;
Ferreira, H. H. D. A. 2
1
FARMACOLOGIA, UNICAMP
2
UNIVERSIDADE SÃO FRANCISCO, USF
Objectives:
Hydrogen sulfide (H2S), like nitric oxide (NO), is a newly found gasotransmitter recently demonstrated to play an important role
in inflammatory diseases. H2S may react with reactive oxygen and/or nitrogen species limiting their toxic effects. It has also been
suggested that H2S may scavenge the excess of NO produced in the inflammatory state. Thus, endogenous H2S might be an
antioxidant in our organism, but its contribution to the pathophysiology of lung allergic diseases, such as asthma, is unclear. The
aim of this study was to investigate the effect of H2S on antioxidant enzymes and on nitric oxide inducible isoform (iNOS)
expression. We also compare the consequence of H2S exposition with the anti-inflammatory effect of iNOS inhibition on the
influx of eosinophils to the lungs of allergic mice.
Methods and Results:
Methods: BALB/c mice, previously sensitized with ovalbumin (OVA; 100 mg), were treated intraperitoneally (i.p.) with a H2S
donor, NaHS (14 µmol/kg), or iNOS inhibitor, 1400W (1.0 mg/kg), 30 minutes and 2 hours before OVA challenge (10 µg),
respectively. Control (non-treated) mice received only saline i.p. Twenty-four, 48, 96, 120 and 144 hours after challenge, the
mice were sacrificed, bronchoalveolar lavage (BAL) was collected and the lungs were removed and homogenized. The total cell
numbers in BAL were determined and differential leukocyte counts were carried out using cytospin preparation of the cell
suspension, stained with Diff-Quick. The expression of enzymes MnSOD, Cu/Zn SOD and iNOS in the lung homogenates were
analyzed by Western blotting. All experiments were approved by the EAC/USF Protocol (002.11.08). Results: The analyses of
leukocyte population present in the BAL of the control group showed that eosinophil infiltration had started by 24 h and that a
marked increase beginning at 48 h had peaked at 96 h. This effect was not sustained since the eosinophil number in the BAL at
120h was similar to that seen at 48 h, but another peak of this cell influx to lungs was observed at 144h after OVA-challenge.
Treatment of mice with 1400W or NaHS resulted in an approximately 50% decrease in eosinophil infiltration at 48h and 144 h.
At other times after challenge, the eosinophil numbers in the BAL were not significantly different from the control. The analysis
of lung enzymes by Western blotting showed that the MnSOD expression was not modified by OVA challenge neither by NaHS
treatment. In addition, the expression of Cu/Zn SOD was significantly (p
Conclusions:
Our results suggest that the beneficial action of H2S on the mice allergic response by inhibiting the migration of eosinophils to
the lungs is similar to those resulted from iNOS inhibition. This effect may not depend on the antioxidant enzyme, MnSOD or
Cu/Zn SOD. The involvement of iNOS in this process needs to be better clarified by examination of iNOS activity in the
presence of NaHS
Keywords: ASTHMA, HYDROGEN SULFIDE, NITRIC OXIDE
Financial Support: FAPESP and CNPq
Resumo:17-193
TREATMENT WITH H2S DONOR AND INOS INHIBITOR ATTENUATES THE INFLAMMATORY RESPONSE IN
THE LUNG OF ALLERGIC MICE
Guedes, C. E. V. ; Rocha, T. ; Almeida, M. F. D. ; Ferreira, H. H. D. A.
UNIVERSIDADE SÃO FRANCISCO, USF
Objectives:
Pathological features of bronchial asthma are characterized by an increase in inflammatory cells, mainly eosinophils, associated
with plasma exudation, oedema, smooth muscle hypertrophy, mucus plugging and epithelial damage and increases in expression
of nitric oxide inducible isoform (iNOS). Unlike other chronic obstructive lung diseases, asthma can be controlled and reversible.
The objective of this study was to analyze the effect of sodium hydrosulfide (NaHS), a H2S donor, and N-3-aminomethyl-benzylacetamidine-dihychloride (1400W), an iNOS inhibitor, on the inflammatory response in the lungs of allergic mice.
Methods and Results:
Methods: All experiments were approved by the animal ethics committee of USF (protocol 0021108). BALB/c mice were
sensitized with subcutaneous injection of ovalbumin (OVA) bound to aluminum hydroxide, followed by a booster injection 7
days after. At days 14 to 17, the mice received an OVA intranasal challenge, twice a day. The non-challenged group (NC)
received only saline intranasally. The challenged mice were subdivided into: 1) group treated with 1400W (1mg/Kg), i.p., 2 h
before each OVA challenge; 2) group treated with NaHS (14µmol/Kg), i.p., 30 min before each OVA challenge and 3) control
group (non-treated) that received only sterile saline i.p. At 24h, 48h and 96h after the first OVA-challenge, mice were sacrificed
and the left lungs were fixed in 10% formalin overnight and embedded in paraffin. Tissue slides (5µm) were stained with HE to
assess the inflammatory cell infiltrate, which was defined as the average of the peribronchial and perivascular inflammation
scores, determined on a subjective scale of 0 (no detectable inflammation) to 3 (most bronchi or vessels surrounded by a thick
layer of inflammatory cells). To quantify the mucin production, the slides were stained with periodic acid –Schiff (PAS) and the
goblet cells were counted. The percentage of PAS-positive cells was determined in bronchus transversal sections. Mucus
plugging was graded as 0 (≤1% positive cells) to 5 (≤1% negative cells). Results: Inflammatory cell infiltrate was not observed in
the NC group. OVA-challenged groups showed airway infiltration of neutrophils, eosinophils, mast cells and macrophages.
However the degree of histopathologic inflammation was qualitatively decreased in NaHS and 1400W-treated animals, as
compared to control, especially at 48h after OVA-challenge. At 96h, the effect of 1400W was reduced although the effect of
NaHS remained active. Percentages of goblet cells were increased in control mice, with the presence of mucus pellet into
bronchus lumen. An expressive reduction in the percentage of goblet cells was observed in the airways from the NaHS or
1400W-treated groups and no mucus pellet was observed. No increase in the number of goblet cell or mucus pellet was present in
the airways of NC mice.
Conclusions:
NaHS and 1400W both efficiently decrease the inflammatory infiltrate. As mucin production is an indicator of airway
obstruction, the reduction in mucus plugging observed in the NaHS and 1400W groups suggest the efficiency of both treatments
at attenuating asthmatic symptoms.
Keywords: H2S, iNOS, LUNGS, INFLAMMATION, MUCUS
Financial Support: CAPES, FAPESP
Resumo:17-194
DUAL EFFECT OF H1 AND H3 HISTAMINE RECEPTORS IN THE TRAFFIC OF THE NOCICEPTIVE PATHWAY
Stein, T. ; Souza-silva, E. ; Tonussi, C. R.
Farmacologia/Universidade Federal de Santa Catarina, UFSC
Objectives:
Spinal cord is known to be a center that controls ortodromic (nociception) as well as antidromic traffic (vasodilation) in the
nociceptive pathway. Histamine receptors are known to participate in the spinal cord nociceptive transmission, thus it is
conceivable that histamine may also play a role in the spinal cord control on the peripheral vasodilation. Aim: Evaluate the role of
spinal cord histamine in the articular incapacitation, edema and cell migration induced by carrageenan injection into rat kneejoints.
Methods and Results:
Male Wistar rats (300 - 400 g), received intrathecal injection of histamine (0.002; 0.2; 2; 20 nmol), the H1 agonist 2pyridylethylamine dihydrochloride (2-PEA; 0.01; 0.1; 1; 10 nmol), the H3 agonist immepip (4; 8; 16; 32 nmol), and the H3
inverse agonist thioperamide (0.0004; 0.04; 4 nmol) were given 20 min before knee-joint carrageenan injection (50 µg). Articular
incapacitation was measured by counting the paw elevation time (PET; s) during 1-min periods of forced walk hourly, until 6
hours after carrageenan injection. Edema was accessed by the metering of the articular diameter (mm). After 6 h, synovial fluid
was collected for the evaluation of leukocyte infiltration (CEUA/PP00368/2009). Histamine decreased the incapacitation (20
nmol; p
Conclusions:
Histamine may act in the spinal cord by H1 and H3 receptors to cause hyponociception, but this action may also increase
peripheral inflammation.
Keywords: edema, histamine, intrathecal, pain, nociception
Financial Support: CNPq; CAPES; FAPESC
Resumo:17-195
EFFECT OF TALIDOMIDE ANALOGUE, LASSBIO-468, ON EXPERIMENTAL SILICOSIS IN MICE.
Ramos, T. J. F. 1; Trentin, P. G. 1; Ferreira, T. P. T. 1; Arantes, A. C. S. 1; Pires, A. L. A. 1; Lima, L. M. 2;
Barreiro, E. J. 2; Martins, M. A. 1; Silva, P. M. R. 1
1
Laboratório de Inflamação / Instituto Oswaldo Cruz, IOC/Fiocruz
2
Laboratório de Avaliação e Síntese de Substâncias Bioativas, LASSBio/UFRJ
Objectives:
Silicosis, one of the oldest occupational diseases in the world, is consequence of a long-term exposure to inhaled dust containing
silica in its free and crystalline form. Lung interstitial inflammation and fibrosis are the main features of the disease, involving a
wide range of chemical mediators such as TNF-&alpha. This is a pleiotropic molecule which exerts its effects on many cell types.
LASSBio-468 is a thalidomide analogue which modulates TNF-&alpha production and inhibits endotoxic shock and arthritis in
animal models. In this study we investigated the potential effect of LASSBio-468 on the experimental model of silicosis in mice.
Methods and Results:
Anesthetized male Swiss-Webster mice (18-20g) received intranasal (i.n.) instillation of silica (10 mg/50 µL) and vehicle (saline).
Treatment consisted of oral administration of the LASSBio-468 (50 and 100 mg/kg) during 7 consecutive days, from day 21 to 28
post-silica. The analyses included lung function (resistance and elastance) and airways hyperreactivity to aerosolized metacholine
(3 -27 mg/mL) were measured by whole body invasive plestimography (Finepoint, Buxco System). Morphological and
morphometrical analyses included classical dyes such as Hematoxylin-Eosin and Picrus-Sirius. Collagen content and
cytokine/chemokine generation were quantified by Sircol technique and ELISA, respectively. All experimental procedures were
performed in accordance with the guidelines of the Committee on Use of Laboratory Animals of the Oswaldo Cruz Foundation
(L-034/09. We showed that silicotic mice exhibited increased basal levels of lung resistance and elastance as well as airways
hyperreactivity to methacholine aerosolization. Tissue inflammatory response, extensive collagen deposition, granuloma
formation and chemokine (KC and MCP-1)/cytokine (TGF-&beta and IFN-&gamma) generation were also detected in the
silicotic lungs. Administration of LASSBio-468 into silicotic mice reduced the lung function compromise and airways
hyperreactivity, tissue collagen deposition (from 198,3 ± 3,5 to 153,5 ± 7,7 μg/right lung in non-treated and treated silicotic mice,
respectively) (mean ± SEM; n=7) and granulomatous area (from 37,8 ± 2,4% to 18,0 ± 2,6% in non-treated and treated silicotic
mice, respectively; n=6). The generation of cytokines and chemokines was also suppressed by the drug.
Conclusions:
Altogether our findings show that the treatment of silicotic mice with LASSBio-468 reduced curatively the lung function
compromise and granulomatous response, indicating this compound seems to constitute a promising tool for the treatment of
chronic fibrotic pulmonary diseases such as silicosis.
Keywords: Silicosis, Inflammation, Lung, Fibrosis, Therapy
Financial Support: FIOCRUZ, CNPq, FAPERJ and CAPES
Resumo:17-196
EFFECT OF ANETHOLE AND ESTRAGOLE IN MICE PAW EDEMA INDUCED BY BRADYKININ, SUBSTANCE P
AND SODIUM NITROPRUSSIDE
Sousa, P. L. 1,2; Ponte, E. L. 1,2,3; Rocha, P. A. M. V. 1,2; Coelho-de-sousa, A. N. 1,2; Leal-cardoso, J. H. 1,2;
Assreuy, A. M. S. 1,2
1
Instituto Superior of Biomedical Sciences , ISCB
2
State University of Ceará, UECE
3
Faculty Christus, FC
Objectives:
Anethole and estragole are position isomers belonging to the terpenoid group and natural constituents of essential oil of aromatic
plants. Preliminary results from our laboratory demonstrated that the essential oil of Croton zehntneri and its major constituents,
anethole and estragole, present anti-inflammatory activity inhibiting the paw edema induced by carrageenan (Cg), histamine,
compound 48/80 and serotonin. The aim of this research was to evaluate the potential anti-inflammatory effect of anethole and
estragole in the model of paw edema induced by bradykinin, substance P and sodium nitroprusside.
Methods and Results:
Swiss mice (25-30g) were manipulated in accordance with the principles of our Ethic Comitee in Research (UECE No.10 13
0208-8/40). The paw edema was induced by subcutaneous (s.c.) intraplantar injection of 50 microL bradykinin (3 nmol),
substance P (20 nmol) and sodium nitroprusside (10 micromol). Animals were treated by gavage with anethole or estragole (10
mg/kg) 60 min before inflammatory stimuli. Positive controls received stimuli and negative controls, sterile saline (NaCl 0.9%;
100 microL/10g). Edema was evaluated by hydroplethysmometry measuring the volume displaced (microL) by paws before (time
zero) and from 30 min to 240 min after stimuli and calculated by the difference between measures. Data were expressed in
microL or area under curve (AUC - arbitrary units). Results: The edema induced by bradykinin was inhibited by anethole at 30
and 60 min, about 55% (BK: 1256 ± 179; anethole: 563 ± 93 AUC). However, estragole inhibited the edematogenic effect only at
30 min about 56% (BK: 4830 ± 703; estragole: 2138 ± 518 AUC). The edema induced by substance P was inhibited by anethole
and estragole, respectively at 30, 60 and 120 min. about 38% by anethole (substance P: 7286 ± 370; anethole: 4543 ± 850 AUC)
and about 63% by estragole (substance P: 6666 ± 373; estragole: 3949 ± 225 AUC). The edema induced by a nitric oxide donor
sodium nitroprusside was reduced by estragole at 120 min. about 30% (sodium nitroprusside: 19838 ± 1460; estragole: 13463 ±
1412 AUC), but not by anethole.
Conclusions:
Anethole and estragole present antiedematogenic activity following a common mechanism that involves inhibition of
inflammatory peptide mediators. However, the mechanism of the inhibitory effect of estragole includes nitric oxide, suggesting
that the structural difference between these molecules influence the response.
Keywords: ANETHOLE, ESTRAGOLE, PAW EDEMA
Financial Support: CAPES, FUNCAP; CNPq and College Christus. The laboratory technique Gabriela F. O
Resumo:17-197
EVALUATION OF THE ANALGESIC EFFECT OF VENLAFAXINE, A SEROTININ AND NORADRENALINE
REUPTAKE INHIBITOR.
Magalhães, R. A. 1; Felix, J. A. 1; Albuquerque, J. C. 1; Bastos, M. V. R. 2; Viana, G. S. D. B. 2; Fonteles,
M. M. D. F. 1; Félix, F. H. C. 3; Fontenele, J. B. 1
1
Farmácia / Faculdade de Farmácia, Odontologia e Enfermagem, UFC
2
Departamento de Fisiologia e Farmacologia, UFC
3
Hospital Infantil Albert Sabin, HAIS
Objectives:
The aim of this study was to test the analgesic effects of the antidepressant venlafaxine in several experimental models of
nociception.
Methods and Results:
Acetic acid-induced writhing: Groups of six to twelve mice were administered i.p. 0.6% acetic acid (10 mL kg−1, i.p.), and the
number of abdominal contractions was registered for 20 min, starting 30 min after the injection. Animals were pre-treated with
venlafaxine (10, 20, 40 and 60 mg kg−1, i.p.), 30 min before acetic acid administration. DMSO 5% was used as vehicle (control
group). With the exception of 10 mg kg−1, all other doses of venlafaxine (20, 40 and 60 mg kg−1, i.p) produced significant
inhibition of writhing (p
Conclusions:
These results corroborate researche that reported the inhibitory effect of venlafaxine on the signs of spontaneous pain and
hindpaw mechanical and thermal hypersensitivity in animal models of tonic nociception and neuropathic pain. Studies continue
in an attempt to elucidate the action mechanisms of these effects.
Keywords: venlafaxina, inibidores da recaptação de serotonina e, dor, antidepressivos, analgesia
Financial Support: UFC
Resumo:17-198
DIFFERENTIAL REGULATION BY THALIDOMIDE OF SILICA-ACTIVATED MACROPHAGES IN VITRO
Sant´anna, E. S. ; Ciambarella, B. T. ; Pires, A. L. A. ; Cordeiro, R. S. B. ; Martins, M. A. ; Silva, P. M. R.
E.
Laboratory of Inflammation / Oswaldo Cruz Institute, FIOCRUZ
Objectives:
Silicosis is a chronic lung disease induced by the inhalation of crystalline silica. Prolonged deposition of particles in the alveoli or
bronchioles leads to lung inflammation and the formation of fibrotic scar tissue. Silica particles can activate different structural
and inflammatory cells including epithelial cells and macrophages. We previously showed that curative treatment with
thalidomide reduced lung function compromise and granuloma formation in experimental silicosis in mice. In this study we
aimed to investigate the potential direct effect of thalidomide on silica-induced macrophage activation in vitro.
Methods and Results:
Male Swiss-Webster mice (18 – 20g) were used. Cells were recovered from the peritoneal cavity after gently washing with RPMI
1640 complete media. Before particle exposure, cells were plated at 5 × 10 5 cells/cm2 on round glass coverslips in 24-well dishes
in RPMI 1640 and allowed to adhere for 1 h at 37°C. The cells were then incubated with varying non-toxic concentrations of
silica (6.25 - 50 µg/mL), for 6 h at 37°C, and the dishes centrifuged at 1000 rpm for 10 min. The supernatant was recovered and
kept at -20oC for further analyses. We evaluated nitric oxide (NO) and IL-6 production by Greiss and ELISA techniques,
respectively. The cells were stained with Giemsa and phagocytosis was quantified under light microscope coupled to an image
analyzer system. The number of cells with internalized particles were counted and then divided by the total number of cells to
derive the percent phagocytosis. Survival rate was based on Trypan blue dye exclusion method. Macrophages were incubated
with different concentraitons of thalidomide (10-6 and 10-4 M), 1 hour before silica stimulation. All experimental procedures
were performed in accordance with the guidelines of the Committee on Use of Laboratory Animals of the Oswaldo Cruz
Foundation (L-034/09). Results: Exposure of macrophages with silica particles induced cell activation evidenced by the
morphological features and by the release of NO and IL-6. A concentration-dependent increase in the number of macrophages
with internalized particles was also detected. Pre-incubation of macrophages with thalidomide significantly reduced the levels of
IL-6 release, at the highest concentration, without affecting NO production. Interestingly, the phagocytosis rate increased under
condition of drug treatment. No cell death was noted under condition of silica stimulation or thalidomide treatment.
Conclusions:
Altogether our data show that thalidomide was able to differentially inhibit silica-induced chemical mediator production by
macrophages in vitro, under conditions where it potentiated phagocytosis. This indicates that cell activation and particle
internalization are not associated events. These findings also point out that macrophages seem to be potential pharmacological
targets for the anti-silicotic effect of thalidomide in mice.
Keywords: Silica particles, macrophages, IL-6, thalidomide
Financial Support: FIOCRUZ, CNPq, FAPERJ
Resumo:17-199
EFFECT OF NEW N-ACYLHYDRAZONES DERIVATIVES ANALOGOUS TO LASSBIO-294 ON
FORMALINÏ¿½INDUCED HYPERNOCICEPTION IN MICE.
Silva, R. V. ; Veloso, R. R. ; Nogueira, M. C. O. ; Maia, R. C. ; Lima, L. M. ; Barreiro, E. J. ; Miranda, A.
L. P
Departamento de Fármacos Faculdade de Farmácia - LASSBio, UFRJ
Objectives:
The pharmacophoric group N-acylhydrazone has shown an important anti-inflammatory and analgesic activity. The compound
LASSBio-294 was identified as an important cardiotonic prototype also presenting anti-inflammatory and analgesic profile
(Quim. Nova 25; 1172, 2002). A previous screening study has demonstrated the antinociceptive potential of a new series of Nacylhydrazone (NAH) derivatives modified from LASSBio-294. Some of them (LASSBio-1499 e LASSBio-1532) showed an
antinociceptive potency of about 3 �mol/kg (approx. 1 mg/kg) (SBFTE Congress, 2010). The aim of this study was to evaluate
this new series of NAH derivatives in inflammatory pain.
Methods and Results:
The antinociceptive activity was evaluated using the formalin-induced hipernociception test in mice. LASSBio derivatives (100
�mol/kg; p.o.) and the standard AINE celecoxib were administered 1h before the intra-plantar injection of formalin 2.5% (20
�l/paw). The time that mice spent licking or biting the injected paw was recorded and separated in two distinct periods: the first
period (earlier or neurogenic phase) was recorded 0-5 min after formalin injection and the second period (later or inflammatory
phase) was recorded 15-30 min after injection (Pain 51; 5, 1992). Results were expressed as the percentage of inhibition
compared to the vehicle control group (gum arabic 5%) (n = 6-12 animals; *p
Conclusions:
These findings suggest in part an anti-inflammatory profile for these compounds, since the hipernociception in the second phase
is a well characterized inflammatory response by the presence of neutrophils and of inflammatory mediators as TNF-α and PGs,
which can also be modulated by the activation of the primary sensor neurons by formalin. The involvements of others
mechanisms of nociception as the activation of vanilloid (TRPV1, TRPA1) and glutamate receptors must be investigate.
Therefore, we highlight LASSBio-1476 as a potent and promising analgesic and anti-inflammatory compound useful for chronic
inflammatory diseases like arthritis.
Keywords: formalin, hypernociception, inflammation, N-acylhydrazone
Financial Support: CNPq, FAPERJ, PIBIC-UFRJ, INCT-Inofar.
Resumo:17-200
PHOSPHODIESTERASE-5 INHIBITION BY TADALAFIL PROVIDES TNF-DEPENDENT ANALGESIA IN
EXPERIMENTAL ARTHRITIS
Nunes, R. D. M. 1,1,1,1; Leite, A. C. R. D. M. 1; Rocha, F. A. C. D. 1
1
Departamento de Biomedicina/Faculdade de Medicina, UFC
2
Departamento de Biomedicina/Faculdade de Medicina, UFC
3
Departamento de Biomedicina/Faculdade de Medicina, UFC
Objectives:
We investigated the effect of the phosphodiesterase (PDE)-5 inhibitor tadalafil (TD) in the acute hypernociception in the
zymosan (Zy) and anterior cruciate ligament transection (ACLT) arthritis models.
Methods and Results:
Rats were subjected to either intra-articular (i.art.) injection of 1mg Zy or surgical ACLT, used as an osteoarthritis (OA) model.
Controls received saline i.art. or sham operation, respectively. Joint pain was evaluated using the articular incapacitation test
measured over 6 h following Zy or between 4 and 7 days after ACLT. Cell counts, tumor necrosis factor-á (TNF), interleukin-1
(IL-1), and CINC-1 levels were measured in joint exudates 6 h after Zy. Groups received TD (0.02 – 0.5 mg kg-1 per os) or saline
2 h after i.art. Zy. Other groups received the µ-opioid receptor antagonist naloxone or the cGMP inhibitor 1H- [1,2,4] oxadiazolo
[4,3-a] quinoxalin-1-one (ODQ) prior to TD.Administration of TD dose-dependently inhibited hypernociception in Zy and OA
models. TD significantly decreased cell influx and TNF release in Zy arthritis while not altering IL-1 or CINC-1 levels. Pretreatment with ODQ but not with naloxone prevented the anti-inflammatory effects of TD.
Conclusions:
Therapeutic oral administration of TD provides analgesia mediated by guanylyl cyclase inhibition that is independent of
endogenous opioids release. This effect of TD is associated to a decrease in neutrophil influx and TNF release into inflamed
joints.
Keywords: Tadalafil, Phosphodiesterase, Arthritis, Pain, Cytokines
Financial Support: CNPq,CAPES
Resumo:17-201
EVALUATION OF THE PHARMACOLOGICAL PROPERTIES OF PARTITIONS OF THE CRUDE EXTRACT OF
MYRSINE CORIASEA
Carrasco, L. P. S. 1; Carvalho, M. F. 1; Leite, M. M. D. S. 1; Souza, P. A. D. 2; Cortes, W. S. 1
1
Dept de Ciências Fisiológicas/Instituto de Biologia, UFRRJ
2
Instituto de Química, UFRJ
Objectives:
To evaluate the pharmacological properties of of the partitions ethyl acetate (EAE), hexane (EH)and buthanolic (EB) of crude
ethanol extract (EE) of Myrsine coriacea, in order to justify its potential use in human and veterinary medicine.
Methods and Results:
Antinociceptive activity was evaluated by the methods of formalin (J. Neurosci. Meth. 14, 69, 1985), peritonitis (Agents and
actions 32, 283, 1991) and pleurisy. In the method of formalin was investigated the mechanism of antinociceptive action. In this
method of solution 3% formalin is injected into the plantar hind paw of the hind paws of a mouse and observe the appearance of
two phases of the genesis of pain, phase 1 (F1) and 2 (F2) (F1, neurogenic pain and F2, inflammatory pain). We used the
following treatment groups: tap water, indomethacin (10 mg/kg) and the test group EB at a dose of 1 g/kg. The group treated with
EB showed no inhibition of F1 compared to control water. In the F2 this group inhibited by 56% (92.17 ± 22.95 seconds, P
Conclusions:
Therefore, our results show an antinociceptive action of hexane, ethyl acetate and butanol partitions of M. coriacea. But there
was no central antinociceptive and anti-inflammatory mechanism seems to be involved in the antinociceptive action of these
extracts.
Keywords: antinflamatory, antinociception, Myrsine coriacea, pain
Financial Support: PIBIC/CNPq, DCF/IB/UFRRJ
Resumo:18-039
STUDY OF ANTIMICROBIAL AND ANTI-INFLAMMATORY ACTIVITY OF AN ANALOG OF PYRAZINAMIDE
Mendonça, M. S. A. ; Candéa, A. L. P. ; Lima, C. H. S. ; Souza, M. V. N. D. ; Henriques, M. G. M. O.
Laboratório de Farmacologia Aplicada, Fiocruz
Objectives:
Tuberculosis is the most important cause of mortality due to a single infectious agent worldwide. The treatment for this infection
is based on drugs developed in the 60´s. The pyrazinamide analogs synthesis has been an important field of research because of
its wide spectrum of pharmacological activities. In this context, the synthesis of pyrazinamide analogs has been an important area
of research, due to the broad spectrum of pharmacological activities. Recently we have selected by in vitro and in vivo pleurisy
studies three potential analogs, and among them the analogue 55 (A55) was selected for the experiments. The aim of this study
was to evaluate the anti-mycobacterial and immuno-modulatory activity of a pyrazinamide analog, A55, in macrophages infected
with BCG in vitro.
Methods and Results:
J774 cells (macrophages cell line) were infected for a period of 5 hours and subsequently treated with different concentrations of
the A55 and pyrazinamide (0.1 M, 0.01 M, 0.001 M, 0.0001 M, 0.00001 M) for 24 hours. The cytotoxicity of the analog was
analyzed by the Alamar blue method and the cell supernatant was collected determine nitrite concentration. Initially we observed
that the analog is cytotoxic for the cells when they are infected, in higher doses (0,001 M; 0,01 M;), while pyrazinamide
possessed little or no effect. This analogy in higher concentrations (0,001 M; 0,01 M; 0,1 M), can induce mycobacteria killing
(>20%). Regarding the release of nitric oxide in the supernatants, we observed that the analog was able to induce an increased
release (>40%) of this mediator in the highest concentration used (0.1 M), while pyrazinamide induced no change.
Conclusions:
Our results indicate that the analog has better in vitro antimicrobial profile in relation to pyrazinamide, due to its higher cytotoxic
activity to mycobacteria and increased production of nitric oxide.
Keywords: Tuberculosis, Pyrazinamide, Inflamation, Mycobacteria
Financial Support: PIBITI/Fiocruz, CNPq, FAPERJ, Farmanguinhos
Resumo:18-040
IN VITRO EFFECTS OF HYDROCORTISONE AND CYCLOSPORINE A IN THE DEVELOPMENT OF MURINE
THYMOCYTES
Costa, K. M. 1; Paiva, L. S. 2; Rumjanek, V. M. 1
1
Instituto de Bioquímica Médica, UFRJ
2
Departamento de Imunobiologia/Instituto de Biologia, UFF
Objectives:
P-glycoprotein is a transmembrane protein involved in the transport of several lipophilic compounds. Although this glycoprotein
was first described in the phenomenon of multidrug resistance, its expression also occurs in normal cells, as in thymocytes. The
expression of P-glycoprotein in these cells seems to be regulated during development and the subpopulation that does not show
activity related to P-glycoprotein is the most sensitive to exposure to glucocorticoids. Furthermore, corticoids are P-glycoprotein
substrates. This suggested the involvement of this transporter protein on the control of intracellular levels of glucocorticoids.
There are evidences that glucocorticoids play a regulatory role on the thymus, more specifically, on the thymic selection. To
understand the role of this glycoprotein on susceptibility to glucocorticoids, we treated thymocytes with hydrocortisone, a
glucocorticoid, and cyclosporine A, a known inhibitor of P-glycoprotein and evaluated the amount of viable cells.
Methods and Results:
The suspension of thymocytes extracted from C57BL/6 females mice of 4-6 weeks old or 3-5 months old were treated with
hydrocortisone (0.01 - 0.1 µM) and / or cyclosporine A (0.2 or 0.4 µg/ml) for 17-18 hours. After treatment, we counted the
number of viable cells by Trypan Blue dye exclusion. For analysis of P-glycoprotein activity, cells were incubated with 0.2 µg/ml
rhodamine 123, fluorescent substrate of P-glycoprotein, in the presence or absence of 0.4 µg/ml of cyclosporine A for 30 minutes.
The cells were washed and incubated with medium in the presence or absence of cyclosporine A for 30 minutes. After that time,
cells were stained with anti-mouse CD4/CD8 and analyzed by flow cytometry. The in vitro treatment with hydrocortisone caused
a dose-dependent decrease in the amount of thymocytes (n=3). The most immature subpopulations were more sensitive to
treatment, whereas mature cells were more resistant. Treatment with cyclosporine A showed a tendency to dose-dependent
reduction in the number of viable thymocytes (n=3). The effect of treatment with hydrocortisone and cyclosporine A was
predominantly additive only in CD4-CD8- and CD4+ CD8+ cells from mice of 4-6 weeks old (n=5). In the other subpopulations
and in mice of 3-5 months old, there were no significant differences between treatment with hydrocortisone alone and in the
presence of cyclosporine A. Finally, we found that treatment with hydrocortisone did not increase the percentage of cells with Pglycoprotein activity compared to control (n=6), demonstrating that the activity of this glycoprotein did not affect treatment with
glucocorticoid.
Conclusions:
Treatment with hydrocortisone reduced the amount of cells independent of P-glycoprotein activity, suggesting that susceptibility
of thymocytes to glucocorticoid exposure is not directly related to the activity of this glycoprotein.
Keywords: Thymocytes, Glucocorticoids, P-glycoprotein, Cyclosporine A
Financial Support: CNPq, FAPERJ.
Resumo:18-041
EVALUATION OF LIPID AND PROTEIN OXIDATION AND BUTYRYLCHOLINESTERASE ACTIVITY IN
SERUM OF PATIENTS WITH INDETERMINATE FORM OF CHAGAS’ DISEASE
Schlemmer, J. B. ; Souza, V. D. C. G. ; Thorstenberg, M. L. ; Castilhos, L. G. ; Rocha, B. C. ; Schlemmer,
K. B. ; Bertoldo, T. M. D. ; Gonçalves, J. F. ; Costa, P. ; Leal, D. B. R.
Departamento de Microbiologia e Parasitologia, UFSM
Objectives:
Aim: Chagas¡¯ disease is caused by Trypanosoma cruzi and is characterized by a chronic inflammation. It is known that changes
in the redox state and regulation of inflammatory process are associated with the progress of several chronic inflammations such
as Chagas¡¯ disease. Therefore, the oxidative profile determined by the lipid peroxidation, protein carbonylation and
butyrylcholinesterase (BuChE) enzyme activity were determined in the serum of patients with indeterminate form of Chagas¡¯
disease.
Methods and Results:
Methods and Results: This study was performed in a control group constituted by 22 T. cruzi serum-negative subjects with an
average age of 47 years old and an infected group constituted by 22 patients with indetermined form of Chagas¡¯ disease (IFCD)
with an average age of 54 years old, selected from the University Hospital of Santa Maria. The protocol was approved by the
Human Ethics Committee from the Federal University of Santa Maria under the number 243/2009 and all the subjects gave
written consent. The lipid peroxidation was determined in serum samples by the thiobarbituric acid method (Free Radic Biol
Med. 20:251-256,1996) with the formation of malondialdehyde (MDA) which it was readed at 532 nm. The results were
expressed as nmoles MDA/mL of serum. The carbonylation of serum proteins was determined by addition of 2,4-dinitrophenylhydrazine to serum as previously standardized (Methods Enzym 186:464-478). The proteins were precipited by 10%
trichloroacetic acid and incubated at 37¨¬C in phosphate buffer, pH 8.0. After this, the color intensity of the supernatant was
measured at 370 nm and the carbonyl content was expressed as nmol/mg proteins. The BuChE enzymatic assay was determined
in serum by a modification of the spectrophotometric method (Biochem. Pharmacol. 7:88-95, 1961). The method is based on the
formation of the yellow anion, 5,5¡Ç-dithio-bis-acid-nitrobenzoic, measured by absorbance at 412 nm during 2-min incubation at
25¡ÆC. The enzyme activity was expressed in ¥ìmol BuSCh/h/mg of protein. All assays were run in duplicate and the data were
analyzed statistically by the Student¡¯s t test. Differences were considered significant when the probability was P<0.05).
Conclusions:
Conclusion: Given the results of TBARS and protein carbonyl, we can conclude that the Chagas¡¯ disease induces a redox
imbalance which leads to alterations in cell function by oxidation and damage of macromolecules, membranes, proteins and
DNA. These results can be explained by a possible ineffective antioxidant response and also because Chagas¡¯ disease develops
an inflammatory process, with the formation of reactive oxygen (ROS), mediated by cytotoxic reactions by the immune system.
The decreased of BuChE activity in the IFCD group suggests an increase in the serum levels of the acetylcholine in this group,
possibly in response to inflammatory damages triggered by the invasion of Trypanosoma cruzi in several tissues, since the
acetylcholine has anti-inflammatory properties.
Keywords: Butyrylcholinesterase, Chagas' Disease, Lipid Oxidation, Protein Oxidation
Financial Support: FIPE, CNPq, CAPES
Resumo:18-042
INTERACTION OF MYCOBACTERIUM LEPRAE WITH HUMAN MACROPHAGES AND DENDRITIC CELLS:
ROLE OF NITRIC OXIDE AND EXPRESSION OF SURFACE MOLECULES.
Souza, I. C. C. ; Castro, H. C. ; Santos, D. O.
Departamento de Biologia Celular e Molecular, UFF
Objectives:
Leprosy is a chronic infectious disease caused by Mycobacterium leprae . The clinical manifestations depend on the patient's
immune response to the bacillus. These findings suggested that differences between the tuberculoid and lepromatous leprosy are
correlated with the presence of different profiles of T cells. However, determining the profile of T cells (Th1 or Th2) primarily
depends on the nature of the antigen-presenting cell (APC), as already demonstrated by our group, demonstrating the reversal of
the inefficiency of the cellular immune response against M. leprae in lepromatous leprosy when dendritic cells (DCs) were used
as APCs.(Clin Exp Derm 32(1):75, 2006). Nitric oxide (NO) and its reactive intermediates (RNI) are microbicides, also
characterizing it as a mediator in the defense against infections. The aim of this study was to investigate the interaction of M.
leprae to macrophages and dendritic cells derived from monocytes isolated from human peripheral blood using the nitric oxide
production and expression of surface molecules as parameters.
Methods and Results:
This project is registered in the Ethics Committee of HUCFF (CAAE 0157.0.197.000-09). Mononuclear cells were purified from
peripheral blood of healthy donors and then monocytes were isolates by ―cold aggregation‖ (J Leprosy 70:15, 2001). Monocytes
were cultured in labteks (2x105/ml) in the presence or not of rhIL-4 (1000U/mL, Innogenetics, Belgium) and rhGM-CSF
(100U/mL, Schering-Plough, Belgium) for 7 days, to differentiate in dendritic cells of macrophages, respectively. After
differentiation, and inhibitor of nitric oxide synthase was additioned (L-NAME 20µM) for 1 hour. After that, cells were infected
with M. leprae (20µg/mL, Colorado University, USA) or BCG (FIOCRUZ). Cells were fixed with paraformaldehyde and stained
by Kynioun method. Part of the cultures was destinated to flow citometry. By light microscopy, we observed a large increase in
infection by M. leprae in the presence of L-NAME, both in macrophages and in dendritic cells, suggesting that nitric oxide is
essential for cell defense against infection. By flow cytometry we have seen an increase in the expression of CD209 when
macrophages are infected by M. leprae , which together with the absence of NO and RNI, facilitate the entry of M. leprae in the
cell. When dendritic cells were stimulated with BCG, there was a significant increase in the expression of CD209 compared to
the macrophages, confirming the importance of DCs in the construction of an effective immune response.
Conclusions:
By light microscopy, we could conclude that macrophages are more likely to be infected by M. leprae than dendritic cells, and
the inhibition of nitric oxide synthesis allows the entry and permanency of M. leprae and BCG inside the cells, macrophages or
dendritic cells, caractherizing nitric oxide as a important mediator in combating infections. By flow citometry, we observed that
M. leprae induce DC-SIGN expression in macrophages, as observed by the CD209 marking.
Keywords: dendritic cells, leprosy, nitric oxide, Mycobacterium leprae
Financial Support: CNPq, FAPERJ, UFF
Resumo:18-043
ASCARIS SUUM EXPERIMENTAL INFECTION MODULATES ACUTE INFLAMMATION VIA
CD4+CD25+FOXP3+ T CELLS.
Titz, T. D. O. ; Macedo-soares M. F; Araujo C. A. A
Instituto Butantan, IBUT
Objectives:
The aim of this study was to investigated the expansion of T cell clones by Ascaris suum infection and the effect of these cells on
LPS-induced acute inflammation
Methods and Results:
Methods: BALB/c mice weighting about 22 g were infected by intragastric route with 2500 Ascaris suum embryonated eggs. A
week later the mesenteric lymph node cells were recovered. The expression of CD4+CD25+FoxP3+ markers in lymph node cells
from non-infected and Ascaris-infected mice was detected using PE-Cy5.5-anti-CD25, FITC-anti-CD4 antibodies and PE-antiFoxP3 antibodies. All samples were acquired using FACSCalibur and analyzed by FlowJo software. The TCD4+CD25- and
TCD4+CD25+ cell population from infected or non-infected mice were purified from lymph node cells by magnetic activatedcell sorter (MACS). These cells were adoptively transferred to recipient mice by intravenous route and 3 days later the air
pouches induced by sterile air injections on the back of the recipient mice were LPS-injected. Control group received only PBS in
their pouches. Three hours after the stimulation, the air pouche exsudates were recovered to the measurement of cytokines levels
by ELISA and quantification of cellular migration. Results: FACS analyses showed the percentage of T cell subpopulations from
non-infected and Ascaris suum-infected mice. The total CD4+ T cell numbers did not differ in Ascaris suum-primed and nonprimed total cells. The number of CD25+FoxP3+ cells gated on CD4+ cell population presented a 4 fold increase in Ascarisprimed total cells compared to non-primed cells. Furthermore, CD4+CD25-FoxP3+ cell numbers were also higher in Ascarisprimed total cells (14.1%) compared to non-primed cells (4.8%). Regarding the suppressive effect of these cell clones our results
demonstrated that adoptive transfer of CD4+CD25+T cells from infected mice resulted in decreasing of leukocyte recruitment
(3.5 fold) and inflammatory cytokines levels (IL-1ƒÒ, IL-6 and TNF-ƒÑ), but increasing of IL-10 and TGF-ƒÒ cytokines. On the
contrary, transfer of TCD4+CD25- cells or TCD4+CD25+ from non-infected mice had no effect on leukocyte influx or cytokines
levels into the air pouches compared to the positive control group
Conclusions:
In our previous studies, the regulatory cytokine IL-10 have been implicated in the down-regulatory mechanisms induced by
Ascaris suum. The results obtained herein indicate that the source of these cytokines may be CD4+CD25+ T cells. Thus, when
these cells, but not other T cell types, were obtained from lymph nodes of Ascaris-infected mice and adoptively transferred to
recipient mice, they prevented the inflammatory response induced by LPS. In conclusion the present study shows that Ascaris
suum infection expands the CD4+CD25+FoxP3+ T cells clones producing IL-10, which are responsible for the anti-inflammatory
activity.
Keywords: Ascaris suum, Infection, immunemodulation
Financial Support: Financial support: CNPq and FAPESP
Resumo:18-044
OLEIC ACID SUPLEMENTATION ATTENUATES INFLAMMATION AFTER A CLP-INDUCED SEPSIS IN SWISS
MICE
Medeiros-de-moraes, I. M. ; Campbell, C. ; Magno, F; Araújo, C. V ; Bozza, P. T. ; Gonçalves-dealbuquerque, C. F; Silva, A. R. ; Castro-faria-neto, H. C
laboratório de Imunofarmacologia/FIOCRUZ, FIOCRUZ
Objectives:
sepsis accounts for a huge number of deaths in intensive care units worldwide. Sepsis describes a complex clinical syndrome that
results from an infection, setting off a cascade of systemic inflammatory responses that can lead to multiple organ failure and
death. Leite et al has showed that mice fed for 6 weeks with an olive oil diet were resistant to endotoxic shock, with 60% survival
at 168 h (Shock 23; 173, 2005). Olive oil is composed by different polyunsaturated fatty acids such as omega 3 and 6 but the
monounsaturated fatty acid omega 9, also known as oleic acid (OA), that is the main component of olive oil - highly consumed in
Mediterranean diet. We aim to investigate the role of OA in an experimental model of sepsis.
Methods and Results:
Swiss mice were given daily doses (orally) of OA, at 282 µg/animal, for 14 days. Control animals received saline. In the sixteenth
day polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Immediately after the procedure all mice received
volemic reposition and after 6 h animals were given imipenem. 24 h after surgery mice were euthanized and the peritoneal cavity
was rinsed with sterile saline. Total leukocyte counts were performed in a Neubauer chamber and differential leukocyte analysis
done in cytosmears stained with May-Grunwald Giemsa. The supernatant and serum were collected for cytokine quantification
and biochemical analysis. In another set of experiments survival rate was determined daily for 7 days in separate groups of at
least 10 animals for each condition. Mice fed with OA were resistant to sepsis, with a 90% survival rate at 168 h compared to
saline treated mice (58%). OA supplementation in CLP-subject animals led to a significantly decrease in the total leukocyte
counts (32.33 x 106 ± 3.93 - mean ± SEM), mainly neutrophils, compared to mice that received saline (46.10 x 106 ± 2.84).
However, in mice that consumed OA the levels of TNF, IL-10, IL-6 and PGE2 were not significantly different from mice fed
with saline submitted to CLP. Moreover, mice fed with OA had a significantly lower level of bacteria (13.6 x 106) in the
peritoneal lavage leukocyte compared to mice submitted to CLP (59.6 x 106). The hepatic and renal functions were assessed by
quantifying the serum levels of aspartate transaminase and creatinine.
Conclusions:
our data suggests that pretreatment with OA reduces mortality in an experimental model of sepsis possibly through immune
response modulation. One mechanism involved may be due to increased bacterial clearance in mice fed with OA. More data are
required to clarify this mechanism of increased survival.
Keywords: oleic acid, sepsis, inflammation
Financial Support: CNPq, FIOCRUZ and FAPERJ
Resumo:18-045
LEUKOTRIENE B4/BLT1 MEDIATES γδ T LYMPHOCYTE MIGRATION DURING INFLAMMATION
Souza-martins, R. 1; Costa, M. F. S. 1; Souza, M. C. 1; Piva, B. 2; Diaz, B. L. 2; Peters-golden, M. 3;
Henriques, M. G. M. O. 1; Canetti, C. A. 2; Penido, C. 1
1
Fundação Oswaldo Cruz - Farmanguinhos, FIOCRUZ
2
Lab. de Inflamação - Universidade Federal do Rio de Janeiro, UFRJ
3
University of Michigan Health System , UMHS
Objectives:
γδ T lymphocytes are unconventional T cells that comprise a minor subset of T cells in lymphoid organs, which are preferentially
distributed in peripheral tissues. These cells exert an early pro-inflammatory role followed by a subsequent regulatory role in an
attempt to restrain the inflammatory response. γδ T lymphocytes increase in number at inflammatory sites during infection and
allergy, a phenomenon mediated via migration towards chemotactic factors and/or local proliferation. In this context, the aim of
this project is to investigate the involvement of the 5-lipoxygenase (5-LO)-derived lipid mediator leukotriene (LT)B4 in γδ T cell
migration and activation.
Methods and Results:
Pleurisy was induced by an intrapleural (i.pl.) injection of LTB4 (0.5 μg/cavity), LPS (250 ng/cavity), OVA (12.5 μg/cavity), or
Mycobacterium bovis BCG (4x105 CFU/cavity). OVA challenge was induced in mice 14 days after prior sensitization by a
subcutaneous injection of 200 μl of a mixture of OVA (50 μg) and aluminum hydroxide (5 mg). Pleural cells were recovered
from thoracic cavities after washing with 500 μl of PBS containing EDTA (10 mM, pH 7.4). The i.pl. injection of LPS triggered
increased levels of LTB4 in C57BL/6 mouse pleural cavities (saline: 12,27+13,56; LPS: 47,74+6,18 pg/mL). The in vivo
inhibition of 5-lipoxygenase (5-LO) activity by zileuton, impaired LPS-induced γδ T cell accumulation into pleural cavities
(saline: 6,33+0,75; LPS:12,14+1,18; zileuton: 8,00+0,72 x 103 cells/cavity). Accordingly, 5-LO knockout (5-LO-/-) mice failed
to recruit γδ T cells into the inflammatory site after LPS i.pl. stimulation (WT-saline: 4,11+1,31; WT-LPS: 19,87+4,19; 5-LO-/- saline: 4,19+1,27; 5-LO-/--LPS: 6,42+1,81 x 103 cells/cavity). The antagonism of high affinity LTB4 receptor, BLT1, by
CP105,696 was also capable to impair γδ T cell accumulation in mouse pleural cavities induced by LPS (saline: 2,45+1,11; LPS:
10,72+0,87; CP105,696: 7,01+0,53 x 103 cells/cavity). Confirming that γδ T lymphocytes can directly respond to LTB4, we
demonstrate that naïve resident γδ T lymphocytes, as well as LPS recruited γδ T cells express BLT1 receptor and isolated γδ T
cells were found to undergo actin cytoskeleton reorganization when incubated with LTB4 in vitro. LTB4/BLT1 also accounted
for γδ T cell migration induced by BCG (saline: 5,53+0,857; BCG: 22,04+5,47; CP105,696: 6,82+1,33 x 103 cells/cavity) or
during antigen challenge in sensitized mice (saline: 3,51+0,98; OVA: 29,42+3,16; CP105,696: 13,29+4,79 x 103 cells/cavity).
Conclusions:
These data show that LTB4/BLT1 pathway mediates γδ T cell migration into the inflammed tissue in response to diverse stimuli.
Keywords: γδ T lymphocytes, Leucotriene B4, CCL2/MCP-1, cell migration, cell activation
Financial Support: CNPq, FAPERJ, FIOCRUZ.
Resumo:18-046
PROLIFERATIVE ACTION INDUCED BY ONIL, A NOVEL LECTIN PURIFIED FROM TILAPIA FISH
Silva, C. D. C. 1; Coriolano, M. C. 1; Melo, C. M. L. D. 3; Silva, M. A. L. D. 1; Carvalho, E. V. M. M. D. 1;
Bezerra, R. D. S. 1; Santos, A. J. G. D. 7; Pereira, V. R. A. 3; Coelho, L. C. B. B. 1
1
Depart. de Bioquímica/UFPE, UFPE
3
Centro de Pesquisas Aggeu Magalhães (CPqAM/Fiocruz), Fiocruz
7
DEPAq/UFRPE, UFRPE
Objectives:
This in vitro study analyzed cellular proliferation and cytokine production in mice splenocytes cultures treated with OniL, a lectin
purified from Oreochromis niloticus serum.
Methods and Results:
Methods: Experimental assays used mice (BALB/c, male, 30 days old, 5/group); all animals were killed and treated in
accordance with the guidelines of the Oswaldo Cruz Foundation Commission for Experiments (Rio de Janeiro, Brazil) with
Laboratory Animals (Ministry of Health, Brazil, 0266/05). OniL is a lectin from serum of tilapia fish (Oreochromis niloticus)
purified by affinity chromatography on Concanavalin A-Sepharose 4B (Sigma). Cancanavalia ensiformis (Concanavalin A – Con
A) was purchased from Sigma Chemical Co. Cellular proliferation assay was performed by [3H]-thymidine incorporation. Cells
of each group were in vitro treated for 24 h with Con A (2.5 µg/mL) and OniL (2.5, 5 and 10 µg/mL) to evaluate the proliferative
activity of lectins. The unstimulated culture plate was used as a negative control. SI values ≥ 3 were considered representative of
positive proliferation. Cytokine production was measured by sandwich ELISA and IL-2 and IL-6 cytokines were quantified. Each
well received Con A (2.5 µg/mL) or OniL (10 µg/mL), and supernatants from cultures stimulated in vitro with lectins were
obtained at 24, 48, 72 h and 6 days. Cells kept in culture medium were also obtained as a negative control. Cell viability assay
was performed by Annexin V conjugated with fluorescein isothiocyanate and propidium iodide. Splenocytes (106 cells) were
stimulated with lectins. Annexin-FITC-⁄PI+ cells were considered necrotic cells and Annexin-FITC+⁄PI-represented splenocytes
in the early stage of apoptosis. Nonparametric assays were used in statistical analysis. All results were expressed as mean values
of groups ±standard deviation and analyzed considering the value of P < 0.05 as statistically significant. Results: A great number
of lectins with distinct characteristics and specificities have been used for their immunomodulatory and proliferative activities,
but few studies show immunological profile induced by fish lectins. Higher and statistical indices of proliferation were induced
by OniL in relation to control cells. OniL showed higher mitogenic activity in relation to control and Con A for all analyzed
concentrations. Higher IL-2 and IL-6 production was promoted by OniL; sometimes this production was also superior to Con A.
Results of viability assay showed that OniL did not promote cell damage in neither analyzed protocols. Beyond, Con A induced
higher apoptosis and necrosis in both 24 and 48 h in relation to control and OniL cultures.
Conclusions:
OniL induces higher proliferative response; this lectin can be used as a mitogenic agent in immunostimulatory assays
Keywords: LECTIN, TILAPIA, PROLIFERATIVE ACTION, IMMUNOMODULATORY, CYTOKINE
Financial Support: CNPq, FACEPE
Resumo:18-047
LECTIN OF COBIA SERUM, RCAL, PROMOTED MITOGENIC RESPONSE IN MICE BALB/C SPLENOCYTES
Coriolano, M. C. 1; Silva, C. D. C. D. 1; Melo, C. M. L. 2; Bezerra, R. S. 1; Santos, A. J. G. 3; Pereira, V. R.
A. 2; Coelho, L. C. B. B. 1
1
DBIOq/UFPE, UFPE
2
CPqAM/FIOCRUZ, FIOCRUZ
3
DEPAq/UFRPE, UFRPE
Objectives:
Aim: The aim of this study was to evaluate the proliferative response and cytokine production in mice splenocytes in vitro
stimulated with a novel Rachycentron canadum lectin of mannose recognition, RcaL.
Methods and Results:
Methods:Experimental assays utilized mice (BALB/c, male, 30 days old, 5/group). All mice were killed and treated in
accordance with the guidelines of the Oswaldo Cruz Foundation Commission for Experiments (Rio de Janeiro, Brazil) with
Laboratory Animals (Ministry of Health, Brazil, 0266/05). RcaL is a lectin isolated and purified from serum of cobia fish (R.
canadum) obtained by affinity chromatography on Concanavalin A-Sepharose 4B (Sigma). Canavalia ensiformis (Concanavalin
A – Con A) was purchased from Sigma Chemical Co. Cellular proliferation assay was performed by [3H]-thymidine
incorporation. Cells of each group were treated, in vitro, for 24 h with Con A (2.5 µg/mL) and RcaL (2.5, 5 and 10 µg/mL) to
evaluate the proliferative activity of these lectins. The unstimulated culture plate was used as a negative control. SI values ≥ 3
were considered representative of positive proliferation. Cytokine production was measured by sandwich ELISA; IL-2 and IL-6
cytokines were quantified. Each well received Con A (2.5 µg/mL) or RcaL (10 µg/mL) lectins, and supernatants from cultures
stimulated in vitro with lectins were obtained at 24, 48, 72 h and 6 days. Cells maintained in culture medium were also obtained
as a negative control. Cell viability assay was performed by Annexin V conjugated with fluorescein isothiocyanate and propidium
iodide. Splenocytes (106 cells) were stimulated with lectins. Annexin-FITC-⁄PI+ cells were considered necrotic cells and
Annexin-FITC+⁄PI-represented splenocytes in the early stage of apoptosis. Nonparametric tests were used in statistical analysis.
All results were expressed as mean values of groups ±standard deviation and analysed considering the value of P < 0.05 as
statistically significant. Results: Mannose binding lectins are known by their immunostimulatory properties, such as cell
proliferation and cytokine production. Higher and statistical indices of proliferation were induced by RcaL in relation to control
cells. Although Con A stimulus promoted statistical values of proliferation in relation to control the stimulation was lower in
relation to RcaL. Furthermore, RcaL and Con A induced higher IL-2 and IL-6 production; results showed similar behavior for
both lectins. In contrast, RcaL and Con A showed similar behavior and induced higher IL-6 production. Only late apoptosis was
promoted by RcaL treatment and it also induced higher necrosis in relation to Con A.
Conclusions:
Conclusions: Results showed that RcaL induces proliferative response and suggested that this lectin can be used as a mitogenic
agent in immunostimulatory assays.
Keywords: CELLULAR PROLIFERATION, COBIA, CYTOKINE, IMMUNOSTIMULATORY PROPERTIES, LECTIN
Financial Support: FACEPE, CNPq.
Resumo:18-048
TSPO LIGAND MODIFY ADHESION MOLECULE EXPRESSION DUE TO BLOCKAGE OF CALCIUM INFLUX
INDUCED BY FMLP
de Lima, C. B. 1; Tamura, E. K. 3; Markus, R. P. 3; Neto, J. P. 2; Farsky, S. H. P. 1
1
Laboratory of Experimental Toxicology, FCF-USP
2
Laboratory of Applied Pharmacology and Toxicology, FMVZ-USP
3
Department of Physiology, IB-USP
Objectives:
Benzodiazepines (BZD) are one of the most commonly used groups of anxiolytic drugs, whose effects are mediated through
binding on neuronal post-synaptic plasma membrane GAGAA receptors. However, a second class of benzodiazepine binding
sites, translocator protein (TSPO), has been found in many peripheral tissues and cell populations, as immune and endothelial
cells. Studies have been shown that TSPO may be related, directly or indirectly, to the modulation of BZD on immune system.
Therefore, these mechanisms involved in the actions of BZD via TSPO are not fully comprehended. This worked investigated
TSPO-binding drugs effects on leukocyte-endothelial interactions, on adhesion molecules expressions and calcium influx.
Methods and Results:
Intravital microscopy assays were performed in the mesentery microcirculation of male Wistar rats (50-60 days old) after topical
application of TSPO ligands (PK 11195 or Ro 5-4864; 100nM) and fMLP (10-8M; topical application). In other set of assay,
circulating leukocytes were in vitro incubated with TSPO ligands (100nM) and 1 hour after incubated with fMLP (10-8M) to
quantify adhesion molecules expressions by flow cytometry. To evaluate calcium influx by confocal microscopy, isolated
neutrophils were incubated with Fluo-3AM previously to TSPO ligands (100nM) and fMLP (10-8M) application All procedures
were performed according to protocols approved by the Brazilian Society of Science of Laboratory Animals for proper care and
use of experimental animals. fMLP stimulation decreased rolling cells and increased adherent cells in control animals, and Ro54864 pre-treatment abolished these effects. In vitro fMLP stimulation reduced and enhanced L-selectin and &beta2-integrin
expression on neutrophils, respectively, and Ro5-4864 treatment inhibited the reduction on L-selectin expression caused by
fMLP. fMLP in vitro stimulation enhanced PECAM-1 expression, but Ro5-4864 abolished this effect. Ro5-4864 inhibited and
PK11195 enhanced maximal response and AUC of calcium influx induced by fMLP.
Conclusions:
Results show that TSPO ligand alters in vivo leukocyte-endothelial interactions evoked by fMLP and Ro5-4864, but not
PK11195, blocks the fMLP effect on neutrophil L-selectin and PECAM-1 expression which may be dependent on inhibition of
calcium influx.
Keywords: adhesion molecules, calcium, TSPO
Financial Support: FAPESP 09/52245-1
Resumo:18-049
PRODUCTS DERIVED FROM LEUKEMIC CELLS MODULATE THE GENERATION OF DENDRITIC CELLS IN
DIFFERENT STAGES
Motta, J. M. ; Rumjanek, V. M.
Instituto de Bioquímica Médica, UFRJ
Objectives:
Dendritic cells (DCs) are professional antigen-presenting cells, specialized in activating T lymphocytes. DCs play an important
role in controlling tumors, since they are able to recognize and capture tumor antigens, initiating an antitumoral immune
response. However, tumor cells have developed mechanisms to inhibit immune responses, favoring tumor progression. Some
products secreted by tumor cells present immunosuppressive characteristics and they can affect different kinds of immune cells,
including DCs. There are many studies involving products released by solid tumors and how they modulate DCs, but there is a
paucity of information about products released by leukemic cells. In this case, leukemic cell factors are secreted in the blood,
where monocytes (DCs precursors) are present; therefore they could present alterations in their development. To investigate this
hypothesis, we decided to analyze a possible influence of leukemic cell products on the development and function of DCs in
different stages.
Methods and Results:
For this, the tumor cell line used was K562, derived from chronic myeloid leukemia. Monocytes, obtained from healthy donors,
were separated by Ficoll-Hystopaque density gradient and cultured in the presence of IL-4 and GM-CSF, stimuli for DC
differentiation, and also in the presence of K562 supernatant. After 5 days, CD14, CD1a expression and dextran phagocytosis
were evaluated in these cells by flow cytometry. In another experiment, looking at the effect of tumor products on DC activation,
immature DCs were cultured with TNF-alpha for a further 2 days. After this period, CD83 expression by DCs was measured by
flow cytometry. Moreover, activated DCs were co-cultured with lymphocytes obtained from a second donor for 24 hours and
lymphocyte‘s expression of CD69 was evaluated. During a control differentiation, monocytes down regulate CD14 expression
and start to express CD1a. In this stage, immature DCs present high phagocytic capacity. In the presence of tumor supernatant,
CD14 expression remained high and CD1a expression was low (N=6). However, the addition of tumor supernatant did not
interfere with dextran phagocytosis by monocytes stimulated to differentiate into DCs (N=4). If activated, DCs considerably
increase CD83 expression and acquire high ability to activate lymphocytes. It was observed that monocytes differentiated in the
presence of tumor supernatant and then stimulated to activation expressed less CD83 (N=3). Moreover, tumor products appear to
inhibit the appearance of CD69 expression on lymphocytes co-cultured with DCs differentiated with tumor supernatants (N=2).
Conclusions:
Finally, these results suggest that soluble products released by leukemic cells affect DC differentiation and activation, suggesting
that, in this condition, these cells become unable to complete their development.
Keywords: dendritic cells, differentiation, leukemic cell products
Financial Support: CAPES, CNPq and FAPERJ
Resumo:18-050
MODULATION OF MONOCYTE SURFACE RECEPTORS BY OUABAIN
Teixeira; M. P. C. ; Valente; R. C. ; Rumjanek; V. M.
INSTITUTO DE BIOQUÍMICA MÉDICA, UFRJ
Objectives:
Ouabain is a steroid produced by hypothalamus and adrenal glands that is capable of binding to and inhibiting Na+K+ATPase.
Also, ouabain is able to trigger different cell signaling pathways. Regarding the immune system, ouabain was shown to modulate
pro-inflammatory cytokines release by mononuclear cells, inhibit mitogen-induced proliferation of lymphocytes and, augment
intracellular calcium levels and CD69 expression also in lymphocytes. However, ouabain effects on monocytes, especially human
monocytes, are still poorly understood. Thus, the aim of the present work was to examine if ouabain was able to modulate the
expression of different surface molecules involved in the activation and effector functions of monocytes.
Methods and Results:
Mononuclear cells from healthy volunteers were separated by Ficoll-Hystopaque density gradient and cultured for 24 hours in the
presence or absence of 10-7M ouabain. After the incubation period, the expression of CD80, CD86, CD40 and CD69 molecules
were assessed using flow cytometry. Our results show that ouabain induced an increase in the expression of CD86, CD80 and
CD69 molecules, although, led only to a small increase of CD40 (n=4). When ouabain is added at the same time as LPS (antigen
capable of activating monocytes), ouabain seems to block LPS-induced CD40 augmented expression (n=5). On the other hand, in
the case of CD69, co-incubation with ouabain and LPS promoted an even higher increase in the expression of this molecule than
the incubation with ouabain or LPS alone (n=8).
Conclusions:
Together, these results showed that ouabain, in the presence or absence of LPS, is able to modulate the expression of different
surface molecules related to the state of activation and function of monocytes, suggesting an immunomodulatory role for ouabain
on these cells.
Keywords: Monocytes, Ouabain, Surface molecules
Financial Support: CAPES, CNPq and FAPERJ.
Resumo:18-051
EVALUATION OF THE PROFILE OF SERUM GLYCOPROTEINS OF HEALTHY INDIVIDUALS AND PATIENTS
WITH DENGUE FEVER AND DENGUE HEMORRHAGIC FEVER USING PURE LECTINS
Melo, M. S. ; Aranda-souza, M. A. ; Paula, R. A. ; Correia, M. T. S.
Departamento de Bioquímica, UFPE
Objectives:
Lectins are proteins or glycoproteins capable of interacting reversibly with carbohydrates through at least two binding sites, plant
cells coalesce and / or animals and precipitate glycoconjugates. The specificity of the interaction between lectins to
glycoconjugates free in solution or present on the cell surface makes these biological properties of biomolecules such as
differential recognition of carbohydrates or glycoconjugates expressed in normal or transformed tissues.The aim of this study was
to use lectins BmoLL, Cramoll-1, 4 and CrataBL for characterization of profile serum glycoproteins of healthy individuals (IH)
and patients with dengue fever (DF) and dengue hemorrhagic fever (DHF).
Methods and Results:
Lectins of seed Cratylia mollis (Cramoll-1,4 specific for D-glucose), leaf Bauhinia monandra (BmoLL specific for D-galactose)
and the bark of Crataeva tapia (CrataBL, specific glycoproteins) were purified. Radial diffusion assay was performed using 1%
(w/v) agarose gel containing 0.15M-NaCl, in Petri dishes. Serum samples (20μL) IH, DF and DHF were applied in a circular
distribution around a central pit containing a lectin (20μL) studied. Diffusion was allowed to take place for 48 h at 4°C in
humidity chambers. The gels were washed with 0.15M-NaCl and distilled water, dried and stained with 0,005% (w/v) Coomassie
Brilliant Blue. The radial diffusion assay in agarose gel showed lines of precipitation for the lectins Cramoll-1,4, BmoLL and
CrataBL showing the recognition and binding of lectins to carbohydrates of glycoproteins of serum samples, justifying the use of
lectins to obtain glycoproteins that can differentiate pathologies in serum from healthy individuals or disease.
Conclusions:
The lectins tested (BmoLL, Cramoll-1,4 and CrataBL) had the potential to promote the isolation of glycoproteins in serum from
healthy individuals and patients with dengue fever or dengue hemorrhagic fever.
Keywords: dengue, glycoprotein, lectin
Financial Support: CAPES, CNPq, FACEPE
Resumo:18-052
LTB4 AS CHEMOATTRACTANT FACTOR IN THE REGULATORY T CELLS MIGRATION
Pecli, C. S. 1; Molinaro, R 5; Peters-golden, M. 2; Kunkel, S. L. 3; Canetti, C. . A. 4; Benjamim, C. F. 1
1
Instituto Ciências Biomédicas, UFRJ
2
Division of Pulmonary and Critical Care Medicine, U M
3
Department of Pathology, U M
4
Instituto de Biofísica Carlos Chagas Filho, UFRJ
5
Instituto Oswaldo Cruz, FIOCRUZ
Objectives:
Leukotrienes are lipidic mediators, among these the leukotriene B4 (LTB4) plays a critical role in leukocyte recruitment and
activation. LTB4 is derived from the metabolism of arachidonic acid by the enzyme 5-LO assisted by FLAP. It exerts its actions
by ligating two G proteins-coupled receptors, BLT1 and BLT2. LTB4 chemoattractant function has also been shown for CD4+,
CD8+ and γδ T cells. Another T cells subset are the regulatory T cells (Tregs). Treg are supressive cells that modulate the
immune system response. The aim of our study is to evaluate the LTB4 effect on Treg cells. As we already shown that Tregs
express BLT1 receptor and it is responsive to LTB4 as a chemoattractant factor, our next goal was to evaluate if the LTB4 can act
synergistically with the CCL17, a Treg cells chemoattractant. Also, as we saw in our data that all Tregs from septic mice express
the BLT1 receptor, we sought to analyze if in vitro activated Tregs show the same profile
Methods and Results:
Splenic cells from C57Bl/6 (B6) were subjected to an EasySep Treg Kit for purification (StemCell, USA), according to the
manufacturer‘s instructions. Purified Treg were placed in the upper well of a Boyden chamber and in the bottom wells 0,1nM
LTB4 and 1nM CCL17 diluted in medium was loaded. As negative control we used RPMI medium only. Migrated cells were
collected after 1h incubation at 37°C and counted by hemocytometer. For activation experiments, Tregs were stimulated with
anti-CD3 (5ug/mL), anti-CD28 (2ug/mL), LPS (100ng/mL) and in some experiment with macrophages (3x105). All experiments
were conducted in accordance with the ethical guidelines of the Institutional Animal Care Committee-CEUA in UFRJ, Rio de
Janeiro, RJ, protocol code: DFBCICB028.
Conclusions:
LTB4 did not show a synergistic activity with the CCL17 chemokine in the chemotaxis assay. BLT1 expression was not upregulated on Tregs activated by different stimulus. We have provided data that Tregs express BLT1 receptor and it is responsive
to LTB4. We also showed that LTB4 can‘t act synergistically with CCL17, and that activated Tregs do not up-regulate BLT1
receptor in vitro. Our next goal is to evaluate the actions of LTB4 in the Treg functions and signaling.
Keywords: chemotaxis, leukotrienes, regulatory T cells
Financial Support: CAPES, CNPq and FAPERJ
Resumo:18-053
THE ROLE OF LAMININ POLYMER IN SIGNALING OF SPLENIC DENDRITIC CELLS
Ladislau, L. 1; Da-fé, A. R. 2; Barja-fidalgo, T. C. 2; Benjamim, C. F. 1
2
DEPTO FARMACOLOGIA E PSICOBIOLOGIA/UERJ, IBRAB/UERJ
1
INSTITUTO DE CIÊNCIAS BIOMÉDICAS/UFRJ, ICB/UFRJ
Objectives:
Dendritic cells (DCs) are considered professional antigen-presenting cell (APCs) and they are very important during immune
response. Laminin (LN) is a major protein, important and biologically active part of the basal lamina, which plays a role in the
cell differentiation, migration, adhesion as well as phenotype and survival of several cells. For APC function, DCs have to
display foreign antigen by the major histocompatibility complex (MHC), express Toll-like receptors and co-stimulatory
molecules on its surface and release cytokines. We showed that LN increases MHC and co-stimulatory molecules expression on
DCs when they were incubated with and without LPS. However we showed that LN did not induce cytokines release from DCs
when they were incubated with and without LPS. It‘s known that DCs express α6β1 integrin, LN receptor, but the effect of LN on
DCs‘s signaling is poorly understood. Our aim is to characterize the effect of LN polymer on signaling of splenic DCs.
Methods and Results:
This animal study was performed in accordance with instructional guidelines, approved by Ethical committee of animal use,
protocol number: DFBCIB 028. C57BL/6 mice were gently donated by National Institute of Cancer (INCA). The LN (50 mM)
was diluted in sodium acetate pH 4,0 or Tris pH 7,0, both with additional calcium chloride (1 mM), and this LN was kept
overnight in 6-well plates at 37oC and 5% CO2. We used BSA (50 mM) for the protein control. DCs were obtained from spleen
with aseptic techniques and subjected to an EasySep CD11c-PE positive selection (StemCell Technologies), according to the
manufacturer‘s instructions. DCs purity, was determined by flow cytometry of CD11c+ cells and showed >90% after selection.
These cells were cultured in 6-well plates pre-coated with LN or BSA. We also used a second hit of LPS (1μg/mL). To analyze
the protein signaling by western blotting, DCs were incubated with stimulus for 30 minutes and 24 hours. We did not observe any
difference at phosphorylation of p38 and ERK in DCs incubated with LN polymer and BSA without LPS in 30 minutes.
However, when DCs were incubated with LN polymer and LPS an increase at ERK phosphorylation was observed, compared
with BSA with LPS group. No difference was observed at p38 phosphorylation. Further, DCs incubated with LN polymer
without LPS for 24 hours presented an increase at p38 and ERK phosphorylation, compared with BSA alone and with LPS
incubation. DCs incubated with LN polymer and LPS presented an increase at p38 and ERK phosphorylation compared with DCs
incubated with LN without LPS. To analyze NFkB activation, EMSA was performed after 1,5 hours of DCs incubation with LN
polymer and BSA with and without LPS. DCs incubated with LN polymer and LPS have more activated NFkB than DCs
incubated with BSA and LPS. In DCs incubation without LPS no difference was observed.
Conclusions:
These results suggest that LN activates phosphorylation of signaling protein and seems to be responsible for long-term
phosphorylation. Also, LN polymer is able to enhance NFkB activation by LPS.
Keywords: DENDRITIC CELLS, LAMININ, SIGNALING, LPS
Financial Support: FAPERJ AND CNPQ
Resumo:18-054
EFFECT OF POLYMER-COATED GOLD NANOPARTICLES ON MACROPHAGE RESPONSE IN VITRO
Albuquerque, A. L. 1; Santos, R. V. 1; Silva, J. P. N. 1; Giacomelli, C. 2; Hickmann, J. M. 1; Santos, C. E. A.
1
; Brito, F. D. A. 1; Martins, M. A. 3; Cordeiro, R. S. B. 3; Barreto, E. O. 1
1
Laboratório de Biologia Celular, UFAL
2
Departamento de Química, UFSM
3
Laboratório de Inflamação, IOC-Fiocruz
Objectives:
Multiple research groups have tried to apply nanoparticles in a variety of fields such as biological research, medicine and
cosmetics. Among them, the majority were focused on gold nanoparticles as it has been reported to be neither cytotoxic nor to
elicit the activation in macrophage cells. In addition, little is known about the effects of such polymer-coated gold nanoparticles
in biological systems. Therefore, the aim of this study was to evaluate the influence of polymer-coated gold nanoparticle on
macrophages in vitro.
Methods and Results:
Characterization of the gold nanoparticles (AuNP) with or without polymer-coated (AuNPc) was performed by using UV-VIS
spectroscopy. Macrophages were obtained by intraperitoneal lavage from Swiss mice (18-25g). Peritoneal fluid was cultured for
2 h to allow the macrophage adherence on plastic wells. The supernatants were harvested to remove non-adherent cells.
Peritoneal macrophages were maintaining in RPMI-1640 medium supplemented with Fetal Cow Serum (10%), ciprofloxacin (2
mg/mL) and glutamine (1%). Macrophages were incubated for 12, 24 and 48h with AuNPc or AuNP and phagocytic activity,
morphological analyses, cell viability and reactive oxygen species generation (ROS) were evaluated. The amount of nanoparticles
incubated per well was 100, 101, 102 and 103 ng. Results were expressed as mean ± SEM and analyzed statistically by the test t
of Student. P values 95%) and uptake of zymosan by macrophages in all doses tested. Moreover, in different points analyzed (12,
24 and 48 h) the nanoparticles did not interfered on cell viability and or phagocytosis. Curiously, AuNP and AuNPc in the high
concentration (103 ng) induced significant cytoplasm vacuolization in the macrophages.
Conclusions:
These results suggest that gold nanoparticle or polymer-coated gold nanoparticle show lower propensity in inducing cell toxicity,
suggesting their bio-compatibility.
Keywords: GOLD NANOPARTICLES , MACROPHAGE , POLYMER-COATED
Financial Support: CAPES and CNPq
Resumo:18-055
EXOSOMES AND WASP IMMUNODEFICIENCY
Sunahara, K. K. S.
Medicine SOlna Allergy and Immunology department KI, KI
Objectives:
To verify how changes in the actin machinery can affect exosome production. To characterize and compare exosomes
derived from B cells and bone-marrow derived dendritic cells from WT and WASP-KO mice
Methods and Results:
Spleen and bone marrow from 6 to 8 weeks BALB-c, WT and WASP-KO mice were processed. B cells were obtained by
negative selection of splenocytes using magnetic beads and stimulated with anti-CD40 antibodies and IL-4 to induce
immunoglobulin class switching. Bone marrow-derived dendritic cells (BMDDCs) were generated using GM-CSF and IL-4, and
on the 6th day of culture, cells were stimulated with LPS for 48 hours to induce DC maturation. WASP-deficient B cells show
higher exosome production when compared to activated bone marrow-derived dendritic cells. Exosomes were phenotyped for
different markers such as tetraspanins (CD9 and CD81), co-stimulatory molecules (CD80 and 86), and the adhesion molecule
CD54. Substantial differences between WT and WASP deficient cells were not observed. However, MHC II expression was
strikingly diminished on WASP deficient activated B cells derived exosomes, while on activated BMDDCs, it was slightly
increased
Conclusions:
WASP deficiency differently affects exosome production and phenotype in B cells and DCs. It would be possible then to
speculate that WASP deficient cells release exosomes that show impaired function, what could compromise the crosstalk among
immune cells
Keywords: exosomes, WASP, immundeficency, auto-immunity
Financial Support: karolinska INstitutet
Resumo:18-056
S100B SECRETION IS STIMULATED BY CYTOKINES IN GLIAL CULTURE AND HIPPOCAMPAL SLICES OF
RATS: LIKELY INVOLVEMENT OF WAY MAPK.
de Souza, D. F. ; Hansen, F. ; Gonçalves, C. A.
Departamento de Bioquímica/ICBS, UFRGS
Objectives:
S100B is a cytokine produced and secreted by astrocytes, has trophic or toxic depending on concentration, is involved in
neuroinflammatory processes, present in neurodegenaratives diseases such as Alzheimer's disease, schizophrenia, among others.
However, its mechanism of secretion has not been elucidated. In this study, we investigated S100B secretion in C6 glioma cells
and acute hippocampal slices exposed to IL-1â, IL-6, IL-8 and TNF-á.
Methods and Results:
We used C6 glioma cells and hippocampal slices from rats exposed or not to cytokines and / or specific inhibitors of MAPK
pathways. Quantification of S100B was performed by ELISA. We used t test or one-way ANOVA assuming p
Conclusions:
Both PD98059, an inhibitor of ERK, and SB203580, p38 kinase inhibitor, were able to prevent the increase in S100B secretion
stimulated by cytokines, suggesting that this effect, observed in two different preparations in vitro, is mediated by signaling
pathway MAPK- ERK. These results contribute to the inference that S100B secretion induced by cytokines, is a component of
the neuroinflammatory response, which would support the involvement of S100B in the genesis of neurodegenerative diseases.
Keywords: S100B, Cytokines, MAPK, Neurodegenerative disease
Financial Support: CNPq, FINEP, CAPES
Resumo:18-057
TLRS ENHANCE FCR-MEDIATED PHAGOCYTOSIS VIA 5-LO PRODUCTS IN MACROPHAGES
Pinheiro, C. S. 1; Teixeira, A. P. M. 1; Benjamim, C. F. 2; Peters-golden, M. 4; Bozza, M. T. 3; Canetti, C. 1
1
Instituto de Biofisica Carlos Chagas Filho, IBCCF
2
Instituto de Ciencias Biologicas, ICB
3
Instituto de Microbiologia Professor Paulo Goes, IMPPG
4
University of Michigan, UM
Objectives:
Toll-like receptors (TLRs) are pattern recognition receptors involved in microorganisms recognition, which are engulfed by a
mechanism known as phagocytosis. Leukotrienes (LTs), lipid mediators produced from 5-lipoxygenase (5-LO) pathway, are able
to increase phagocytosis in FcgR dependent process. They are produced from arachidonic acid metabolism in a two-step process
to yield the epoxide intermediate, LTA4 which is then converted into either LTB4 by LTA4 hydrolase or conjugated with
glutathione by LTC4 synthase to form the cysteinyl LTs (cys LTs). The aim of this study was to evaluate the participation of
TLRs ligands on FcgR-mediated phagocytosis and the role of 5-LO products.
Methods and Results:
Methods: Rat alveolar macrophages (AMs) or mouse (SV129 or 5-LO-/-) peritoneal macrophages (PMs), and bone-marrow
derived macrophages (BMDM) were harvested, cultured, and treated with TLR ligands and then challenged with IgG opsonised
sheep red blood cells (IgG-sRBCs). Zileuton, a 5-LO inhibitor, was incubated before TLR ligands addition. After 90 min, the
phagocytic targets were removed and phagocytosis evaluated through a colorimetric assay. Lipid mediators (LTB4 and cys LTs)
were measured by EIA. Western blotting, was used to detect activation and expression of mitogen-activated protein kinase
pathway. Results: Except for CpG, all TLR ligands tested (i.e ligands for TLR2, TLR3, and TLR4) were able to amplify
phagocytosis of IgG-sRBC in AM and BMDM. The phenomenon occurred in a concentration-dependent manner. Futhermore,
phagocytosis amplification induced by TLR3 ligand was a time-dependent event, fact not observed for TLR2 and TLR4 ligands.
AMs treatment with zileuton also impaired TLR4 ligand-induced phagocytosis amplification, suggesting the participation of 5LO products in this phenomenon. To confirm LTs involvement in phagocytosis mediated by FcgR, PMs from 5-LO-/- mice did
not presented any difference on phagocytosis when stimulated with TLR2, 3, and 4 ligands. LTB4 production induced by IgG
engagement was amplified by TLR2 and TLR4 ligands, but not by TLR3. Similar results were obtained for cys-LTs, whose
production was increased by TLR2, TLR3, and TLR4. ERK1/2 and p38, mitogen-activated protein kinase (MAKs) pathways
were phosphorilated under TLRs agonists. This phenomenon was exacerbated under IgG-sRBC treatment.
Conclusions:
Our data suggest that TLR ligands amplify phagocytosis in a time and concentration dependent manner. Furthermore, 5-LO
products pathway seems to be responsible for this phagocytosis amplification. ERK1/2 and p38 presented a greater activation
when cells were incubated with TLRs agonists and challenged with IgG-sRBC, suggesting a possible interaction between FcgR
and TLR signaling pathway.
Keywords: phagocytosis, TLRs, Leukotrienes
Financial Support: FAPERJ, CNPq, and FUJB
Resumo:20-001
INHIBITION OF RT HIV-1 BY DITERPENES ISOLATED FROM BRAZILIAN BROWN SEAWEEDS: A
STRUCTURE-ACTIVITY RELATIONSHIP STUDY
Miceli, L. A. 1; Souza, A. M. T. 1; Teixeira, V. L. 1; Paixão, I. N. P. 1; Rodrigues, C. R. 2; Castro, H. C. 1
1
Departamento de Biologia Geral (GBM), UFF
2
Faculdade de Farmácia da UFRJ, UFRJ
Objectives:
The marine organisms are source of numerous molecules presenting distinct and important roles such as chemical defense against
herbivores. Diterpenes isolated from Brazilian brown seaweeds of the genus Dictyota have a promising and potential activity as
antiviral against HIV. Interestingly, the diterpene 8,10,18-trihydroxy-2,6-dolabelladiene (THD), (6R)-6-hydroxydichotom-3,14diene-1,17-dial (HDD) and (6R)-6-acetoxydichotom-3,14-diene-1,17-dial (ADD) showed a significant antiviral activity against
reverse transcriptase (RT-HIV-1) of HIV-1 (IC50=16.5uM, 10µM and 35µM, respectively) and low cytotoxicity (CC50 >200
µM). The aim of the present work is to study the Structure-Activity relationship (SAR) of these diterpenes as anti RT-HIV-1
agents to identify important parameters related to this biological profile, using a molecular modeling approach.
Methods and Results:
Stereoelectronic parameters from diterpene compounds were obtained from Single-Point HF/6-31G* calculations using
SPARTAN‘08 program. Among all ten parameters calculated, the overall analysis revealed a feasible relationship of the anti RTHIV-1 activity with GAP (ELUMO-EHOMO), which represents the molecule reactivity. Higher values of GAP led to higher
antiviral profile in contrast to the polarizability and dipole moment values where higher values negatively affected it. Since ADD
presents the highest molecular weight (366 Da) and the lowest antiviral activity profile, probably the size of the molecule may
also influence the derivative interaction with the RT-HIV-1 active site. In this work we calculated the theoretical toxicology of
these compounds. Interestingly, the theoretical toxicity results were analogous to the previous experimental data where THD
showed a lower cytotoxicity (CC50=500 µM) than HDD and ADD (CC50 = 200 µM) and nevirapine (CC50>100 µM). Finally,
we calculated the theoretical lipophilicity (cLogP) and tested the fulfillment of the Lipinski ―rule-of-five‖ for all compounds
using Osiris Property Explorer server. Our results suggested that the compounds may have a good oral availability as they
fulfilled the Lipinski ―rule-of-five‖ and presented higher cLogP values.
Conclusions:
According to our SAR study, dipole moment, polarizability, GAP and molecular weight should be carefully considered when
designing new anti RT-HIV-1 agents since they apparently influence the antiviral profile. Overall THD may be pointed as a
promising lead molecule for further exploration by using a molecular docking approach.
Keywords: HIV-1, Reverse Transcriptase, Diterpenes, Molecular Modeling, Antiviral
Financial Support: UFF-PROPPI, CNPq, FAPERJ
Resumo:20-002
CRYPTOCOCCUS NEOFORMANS CYP51 ENZYME: HOMOLOGY MODELING AND INTERACTION WITH
AZOLE ANTIFUNGALS
Carvalho, K. L. 1; Abreu, P. A. 3; Castro, H. C. 1; Rodrigues, C. R. 2
1
Depto de Biologia Celular e Molecular / Inst. de Biologia , UFF
2
Faculdade de Farmácia, UFRJ
3
Faculdade de Farmácia, UFRJ / Campus Macaé
Objectives:
Cryptococcus neoformans is an opportunistic fungus involved in central nervous system infections, which are fatal if untreated. It
is also extremely important in affecting immunocompromised individuals. Fluconazole and other azoles antifungal are one of the
main treatments that inhibit lanosterol 14α-demethylase, a CYP51 family protein. In fungi, CYP51 participates in ergosterol
biosynthesis essential for fungal viability. Currently, the emergence of resistant strains and the cross-binding of known azoles
with human CYP51 demand the understanding of CYP51 structure and the search for more selective compounds. Since the
experimentally 3D-structure of the CYP51 of C. neoformans is not described in the literature, the aim of the present study is to
construct an homology model of this enzyme and dock it with known azoles to evaluate their interactions. We also docked these
inhibitors into the human CYP51 crystal structure for further comparison.
Methods and Results:
The multiple sequence alignment of the C. neoformans CYP51 and other CYP51 primary sequence using CLUSTALW revealed
low overall identity percentage ranging from 25 to 46%. Nevertheless, the secondary structure was conserved for all sequences
that also presented a similar folding prediction or crystal structure. The Swiss-model program was used to construct the C.
neoformans CYP51 homology model using the human crystal structure as template due to its higher degree of similarity (36%)
compared with other 3D-structures currently available (i.e. Mycobacterium tuberculosis CYP51 = 28%). The model was refined
using molecular mechanics and the reliability was assessed by Ramachandran plots and Profile-3D that confirmed the feasibility
of the structure. The C. neoformans model was similar to the template with 15 α-helix and 5 β-sheet. However, the electrostatic
potential map analysis revealed a more positive profile for the human CYP51 compared with the C. neoformans CYP51
homology model. The theoretical studies with ketoconazole and fluconazole, constructed and minimized using the program
Spartan 8.0, and docked into human and Cryptococcus enzymes using the Autodock program, showed the same binding mode
involving the iron of the CYP51 Heme group and the cysteine 490. Interestingly, the study pointed to a higher binding energy of
the ketoconazole and fluconazole to the C. neoformans CYP51 compared with the human enzyme. In addition, the comparison of
each enzyme active site (within 6Å from the inhibitors) revealed different residues interacting and/or near to the compounds
including Y77F, L100M, R103K, I105F, A130V, V144A, I159L, P242H, F245W, M246L, A313G, M316L, S388I, I389M,
Y390M.
Conclusions:
Compared with the human enzyme, the CYP51 from C. neoformans presents differences in the electrostatic charge distribution
and in residues involved in the inhibitor interaction. Our study pointed to new regions to be further explored on designing new
more effective antifungals.
Keywords: Homology modeling, Cryptococcus neoformans, 14α-lanosterol demethylase
Financial Support: FAPERJ, CNPq, CAPES, UFF-PROPPi
Resumo:20-003
MOLECULAR DOCKING APPROACH FOR STUDYING PIPERAZINE DERIVATIVES: A NEW SERIES OF NMDA
RECEPTOR ANTAGONISTS
Santana, M. V. D. S. 1; Abreu, P. A. 2; Castro, H. C. 1
1
Depto. de Biologia Celular e Molecular/Instituto de Biologia, UFF
2
Faculdade de Farmácia/Universidade Federal do Rio de Janeiro, UFRJ
Objectives:
The N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate-gated channels that are essential to synaptic plasticity,
neuronal development, memory and learning. The NMDARs are composed of different subunits as NR1, NR2 (A-D) and NR3 (A
and B). The excessive stimulation of the NMDAR is associated with some pathological conditions as Parkinson, Huntington and
Alzheimer diseases. Because of its role in neuronal death, NMDA receptor antagonists is the focus of intense research, especially
involving selective NR2B subunit antagonists. As NR2B is the only subunit absent in the cerebellum, the block of this subunit
results in less adverse effects as they may not interfere with the motor function. In this context, the aim of this work is to analyze
the interactions and the binding mode of 17 piperazine-2,3-dicarboxylic acid derivatives (13, 17, 23, 16a-n) within NR2B subunit
of NMDA receptor.
Methods and Results:
For docking the piperazine derivatives we used Autodock 4.2 software that calculates ligand interactions within the receptor
active site. On that purpose three steps were performed including preparation of the structure files, pre-calculation of the possible
interacting atoms in the ligand (AutoGrid) and the docking of the molecules based on the information of the grid. The results of
docking analysis showed a region within the receptor active site characterized by a hydrophobic pocked compose of the residues
Tyr212, Asp213, Thr241, Thr172, Ser171 and Glu13, which interact with the hydrophobic core of the ligands. Compounds
bearing bulky hydrophobic groups (16e, 16g, 16h and 16n) were the most potent compounds. They interact with the same
residues in the active site with a similar binding mode. The most active ligands presented hydrogen bonds with Lys85 and His86,
as most of the other derivatives. 16h derivative showed a different interaction pattern and a slightly different conformation
compared with 16e, 16g and 16n, that did not affect the biological activity. Interestingly, compound 16g, with the best Ki value,
also presented the lower binding energy. Analogously, 16a, with the worst Ki value, also presented the higher binding energy,
which may be related to its completely different conformation from the most active molecules.
Conclusions:
Our results suggest that interactions of bulky hydrophobic groups of the ligands with the NMDA receptor are determinant for the
biological activity as the most potent molecules of this series presented a phenanthrene ring as substituent that directly interact
with the receptor. The hydrogen bonds detected within the active site were almost the same for the compounds pointing to the
importance of van der Walls interactions of the hydrophobic groups of 16e, 16g, 16h and 16n, the best antagonists present in the
study. This study may help in the rational design of more potent and selective drugs for neurodegenerative diseases.
Keywords: Autodock, Piperazine derivatives, Molecular docking, NMDA, NR2B
Financial Support: CNPq, FAPERJ, UFF-PROPPi, CAPES
Resumo:20-004
A SYSTEM BIOLOGY APPROACH ON THE EVOLUTION OF CHEMOSENSORY RECEPTORS IN MICE, RATS
AND HUMANS
Albanus, R. D'o. ; Dalmolin, R. J. S. ; Moreira, J. C. F.
Instituto de Bioquímica, UFRGS
Objectives:
The aim of the present study is the use of System Biology analysis tools in the assessment of evolutionary parameters of the
chemosensory networks, namely taste, olfactory and vomeronasal reception, on Mus musculus, Homo sapiens and Rattus
norvegicus.
Methods and Results:
Using data gathered from The Gene Ontology Project (GO), we were able to amass the genes associated with each type of
sensory perception. The GO ID‘s used for this work were 0004984 and 0007608, 0050909 and 0008527, 0016503 and 0019236,
for olfactory, gustatory and vomeronasal ontology groups, respectively. We sorted these genes in groups by receptor modality,
with one further division of the Taste Group into the Taste-1 and Taste-2 receptors types groups. We acquired the network
parameters of the proteins translated from these genes groups using data from STRING Database. We used as criterions for our
analysis the connectivity and clusterization indexes (i.e. k(i) and c(i), respectively). Also used in our analysis was the
Evolutionary Plasticity Index (EPI) of proteins‘ orthologous group, an indicator that is able to infer the evolutionary constraints
acting on its evolution. We subsequently divided the whole dataset into two more groups: those of proteins directly involved in
the chemical binding of the stimuli, named Receptor Group (RG); and those involved into signal transduction and further
chemical pathways, named Accessory Group (AG). We compared the mean EPIs of all the RGs and AGs using One-Way
ANOVA and Tukey test and found that the AGs‘ means were significantly lower than the RGs‘ (p<0,05).
Conclusions:
Our findings suggest that the evolutionary plasticity in the RGs is higher than that on the AGs. This means that the selective
pressures among the chemoreceptors are weaker than that on the transduction machinery, resulting in a greater diversity for these
receptors. The peaks of clusterization and connectivity for the Olfactory RGs are probably due to the fact that the expression of
one olfactory receptor apparently inhibits the others, in an one-neuron-one-receptor model. Put together, these results support the
Birth-and-Death Evolution model proposed for chemosensory receptors and others genetic families with notably high degree of
variability among its members. This work also demonstrate the value of System Biology as an efficient and cost-compatible
analysis tool to study complex interactions between proteins networks, being able to infer evolutionary relationships based on the
mathematical (and therefore, comparable) data generated by this kind of approach.
Keywords: Chemoreception, Evolution, System Biology
Financial Support: This work was funded by CNPQ and Capes.
Resumo:20-005
ANALYSIS OF THEORETICAL MODELS FOR ATP PRODUCTION: CONFLICTS AND CORRELATIONS
AMONG THE MODELS AND WITH EXPERIMENTAL DATA
Siqueira, K. M. ; Saa, A.
Dept. of Applied Mathematics / IMECC, UNICAMP
Objectives:
In 1997, Magnus and Keizer (Am. J. Physiol. 273, C717, 1997) first proposed a theoretical dynamic model for ATP production in
the mitochondria (MK model) based on the biophysical properties of the enzymes and transporters involved in the process. This
model is very complete and takes into account most of the processes thought to be important for mitochondrial oxidative
phosphorylation. However, some biological properties are difficult do derive from the MK model due to its complexity. A few
years later, a simplification of this model was proposed by Bertram et al. (J. Theor. Biol. 243; 575, 2006), and, although this
model has been used in several works since its publication, is hadn‘t yet been fully tested as to it‘s correlation with the MK model
and with current biological data. Therefore, the purpose of this work was to evaluate the validity of the modifications proposed by
Bertram et al., by comparing the predictions of this model with the MK model and with current biological data.
Methods and Results:
MK model and Bertram‘s model are composed of a group of equations that describes the dynamics of the citric acid cycle, the
proton pump as well as ATP and calcium transporters at the inner mitochondrial membrane. We‘ve studied each of Bertram‘s
equations in comparison to the MK model. By graphical analysis, the general behavior of Bertram‘s equations was compared
both with the MK model and with current biological data from the literature. For three equations, we found that the Bertram
Model had a significantly different comportment (even qualitatively) from MK model. These equations described the rate of
production of NADH by the citric acid cycle, of the adenine nucleotide translocator‘s activity and of the calcium uniporter
transporter. For these three rates, Bertram‘s model didn‘t agree with the biological data, while the MK model described it
accurately. Here, we proposed some modifications of the MK model, that provided simpler equations and still presented a good
correlation with MK‘s. The other equations from Bertram‘s model agreed with the MK model as well as with the biological data.
Conclusions:
We found that three equations from Bertram‘s model didn‘t agree with biological data. Apart from that, both models (MK and
Bertam‘s) presented a good correlation with biological data, suggesting, therefore, that they can be powerful tools in the study of
ATP production dynamics.
Keywords: ATP production, Biophysics, Mathematical Model, Mitochondria
Financial Support: CNPq
Resumo:20-006
ALTERNATIVE METHODS OF TEACHING PHARMACOLOGY: FULFILLING AROUCA’S LAW.
Chaves, H. V. ; Saraiva, T. V. ; Cruz, M. P. ; Vieira, A. M. ; Silva, W. C. B. ; Oliveira, I. N. S. ; Camelo, J.
R. R. ; Val, D. R. ; Silva, A. A. R. ; Bezerra, M. M.
Universidade Federal do Ceará, UFC
Objectives:
The practical activities in pharmacology teaching had to suit the new legislation enacted in 2008 as the Arouca‘s Law, which
states that "whenever possible, teaching practices should be photographed, filmed or recorded, allowing its reproduction for
future practices, avoiding the unnecessary repetition of procedures teaching with animals." In this regard, the aim of this study
was to develop software that will be used for teaching practical classes in pharmacology in accordance with Arouca‘s Law,
stimulating the interdisciplinarity between the faculties of Medicine and Computer Engineering from the UFC - Campus of
Sobral.
Methods and Results:
We observed the classroom practice of routes of administration of drugs held in the ―Principles of Pharmacology‖ module at
Faculty of Medicine of Sobral. Chloral Hydrate (10mg/kg), a sedative-hypnotic agent, was injected in six female Wistar rats (200
– 220g) via oral, subcutaneous and intramuscular, and the time to onset of drug effect was recorded. For inhalation and
intravenous routes a swab of cotton wet with ether and Evans blue (1%), has been used, respectively. To record the videos we put
the animals in a glass box (40x40x30cm) and used a high definition camera and fluorescent lamps tilted 45 degrees from the
sidelines. There is a menu at the initial screen of the software containing the presentation, objectives, methodology, videos and
results. There is also an area with graphics and issues related to practice with the purpose of exercising the content worked.
Conclusions:
The software is an alternative method of teaching pharmacology. Moreover, its development has encouraged the interdisciplinary
between exact and life sciences.
Keywords: Alternative Methods , Arouca's Law, Computer Engineering , Software , Teaching Pharmacology
Financial Support: UFC
Resumo:20-007
DIPHENYL DISELENIDE INHIBITS MAMMALIAN AND DROSOPHILA MELANOGASTER BUT NOT PLANT
&DELTA;-AMINOLEVULINIC ACID DEHYDRATASE ENZYME: A MOLECULAR MODELING APPROACH.
Bueno, D. C. ; Nogara, P. A. ; Saraiva, R. A. ; Rocha, J. B. T.
Depto de química - Universidade Federal de Santa Maria, UFSM
Objectives:
In the last years, diphenyl diselenide [(PhSe)2] has been suggested as a potential therapeutic agent, with neuroprotective,
analgesic and antioxidant effects both in vivo and in vitro. Delta-aminolevulinic acid dehydratase (δ-ALAD) is a metalloprotein
that catalyzes porphobilinogen (PBG) formation. Since δ-ALAD is important for heme synthesis, its inhibition can be toxic.
Previous studies have demonstrated that (PhSe)2 inhibits human, murines and fly (Drosophila melanogaster) δ-ALAD by
oxidation of its sulfhydryl residues. On the other hand, cucumber (Cucumis sativus) δ-ALAD is not inhibited by (PhSe)2. So, we
compared δ-ALAD structures from mammals, fly and cucumber and performed molecular modeling studies using protein-ligand
docking tools, in order to get a better understanding of in vitro effects observed.
Methods and Results:
FASTA sequences from Homo sapiens δ-ALAD (Hsδ-ALAD), Mus musculus δ-ALAD (Mmδ-ALAD), D. melanogaster δALAD (Dmδ-ALAD) and C. sativus δ-ALAD (Csδ-ALAD) were obtained from BLAST. The crystal structure from Hsδ-ALAD
was obtained from Protein Data Bank (PDB ID: 1E51) and Dmδ-ALAD was modeled using Swiss-MODEL server. Virtual
docking was performed by AutoDock Vina 1.1 program. Avogadro program was used to construct the ligands, and they were
optimized using UFF method. The grid box was centered in the following coordinates: x = 37.1, y = 74.9 and z = 58.01, and box
size was defined as 20 Å x 20 Å x 20 Å (spacing = 1 Å). Docking simulations with Hsδ-ALAD showed that the aromatic rings of
(PhSe)2 make ζ-π interactions with Phe79, π-π interactions with Phe208, and cation-π interactions with Lys199 and Arg209,
allowing an approximation between Se atoms and thiol group of Cys124, with distances ranging between 3.3 Å and 3.4 Å.
Docking simulations with Dmδ-ALAD showed that the aromatic rings of (PhSe)2 make π-π interactions with Phe204 and cation-π
interactions with Lys195 and Arg205 also allowing an approximation between Se atoms and thiol group of Cys124, with
distances ranging between 3 Å and 4 Å. In spite of some modifications between Dmδ-ALAD, Mmδ-ALAD and Hsδ-ALAD
active site are observed, the multiple sequence alignment show that the residues related to the activity are conserved. However,
when comparing Hsδ-ALAD with Csδ-ALAD, tyrosyl, alanyl and aspartyl residues in place of cysteyl residues are observed.
Conclusions:
The results obtained are consistent with experimental data. The approximation between Se atom from (PhSe) 2 and Cys124 allows
the oxidation of thiol groups, inhibiting porphobilinogen synthesis. According to the analysis of multiple sequence alignments,
the substitution of cysteyl residues in Csδ-ALAD could explain why (PhSe)2 does not inhibit it, suggesting the importance of
cysteyl residues in Hsδ-ALAD and Dmδ-ALAD inhibition by (PhSe)2.
Keywords: diphenyl diselenide, molecular modeling, phorphobilinogen synthase, thiol oxidation
Financial Support: CNPq
Resumo:20-008
MOLECULAR VIRTUAL DOCKING AND IN VITRO STUDIES OF SYNTHETIC ALKALOIDS WITH
QUATERNARY AMMONIUM AS ACETYLCHOLINESTERASE INHIBITORS
Saraiva, R. A. 1; Nogara, P. A. 1; Bueno, D. C. 1; Pereira, R. P. 1; Appel, A. S. 1; Paixão, M. W. 2; Rocha, J.
B. T. 1
1
Departamento de Química / Bioquímica Toxicológica, UFSM
2
Departamento de Química, UFSCar
Objectives:
Alzheimer's disease (AD) is a neurodegenerative disorder associated with loss of brain neurons and affects a large number of
people around the world. The use of acetylcholinesterase (AChE) inhibitors has been considered one of the strategies to treat AD
since these compounds increase the concentration of acetylcholine (ACh) in the brain. Among the AChE inhibitors, some natural
and synthetic alkaloids with quaternary ammonium (i.e., edrophonium) are largely employed. Despite their benefits, these
compounds present some limitations. Thus, further researches into the development of new AChE inhibitors with less adverse
effects are need. In line with this, we aimed to evaluate the AChE inhibitory activity from eight new compounds with quaternary
ammonium derivatives from the alkaloid cinchonidine (compounds A to H) as well as to understand the interactions involved in
their AChE inhibition by molecular docking in silico.
Methods and Results:
IC50 from compounds was estimated by AChE activity by Ellman's method in vitro (Biochem Pharm 7: 88, 1961). The
compounds (at concentrations ranging from 0.1 to 100 μmol/L) or vehicle 2% DMSO were pre-incubated for 20 min with AChE
prior to 2-min incubation with acetylthiocholine substrate at 25 °C. Molecular docking studies were carried out using AutoDock
Vina 1.1 program. The crystal structure of AChE was obtained from Protein Data Bank (PDB id: 2XUF). Ligands were drawn in
Avogadro 0.9 program and their energies were minimized using MMFF94 force field. The active center gorge was centered at
coordinates x = 32.81, y = 24.17, z = 10.59. The analyses of results were performed using PyMOL and Accelrys Discovery
Studio Visualizer 2.5 programs. The pose with lowest binding free energy was accepted as the most probable interaction model
for each docking procedure. According to in vitro assay, compounds B (0.80 μmol/L), F (2.80 μmol/L), D (7.90 μmol/L) and C
(18.10 μmol/L) showed the lowest IC50 when compared to other compounds (IC50 > 40 μmol/L). Taking into account the
compounds B, C, D and F, together, molecular docking analysis demonstrated an approximation of quaternary ammonium close
to residues from anionic subsite (Trp86, compound F) or peripheral anionic subsite (Trp286, compounds B, C and D) of AChE by
cation-π stacking interactions. Π-π stacking interactions between aromatic groups from these compounds and aromatic residues
(Trp286, Tyr 72 and Tyr341) was also observed. The compounds D and F make H-bond involving the residue Asp74.
Conclusions:
The proposed molecular models suggest an important role of peripheral aromatic residues (Trp, Phe and Tyr) from AChE active
site gorge in the interaction involving both the aromatic moieties and the quaternary ammonium from compounds that could lead
to blocking the entrance of ACh in the enzyme. These data also point that the compounds B, C, D and F could be used as drugs in
the treatment of AD and more pre-clinical studies should be conducted in order to understand their in vivo effects.
Keywords: acetylcholinesterase, virtual docking, Alzheimer's disease, quaternary ammonium
Financial Support: CNPq, FAPERGS
Resumo:20-009
CIS-REGULATORY CONTROL OF PATTERN FORMATION IN DROSOPHILA EMBRYONIC DEVELOPMENT.
Lopes, F. J. P. 1; Cardoso, M. A. 2; Vieira, F. D. M. C. 3; Holloway, D. M. 4; Spirov, A. V. 6; Bisch, P. M. 2
1
Instituto de Biofisica Carlos Chagas Filho, UFRJ Xerém
2
Instituto de Biofísica Carlos Chagas Filho, UFRJ
3
Instituto de Química, UnB
4
Mathematics Department, BCIT
6
Center for Developmental Genetics, SUNYSB
Objectives:
During Drosophila embryonic development the concentration gradients of maternal factors, like the Bicoid protein, establish
positional information along the embryo. A cascade of developmental genes reads out this positional information by exhibiting
different activation levels according to their position along these gradients. One of the first genes of this cascade, the gap gene
hunchback (hb) has strong anterior expression and a sharp on-to-off gradient which is essential for regulating pattern in the midembryo. It has been shown that Bcd binds to hb promoter cooperatively and that hb activates its own regulation. The role of Bcd
cooperative binding for Hb pattern positioning has been demonstrated already, but the mechanism that allows the shallow Bicoid
gradient to regulate the sharp Hb border remains an unsolved problem.
Methods and Results:
We used immunohistochemistry to quantify the dynamics of hb gene expression in flies that were wild-type, were mutant for hb
self-regulation or Bcd binding, or contained an artificial promoter construct consisting of six Bcd and two Hb sites. In addition to
these experiments, we developed a reaction–diffusion model of hb transcription, with Bcd cooperative binding and hb selfregulation. We found that bistability stemming from hb self-regulation produces the sharp Hb border; the loss of sharpness for the
hb14F self-regulating mutant and the shallow pattern of a Bcd-dependent lacZ artificial construct support this result. In addition,
our results indicate that Bcd cooperative binding determines the position of Hb pattern by determining the position where
bistability occurs; our Bcd cooperative mutant data support this conclusion PLoS Comput Biol 4(9), 2008.
Conclusions:
The ability to produce sharp borders is a central step for the expression of developmental genes in Drosophila and other
organisms, and we show that spatial bistability can play a key role in this process.
Keywords: Development, Pattern, Drosophila, Embryo, Morphogen
Financial Support: CNPq, NIH (2-ROIRR07801) , NSF/NIGMS (1-R01-GM072022) and FAPERJ
Resumo:20-010
COMPUTATIONAL MODELING OF NEW DENTATE GYRUS GRANULE CELLS BORN AFTER STATUS
EPILEPTICUS INDUCED BY PILOCARPINE
Tejada, J. 1; Arisi, G. M. 1; Cairasco, N. G. 1; Roque, A. C. 1
1
Depto. de Física - FFCLRP, USP
2
Depto. de Fisiologia - FMRP, USP
Objectives:
Status Epilepticus (SE) provokes changes in the morphology of dentate gyrus (DG) granule cells (GCs) (Epilepsia, 50:2638,
2009; J. Neurosci. 17:3727, 1997; Brain Res 1165:126, 2007.). The new DG GCs have shorter dendrites, narrower arborizations
with greater number of branches (Brain Res 1165:126, 2007) and, in some cases, a basal dendrite that is projected towards DG
hilus (J. Neurosci 27:9400, 2007). The aim of this work is to evaluate how these morphological differences affect the
electrophysiological properties of DG GCs, using as tool computer simulations of biophysically detailed multicompartmental
models.
Methods and Results:
We worked with a sample of 40 tridimensional reconstructions of DG GCs including 20 from animals treated with pilocarpine
(PILO) and 20 from control rats (Brain Res 1165:126, 2007). Whole dendritic arborizations of each cell were reconstructed using
Neurolucida© (Microbrightfield), available on the internet site of the NeuroMorpho project (http://neuromorpho.org/).
Biophysical information came from models of DG GCs(J. Physiol. (Lond.) 538: 227, 2002) avaliable at NeuronDB
(http://senselab.med.yale.edu/neurondb/). The cell models were constructed in NEURON (Neural Comput 9:1179, 1997). The
ionic channels and their maximum conductance distributions were the same for both control and PILO cell models, and the
objective for this was to evaluate the effects of differences in morphology on the electrophysiological behavior. Modeled cells
were submitted to voltage clamp simulations using 14 different current values from 0.005 nA to 0.075 nA. Currents were injected
in sequence with intervals of 500 ms. A 500 ms delay was used for the first current injection. Currents were injected using
different numbers of electrodes connected randomly to one up to five dendrites. The electrodes were placed in three different
areas of the dendritic tree, which correspond to areas into which DG molecular layer is divided: inner (IML), middle (MML) and
outer (OML) molecular layer (J. Neurosci. 31:105, 2011). Membrane voltages were recorded to generate spike counts and
interspike intervals. Simulation results show that PILO cell models are less excitable, presenting smaller number of spikes and
longer interspike intervals, mainly when stimulated at IML.
Conclusions:
Dendrites of DG new cell models exhibited lower response precisely in the area in which mossy fiber sprouting after SE
generates reverberant circuits, which stimulate DG cells. The smaller IML excitability of PILO cell models, if confirmed
experimentally, can be interpreted as a protective mechanism that allows cells to cope with increased stimulation resulting from
mossy fiber sprouting after SE.
Keywords: Computational neuroscience, Dentate gyrus, Detailed single-neuron modeling, Morphology-function relationship,
Mossy fiber sprouting
Financial Support: FAPESP, FAPESP-CInAPCe, CNPq, PROEX-CAPES, FAEPA
Resumo:20-011
THE ROLE OF MICRORNAS IN THE CONTROL OF APOPTOSIS IN NEURODEGENERATION – AN IN SILICO
APPROACH
Sousa, É. ; Kihara, A. H.
Universidade Federal do ABC, UFABC
Objectives:
Neurodegenerative diseases are some of the most common disorders that affect people in every age. Many of these disorders do
not have an effective therapy, and are extremely debilitating for the patients. In the last decade, the study of short RNAs increases
exponentially and have provide important information about pathogenesis and prognostic of several diseases. Nowadays, it is
well defined a new platform for drug development, based on short RNAs, especially microRNAs (miRNAs). These are noncoding oligonucleotides whose activity regulates negatively the translation of target mRNAs, by pairing a complementary
sequence at 3‘UTR. Each miRNA has potential to regulate many genes, and its function may be different depending on the tissue
and pathophysiological process. Thus, miRNAs analyses are complex because involves gene networks that govern several
activities at the same time. Bioinformatics provides many tools for these analyses, and are essentials for the success of miRNA
research. The aim of this study was to find potential targets for eighteen selected miRNAs through bioinformatics tools and infer
their putative role in neurodegenerative process.
Methods and Results:
Eighteen miRNAs (miR-7-1, miR-9-1, miR-21, miR-25, miR-96, miR-103, miR-107, miR-124-1, miR-146a, miR-150, miR-191,
miR-204, miR-210, miR-222, miR-223, miR-300, miR-335, and miR-494) were selected by previous described association with
neurodegeneration. Subsequently, we used miRGator (available at http://genome.ewha.ac.kr/miRGator/miRGator.html) and PITA
(available at http://genie.weizmann.ac.il/pubs/mir07/mir07_dyn_data.html) algorithms for prediction of potential targets for these
miRNAs. From this list of target genes, we selected genes expressed at neural tissue using the TiGER algorithm (available at
http://bioinfo.wilmer.jhu.edu/tiger/). The putative processes which these genes are involved were analyzed at Gene Ontology
(available at http://www.geneontology.org/). From this method, we were able to obtain a list of 64 genes involved in apoptosis
which are possible targets of the selected miRNAs. Since criterions for miRNA-mRNA interaction are still in discussion, the
number of target genes ranges from approximately 40 to 60, according to the target prediction method. This data, when analyzed
in parallel to published data, elucidate some cellular processes that, when deregulated, deflagrate apoptosis in neurodegeneration.
Conclusions:
Our in silico analysis permitted the visualization of gene interactive networks and provided data that may trigger studies using
experimental approaches. In doing so, it will be possible to find new ways to intervene at the molecular level to prevent damage
caused by neurodegeneration.
Keywords: microRNA, Apoptosis, Neurodegeneration, Retina, Bioinformatics
Financial Support: FAPESP, CNPq and UFABC
Resumo:20-012
ROLE OF ACTIVE DENDRITES IN SHAPING THE RESPONSE FUNCTION OF RETINAL GANGLION CELLS
Publio, R. ; Carlos, A.
Departamento de Fisica e Matematica/FFCLRP, USP
Objectives:
A central problem in contemporary neuroscience is to understand the functional role of voltage-dependent conductances in
neuronal dendritic trees (Annu Rev Neurosci 28:503, 2005). A recent hypothesis for the functional role of active dendrites is that
they enhance the dynamic range of neurons (PLoS Comput Biol 5:e1000402, 2009). The objective of this work is to investigate
this hypothesis using computational models of retinal ganglion cells.
Methods and Results:
We used a data set of 30 morphologically reconstructed ganglion cell models (J Neurophysiol 81:1685, 1999). The models
received four voltage-dependent (Na, K, Ca, KA), one calcium-dependent (KCa) and a leakage conductance, which were
distributed over their dendrites according to realistic patterns (J Neurophysiol 103:1357, 2010). The number and distribution of
ribbon synaptic locations in a dendritic tree were determined as in (J Neurophysiol 81:1685, 1999). Dendritic sites were
stimulated in various distributions with total excitatory currents in the range of 15-320 pA (J Neurophysiol 81:1685, 1999). We
fitted the experimental EPSP waveform with a double-exponential function. By varying the stimulus current we constructed
input-output response curves that related the firing frequency of the cell with the stimulus current (F-I curve). The dynamic range
was determined from the F-I curve as in (PLoS ONE 4:e6970, 2009). Our results show that the dynamic range depends on the
total dendritic area and the number of branches in the dendritic tree.
Conclusions:
Our results show a strong nonlinear effect of the active dendrites on the F-I curve of retinal ganglion cells. The dynamic range of
our model neurons is affected by the total dendritic area and the number of branches in the dendritic tree.
Keywords: Retina, Ganglion cell, Dynamic Range, Dendrite, Branch
Financial Support: FAPESP, CNPq, INCEMAQ
Resumo:21-106
NEURONAL NITRIC OXIDE SYNTHASE (NNOS) AND CONSOLIDATION OF CONTEXTUAL FEAR
CONDITIONING: EFFECTS OF 7-NITROINDAZOLE, A SELECTIVE NNOS INHIBITOR.
Faria, L. O. M. 1; Canova, F. 1; Vieira, A. S. 1; Teixeira, S. A. 2; Muscará, M. N. 2; Ferrari, E. A. D. M. 1
1
Anatomia, Biol. Celular, Fisiologia e Biofísica - IB/UNICAMP, UNICAMP
2
Departamento de Farmacologia - ICB/USP , USP
Objectives:
This study examined the effects of the intracerebroventricular administration of 7-nitroindazole (7-NI), a selective nNOS
inhibitor, in the consolidation of contextual fear memory in pigeons. The enzymatic activity of Ca2+-dependent and independent
NOS and protein expression of nNOS in the hippocampus were analyzed.
Methods and Results:
We used adult male pigeons with an average 350 g weight, distributed in 5 groups: 7-NI group (7-NI, 100nmol/0.5μ/l., i.c.v., n =
8), vehicle group (VEIC, DMSO 5% and PBS, n = 8), conditioning-untreated group (COND, n = 8), context-untreated group
(CONT, n = 8) and naive (NAIVE, n = 8). The pigeons were submitted to surgery for implant of an intracerebroventricular (i.c.v.)
cannula. Administration of 7-NI or vehicle followed immediately the 20 min training session which had three context-shock
pairings. After 24 hours, the animals were tested in a session of re-exposure to the conditioning context during 5 min. CONT
group was exposed to context without shock presentations and the NAIVE pigeons were only daily weighed. The sessions were
digitally recorded for posterior transcription and behavioral analysis. The hippocampal tissue was analyzed for enzymatic activity
of Ca2+-dependent and independent NOS which was expressed as pmols L-citrulline produced per minute and per mg of protein.
Protein expression of nNOS was analysed by the Western Blotting method and the values of optical densitometry of the
immunoreactive nNOS bands were normalized for the total protein content of the samples as determined by Ponceau S solution
for histochemical staining. In the training session of 7-NI, VEIC and COND groups there was greater expression of freezing in
the last five blocks of 1 min than in the first five blocks of 1 min (p < 0.05) and between 1 min interval blocks during the sessions
(p < 0.05). The enzymatic activity of Ca2+-dependent NOS showed marginally significant difference between groups (p = 0.06;
ANOVA). There was no significant difference in the enzymatic activity of Ca2 +-independent NOS (p > 0.05). Values of optical
densitometry of the immunoreactive nNOS bands in 7-NI, VEIC, COND and CONT groups were greater than the observed for
NAIVE group (p <0.05).
Conclusions:
The behavioral data indicate that inhibition of nNOS with i.c.v. administration of 7-NI immediately after training disrupted the
consolidation of aversive contextual memory in pigeons. The alteration of both the enzymatic activity of NOS and the
quantitative nNOS protein expression in the hippocampus suggests the participation of nitric oxide in the retrieval of aversive
contextual.
Keywords: contextual fear conditioning, hippocampus, neuronal nitric oxide synthase, 7-nitroindazol, freezing
Financial Support: FAPESP process – 2009/13213-7
Resumo:21-107
CARDIOVASCULAR RESPONSES EVOKED FROM THE PERIAQUEDUCTAL GREY REQUIRE NEURONAL
ACTIVITY IN THE AMYGDALA.
de Abreu, A. R. R. ; Abreu, A. R. R. ; de Menezes, R. C. A
Departamento de Ciências Biológicas, UFOP
Objectives:
The periaqueductal gray matter (PAG) is involved in the integration of specific cardiovascular changes associated with emotional
behaviors. Moreover, microinjection of excitatory aminoacids (EEA) into the lateral/dorsolateral column of the PAG (l/dlPAG)
evokes increases in the heart rate (HR) and in the mean arterial pressure (MAP) in a pattern similar to those observed during
emotional stress. This pattern of cardiovascular changes can also be attained by activation of the basolateral amygdala (BLA)
neurons. Therefore, the aim of our study is to evaluate the role of the BLA in the cardiovascular responses evoked by the
stimulation of PAG.
Methods and Results:
Methods: Male Wistar rats (300 ± 20g) were anesthetized with ketamine-xylazine (80 mg/kg – 11.5 mg/kg, i.p), and ipsilateral
guide cannulae were implanted into the l/dlPAG and into the BLA. In another set of rats we implanted guide cannulae into the
l/dlPAG and into a region at the vincinity of the BLA (NBLA), but outside of it (Negative Control). Six days latter, the rats were
anesthetized again (Isoflurane 2% in 3l of O2) and a catheter was inserted into the femoral artery for recording of MAP and HR.
Experiments were performed 48 hr later. Each rat was subjected to two different trials, 24h apart, and in random order, in which
either saline vehicle (100 nl) or muscimol (100 pmol/100 nl) was microinjected into the BLA/NBLA followed, 5 minutes later,
by the microinjection of a EEA, NMDA (6pmol/100nl), into the l/dlPAG. Results: Microinjection of muscimol into the BLA
attenuated the increase in HR evoked by the microinjection of NMDA into the l/dlPAG when compared with the saline treatment
(24 ± 9 vs 79 ± 10 bpm after saline, n=6, p=0,0003, by student paired t-test). Likewise, the microinjection of muscimol into the
BLA reduced the increase in MAP produced by the microinjection of NMDA into the l/dlPAG (6 ± 2 vs 16 ± 4 mmHg after
saline, n=6, p=0,021, by student paired t-test). In negative control animals, microinjection of muscimol into the NBLA did not
attenuate the increase in HR evoked by the microinjection of NMDA into the l / dlPAG compared with saline treatment (71 ± 16
vs 88 ± 5 bpm, n=4, p=0,3248, by student paired t-test). Similarly, the microinjection of muscimol into the NBLA did not reduce
the increase in MAP produced by the microinjection of NMDA into the l / dlPAG (13 ± 3 vs 22 ± 7 mmHg after saline, n=4,
p=0,3554, by student paired t-test).
Conclusions:
These results indicate that the neuronal activity in the region of the BLA plays a role in the generation of physiological response
induced by activation of the l/dlPAG neurons.
Keywords: Amygdala, Muscimol, NMDA, Periaqueductal, Stress
Financial Support: CNPq, FAPEMIG e UFOP
Resumo:21-108
BDNF AND ESTROGEN LEVELS ARE CORRELATED IN WOMEN WITH BIPOLAR DISORDER
Colpo, G. D. 1,2,3; Sulzbach, M. 2,3; Brietzke, E. 2,3; Tramontina, J. 2,3; Migliavacca, F. 2,3; Goi, P. D. 2,3;
Wollenhaupt - Aguair B. 1,2,3; Ceresér, K. M. M. 2,3; Sant'anna, M. K. 2,3; Kapczinski, F. 1,2,3
1
Programa de Pós Graduação Ciências Médicas/ UFRGS, UFRGS
2
Programa de Transtorno Bipolar/ HCPA, HCPA
3
INCT Translacional em Medicina, INCT-TM
Objectives:
Hormonal changes throughout life are associated with mood changes in healthy women. Estrogen (E) seems to have a positive
correlation with the levels of brain-derived neurotrophic factor (BDNF) in healthy volunteers. However, this correlation was not
investigated in patients with bipolar disorder (BD). The study aims to investigate the relationship between brain-derived
neurotrophic factor (BDNF) levels and hormones involved in hypothalamus-pituitary-gonadal axis in women with bipolar
disorder (BD), during reproductive years and also in postmenopausal group. In addition, differences across the two phases of
menstrual cycle were also evaluated in the group during reproductive years.
Methods and Results:
Thirty two euthymic women with BD were included. Patients in reproductive years had regular menstrual cycles without
hormonal contraceptive and postmenopausal women were not under hormonal replacement therapy. Endocrine instable
conditions were considered an exclusion factor. Blood samples were withdrawn for measures of BDNF, estrogen (E),
Progesterone (P), LH and FSH levels, being collected in follicular and lutheal phases of menstrual cycle. Menopausal women
were held only one blood sample. Diagnoses were assessed using SCID-I. Results: Considering the whole sample, BDNF
presented a positive correlation with E levels (Pearson Correlation, coefficient= 0.36, P= 0.043). In patients in reproductive
phase, there was a negative correlation with FSH (Pearson Correlation, coefficient= 0.831, P= 0.040). A similar result was found
with LH levels (Pearson Correlation, coefficient= 0.908, P=0.012) in the second phase of menstrual cycle.
Conclusions:
There was a statistically significant positive correlation between BDNF and E levels, with higher BDNF levels being associated
with o estrogenic stimulation. The results of this study open new avenues of investigation in pathophysiology of mood
abnormalities related to hormonal variation as well as in their treatment.
Keywords: bipolar disorder, women, brain-derived neurotrophic factor , estrogen, hypothalamus-pituitary-gonadal axis
Financial Support: HCPA, CNPq
Resumo:21-109
RENOVASCULAR HYPERTENSION INFLUENCES THIRST AND SODIUM APPETITE IN RATS.
Blanch, G. T. 1; Freiria-oliveira, A. H. 1; Amaral, A. A. 1; Sumners, C. 2; Menani, J. V. 1; Colombari, E. 1;
Colombari, D. S. A. 1
1
Department of Physiology and Pathology, FOAr-UNESP
2
Department of Physiology and Functional Genomics, Univ. of Florida
Objectives:
Previous studies have shown that daily 2% NaCl intake was greater in rats with renovascular 2 kidney 1 clip hypertension [2K1C;
Schomig et al, Clin. Exp. Pharmacol. Physiol., 7(2): 169, 1980]. However, whether 2K1C hypertension affects induced water
intake and/or sodium intake is unknown. Thus, we aimed to study the effect of 2K1C hypertension on water intake induced by
increasing plasma osmolality (intragastric 2 M NaCl) and on sodium appetite induced by the treatment with furosemide (FURO)
along with a low dose of captopril (CAP).
Methods and Results:
Male Holtzman rats (150-180 g) maintained with water and 0.3 M NaCl ad libitum were anesthetized, and the left renal artery
was partially obstructed with a silver clip of 0.2 mm width. Sham animals were submitted to the same surgical procedure without
partial renal artery occlusion. After 5 weeks, on the day of the experiments, sham (n = 7) and 2K1C (n = 8) rats had water, NaCl
and food removed from the cages. The rats received, sequentially, 5 days apart, an intragastric (ig) load of 2 M NaCl (2 ml) and a
subcutaneous (sc) injection of FURO (10 mg/kg) + CAP (5 mg/kg). In both cases, urine output was collected over the next hour
after the treatments. After this 1 h period, the fluids were returned to the cage and intakes/outputs were recorded for 2 h. Urine
volume and natriuresis after hypertonic saline were similar amongst the groups (sham: 1.9 ± 0.4, vs. 2K1C: 2.6 ± 0.6 ml/1 h and
sham: 1874 ± 179 vs. 2K1C: 1941 ± 293 µEq/1 h). The water intake induced by ig administered 2 M NaCl was smaller in 2K1C
rats (sham: 9.5 ± 0.8, vs. 2K1C: 6.2 ± 1.1 ml/2 h, p< 0.05). In the FURO + CAP protocol diuresis (sham: 7.8 ± 0.8 vs. 2K1C: 7.3
± 1 ml/1h) and natriuresis (sham: 853 ± 80 vs. 2K1C: 752 ± 124 µEq/1 h) values were not different between the groups.
Nevertheless, sodium intake induced by FURO + CAP was smaller in 2K1C rats compared to shams (sham: 5.9 ± 0.9 vs. 2K1C:
3.0 ± 0.4 ml/2 h, p<0.05).
Conclusions:
The results suggest that renovascular hypertension impairs water intake induced by hyperosmolality and sodium intake induced
by sodium depletion. Further studies are necessary to disclose the mechanisms involved with these responses in 2K1C rats.
Keywords: 2K1C, water intake, extracellular dehydration, cellular dehydration, diuresis
Financial Support: CAPES-PNPD, CNPq, FAPESP, NIH HL-076803
Resumo:21-110
A2 NEURON LESION INCREASES HYPOTHALAMIC MAGNOCELLULAR PARAVENTRICULAR NUCLEUS
ACTIVATION INDUCED BY PLASMA HYPEROSMOLALITY.
Freiria-oliveira, A. H. ; Blanch, G. T. ; Menani, J. V. ; Colombari, E. ; Colombari, D. S. A.
Dept of Physiology and Pathology, FOAr-UNESP
Objectives:
Plasma hyperosmolality activates the paraventricular (PVN) and the supraoptic nuclei (SON) of the hypothalamus and increases
vasopressin secretion. Previously we have shown that hyperosmolality causes vasopressin-dependent pressor response in rats
with specific lesions of the A2 noradrenergic group of the commissural nucleus of the solitary tract (A2/cNTS). In the present
study, using c-fos immunohistochemistry labeling, we aimed to investigate the neuronal activation in the hypothalamic areas
activated by plasma hyperosmolality in A2/cNTS-lesioned animals.
Methods and Results:
Male Holtzman rats (280-320 g) were submitted to lesions of dopamine-beta-hydroxilase (DβH)-containing neurons in the
A2/cNTS achieved by injections of anti-DβH-saporin (12.6 ng/60 nl, A2 lesion group, n=4) or sham lesions (injection of IgGsaporin, 12.6 ng/60 nl, n=4). Plasma hyperosmolality was induced by intragastric (ig) 2 M NaCl load (2 ml/rat). For training, the
animals were submitted to one ig 2 M NaCl load 7 days before the experiments and in the next 6 days, the animals received one
ig 0.15 M NaCl load (2 ml) daily. In the day of the experiments, half of the A2 lesioned and half of sham rats received ig load (2
ml) of 2 M NaCl and the remaining animals received ig load of 0.15 M NaCl. Two hours later the animals were deeply
anesthetized (pentobarbital – 50 mg/kg ip) and perfused with 4% paraformaldehyde. C-fos imunohistochemistry procedure was
performed using DAB staining. Intragastric 0.15 M NaCl load promotes no c-fos expression in SON, magnocellular PVN
(mPVN) and parvocellular PVN (pPVN) in both sham and A2 lesion groups. Intragastric 2 M NaCl load similarly increased c-fos
expression in both SON and pPVN in sham animals (SON: 105 ± 27 and pPVN: 22 ± 9 cells/section) and in A2 lesioned animals
(SON: 88 ± 7 and pPVN: 37 ± 6 cells/section,). However, ig 2 M NaCl load increased more the c-fos expression in the mPVN of
A2 lesioned animals (90 ± 13 cells/section, p < 0.05) compared to sham (47 ± 20 cells/section).
Conclusions:
These results suggest that the activation of the mPVN by hyperosmotic stimuli is reduced or limited by an inhibitory mechanism
dependent on the cNTS A2 noradrenergic neurons.
Keywords: PVN, osmoreceptors, NTS, c-fos
Financial Support: FAPESP, CNPq
Resumo:21-111
CANNABINOID CB1 RECEPTOR EXPRESSION IN THE RAT BASAL GANGLIA AND THE EFFECTS OF ACUTE
CANNABINOID TREATMENT IN RATS WITH 6-HYDROXYDOPAMINE-INDUCED HEMIPARKINSONISM
Chaves, G. P. ; Mazucanti, C. H. Y. ; Real, C. C. ; Britto, L. R. G. ; Torrão, A. S.
Department of Physiology and Biophysics, ICB - USP
Objectives:
Although pharmacological evidence indicates the occurrence of cannabinoid CB1 receptors in the basal ganglia, detailed
morphological and functional data are scarce on this issue. Moreover, in Parkinson´s Disease (PD) the function of CB1 and its
relation to other neurotransmitter systems (such as the GABAergic system) are both unclear. Yet the cannabinoid system seems
to have an important role in neurodegeneration and/or neuroprotection processes. We investigated here the possible colocalization of CB1 with markers for glial cells and structural elements of neurons and with tyrosine hydroxylase (TH) in the
basal ganglia of rats. We also analyzed, in 6-hydroxy-dopamine (6-OHDA)-induced PD rats, the GABA, GAD and CB1 levels in
the basal ganglia, the acute action of AM 404 (an inhibitor of anandamide reuptake) on the expression of TH and glial cell
markers, and the acute effects of different doses of ACEA (cannabinoid agonist) on the TH expression in the basal ganglia.
Methods and Results:
Double immunostaining of CB1 with markers for astrocytes (GFAP), oligodendrocytes (OD), microglia (OX-42), axons
(neurofilaments), dendrites (MAP-2) and TH was performed in the basal ganglia of normal rats. Other rats received injections of
6-OHDA (12µg in 1µl) in the right striatum, and the left striatum was used as a control. The 6-OHDA-lesioned animals were then
divided into five groups: (1) time course evaluation of the lesion after distinct survival times (3, 7, and 10 days); (2) acute
treatment with 2 mg/kg of AM 404 or (3) vehicle; (4) acute treatment with 0.5, 1, 2, and 4mg/kg ACEA or (5) vehicle. Brains
from the time course group were subjected to immunohistochemistry for TH, CB1, and GABA, and immunoblotting for GAD.
Brains of AM 404 group was processed for TH, GFAP, and OX42, and of the ACEA group was processed for TH
immunohistochemistry. The results were analyzed by one-way ANOVA or unpaired t test. We found scarce co-localization of
CB1 with markers of glial cells, but a marked co-localization with MAP-2, especially in the substantia nigra (SN) and the
external globus pallidus. The co-localization of CB1 with axon markers occurred primarily in the SN. Co-localization of CB1
with TH was not clearly detected in the basal ganglia. In PD-induced rats, no change was found in the expression of GABA or
GAD in basal ganglia at all survival times analyzed (N=3, p>0.05). On the other hand, a gradual decrease in CB1 staining of
about 30% and 40% in the SN was observed after 7 (N=4, p< 0.01) and 10 days (N=3, p< 0.05) after the lesion, respectively. The
treatment with AM 404 decreased the TH expression in SN by 10% (N=5, p
Conclusions:
This study adds information about the cellular localization of CB1 in the basal ganglia and indicates that CB1 receptor expression
decreases in PD by an event that is unrelated to the GABAergic system. It is possible that this event is related to some indirect
effect of the loss of dopaminergic neurons. Nevertheless, acute activation of CB1 appeared to be deleterious in the 6-OHDA rat
model of PD.
Keywords: Cannabinoid System, CB1 receptor, Neurodegeneration, Parkinson´s Disease
Financial Support: FAPESP and CNPq.
Resumo:21-112
REACTIVE OXYGEN SPECIES AND ASCORBIC ACID ARE MODIFIED IN SPINAL CORD OF RATS WITH
NEUROPATHIC PAIN
Goecks, C. S. B; Horst,a. ; Moraes, M. S. ; Scheid, T ; Kolberg, C. ; Partata, W. A.
Fisiologia/ICBS, UFRGS
Objectives:
Reactive oxygen species (ROS) have been implicated in the development of persistent pain states, including neuropathic pain
(one ocurring as a result of peripheral or central nervous system injury). While the involvement of ROS in this condition is
becoming clear, the detailed changes to these species in different models of neuropathic pain are still unclear. Thus, this study
proposes to correlate hyperalgesia and hydrogen peroxide (H2O2), superoxide dismutase (SOD) and catalase activities, and
ascorbic acid (AA) measures in the spinal cord of rats submitted to chronic constriction injury (CCI) of the sciatic nerve, a useful
experimental model of neuropathic pain.
Methods and Results:
After approval of the trial by the Ethical Committee of Animal Research of the Federal University of Rio Grande do Sul (No.
2008052), adult male rats weighing 200-250 g were divided in three experimental groups (six animal/group): Näive (animals
which did not undergo surgical manipulation), Sham (animals in which all surgical procedures to expose the sciatic nerve were
used except the constriction of this nerve) and CCI (animals in which the sciatic nerve was exposed and 4 ligatures were placed
around the nerve at the proximal part of the trifurcation with a distance of 1 mm between each ligature). The animals were
sacrificed 3 and 10 days after the surgical procedure. The mechanical threshold with von Frey hairs and thermal latency with hot
plate were measured just before the surgery and 3 and 10 days after it. H2O2 determination was based on horseradish peroxidase-
mediated oxidation of phenol red. SOD activity was determined by the inhibition of superoxide radical reaction with pyrogallol.
Catalase activity was determined by following the decrease in H2O2. AA level was determined through the 2,4-dinitrophenylhydrazine derivative of dehydroascorbic acid. Data were expressed as means and errors and compared by two-way
ANOVA followed by the Bonferroni test. Differences were considered statistically significant when p
Conclusions:
Overall, these data demonstrate that hyperalgesia attributed to the CCI model of the sciatic nerve coincides with changes in spinal
cord antioxidant enzymes and AA. These modifications should be occurring to maintain the reduced levels of H2O2. However,
such reduction did not attenuate the hyperalgesia signs caused by CCI of the sciatic nerve.
Keywords: chronic constriction injury , sciatic nerve, superoxide dismutase, catalase, hydrogen peroxide
Financial Support: CNPq, FAPERGS
Resumo:21-113
PROFILE FUNCTIONALITY AND DEPRESSIVE STATE IN PATIENTS WITH PARKINSON'S DISEASE
Freire, Q. C. 1; Filho, H. D. A. G. 1; Lima, A. L. S. D. 1; Aragão, C. D. A. 1; Campêlo, C. L. D. C. 1; Neto,
E. R. 1; Campelo, M. D. G. L. D. C. 2; Franco, C. I. F. 1
1
Depto. de Fisioterapia/ Universidade Estadual da Paraíba, UEPB
2
Depto. de Medicina/ Universidade Federal de Campina Grande, UFCG
Objectives:
Individuals with Parkinson's Disease (PD) may present cognitive decline, causing losses in thinking, perception and trial. Thus,
difficulties with memory, calculation and activities that require spatial orientation end up occurring with greater frequency. This
study aimed to evaluate motor and cognitive changes as well as depressive symptoms present in patients with PD.
Methods and Results:
METHODS: The sample consisted of eight individuals of both sexes with a clinical diagnosis of PD, assisted in Physical Therapy
Clinic of UEPB. The instruments used were the Neurological Assessment Protocol for socio-demographic, Rating Scale Unified
Parkinson's disease (UPDRS) for analysis of motor exploration activity, and the Beck Depression Inventory (BDI), to survey the
intensity depressive symptoms. Data were analyzed using Graph Pad Prism 4.02, and values are expressed in percentage, mean
and standard deviation, considering significant values with p
Conclusions:
Based on data obtained, it is possible to suggest that changes in PD motor precedence over the cognitive, as well as most of the
sample showed signs of depression.
Keywords: Cognition, depressive state, motor changes, Parkinson's disease
Financial Support: PROBEX / UEPB
Resumo:21-114
STROKE: ANALYSIS OF CORRELATION OF TRANSFER FUNCTION BETWEEN TWO INSTRUMENTS FOR
MOTOR EVALUATION.
Silva, M. J. C. ; Franco, C. I. F. ; Monteiro, D. L. ; Brito, A. S. S. ; Tavares, C. D. ; Araújo, D. P.
Depto. de Fisioterapia/Universidade Estadual da Paraíba, UEPB
Objectives:
The hemiparesis is the classic clinical signs resulting from an ischemic or hemorrhagic cerebrovascular disease, involving cortex
or brainstem, which compromises the motor control of the affected hemisphere. Spasticity leads to loss of functional capacity and
personal achievement of these individuals. The aim of this study was to investigate the correlation between the transfer function
of two motor evaluation instruments in chronic hemiparetic.
Methods and Results:
This is an exploratory, descriptive and cross-sectional study involving 17 individuals assisted in the extension project "Group of
Interdisciplinary Assistance of the hemiparetic patient - GAIPH" conducted at the School of Physiotherapy, State University of
Paraíba - UEPB. It was used as an evaluation tool, Neurological Assessment Protocol for characterization socio-demographic
data, the category transfer of Motor Functional Independence Measure (MIFm) and the Berg Balance Scale (BBS). The data were
analyzed using the software Graph Pad Prism 4.02, and values are expressed in percentage, mean and standard deviation,
considering significant values with p <0.05).
Conclusions:
Based on the results we suggest that there was no correlation between the transfer function of FIM and command of BBS, except
for field stationary tests.
Keywords: Berg Balance Scale, Cerebrovascular Disease, Hemiparesis, Motor Functional Independence Measure, Physiotherapy
Financial Support: PROBEX-UEPB
Resumo:21-115
SOCIAL ISOLATION IMPAIRED OLFACTION AND SOCIAL DISCRIMINATION MEMORY, BUT NOT
NEUROGENESIS IN THE MOUSE OLFACTORY BULB.
Monteiro, B. M. D. M. 1; Raslan, A. C. S. 1; Moreira, F. A. 2; Massensini, A. R. 1; Moraes, M. F. D. 1;
Pereira, G. S. 1
1
FISIOLOGIA/UNIVERSIDADE FEDERAL DE MINAS GERAIS, UFMG
2
FARMACOLOGIA/UNIVERSIDADE FEDERAL DE MINAS GERAIS, UFMG
Objectives:
To evaluate the effect of social isolation on olfaction and social discrimination memory, as well as on olfactory bulb
neurogenesis.
Methods and Results:
Methods: twenty C57/Bl6 male mice (8-12 weeks old) were divided in two groups: social isolated (SI) and group-housed (GH)
for 6 days. Then, each animal was placed in a box containing two empty cylinders with around 60 holes. After 30 min, one Swiss
juvenile (around 21 days old) was introduced into each cylinder and the time spent in social exploration (when the resident
animal introduced its nose and/or whiskers inside any of the cylinder‘s holes) was quantified during 5 min. Twenty-four hours
later, the animal was re-introduced in the same box, now containing one familiar and one new juvenile. The social behavior was
measured during 5 min. As a control for olfactory perception, each mouse had one piece of chocolate introduced in its home cage
(7g/day during 3 days). Then, the food and chocolate were withdrawn and, 12 hours later, the animals were introduced in a new
box, where the latency to find a hidden piece of chocolate was measured. A second set of animals was used to measure
neurogenesis in the olfactory bulb. Ten Swiss male mice (8-12 weeks old) were divided in SI and GH groups and received a daily
injection of BrdU (75 mg / kg) during 7 days. Twenty-four hours after the last injection, they were perfused with 4%PFA and the
brains were processed for BrdU and NeuN immunofluorescence. Images were captured using a microscope and a Z-stack analysis
was performed at 1um intervals. New neurons were quantified using Image J software. The data were analyzed by Student‘s t
test. Results: Both groups present similar social recognition indexes in the day 1 (GH=0.37±0.10; SI=0.39±0.11; p=0.74), but
only the GH group was able to discriminate juveniles in the day 2 (GH=0.66±0.1; SI=0.39±0.14; p=0.0002). The SI group took
longer to find the hidden chocolate compared to GH group (GH =20.16±6.74s;SI=46.56±15.05s; p=0.0001). There was no
significant difference between groups in the number of BrDu/NeuN positive cells in the olfactory bulb.
Conclusions:
Social isolation impaired social discrimination memory, probably by reducing the animal‘s olfactory capacity. However, the
decrease in the sensory input did not impaired cell proliferation in the olfactory bulb. Thus, it seems that the mnemonic and
sensorial changes caused by social isolation cannot be attributed to a decrease in neurogenesis.
Keywords: NEUROGENESIS, OLFACTION MEMORY, SOCIAL ISOLATION , SOCIAL DISCRIMINATION MEMORY
Financial Support: CAPES, CNPq, FAPEMIG
Resumo:21-116
ABSENCE OF TYPE I AND II INTERFERON SIGNALING AND THE EXPRESSION OF ANXIETY-LIKE
BEHAVIORS IN MICE
Saito, V. M. ; Rodrigues, D. H. ; Miranda, A. S. ; Teixeira, A. L.
Laboratório de Imunofarmacologia, UFMG
Objectives:
The objective of this study was to evaluate the role of interferons (IFN) in the expression of emotional behaviors by submitting
naïve female knockout IFN-α/β receptor mice (IFNAR(-/-)) and IFN-γ deficient mice (GKO) and their wild type (WT) littermates
to behavioral paradigms commonly used to study depressive- and anxiety-like traits, i.e., the Elevated Plus Maze (EPM),
Porsolt‘s Forced Swim Test (FST) and Open Field Test (OFT).
Methods and Results:
Female IFN-γ knockout mice (GKO; n=13), IFN-α/β receptor knockout mice (IFNAR(-/-), n=8) and their wild type littermates
(WT; n=22) raised on a C57BL/6J genetic background (7 to 10 weeks old) were tested on Day 1 on the EPM, where the number
of open arm entries and time spent on open arms during a period of 5 minutes were recorded. On Day 2, all groups were tested on
the OFT, which consisted of a circular arena, divided into 4 central portions and 8 peripheral sectors where animals were allowed
to explore freely for 5 minutes and their locomotion score was recorded; and in the FST, in which mice were placed individually
in a 3-liter glass beaker filled with water and motion recorded for 6 minutes. Immobility time was measured during the last 4 min
of the session. Behavioral analyses were performed by a trained observer. Data were analyzed using one-way ANOVA followed
by Bonferroni‘s post test. Values are expressed as Mean ± Standard Error of the Mean. A p value less than 0.05 was considered
statistically significant. In the EPM, the percent time spent in open arms averaged 34.09 ± 5.2 in the control group (n=22).
IFNAR(-/-) mice spent significantly more time in the open arms (65.1 ± 6.0, n=8, p<0.05).
Conclusions:
These findings suggest that IFN-gamma-deficient mice may have alterations in brain pathways that regulate behavior. The
complex phenotype displayed by KO mice indicates that these animals cope differently with stressors, and further studies are
necessary to investigate the biochemical aspects of this phenomenon.
Keywords: Anxiety, Behavior, Cytokine, Genetic knockout, Neuroimmunology
Financial Support: CAPES/FAPEMIG
Resumo:21-117
THE CO-CHAPERONE BAG2 REVERSES NICOTINE-INDUCED UP-REGULATION OF TOXIC TAU IN SH-SY5Y
CELL LINE - IMPLICATIONS FOR ALZHEIMER'S DISEASE.
Oliveira, A. S. A. D. 1,2; Costa, M. A. 2; Fior-chadi, D. R. 2; Carrettiero, D. C. 1
1
Universidade Federal do ABC, UFABC
2
Departamento de fisiologia - IB/Universidade de São Paulo, USP
Objectives:
Alzheimer's disease (AD) is well characterized by the presence of histopathological hallmarks like neurofibrillary tangles (PHF)
composed mainly by intracellular hyperphosphorylated microtubule-associated protein Tau. A decrease in nicotinic receptor on
cellular membrane is also reported in AD patients. The co-chaperone BAG2 up-regulate the clearance of toxic Tau by increasing
cellular degradations process. In light of this, the aim of the present study is to investigate the relationship between nicotinic
receptor activation and the modulatory effect of BAG2 activity on hyperphosphorylated Tau clearance in SH-SY5Y cell line.
Methods and Results:
Human neuroblastoma SH-SY5Y cells were grown in a 1:1 mixture of essential growth medium DMEM and F12 supplemented
with 15% of fetal bovine serum, antibiotic and allowed it to grow at 37ºC. 24 h after plating, SH-SY5Y cells were treated with
different concentrations of nicotine (10, 50 and 100μM) by 6h in the presence and absence of pEGFP-C1 expression vector
containing the cDNA sequence of BAG2. After treatment, the proteins were extracted and subjected to Western blotting
technique in order to investigate the levels of hyperphosphorylated Tau using P-Tau Ser 199/202 antibody. Superexpression of
BAG2 promoted a decrease in phosphorilated Tau levels (68.67 ± 9.26*, % of control). In absence of exogenous BAG2
expression, nicotine promoted an increase in phosphorilated Tau levels (10μM, 111.20 ± 11.28; 50μM, 137.90 ± 9.30*; 100μM,
153.40 ± 10.68**, % of control). However, in the presence of superexpressed exogenous BAG2, nicotine promoted a decrease in
phosphorilated Tau levels (10μM, 40.33 ± 9.02**; 50μM, 29.33 ± 4.91**; 100μM, 15.00 ± 4.04**, % of control). Statistical
analyses were performed using one-way ANOVA followed by Dunnett‘s multiple comparison test. Values are shown as mean ±
S.E.M., *p < 0.05, **p < 0.01, n=3.
Conclusions:
The relationship among nicotinic receptor, hyperphosphorylated Tau and BAG2 is not yet characterized in the scientific
literature. We demonstrated in the present study that the co-chaperone BAG2 indeed up-regulate the clearance of toxic Tau.
However, the main point of the present work is that the co-chaperone BAG2 reverses nicotine-induced up-regulatoins of toxic
Tau. In other words, nicotinic receptor activation might increase BAG2 activity and consequentially up-regulate
hyperphosphorylated Tau clearance in SH-SY5Y cell line. These data suggest a possible pharmacological approach for
Alzheimer's disease.
Keywords: Alzheimer, BAG2, Degradation, Nicotinic receptor, Tau phosphorylation
Financial Support: UFABC, FAPESP
Resumo:21-118
MEMORY EVALUATION AND ANTIEPILEPTIC ACTION OF INTRA-HIPPOCAMPAL INJECTION OF
ADENO−ASSOCIATED VIRAL (AAV) GENE OF THE NEUROPEPTIDES Y (NPY) IN THE PILOCARPINE MODEL
IN MARMOSETS (CALLITHRIX JACCHUS)
Blanco, M. M. 1; Kohek, S. R. B. 1,1; Baldaia, M. A. 1; Pontes, J. C. C. 1; Cinini, S. M. 1; Mendes, P. P. 1;
Bland, Rj2; Fitzsimons, H. L. 2; During, M. J. 3; Vezzani, A. 4; Mello, L. E. 1
1
Fisiologia, unifesp
2
Neurology, Neurologix
4
Neuroscience, Laboratory Exp. Neurol., Mario Negri Inst
3
Molecular Virology,Immunology and Med Genetic, OSU
Objectives:
Here we evaluated the role of overexpression of NPY as an anticonvulsant and memory effects based on the administration of a
recombinant adeno-associated viral vector (rAAV) in marmoset.
Methods and Results:
Marmosets were subjected to stereotaxic surgery for injection of the rAAV in the hippocampus and implanted with a sensor for
telemetry recordings of electrocorticography (ECoG).The titer of the injected solution was 2.7x1013 vg/mL (rAAV1-CBANPY). In addition we also had a control non-injected group. All animals were submitted to memory tests in three periods
(baseline [after virus/before pilocarpine], latent [short after pilocarpine] and chronic [long after pilocarpine]) and to the induction
of status epilepticus (SE) by means of pilocarpine administration (250mg/kg, i.p.). ECoG and histological analysis were
evaluated. RESULTS: The baseline memory test for the T5 (hippocampus-dependent) task showed an improvement in the
performance of the rAAV1-NPY group when compared to control groups. The induction of SE in rAAV1-NPY did not differed
in the characteristics of SE except for phase IV, where rAAV-NPY animals had a shorter duration (60+28 min) than controls
(160+27 min). Memory tests in rAAV1-NPY groups at the latent and chronic periods showed an improved performance when
compared to baseline period. The onset of SRS (21+3.5 and 9+3.7 weeks) and its frequency (0.02+0.01 and 0.50+0.01
seizures/day) were markedly different between rAAV-NPY and control animals, respectively. In the rAAV1-NPY group there
was an increase in the expression of NPY in the hippocampal region as compared to controls.
Conclusions:
Memory tests demonstrated improved performance that was specific for hippocampus-dependent-task in rAAV1-NPY injected
animals. The rAAV-NPY in the marmoset model of pilocarpine did not lead to clear anti-SE effects. In the chronic period
however there was a long-lasting NPY over-expression in neurons and a marked decrease in the frequency of spontaneous
seizures.
Keywords: neuropeptides y, marmoset, epilepsy, memory, pilocarpine model
Financial Support: FAPESP and CNPq (Brazil)
Resumo:21-119
PERINATAL UNDERNUTRITION INDUCES A LONG-LASTING DEFICIT OF CELL PROLIFERATION IN THE
HIPPOCAMPUS.
Pinho, M. C. 1; Matos, R. J. B. 2; Orozco-sólis, R. 3; Lopes-de-souza, S. 4; Manhães-de-castro, R. 5;
Bolaños-jimenez, F. 6
1
Depto. de Fisioterapia/Universidade Federal de Pernambuco, UFPE
2
Núcleo de Educação Física e Ciências do Esporte, UFPE/CAV
3
Irvine University, IU
4
Departamento de Anatomia/Universidade Federal de Pernambuco, UFPE
5
Departamento de Nutrição/Universidade Federal de Pernambuco, UFPE
6
UMR1280-Physiologie des Adaptations Nutritionnelles, INRA
Objectives:
Maternal malnutrition alters various maturational events in the brain of the offspring resulting in learning and memory deficits
that extend into adulthood. At the cellular level, learning and memory relay on the production of new neurons in the hippocampal
dentate gyrus (DG), but whether adult neurogenesis is affected by early malnutrition has not been completely explored. To get a
better insight into the cellular mechanisms that might underlie the deleterious effects of perinatal undernutrition on learning and
memory, here we examined the neurogenesis processes in the hippocampus of adult rats exposed to prenatal and/or neonatal
nutrient restriction.
Methods and Results:
Pregnant Spraguey-Dawley rats were fed either ad libitum (C) or were undernourished by reducing their daily food intake by
50% in relation to the C group during gestation and lactation (FR) or during the lactation period only (AdLib/FR). At 60 days of
age, the offspring from each group was injected with bromodeoxyuridine (BrdU), and sacrificed after 2 h, 1 week or 3 weeks.
The number of BrdU-labeled cells in the DG was significantly reduced in the offspring of FR dams in relation to controls at all
the time points examined. However, the proportion of new cells exhibiting a neuronal phenotype was higher in FR rats than in
controls as revealed by the co-labeling at 3 weeks of the BrdU-labeled cells with NeuN. AdLib/FR animals, exhibited also a
reduction in cell proliferation at 2h and 1 week. However, we found no significant differences at 3 weeks in either the number of
BrdU-labelled cells or in the proportion of new neurons between controls and AdLib/FR rats.
Conclusions:
These results indicate that undernutrition during perinatal development induces a long-lasting deficit of cell proliferation in the
hippocampus without affecting the neurogenesis process per se.
Keywords: cell proliferation , hippocampus, neurogenesis, Perinatal undernutrition
Financial Support: INRA-Université de Nantes (France) and FACEPE/CNPq.
Resumo:21-120
SELECTIVE LESION OF RETROTRAPEZOID PHOX2B-EXPRESSING NEURONS ATTENUATES THE CENTRAL
CHEMOREFLEX IN CONSCIOUS RATS.
Ragioto, D. A. M. T. 1; Falquetto, B. 1; Colombari, E. 3; Moreira, T. S. 1; Takakura, A. C. 1
1
Department of Physiology and Biophysics, ICB USP
2
Department of Pharmacology, ICB USP
3
Department of Physiology and Pathology, FOAr UNESP
Objectives:
The retrotrapezoid nucleus (RTN) contributes to an unknown extent to the central chemoreflex (the activation of breathing by
elevation of CNS PCO2). Neurophysiological and genetic evidence suggests that the RTN neurons involved in this reflex are a
group of chemosensitive glutamatergic interneurons that express the transcription factor Phox2b and lack tyrosine-hydroxylase
(henceforth called RTN Phox2b+TH− neurons). In the present study, we ask whether the selective destruction of Phox2b+THRTN neurons could affect breathing in conscious rats.
Methods and Results:
Male Wistar rats (280-300 g) with bilateral injections of the toxin [Sar9,Met(O2)11]-substance P (SSP-SAP) were used. We
showed that RTN contains around 2000 Phox2b+TH- cells and bilateral injections of SSP-SAP into RTN destroyed Phox2b+THneurons but spared facial motoneurons, catecholaminergic and serotonergic neurons and the ventral respiratory column caudal to
the facial motor nucleus. Bilateral inhibition of RTN neurons with SSP-SAP (0.6 ng/30 nl) reduced resting ventilation
(1029±142, vs. saline: 1663±137 ml/min/kg) and the increase in ventilation produced by hypercapnia (7% CO2) (2916±162, vs.
saline: 3736±243 ml/min/kg). In anesthetized rats with bilateral lesions of around 80% of the Phox2b+TH- neurons, acute
activation of the Botzinger or the preBotzinger complex with NMDA (5 pmol/50 nl) elicited normal phrenic nerve activity.
Conclusions:
In conclusion, the destruction of the Phox2b+TH- neurons is a plausible cause of the respiratory deficits caused by injection of
SSP-SAP into RTN. Our results also suggest that RTN neurons activate facilitatory mechanisms important to the control of
ventilation in resting or hypercapnic conditions in conscious rats.
Keywords: hypercapnia, retrotrapezoid nucleus, Phox2b, central chemoreceptors, breathing
Financial Support: FAPESP
Resumo:21-121
CORRELATION OF FEAR OF FALLING ON THE BALANCE OF INDIVIDUALS WITH CHRONIC
CEREBROVASCULAR ACCIDENT (CVA).
Silva, C. A. D. ; Monteiro, K. S. ; Souza, C. G. D. ; Pontes, I. E. D. A. ; Lima, J. D. ; Silva, P. G. E. ;
Franco, C. I. F.
Depto.de Fisioterapia/Universidade Estadual da Paraíba, UEPB
Objectives:
The aim of this study was to correlate the fear of falling and the balance in individuals with chronic CVA.
Methods and Results:
17 individuals of both sexes with a clinical diagnosis of chronic CVA (= or> 6 months) assisted in the Clínica Escola de
Fisioterapia da UEPB were part of this research. Three instruments were used for methodological purposes: the Neurological
Evaluation Protocol for socio-demographic and clinical characterization, the Fear of Falling Scale (RMS) to assess fear of falling
in individuals and the Berg Balance Scale (BBS) to evaluate balance. Data were analyzed using the Graph Pad Prism 4.02, and
values are expressed in percentage, average and standard deviation, considering significant values with p
Conclusions:
Based on the results it is possible to suggest that the greater the fear of falling, lower is the balance in individuals with CVA.
Keywords: Cerebrovascular Accident, Balance, Fear Of Falling
Financial Support: UEPB
Resumo:21-122
THE EFFECTS OF AMBIENT TEMPERATURE ON SOCIAL INTERACTIONS IN RATS
Ishikawa, D. ; Almeida, M. C.
CCNH / Universidade Federal do ABC, UFABC
Objectives:
The mechanisms responsible for ambient temperature (Ta) detection although known to be essential for adequate
thermoregulation, are still not completely understood. The temperature influences not only physiological mechanisms, but is also
capable of influencing psychological aspects, behavior and wellness (J Pers Soc Psychol 17: 92-98, 1971; Am J Physiol Regul
Integr Comp Physiol 00377.02007, 2007.; J Pers Soc Psychol 15: 240-244, 1970; Science 322: 606-607, 2008). This study aims
to investigate the effect of different Ta on social behavior in rats.
Methods and Results:
We used 16 male Wistar rats weighing 290-400g. Rats were acclimated to different Tas [divided in cold (18°C), cool (22°C),
termoneutral (26°C), warm (29°C) and hot (32°C)] for 2 hours. Thereafter they were placed in pairs and the behavior was
registered. The time spent on social interactions (sniffing, approaching or following the other animal) as well as the grooming (a
behavior associated to thermoregulation) were analyzed. Rats exposed to 22°C (cool Ta) spent 49.8 ± 4% of their time in social
interaction (n = 4 pairs). This time was slightly bigger in rats exposed to neutral (26°C, n = 8 pairs) or warm Ta (29°C, n = 3
pairs) (~ 52% in both groups). Time spent in social interaction at cold and hot Ta was 56 ± 4 and 45.6 ± 2%, respectively (n = 8
pairs for each group). While rats at neutral Ta spent only 3% of their time in grooming behavior, rats exposed to hot Ta spent
almost 10%.
Conclusions:
Our data suggests that warmer temperatures increases social interaction in comparison to cool Tas. The result that cold and hot
temperature respectively increases and decreases social interaction seems controversial, however, at these temperatures, animals
spent time in behavioral thermoregulation such as grooming (hot Ta) and most probably grouping (cold Ta). In fact, the
development of these behaviors (social interaction and thermoregulatory behaviors) are competitive, and from the moment that
maintaining the temperature becomes the most important motivation for the individual, the thermoregulatory behavior prevails at
the expense of social behavior, which explain the apparently controversial result.
Keywords: Ambient temperature, Grooming, Social interaction, Thermoregulation
Financial Support: PIC/UFABC (to D.I.), CNPq Universal (to M.C.A.)
Resumo:21-123
EVALUATION OF THE NEUROPROTECTIVE EFFECT OF CAFFEIC ACID IN MICE AFTER PERMANENT
BRAIN FOCAL ISCHEMIA.
Fernandes, F. D. P. ; Carmo, M. R. S. ; Neves, J. C. S. ; Fonteles, A. A. ; Nunes, A. C. L. ; Andrade, G. M.
Departamento de Fisiologia e Farmacologia, UFC
Objectives:
According to the World Health Organization, occurs about 6 million deaths per year related to brain ischemia, representing the
third leading cause of death after coronary heart disease (7,2 million) and cancer (7,1 million). Brain stroke is the leading cause of
neurological disability that requests rehabilitation and care in industrialized countries (BROUNS, 2009). Studies of phenomena
related to the mechanisms of ischemia and hypoxia aimed at improving the quality of life of these patients.
Methods and Results:
There were 3 groups of animals: Sham-operated, Ischemic induced and Ischemic induced treated with caffeic acid. The shamoperated and the ischemic induced groups were were treated with drug vehicle (DMSO 20%) in the same way used in the
treatment of animals which were treated with the caffeic acid. The treated animals received two doses of 30mg/kg,
intraperitoneally, the first one 30 minutes before surgery, and the second one 1 hour after it. They were treated with this dose
twice daily for 3 days. The analyzed parameters were sensorimotor, acquisition and retention of memory and damaged cerebral
area by stroke. After ischemia, it was found through neurological assessment, a significant decrease in sensory function and
motor performance of animals. The percentage of infarct area in the treated animals was significantly lower than those submitted
to ischemia (SO = 0.89 ± 0.18%; ISC = 9.06 ± 1.2%; ISC + Caf.Ac. = 2.46 ± 0.2%). It was also observed an increase in vertical
exploratory activity (number of Rearings) in treated animals compared to animals in group ischemic (ISC: 9.5 ± 1.8, ISC +
Ac.Caf = 15.8 ± 0.5). The treatment significantly improved the deficits in working memory induced by ischemia, assessed at Ymaze test (SO = 73.8 ± 1.9%; ISC= 56.7 ± 2.9%; ISC + Ac.Caf = 70.7 ± 3.6%). Another similar results were observed in the
passive avoidance test in which the treatment improved acquisition of short memory and significantly long duration (SO = 235.0
± 33.3 s; ISC = 92.5 ± 25.0 s; ISC + Ac.Caf = 296.7 ± 1.85 s).
Conclusions:
The results demonstrate the caffeic acid‘s antioxidants properties in the pathophysiology of cerebral ischemia, which showed a
significant effect neuroprotective on neuronal damage, motor behavior and memory after ischemia and indicate that this effect
may be related with the caffeic acid‘s antioxidant properties.
Keywords: Caffeic Acid, Brain ischemia, Memory, Antioxidant effect, Neuroprotection
Financial Support: CNPq
Resumo:21-124
NEUROTOXIC EFECTS OF 3-ACETYL-PYRIDINE ON HIPPOCAMPAL DENTATE GYRUS CITOLOGY AND
SPATIAL BEHAVIOR.
Araujo, V. M. 1; Rocha, E. D. 1; Freitas, F. S. 2; Rocha, M. S. 2; Allodi, S. 1; Cavalcante, L. A. 1
1
Instituto de Biofísica Carlos Chagas Filho/UFRJ, IBCCF
2
Instituto de Ciências Biomédicas/UFRJ, ICB
Objectives:
The hippocampal dentate gyrus is a region of the brain where the production of neurons throughout adult life, even in normal
conditions, has been best characterized. However, little is known about the properties of hippocampal glial cells or their response
to lesion. The objectives of the present work are to study the chronology of the morphological and phenotypical markers of
neuron and glial cells of the dentate gyrus in response to lesions induced by the neurotoxin 3-acetyl-pyridine (3AP), and to
investigate possible changes in spatial memory.
Methods and Results:
Thirty to thirty five day-old Wistar rats were submitted to a single 3AP intraperitoneal injection and euthanized 24 hours (h) or 4,
10, 21 or 36 days (d) after the lesion. Animals with 11, 21, and 36 d after 3AP and aged-matched controls were tested for changes
in spatial learning and memory using the Morris water maze. Coronal sections of the hippocampus from animals with 24 h to 21
d survival and their respective controls were processed for immunohistochemical reactions and the images were analyzed by
conventional microscopy. Digital images were collected and used for counting the cells in the hylus of the dentate gyrus and
performance in the Morris aquatic maze was evaluated. There was a decrease in microtubule-associated protein (MAP2)
immunoreactivity at 24 h and 4 d after 3AP treatment, as reported in the early stage of various hippocampal lesions. The most
significant changes were an increase in the number of hylus cells expressing GFAP at 24 h, suggesting that either differentiated
astrocytes are involved, together with microglia, in central nervous system responses from injury or that GFAP-expressing
neuroblasts are increased. The latter is reinforced by an increase in cells expression Beta 3-tubulin at 4 days. Spatial behavior was
significantly impaired at 11 d when compared with controls. This tendency still persisted at both 21 d and 36 d but was no longer
significantly different from the controls, suggesting that impairment of spatial learning and memory was reversible with time.
Conclusions:
These evidences suggest that damage to the hippocampus by the neurotoxin 3AP persists after recovery of the locomotory ability
but is, at least, partially reversible in young adult rats. Behavioral recovery is coherent with the time course of the progressive
changes in neurogenesis and synaptogenesis in this species.
Keywords: 3-ACETYL-PYRIDINE, glial cells, hippocampus, spatial behavior
Financial Support: CNPq, FAPERJ
Resumo:21-125
REACTION TIME AND ARTIFICIAL LIFE
Feher-da-silva, C. ; Baldo, M. V. C.
Instituto de Ciências Biomédicas - Universidade de São Paulo, ICB-USP
Objectives:
In a simple reaction time (RT) task, the subject has to respond as fast as possible to the abrupt appearance of a target stimulus
preceded by a cue, which may be valid (when it indicates correctly where the target will appear), neutral (when it doesn't indicate
where the target will appear) or invalid (when it indicates incorrectly where the target will appear). Performance is evaluated by
measuring the time elapsed between the appearance of the target and the subject's response. It is assumed that the cue directs
attention to the location it indicates and speeds up sensory processing there, so that for humans and other animals RT is lowest
when the cue is valid and highest when it is invalid. We used artificial life models to investigate the role of noise, action
selection, and neural processing capacity in the evolution of attentional mechanisms engaged by RT tasks.
Methods and Results:
We performed artificial life simulations wherein populations of artificial animals with limited processing capacity evolved
through a genetic algorithm based on their performance in a simple RT task. The proportion of valid, neutral, and invalid cues
was 8:5:2 or 8:15:2. When the simulation was run for 150 generations, the animals achieved the lowest possible RT (1 unit)
regardless of cue validity and made no errors. When the simulation was cut short at 30 generations, the animals had already
achieved very low RT averages (2.5-4.0 units) and error rates. The lowest RT was achieved for the most frequent cue type (the
frequency effect), whether it was valid or neutral, which indicates the animals were not using the information provided by the
cues. With cue proportion set as 8:15:2, the addition of gaussian noise (mean = 0, standard deviation = 2) to each neuron in the
neural networks caused a large increase in RT averages (RT: 31.9 ± .6, 30.4 ± .4, 34.1 ± .7 for the valid, neutral and invalid cues
respectively) and in errors, but the lowest RT was still achieved for the neutral cue. The task the animals had to perform was
switched to a choice RT task, wherein there were two possible responses, but only one was correct, depending on the location
where the target appeared. Cue proportion was 8:15:2. The results were similar to previous ones without noise, but the addition of
gaussian noise (mean = 0, standard deviation = 2) caused animals to respond fastest for the valid cue, even though the neutral cue
was the most frequent (RT: 37.7 ± .7, 40.4 ± .6, 63.8 ± 4.2; p < .05). By equalling the number of neutral cues to the number of
valid cues and then to the number of invalid cues, the frequency effect was eliminated and the effect of the valid and invalid cue
relative to the neutral cue could be calculated: -5.3 or -12.5% and 10.6 or 21.3% respectively. Increasing the number of neurons
in the neural networks and thus their processing capacity didn't abolish the ―attentional effect‖, but instead increased the effect of
the valid cue.
Conclusions:
In artificial life models with noise and action selection, artificial animals were similar to humans and other animals in a RT task.
Our results suggest that noise and action selection, but not limited capacity, were important environmental features that helped
drive the biological evolution of attentional mechanisms engaged by RT tasks.
Keywords: attention, artificial life, reaction time, neural networks, noise
Financial Support: FAPESP
Resumo:21-126
EXPOSURE TO CIGARETTE SMOKE CONTAINING EITHER HIGH OR LOW LEVELS OF NICOTINE DURING
ADOLESCENCE
Guthierrez, M. C. S. 1; Manhães, A. C. 1; Filgueiras, C. C. 1; Cavina, C. C. 1; Naiif, V. F1; Ribeiro-carvalho,
A. 2; Abreu-villaça, Y1
1
Universidade do Estado do Rio de Janeiro, UERJ
2
Instituto Federal de Educação, Ciência e Tecnologia do Rio d, IFFRJ
Objectives:
Smoking typically begins during adolescence. Nicotine is an important psychoactive substance present in tobacco and, in fact, an
increasing number of animal studies on effects of nicotine in the adolescent brain is made available every year. However, tobacco
smoke contains about 4500 additional components. Accordingly, the possibility that tobacco compounds other than nicotine
participate in the effects of tobacco smoke can be raised. Despite that, there is a lack of experimental studies that investigate the
effects of tobacco smoke exposure during adolescence. The current study was undertaken to investigate effects of distinct levels
of adolescent nicotine exposure when combined to tobacco smoke on the behavior of mice. To do that, we generated smoke from
2 types of cigarettes, one containing a high level of nicotine and another containing a very low level of nicotine.
Methods and Results:
From postnatal day (PN) 30 to 45, Swiss male and female mice were exposed to tobacco smoke (whole body exposure for
8hr/day, 7days/week) generated from one of two reference research cigarettes: type 2R1F (HighNIC group nicotine=1,74mg/cig; n=37) or type 4A1 (LowNIC group - nicotine=0.14 mg/cig; n=19) in a chamber that received the smoke
generated from an automatic cigarette smoking machine. A smoke mixture containing 89% sidestream and 11% mainstream
smoke as a surrogate for active smoking, was generated at the rate of a single 35ml puff of 2s/min. Control animals (CT, n=30)
were exposed to air in a chamber like the chamber of smoke exposure. On PN44, 74 animals were submitted to the Elevated Plus
Maze (EPM) test. The EPM consists of two ―open‖ (no walls, 5cm×29cm) and two ―closed‖ (5cm×29cm×15cm) arms, arranged
perpendicularly, and elevated 50cm above the floor.The percentage of time spent in the open arms of the maze was used as a
measure of anxiety, while the number of entries in the open+closed arms was used as a measure of activity. On PN45, the animals
were tested in the Hole Board which consists of a polyethylene box (30x38x20cm). The floor is divided into 16 rectangles with a
1.5cm diameter hole located on the center of each rectangle. The number of head-dips into the holes was used as a measure to
novelty seeking behavior. On PN45, cotinine (nicotine metabolite) serum levels from HighNIC, LowNIC and CT mice (n=12)
were determined using a kit from Orasure Technologies. (Bethlehem, PA). On PN45, HighNIC cotinine serum levels were 109.1
± 24.0ng/ml while LowNIC and CONT mice presented values below the detection limit of the technique (
Conclusions:
These results indicate that exposure to cigarette smoke alters the novelty seeking behavior, and the pattern of change depends on
the type of cigarette. Since exposure to smoke containing low concentrations of nicotine caused a reduction of the search for
novelty while exposure to smoke containing high concentrations of nicotine caused an increase of the search, we suggest that
among the constituents of cigarette smoke, nicotine is one of the determinants of our results. Additionally, this behavior has been
associated with a greater tendency to use drugs in humans, so, this model may be useful to investigate the susceptibility to drugs
of abuse.
Keywords: CIGARETTE , ADOLESCENCE, BEHAVIOR
Financial Support: Fellowships from CNPq; PIBIC-CNPq and CAPES and a grant from CNPq
(476991/2010-2
Resumo:21-127
EFFECTS OF NEONATAL EXPOSURE TO ORGANOPHOSPHATE PESTICIDES ON THE CENTRAL
CHOLINERGIC SYSTEM OF MICE
Nunes-freitas; A. L. 1; Lima; C. S. 1; Ribeiro-carvalho, A. 1,3; Dutra-tavares, A. C. 1; Nunes F. 1; Filgueiras,
C. C. 1; Manhães, A. C. 1; Meyer, A. 2; Abreu- Villaça Y. 1
1
UNIVERSIDADE DO ESTADO DO RIO DE JANEIRO, UERJ
2
UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, UFRJ
3
INSTITUTO FEDERAL DO RIO DE JANEIRO, IFRJ
Objectives:
In countries in which economy is based on agribusiness, like Brazil, the large utilization of pesticides represents an important
problem of public health. Organophosphates (OPs) are among the most used pesticides worldwide. Classically, OPs act by
irreversibly inhibiting the enzyme acetylcholinesterase (AChE), the enzyme responsible for the breakdown of acetylcholine.
However, they may differ substantially in many of their noncholinesterase effects. Considering that previous studies describe that
OPs elicit more severe effects during development, here, we investigated whether early exposure to chlorpyrifos and
methamidophos at doses well below the threshold for systemic toxicity and that cause only 20% of acetylcholinesterase (ACHE)
inhibition elicit alterations in other biomarkers of cholinergic function.
Methods and Results:
From the 3rd to 9th postnatal day (PN), 80 Swiss mice were exposed to daily injections (s.c.) of either one of two different OPs,
methamidophos (METH; N= 10 females and 10 males) or clorpyrifos (CLOR; N= 10 females and 10 males) at doses (1 and
3mg/kg respectively) which were previously shown to cause low and similar AChE inhibition in brain (¡Ö20%). Control animals
(CONT METH; N= 10 females and 10 males and CONT CLOR; N= 10 females and 10 males) received DMSO (s.c.). All
animals were sacrificed one day after the end of exposure, at PN10. We assessed the binding of [3H]hemicholinium-3 (HC-3) to
the high-affinity presynaptic choline transporter and choline acetyltransferase (ChAT) activity in the cerebral cortex and
brainstem. Results were evaluated by analyses of variance on all factors: treatment and sex. Treatment effects and interactions
were followed by post-hoc analyses using Fisher¡¯s Protected Least Significant Difference (FPLSD). Cholinergic alterations
were sex-dependent, being significant only in females: In the brainstem, methamidophos promoted an increase in HC-3 binding
when compared to its corresponding control group (METH vs CONT METH, 46.7 ¡À 2.1 vs 35.2 ¡À 1.7; P=0,0006) In the
cortex, both clorpyrifos (CLOR vs CONT CLOR, 44.0 ¡À 1.9 vs 34.6 ¡À 2.1, P=0,0027, FPLSD) and methamidophos (METH vs
CONT METH, 49.0 ¡À 2.5 vs 42.1 ¡À 1.2; P=0,022, FPLSD) caused an increase in HC-3 binding. ChAT activity was not
affected.
Conclusions:
Considering that the high-affinity choline uptake system, assessed by HC-3 binding, is regulated by nerve impulse activity,
changes in HC-3 binding observed here represent an increase in cholinergic activity. In addition, since the doses of
methamidophos and chlorpyrifos used in the present study elicit similar levels of ACHE inhibition, the fact that these pesticides
elicited region-dependent effects, suggests that OPs can act through multiple mechanisms, not necessarily related to AChE
inhibition, that culminate in other cholinergic alterations.
Keywords: ORGANOPHOSPHATE, DEVELOPMENT, CHOLINERGIC SYSTEM, METHAMIDOPHOS, CLORPYFOS
Financial Support: CAPES, CNPq, FAPERJ and Mount Sinai school of Medicine
Resumo:21-128
SOMATIC MATURATION AND REFLEX ONTOGENY IN NEONATE RATS FED DURING PREGNANCY AND
LACTATION WITH A HIGH FAT DIET
Santos, T. D. ; Ferreira, A. K. ; Ladislau, H. F. L. ; Pereira-da-silva, M. S. ; Rocha-de-melo, A. P. ; Borba,
J. M. C.
Depto. de Nutrição - Lab. de Fisiologia da Nutrição -LAFINNT, UFPE
Objectives:
Aim: Investigate if a high fat diet intake containing 14% of hydrogenated vegetable fat during pregnancy and lactation influences
the somatic maturation and reflex ontogeny in neonatal rats.
Methods and Results:
Methods and Results: Female Wistar rats were fed a diet containing 7% soybean oil (control group - C, n = 32) or 14%
hydrogenated vegetable fat (Experimental Group - E, n = 47) during gestation and lactation. The pups were weighed at 1 st, 7th,
14th and 21st days of life. Somatic Maturation Indexes - eye opening (AO), ear unfolding (EU), auditory conduit opening (ACO),
eruption of the upper incisors (EUI), eruption of the lower incisor (EUI) and time of appearance of reflexes (palmar grasp - PG,
cliff avoidance - CA, vibrissae placing - VP, negative geotaxis - NG, righting - R, auditory startle response - ASR and free fall
righting- FFR) were evaluated in each litter (6 males and 2 females) daily between 8:00 and 10:00 am from the 1 st to 21st days of
life. Rats of the E group showed a lower weight gain at the 1 st lactation day (6 – 1; median – interquartile) when compared to the
C group (6,82 - 0,852). This fact was also observed at the 7 th day of lactation (E: 14 – 2,75 and C: 15,83 – 4,9925) . The E group
showed a significant delay in the following indices (days): EU – (3 -1, median – interquartile) and ACO (13 – 1) compared to the
C group (EU: 3 – 0 and ACO: 12 – 1). The development of PG, CA and ASR was delayed in the E group (PG: 6 – 1, CA: 7 – 3,
ASR: 13 – 2,5) when compared to the C group (PG: 4 – 2, CA: 5 – 2, ASR: 12 – 0,25). On the other hand, R and FFR appeared
earlier in the E group (R: 4 – 1 e FFR: 15 – 0,5) when compared to the C one (R: 5 – 2 e FFR: 17 – 1).
Conclusions:
Conclusions: The chronic intake of a high fat diet, based on hydrogenated vegetable fat, seems to produce weight changes and
cause alterations in somatic and reflex maturation.
Keywords: High Fat, Hydrogenated Vegetable Fat, Rats, Reflex Ontogeny , Somatic Maturation
Financial Support: UFPE/ PROPESQ
Resumo:21-129
SURVIVAL OF RGC MEDIATED BY IL-2, BUT NOT BY IL-4, IS DEPENDENT OF HEPARAN SULFATE
EXPRESSION IN VITRO
Marra C. ; Moret, D. G. ; Corrêa, A. D. S. ; Sholl-franco, A.
IBCCF - Universidade Federal do Rio de Janeiro, IBCCF - UFRJ
Objectives:
Programed cell death (PCD) during development is widespread in both vertebrates and invertebrates, occurring in virtually all
cell types of the nervous system, including retinal neurons such as retinal ganglion cells (RGC). In addition to physiological
conditions, this phenomenon also occurs in retinal pathologies as observed in optic neuropathies, a group of retinal diseases
characterized by RGC loss visual field reduction and/or blindness blindness. Several factors are involved in the regulation of
RGC death, including cytokines and extracellular matrix (ECM) molecules. Previously, we have demonstrated the
neuroprotective effect of interleukin-2 (IL-2) and interleukin-4 (IL-4) upon retinal ganglion cells in vitro. In this work we: (i)
investigated if cytokines treatment could regulate the expression of heparan sulfate proteoglycans (HSPG) in retinal tissue, and
(ii) analyzed the correlation between the neuroprotective effect of cytokines and the heparan sulfate (HS) expression.
Methods and Results:
Newborn rats were anaesthetized by hypothermia and received injections of rhodamine dextran (RD) in their superior colliculus
for retrograde staining of RGC. After 2 days, retinal explants were obtained and maintained with or without IL-2 (50U/mL), IL-4
(5U/mL) and/or heparitinase for 5 days in vitro (DIV). The total number of nuclei (stained with DAPI) in the ganglion cells layer
and the specific number of RGC labeled with RD were counted using fluorescence microscopy.Immunohistochemical analysis
were performed to verify the HS expression in retinal tissue. The results showed that IL-2 and IL-4 treatment of retinal explants
increased the HSPG expression in all retinal layers after 5 DIV. Moreover the neuroprotective effect of IL-2, but not IL-4 was
completely blocked by pre-treatment with heparitinase.
Conclusions:
Our data demonstrate that IL-2 and IL-4 modulates the expression of HSPG in retinal tissue, and that early presence of this
molecule is essential for RGC survival mediated by IL-2, but not by IL-4.
Keywords: Retina, Retinal Ganglion Cells, Citokines, Extra Celular Matrix, Heparan Sulfate
Financial Support: CNPQ , CAPES, FAPERJ
Resumo:21-130
ETHANOL-INDUCED LOCOMOTOR SENSITIZATION INCREASES ∆FOSB IMMUNOREACTIVITY IN THE
HIPPOCAMPUS AND AMYGDALA OF ADULT SWISS MICE.
Coelhoso, C. C. 1; Filev, R. 1; Varela, P. 1; Engelke, D. 1; Mello, L. E. 1; Santos-junior, J. G. 1,2
1
Departamento de Fisiologia/Universidade Federal de São Paulo, UNIFESP
2
Departamento de Ciências Fisiológicas/FCMSCSP , FCMSCSP
Objectives:
Chronic exposures to drug of abuse induce several neuroadaptations in neuronal circuitry related to addiction. One of them is the
increase in ΔFosB expression, notably in the dorsal striatum. Here, we investigate the ΔFosB immunoreactivity in mice sensitized
and non-sensitized to the stimulant locomotor effects of chronic ethanol exposures.
Methods and Results:
Male Swiss mice (90 days, 30-40 g) were maintained for 15 min in an activity box and the distance traveled during this period
was measured. After that, the animals (N=32) were treated with ethanol (2 g/kg, i.p. 15% v/v) for 21 consecutive days. Control
animals (N=8) were treated with saline. After 4 days of withdrawal, the animals were challenged with ethanol (1.4 g/kg, i.p.) and
again placed in the activity box. In the next day, the animals were perfused and their brains processed for FosB/ΔFosB
immunohistochemistry. According to the locomotor activity in the challenge day, ethanol treated animals were distributed in two
sub-groups: sensitized (1 SD above the mean) and non-sensitized (1 SD below the mean). FosB/ΔFosB immunoreactivity was
measured in the medial prefrontal cortex (mPFC), nucleus accumbens core (NAco) and shell (NAsh), caudate-putamen (CPu),
hippocampus (HPC), ventral tegmental area (VTA), basolateral (BlA) and central nucleus of the amygdala (CeA). The data were
analyzed by one-way ANOVA followed by Tukey post hoc when necessary. The significance was set at PP=0.16]. Moreover,
Et_Sens had a higher locomotor activity after challenge when compared to the other groups [F(2,21)=57.93, PPPPP<0.001].
Conclusions:
These results suggest that increases on FosB/ΔFosB in HPC (DG, CA1 and CA3) and in CeA could play an important role in the
ethanol-induced locomotor sensitization in mice.
Keywords: Ethanol, ∆FosB , Immunohistochemistry, Locomotor Sensitization
Financial Support: CNPq
Resumo:21-131
TEMPORAL BONE MODIFICATIONS FOLLOWING SPINAL CORD INJURY IN RATS.
Medalha, C. C. 1; Amorim, B. O. 1; Rosseti, K. 1; Ribeiro, D. A. 1; Pereira, R. M. R. 2; Rennó, A. C. M. 1
1
Departamento de Biociências, UNIFESP
2
Departamento de Reumatologia, USP
Objectives:
The aim of this work was to investigate the temporal alterations on bone mass, bone biomechanical properties and bone
morphology in spinal cord injured rats 2, 4 and 6 weeks after surgery.
Methods and Results:
Fourty male Wistar rats (aged 8 weeks and weighing 290 ± 6.8 g) were randomly distributed into four groups (n=10 each group):
sham control group (CG: control without spinal cord-injured animal); spinal cord-injured 2 weeks (2W: spinal cord-injured
animals sacrificed 2 weeks post-surgery); spinal cord-injured 4 weeks (4W: spinal cord-injured animals sacrificed 4 weeks postsurgery); spinal cord-injured 6 weeks (6W: spinal cord-injured animals sacrificed 6 weeks post-surgery). The animals were
anesthetized by an intraperitoneal injection of ketamine (90 mg/kg) and xilasine (10 mg/kg) and a laminectomy was executed at
Th9-10. In injured rats, the duramater was exposed and the spinal cord was completely sectioned with microscissors. To reveal
temporal changes to the locomotor function after SCI, an evaluation was carried out by using the Basso, Beattie and Bresnahan
(BBB) Locomotor Rating Scale. Biomechanical properties of the right tibia were determined by a three-point bending test in an
Instron® Universal Testing Machine (USA, 4444 model, 1 KN load cell). To measure bone mineral content (BMC g/cm2) and
bone mineral density (BMD-g/cm2) of the right femur densitometry (DEXA Hologic Inc Discovery model-Belford, MA, USA)
analysis was carried out by using specific software for small animals. The distribution of all variables was tested for normality by
using Shapiro Wilk´s W test. Data was analyzed by Kruskal Wallis One-Way Analysis of Variance on Ranks followed by the
Student Newman Keuls´s post-hoc test. STATISTICA version 7.0 (data analysis software system - StatSoft Inc.) was used to
carry out statistics analysis. Values of p
Conclusions:
The lesion procedure caused severe degradation in behavioral performance, as measured by the BBB score. Injured animals did
not present any recovery in their general motor behavior and none of them presented plantar placement of the paw with weight
support during the experimental period. Two weeks after surgery, the injured animals showed a statistically significant decrease
in maximal load compared to control animals (P=0.02). Interestingly, the animals sacrificed 4 and 6 weeks after surgery did not
demonstrate any difference in the biomechanical evaluation when compared to control (P=0.42; P=0.44 respectively). BMD had a
significant difference, and a decrease 2 and 4 weeks after SCI (P<0.001).
Keywords: spinal cord injury, bone , osteoporosis
Financial Support: CnPQ and Fapesp
Resumo:21-132
ACUTE RESISTANCE EXERCISE INCREASE THE EXPRESSION OF SYNAPSIN I IN HIPPOCAMPAL
FORMATION OF RATS
Fernandes, J. 1; Pena, L. F. P. 1; Cassilhas, R. C. 2; Mello, M. T. D. 2; Venancio, D. P. 2; Scorza, F. A. 3;
Silva, S. G. D. 1; Cavalheiro, E. A. 3; Arida, R. M. 1
1
Departamento de Fisiologia/Universidade Federal de São Paulo, UNIFESP
2
Dept. de Psicobiologia/Universidade Federal de São Paulo, UNIFESP
3
Dept. de Neurologia/Universidade Federal de São Paulo, UNIFESP
Objectives:
Aerobic exercise has been shown to enhance memory and cognition, facilitate functional recovery following brain injury,
promote neurogenesis in the adult brain, and even ameliorate the mental decline associated with aging. However, the influence of
resistance exercise on brain function and neuroplasticity has not been well estabelished. Therefore, the purpose of the present
study was to analyze the impact of one session of resistance exercise on expression of synapsin I – phosphoprotein involved in
vesicle clustering, neurotransmitter release and synaptic transmission.
Methods and Results:
Twelve adult male wistar rats were distributed in two groups: sedentary control group (CTRL=6) and resistance exercise group
(RES=6). Animals from resistance exercise group were submitted to one session of resistance exercise where its climbed a 1.1-m
vertical (80o incline) ladder with incremental weights secured to their tails. The session consisted of 8 climbs with 8-12 dynamic
movements per climb. Exercise increased significantly synapsin I levels (p>0.05) in the hippocampal formation.
Conclusions:
The present result demonstrate that acute resistance exercise promotes changes in a protein marker of synaptic plasticity,
suggesting that some of the neural adaptations by resistance exercise may be observed in early phase of the trainig program.
Keywords: RESISTANCE EXERCISE, SYNAPSIN I, SYNAPTIC PROTEINS, NEUROPLASTICITY, HIPPOCAMPAL
FORMATION
Financial Support: CNPq, CAPES, FAPESP and CInAPCe-FAPESP
Resumo:21-133
DIFFERENTIAL RESPONSE OF SYNAPTIC AND STRUCTURAL PROTEINS IN THE NERVOUS SYSTEM OF
RATS SUBJECTED TO DIFFERENT TYPES OF MOTOR TRAINING
Garcia, P. C. 1; Real, C. C. 2; Britto, L. R. G. 2; Pires, R. S. 1
1
Universidade da Cidade de São Paulo, UNICID
2
Universidade de São Paulo, USP
Objectives:
A number of studies performed in rodents have revealed cellular and molecular exercise-induced changes in several brain
structures. Motor skill learning is an essential aspect of development, adult life, and recovery after brain lesions. Learning
requires protein synthesis and is likely that some of the proteins synthesized are involved in structural plasticity. Since different
types of exercise may induce distinct changes in different brain areas, it is important to study the plastic responses generated by
the training of complex motor tasks (acrobatic exercise) and compare them with those involved in rhythmic tasks (forced exercise
on a treadmill). The aim of this study was to evaluate the protein expression of synapsin I (SYS), synaptophysin (SYP), MAP2
and neurofilament (NF68) in the striatum and cerebellum of adult rats subjected to forced and acrobatic exercise.
Methods and Results:
We used adult male Wistar rats, divided into 3 groups based on types of exercise training, namely control-sedentary (n = 15),
forced exercise (FE) (n = 20) and acrobatic exercise (AE) (n = 20). The FE rats were trained on a treadmill with a maximum
speed of 0.6 Km/h, for 40 minutes, 3 times per week for 30 days. In the AE group, the rat had to move through circuits consisting
of various obstacles during the same period of time as the other group. The methods used for the analyses were
immunohistochemistry and Western blotting. Our results show that the present exercise protocols induced specific plastic
changes in both regions studied, which varied depending on the protein studied. Western blot analysis showed that trained
animals, regardless of the type of exercise, showed a higher expression of structural proteins (MAP2 and NF68) than synaptic
proteins (SYS and SYP). The rats of the FE group showed changes only in expression of the structural proteins, exhibiting a
significant increase of MAP2 (p<0.02).
Conclusions:
Acrobatic exercise induced changes in the expression of synaptic and structural proteins mainly in the striatum, which may
underlie part of the learning of complex motor tasks involved in the protocol. On the other hand, forced exercise promoted
changes only in structural proteins in both the cerebellum and the striatum. Taken together, these results suggest that moderate
physical exercise and acrobatic exercise modulate synaptic and structural proteins in motor brain areas, which may play an
important role in the exercise-dependent brain plasticity.
Keywords: motor training, plasticity, structural protein, synaptic proteins
Financial Support: FAPESP, CNPQ
Resumo:21-134
CHRONIC TREATMENT WITH ASCORBIC ACID ENHANCES CORTICAL SPREADING DEPRESSION IN
DEVELOPING WELL NOURISHED AND MALNOURISHED RATS
Alves, E. V. S. ; Guedes, C. K. R. D. M. ; Viana-da-silva, E. ; Guedes, R. C. A.
Depto de Nutrição, UFPE
Objectives:
Ascorbic acid (AA) is an antioxidant that has been suggested as being important for brain development. However, under certain
conditions AA has also been shown to exert biphasical modulating action on excitability-dependent brain phenomena
(Toxicology 249; 35-39, 2008; Neuroscience 128; 721–728, 2004). Here we explore the question of whether AA chronic
administration alters cortical excitability as indexed by the cortical spreading depression (CSD).
Methods and Results:
Well-nourished (W) and malnourished (M) Wistar rats pups received per gavage AA (60mg/kg/d; n=11), or saline (n=11), or no
treatment (naïve group; n=10) during postnatal days 7-28. When they became 30-40days old, under anesthesia, CSD was elicited
at 20 min intervals by KCl-stimulation and electrophysiologically recorded during 4h on 2 points of the right parietal cortex.
ANOVA plus Tukey test showed that M rats presented higher (p
Conclusions:
Chronic AA treatment of developing animals facilitates CSD propagation, and this effect is not modified by early malnutrition.
Data are consistent with the hypothesis that, at the dose of 60 mg/k/d, AA can have a prooxidant effect, as previously postulated
(Toxicology 249; 35-39, 2008).
Keywords: CORTICAL SPREADING DEPRESSION, ASCORBIC ACID, WELL NOURISHED, MALNOURISHED, RATS
Financial Support: CAPES, CNPq, FACEPE, INNT-Rede Glial, IBN-Net.
Resumo:21-135
ROLE OF P2X7 RECEPTOR IN NEURODEGENERATION AND MICROGLIOSIS AFTER FOCAL CEREBRAL
ISCHEMIA IN MICE
Amorim, F. E. 1; Navarro-martins, A. 2; Coutinho-silva, R. 3; Cintra, W. M. 1; Mendez-otero, R. 2
1
Laboratório de Farmacologia Molecular, ICB/UFRJ
2
Laboratório de Neurobiologia Celular e Molecular, IBCCF/UFRJ
3
Laboratório de Imunofisiologia, IBCCF/UFRJ
Objectives:
Strokes are second cause of death of world and the first in Brazil. It‘s shown that the damage caused by ischemia may be
aggravated by secondary events such as the inflammatory response. It‘s known that the purinergic P2X7 receptor (P2X7R) is able
to activate microglia inducing it to release pro-inflammatory factors. The aim of this study was to investigate the effect of
blocking P2X7R in microglia and neurodegeneration after permanent middle cerebral artery occlusion (MCAo) by
electrocauterization.
Methods and Results:
C57Bl6 females mice received three doses (45.5 mg/kg - 3 mg/ml, via ip, in PBS containing 0.2% DMSO) of Brilliant Blue G (a
P2X7R antagonist) or vehicle at intervals of 48 hours. The first dose was one day before MCAo. For analysis of lesion volume,
Triphenyl Tetrazolium Chloride (TTC) reaction was made on coronal sections (1 mm thickness) of ischaemic mice 4 days after
MCAo. For immunohistochemical reactions, animals (4 days post-MCAo) were perfused with 4% paraformaldehyde,. Brains
were removed and coronal sections were taken (20 μm) in a criostat. To determine lesion area and density of degenerating
neurons we used the Fluoro-Jade C staining, a marker of early neuronal degeneration. Inflammatory infiltrate was analyzed by
double labeling with GSA I-B4 and anti-ED1 (respectively, total and activated microglia). All evaluations were done by t test (p
Conclusions:
There was no difference in rate of microglial activation (activated microglia / microglia total) in both groups, experimental (96.24
± 0.54) and control (96.07 ± 0.46), P = 0.812. This study demonstrated P2X7R blocking after focal cerebral ischemia by MCAo
in mice decreased neuronal death in ischemic penumbra.
Keywords: Focal Ischemia, Middle Cerebral artery, Oclussion, P2X7, Stroke
Financial Support: CAPES
Resumo:21-136
OXYTOCINERGIC CONTROL OF FOOD INTAKE IS MODULATED BY ESTROUS CYCLE.
Lima, A. A. 1; Xavier, S. D. O. 1; Feitosa, V. L. C. 1; Reis, F. P. 2; Aragão, J. A. 1; Lucca Jr, W1
1
Dept. de Morfologia da Universidade Federal de Sergipe, DMO-UFS
2
Faculdade de Medicina da Universidade Tiradentes, FM-UNIT
Objectives:
To evaluate estrous cycle modulations on the oxytocinergic control of food intake.
Methods and Results:
After seven days of recover from the cannula implantation surgery, the metestrus and proestrus phases animals were fastened for
48 hours and received at 7:00 h a microinjection (5 uL) of OT (1 g/L - Novartis) in the lateral ventricle. Immediately after the
infusions, it was offered access to balanced ration, and the amount of ingested food was measured during 90 minutes after the
beginning of the food access. Following, the animals were anesthetized with an intraperitoneal injection (1 mL / 100 g of body
weight) of ketamine (80 mg/kg) and xylazine (8 mg/kg) and 30 mL of 0.01M sodium phosphate buffer (pH 7.4) were perfused
through the ascending aorta followed by 300 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4).The other
were sacrificed and wasted, according to the protocol. It was observed that 11% of the animals submitted to the cannula
implantation surgery, altered the cycle regularity and remained in metestrus phase, presenting sometimes a diestrus phase.17 of
31 rats submitted to cannula implantation in the lateral ventricle received isotonic saline microinjection (5 uL) and 14 received
OT microinjection (5 uL). After 48 hours of fast, 9 rats subjected isotonic saline icv microinjection presented food intake of 2,25
(± 0,1897 ) g/100g body weight during the 90 minutes that ration was available. The 14 rats subjected OT icv microinjection
presented food intake of 1,45 (± 0,1727) g/100g body weight during the 90 minutes that ration was available. 9 of 16 rats in
metaestrus phase submitted to cannula implantation in the lateral ventricle received isotonic saline microinjection (5 uL) and 7
received OT microinjection (5 uL). After 48 hours of fast, 9 rats subjected isotonic saline icv microinjection presented food
intake of 2.07 (± 0.2536) g/100g body weight during the 90 minutes that ration was available. The 7 rats subjected OT icv
microinjection presented food intake of 1,22 (± 0,1562) g/100g body weight during the 90 minutes that ration was available. 8 of
15 rats in proestrus phase submitted to cannula implantation in the lateral ventricle received isotonic saline microinjection (5 uL)
and 7 received OT microinjection (5 uL). After 48 hours of fast, 8 rats subjected isotonic saline icv microinjection presented food
intake of de 2,46 (± 0,2849) g/100g body weight during the 90 minutes that ration was available. The 7 rats subjected OT icv
microinjection presented food intake of 1,67 (± 0,2957) g/100g body weight during the 90 minutes that ration was available. The
difference between groups wasn‘t considered statistically significant at p <0.05.
Conclusions:
The OT centrally administered lowers the ration intake of Wistar female previously submitted to a fast and the gonadal steroids
modulate this oxytocinergic inhibitory action in the food intake control.
Keywords: Ocitocina, Ingestão, Esteróides gonadais
Financial Support: FAPITEC
Resumo:21-137
ACTIVATION OF C-JUN N-TERMINAL KINASE (JNK) DURING MITOSIS IN RETINAL PROGENITOR CELLS.
Ribas, V. T. ; Gonçalves, B. S. ; Linden, R. ; Chiarini, L. B.
Instituto de Biofísica Carlos Chagas Filho, IBCCF/UFRJ
Objectives:
During development of rat retina, cells are found in various stages of development: proliferation, differentiation and programmed
cell death. In this study, we investigated the behavior of c-Jun N-termianl Kinase (JNK) and two of its targets, ATF-2 and c-Jun,
during mitosis of progenitor cells in newborn rat retina.
Methods and Results:
Retinal explants from newborn rats were kept in vitro for various intervals and under distinct treatments. Sections of retinal
explants were used to detect of JNK, ATF-2 and c-Jun phosphorylation by immunohistochemistry, and examined through both
fluorescence and confocal microscopy. Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and
localization in the retinal tissue. The subcellular localization of phosphorylated proteins was analyzed by double staining with
both DAPI and an antibody to each phosphorylated protein. Phosphorylation of JNK was also examined by Western blot. The
results showed that in the retina of newborn rats (P1) JNK is phosphorylated during mitosis of progenitor cells, mainly during the
early stages of mitosis. It was shown that JNK1/2, but not JNK3, are phosphorylated in mitotic cells. The transcription factor
ATF-2 is not phosphorylated in mitotic cells, whereas transcription factor c-Jun is phosphorylated on both residues serine 63 and
serine 73 at all stages of mitosis. Phosphorylated c-Jun was detected also in the centrosome in mitotic cells. Inhibition of JNK
induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but
did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrant chromatin
morphology were found after treatment with the JNK inhibitor. Inhibition of JNK, p38, PKC, ERK or PI-3K did not block the
phosphorylation of c-Jun. Finally, inhibition of JNK induced programmed cell death in progenitor cells in newborn rat retina.
Conclusions:
These data indicate that JNK may play an important role during mitosis of progenitor cells in newborn rat retina.
Keywords: c-Jun N-terminal Kinase, Mitosis, Retina
Financial Support: Capes, CNPq, FAPERJ, PRONEX-RJ.
Resumo:21-138
EFFECTS OF ACETYLCHOLINE INJECTED INTO THE NUCLEUS OF THE SOLITARY TRACT ON
SYMPATHETIC AND PHRENIC NERVE ACTIVITIES
Furuya, W. I. 1; Zoccal, D. B. 2; Menani, J. V. 1; Colombari, E. 1; Colombari, D. S. A. 1
1
Universidade Estadual Paulista - Faculdade de Odontologia, UNESP
2
Universidade Federal de Santa Catarina, UFSC
Objectives:
A cholinergic system was identified within the nucleus of the solitary tract (NTS) of the rat. Accordingly, it has been shown that
acetylcholine (ACH) microinjected into the nucleus of the solitary tract (NTS) induces hypotension and bradycardia (da Silva, L.
G. et al, 2008, AJP, 295, p R1774). However, the effects of ACH in the NTS on sympathetic nerve activity and phrenic discharge
have not been evaluated yet. Thus, the aim of this study was to test the effects of ACH microinjected into the medial NTS
(mNTS) on sympathetic and phrenic nerve activity.
Methods and Results:
Decorticated arterially-perfused in situ preparations were made from male Holtzman rats (60-80 g, n=5) to record the activities of
thoracic sympathetic (tSNA) and phrenic nerves (PNA). In these preparations, microinjections of saline (60 nl) and ACH (0.1, 1.0
and 10 nmol/60 nl) were performed randomly into the mNTS and the alterations in tSNA and PNA were evaluated. It was
observed that increasing doses of ACH (0.1, 1.0 and 10 nmol/60 nl) into the mNTS significantly inhibited SNA, whose peak of
response was comparable among the doses (respectively, -51 ± 5.7%, -64 ± 4.2%, -60 ± 1.4%, vs. saline: -8 ± 8% of baseline, p <
0.05). However, the decreases in SNA measured over 30 s after ACH were dose-dependent (-11 ± 5.3%, -22 ± 5.8%, -60 ± 1.4%,
vs. saline: 0 ± 4% of baseline, p < 0.05). Conversely, ACH microinjections into the mNTS did not produce significant reductions
in PNA frequency (-3.5 ± 1.7, -5.2 ± 0.9, -7.3 ± 1.3, vs. saline: -1.4 ± 1.8 bursts per minute, p > 0.05).
Conclusions:
These data suggested that cholinergic system at the mNTS level is involved in the regulation of SNA, possibly acting on neurons
involved in the baroreflex pathways. On the other hand, ACH in the mNTS, at least in the doses studied, may not influence the
generation of PNA.
Keywords: acetylcholine, nucleus of the solitary tract, phrenic nerve activity, sympathetic nerve activity
Financial Support: FAPESP, CNPq
Resumo:21-139
CIGARETTE SMOKE AND/OR ETHANOL DURING ADOLESCENCE OF MICE: MEMORY AND LEARNING
EFFECTS DURING EXPOSURE AND WITHDRAWAL.
Graça, A. C. C. ; Naiff, V. F. ; Carvalho, A. R. ; Manhães, A. C. ; Filgueiras, C. C. ; Villaça, Y. A.
Departamento de Ciências Fisiológicas/ UERJ, IBRAG/UERJ
Objectives:
AIM: Adolescents often associate tobacco smoking and consumption of alcoholic beverages. In spite of this frequent association,
little is known about the basic neurobiology of the dual exposure in the adolescent brain. This study sought to evaluate the effect
of tobacco smoke, ethanol and the double exposure tobacco smoke+ethanol on memory/learning of mice during exposure and
after a short period of withdrawal.
Methods and Results:
METHODS: Four groups of Swiss mice were used to assess the impact of cigarette smoke and/or ethanol consumption on
memory/learning: 1) SMK+ETOH - animals exposed to smoke generated from burning research cigarettes (Kentucky University,
Louisville, KY, USA) containing 0.73mg of nicotine in a cigarette smoking machine (Teague Enterprises, Davis, CA) and i.p.
injection of ethanol solution (2g/kg, 25%); 2) SMK - cigarette smoke and i.p. saline; 3) ETOH - air and i.p. ethanol; 4) VEH - air
and i.p. ethanol. Smoke exposure was carried out from postnatal day (P) 30 to P45 for 8h per day in order to mimic the pattern of
consumption of regular smokers that discontinue consumption during sleep. Ethanol exposure was carried out every other day to
mimic binge consumption. At P44, animal behavior associated with memory/learning was evaluated in the step down passive
avoidance test (SD). Animals were placed on a platform and were allowed 3 min to descend from it, at which time they received
a foot shock (0.2 mA, 3 s). Three and 24h after the initial testing (AIT), animals were retested in the SD (shock was not
administered). The latency to leave the platform was the measured variable. Data were analyzed using ANOVAs. RESULTS: Our
results showed a treatment effect on P45 (P=0.007) 3 h AIT. This can be explained by the fact that SMK, SMK+ETOH e ETOH
animals presented a shorter latency to leave the platform when compared to VEH animals (Pairwise comparisons: ETOH and
VEH: P=0.009; SMK and VEH: P=0.024; SMK+ETOH and VEH: P=0.007). Twenty-four h AIT, we found a treatment effect
(P=0.028) as well as a sex effect (P=0.055), which can be explained by the fact that female SMK e SMK+ETOH presented
shorter latency to leave the platform when compared to VEH females (SMK and VEH: P=0.049; SMK+ETOH and VEH
P=0.027). At P50, three h AIT we did not obtain significant differences between groups. Twenty-four hours AIT, we observed an
interaction between treatment and sex (P=0.045) showing that male ETOH e SMK+ETOH presented shorter latency when
compared to male VEH (ETOH and VEH: P=0.004; SMK+ETOH and VEH: P=0.002).
Conclusions:
CONCLUSION: Exposure tobacco smoke, ethanol or the combined exposure during mice adolescence affects memory
performance in the step down passive avoidance test.
Keywords: Cigarette smoke , Ethanol , Memory and learning , Mice, Adolescence
Financial Support: FAPERJ, CNPq, CAPES and SR2-UERJ
Resumo:21-140
DYNAMICS OF SPONTANEOUS AND REFLEX BLINKS IN THE BURROWING OWL (ATHENE CUNICULARIA).
Vieira, P. G. 1; Almeida, F. 1; Souza, J. K. S. 2; Pinto, M. A. S. 2; Sousa, J. P. M. D. 2; Tierra-criollo, C. J. 2;
Baron, J. 1,2
1
Fisiologia e Biofísica / Instituto de Ciências Biológicas, UFMG
2
Engenharia Elétrica / Escola de Engenharia, UFMG
Objectives:
Several times a minute, eyeblinks produce transient visual disruptions that are usually not registered consciously. As a
preliminary step towards understanding the central mechanisms underlying the perceptual filling-in of such temporal scotomas,
we presently report kinematics data of spontaneously occurring and air-puff evoked blinks in the animal species that we selected
as a model for addressing this issue, namely the burrowing owl (Athene cunicularia).
Methods and Results:
Methods: We recorded 78 spontaneous and 82 induced blinks in two head-restrained, non-anesthetized owls using a computerassisted infrared pupillometer developed by our group. Eyelid and nictitating membrane movements were measured at a
sampling rate of 120 Hz. To induce a blink reflex, we used a customized air-puff device that comprised an electronically
controlled valve unit, an air reservoir and a flexible thin plastic tube for funneling the air to the cornea and periorbital region.
The air-puff pulse duration was set to 20 ms. For data analysis, we used our own Labview-based pupillometric software, Matlab
(MathWorks, Natick, MA, USA) and the public domain image processing program ImageJ 1.44 (NIH,
http://rsb.info.nih.gov/ij/).Population measurements were presented as median, using the 25th and 75th percentile as an
indicative of population dispersion. All experimental and animal care procedures were approved by the University Ethics
Committee for Animal Experimentation (CETEA, license n 106/10). Results: Spontaneous blinks were characterized by a lack of
complete eyelid closure, occluding only 30% (16.6 – 38.2%) of the pupil. In contrast, the nictitating membrane almost always
swept the entire eye globe. Duration of spontaneous blinks was 350 ms (275 – 450 ms) with a down phase duration of 125 ms
(108 – 150 ms) and up phase of 208 ms (167 – 300 ms). Evoked blinks resulted in almost complete covering of the eye globe.
Duration of reflex blinks was 575 ms (460 – 756 ms) with a down phase duration of 158 ms (125 – 217 ms) and up phase of 408
ms (317 – 556 ms). Latency of eyelid response to air puffs was short (92 ms). For both spontaneous and reflex blinks, the down
phase of the nictitating membrane occurred concomitantly with that of the eyelid; the upward phase of the nictitating membrane
was nevertheless much slower.
Conclusions:
Our study reveals that the down phase is fairly constant for both types of blinks, whereas the up phase differs markedly in
duration. At least three factors may explain this finding: (1) differences in palpebral response amplitude increasing the traveling
distance for fully reopening the eye; (2) variation in the animal central state due to corneal e periorbital stimulation as opposed
to absence of stimulation; (3) involvement of different neuronal components involved in mediating the two types of blink. Which
of these factors explain our data remains to be determined.
Keywords: Blink dynamic, owl, temporal scotomas
Financial Support: FAPEMIG, CNPq
Resumo:21-141
AV3V LESION DOES NOT CHANGE THE CARDIOVASCULAR RESPONSES TO CARBACHOL IN THE NTS.
Zanella, D. C. ; Menani, J. V. ; Paula, P. M. D.
Universidade Estadual Paulista "Júlio de Mesquita Filho", Unesp
Objectives:
Studies from our laboratory have shown that lesions of the preoptic periventricular tissue surrounding the anteroventral third
ventricle (AV3V region) reduced the pressor responses to glutamate and adenosine 5'-triphosphate (ATP) into the nucleus of the
solitary tract (NTS). This lesion also reduced the hypotension caused by alpha, beta-methyl ATP (a selective P2X purinergic
receptors agonist) into the NTS. Similar to alpha, beta-methyl ATP, carbachol (cholinergic agonist) when injected into the NTS
in awake rats produced a drop in mean arterial pressure (MAP) and heart rate (HR). Therefore, in the present study, we
investigated the effects of acute (1 day) electrolytic lesions of the AV3V region on the hypotension and bradycardic responses
induced by injections of carbachol into the NTS in unanesthetized rats.
Methods and Results:
Male Holtzman rats (n=6/group) with sham or electrolytic (2 mA x 10 s) AV3V lesions and a stainless steel cannula implanted
into the NTS were used. The hypotension and bradycardic responses produced by unilateral injections of carbachol (2.5 nmol/100
nl) into the NTS (-25 ± 1 mmHg and -154 ± 8 bpm) did not change in 1 day AV3V-lesioned rats (-29 ± 3 mmHg and -134 ± 7
bpm, p>0.05).
Conclusions:
The results show that the integrity of the AV3V region is not important for the cardiovascular responses produced by cholinergic
activation into the NTS but is essential for the cardiovascular responses by purinergic and glutamatergic activation into the NTS.
Keywords: arterial pressure, AV3V region, carbachol , glutamatergic , NTS
Financial Support: CNPq and FAPESP
Resumo:21-142
IMPAIRED INSULIN SIGNALING IN A PRIMATE MODEL OF ALZHEIMER`S DISEASE: LINK WITH TYPE-2
DIABETES
Forny-germano, L. 1; Bomfim, T. R. 1; Lyra E Silva, N. M. 1; Christophe-houzel, J. 1; Brito-moreira, J. 1;
Klein, W. L. 3; Munoz, D. P. 4; Ferreira, S. T. 1; de Felice, F. G. 1
1
Universidade Federal do Rio de Janeiro, UFRJ
3
4
Northwestern University, NU
Queen's University, queensu
Objectives:
Alzheimer�s disease (AD) has been linked to defective brain insulin signaling, a proposed third type of diabetes (Curr.
Alzheimer Res. 4:147, 2007; BMB. Rep. 42:475, 2009; FASEB J. 22:246, 2008). Although this intriguing connection between
AD and diabetes has been suggested, a major unknown is the mechanism by which insulin resistance develops in AD brains. In
type 2 diabetes, tumor necrosis factor-� (TNF-�) signaling stimulates c-Jun N-Terminal Kinase (JNK). This results in serine
phosphosphorylation of the insulin receptor substrate (IRS-1), blocking downstream signaling and triggering insulin resistance
(Science 271:665, 1996). One link between diabetes and AD was found in the ability of A� oligomers - toxins that accumulate
in AD brains and instigate synaptic damage - to activate the JNK/TNF-� pathway, leading to phosphorylation of IRS1pSer636. Currently, there is no effective treatment for AD, and the pursuit of novel disease-modifying therapeutics is the object
of intense investigation. Although a few therapeutic drugs (JAMA 304:1903, 2010) and antibodies targeting A� oligomers
(Neurobiol. Aging 30:1728, 2009) have shown some promises in preclinical studies and early clinical trials, unfortunately a
number of trials have failed to show any efficiency in AD patients (JAMA 304:1903, 2010). Part of this discrepancy lies in the
difficulty to translate therapeutic approaches developed in rodent models to the specific conditions of the human disease. In an
attempt to get closer to the complexity of primate brains, here we extend our model of chronic intracerebroventricular injection of
A� oligomers in rodents to macaque monkeys and investigate if Abeta oligomers are capable of triggering diabetes-like effects
on the JNK/TNF-� pathway in primates.
Methods and Results:
Freshly prepared A� oligomers were injected in the cerebral ventriculs of three adult (~9 years of age) cynomolgus monkeys
(Macaca fascicularis, weight 4.7-7.0Kg). Monkeys received 100�g of A� 3 times a week for 3 weeks. One sham-canulated
monkey served as control. Brains were removed and later, immunohistochemistry for pJNK and IRS-1pSer636 was performed on
40 �m thick frozen cut sections. Analysis revealed a marked activation of JNK in the hippocampus in A� oligomers injected
monkeys, when compared to control. In addition, pixels per area quantification using NIH Image Java indicated that levels of
IRS-1pSer636 were significantly increased in the hippocampus (p=0.0002) and in the temporal cortex (p
Conclusions:
Our results reinforce the link between diabetes and AD. Furthermore, considering the dearth of animal models that truly
recapitulates the main features of Alzheimer�s disease, this new non-human primate model offers a potential to provide
insights into central aspects of AD that may be exclusively present in primates.
Keywords: Alzheimer`s disease, c-Jun N-Terminal Kinase, Insulin receptor substrate, Primate model, Type-2 Diabetes
Financial Support: CNPq, FAPERJ, INNT.
Resumo:21-143
PUPILLARY LIGHT REFLEX IN THE BURROWING OWL (ATHENE CUNICULARIA).
Souza, J. K. S. 1; Vieira, P. G. 2; Tierra-criollo, C. J. 1; Baron, J. 1,2
1
Engenharia Elétrica / Escola de Engenharia , UFMG
2
Fisiologia e Biofísica / Instituto de Ciências Biológicas, UFMG
Objectives:
The pupillary light reflex (PLR) refers to the constriction of the pupil produced by an increase in retinal illumination. Here, we
provide PLR data obtained from adult burrowing owls (Athene cunicularia) as a means to gain further insights into the functional
properties of the neural pathway mediating this reflex in birds.
Methods and Results:
Methods: PLR was measured in two head-restrained, non-anesthetized owls using a computer-assisted infrared pupillometer
developed by our group. Pupil diameter was measured at a sampling rate of 120 Hz with an estimated spatial resolution of 0.07
mm. Visual stimuli were presented either mono- or binocularly from a 19-inch CRT monitor placed 35 cm from the eyes of the
animal and consisted of brief flashes (100 ms) of large uniform fields (angular subtense: 39.4 x 26.6 degrees) of achromatic light
that varied randomly in luminance (steps: 15, range: 15 to 162 cd/m2). All recording sessions were initiated after 20 minutes of
dark adaptation. Data were analyzed using our own Labview-based pupillometric software, Matlab (MathWorks, Natick, MA,
USA) and the public domain image processing program ImageJ 1.44 (NIH,http://rsb.info.nih.gov/ij/). Results are presented as
arithmetic means ± SD. All experimental and animal care procedures were approved by the University Ethics Committee for
Animal Experimentation (CETEA, license n 106/10). Results: Mean pupil diameter varied from about 10 mm under near-dark
conditions to just over 5 mm (≅4-fold reduction in retinal illuminance level). Saturation in pupillary response was observed for
stimuli above 65 cd/m2. Response onset latency was fairly constant across all luminance levels (mean value: 54.0±7.6 ms). At the
highest luminance tested, time to reach peak constriction was 136.8±10.0 ms, yielding in a constriction speed of 58.6±4.2 mm/s;
return to baseline level (post-stimulus dilatation) was significantly slower (duration: 813.2±267.4 ms; speed: 20.9±2.0 mm/s). At
the lowest luminance, time to reach peak constriction was 85.1±9.3 ms, resulting in a constriction speed of 12.6±4.1 mm/s; return
to baseline level (post-stimulus dilatation) was also significantly slower (duration: 725.0±310.2 ms; speed: 10.1±3.1 mm/s).
There was no statistical difference in pupillary response between monocular and binocular viewing conditions.
Conclusions:
Our study reveals that PLR is about six times faster in burrowing owls than in primates. Another interesting difference is that, in
the owl, response latency does not vary as function of luminance and no consensual response is observed. Whether such
differences are solely explained by the striated musculature of the owl‘s iris (as opposed to smooth muscles in mammals) remains
an open question.
Keywords: Pupillary light reflex, owl, pupillometry
Financial Support: FAPEMIG, CNPq, CAPES
Resumo:21-144
HUMAN DIFFERENCE LIMENS FOR FLICKER FREQUENCY OF ACHROMATIC LIGHT
Akemy, B. 1; Sousa, J. P. M. 2; Pinto, M. A. D. S. 2; Tierra-criollo, C. J. 2; Baron, J. 1,2
1
Fisiologia e Biofísica / Instituto de Ciências Biológicas, UFMG
2
Programa Pós-Graduação Engenharia Elétrica/Escola Engenharia, UFMG
Objectives:
The temporal visual sensitivity of human perception may be assessed in a number of ways. Here, we aim at estimating differential
thresholds for detecting slight differences in flickering frequency increments and decrements.
Methods and Results:
Experimental sessions took place in a soundproof and light−controlled booth. Subjects (n=14) were accommodated in an
armchair positioned 57 cm away from the stimulation source, provided by flickering white LEDs driven by a portable,
micro−controlled stimulator developed by our group (J.Neurosci. Methods, 197, 82−91; 2011). Perceptual thresholds were
measured using a temporal two−alternative forced choice (2AFC) procedure in combination with a method of constant stimuli.
Each trial consisted in presenting a reference stimulus flickering at 10 Hz (F1) during 2s, followed by a test stimulus (F2)
flickering at a slightly different frequency (n=14; increments⁄decrements: ± 0.2 Hz) or at the same frequency as F1 (n=4) during
the same period. An interval of 4s was left between F1 and F2. Using a response button, subjects were asked to report which
stimulus in the trial sequence had the highest flickering frequency (one press: F1; two presses: F2). No feedback about the
accuracy of the judgments was given. All procedures adopted in this study have been approved by the University Human
Research Ethics Committee (COEP, ETIC 467⁄08). All subjects readily perceived flickering frequency increments and
decrements above 2 Hz. The average hit rate when F2 was higher than F1 was 86,96% (SD: 9,33%) and 76,82% (SD: 9,24%)
when the opposite stimulation condition occurred. Threshold estimates across subjects were fairly constant and approximated 0.9
Hz and 0.4 Hz for frequency decrements and increments, respectively. For most subjects, a robust subject criterion bias was
however observed, which induced higher hit scores for positive increments. Catch trial analysis revealed that almost all subjects
had a tendency to choose F2 higher than F1 in 2⁄3 of the cases.
Conclusions:
Our results are globally coherent with earlier studies that have assessed difference limens for flicker frequency, but using a spatial
2AFC experimental design. Future work will include the testing of other F1 values and the effect of increasing the
inter&minusstimulus interval as a means to assess the retention capability of temporal attributes of basic visual stimuli.
Keywords: temporal visual sensitivity, flickering frequency, estimating thresholds
Financial Support: Brazilian agencies CAPES and FAPEMIG
Resumo:21-145
NUCLEAR FACTOR KAPPA B AS A DUALISTIC MEDIATOR OF CEREBELLAR GRANULE CELL CULTURE
DAMAGE
Franco, D. G. ; Markus, R. P.
Dep. Fisiologia - Inst. de Biociências, IBUSP
Objectives:
In the central nervous system nuclear factor kappa B (NFKB), a transcription factor primarily related to inflammatory responses,
plays an important role in the modulation of plasticity, development and cell survival (Cell & Mol Immuno. 1:425, 2004). As an
example, in cerebellar granule cell culture (CGC), which has a basal NFKB activation, tumor necrosis factor (TNF) increases
NFKB nuclear translocation leading to an inverted-U shaped dose survival curve. Therefore, very high or low concentrations of
TNF result in cells death, while intermediary concentrations are necessary for cell survival(Bioch et Bioph Acta. 1745:287,
2005). Melatonin, the pineal gland hormone, acts as a cytoprotector and anti-oxidant agent impairing the activation of NFKB in
macrophages (FASEB J. 12:685, 1998) and endothelial cells culture (J. Pineal Res. 46:268, 2009). We have previously shown
that in CGC, melatonin inhibits lipopolysaccharide (LPS)-induced NFKB translocation, but has no effect on basal NFKB nuclear
content (Annals XV Congress SBBC 2010, pp 130). This fine tune balance a critical factor involved in cell survival lead us to
investigate the NFKB manipulation effects on cell damage. Therefore, we evaluated cytotoxicity promoted by the inhibition of
basal NFKB by PDTC (pyrrolidine dithiocarbamate), a competitive blocker NFKB dimers binding to DNA kappa B responsive
element, or the NFKB activated by LPS in the presence or absence of melatonin in CGC.
Methods and Results:
Granule cells isolated from rat Wistar (7-8 days-old) cerebellum were cultivated for 8 days in DMEM (10% FBS). At the 7th/8th
day, cells were incubated with LPS (0.1 and 1µg/mL) in the presence or absence of melatonin (0.001, 0.1 and 1µg/mL) or PDTC
(0.001 to 10µg/mL) for 12h. Cellular membrane damage was determined by lactate dehydrogenase (LDH)-leakage into the
medium. Statistical analyses were performed using ANOVA followed by the Newman–Keuls test. Values of p
Conclusions:
In CGC, the balance of NFKB activation is an important factor for cell survival. As we know, melatonin has no effect on basal
nuclear NFKB, so that, it had no effect on membrane damage. In the other hand, PDTC that promotes inhibition of nuclear NFKB
translocation promoted an increase in cytotoxicity. The nuclear translocation of NFKB by LPS did not result in cell toxicity,
however, the concomitant incubation of LPS and melatonin increased cell damage. Before considering that melatonin is not
exerting its cytoprotective effect, it is important to point out that lower concentrations were more deleterious than higher
concentrations. A special and inverse related dose-response regarding the relationship between cell survival and NFKB activity
was also observed with PDTC, since the lower concentrations were also more deleterious than the higher ones. Therefore, this
work shows a new perspective for evaluating NFKB as a pivotal transcription factor involved in the balance between the classical
pro and anti-inflammatory responses in cerebellar neurons.
Keywords: cell damage, cytoprotection, granule cell culture, melatonin, nuclear factor kappa B
Financial Support: FAPESP, CNPq, CAPES
Resumo:21-146
ACUTE DEXAMETHASONE IMPROVES PERFORMANCE AND SEXUAL MOTIVATION IN MALE RATS
Teixeira, A. S. ; Santiago, M. B. ; Giusti-paiva, A.
Instituto de Ciências Biomédicas/Univ. Federal de Alfenas, ICB/ UNIFAL-MG
Objectives:
The study of sexual behavior in rats involves the analysis of motivation and sexual performance. The distinction is between
seeking sexual contact and be able to perform the copula. This behavior is necessary for the preservation of species and not for
individual survival. Thus, it differs considerably from other spontaneous behaviors such as feeding, hydration, respiration or
thermoregulation. Studies show that stress and consequent activation of the hypothalamic-pituitary-adrenal (HPA) axis and
secretion of glucocorticoids can alter various hormonal and behavioral parameters. The aim of this study was to assess sexual
motivation and performance of male rats and its possible modifications before of acute administration of dexamethasone (DEX),
a synthetic glucocorticoid.
Methods and Results:
Methods:Wistar rats were housed in groups of four animals per cage in a room maintained under a light-dark cycle of 12h at 21 ±
1°C. In experiment 1, adult male rats (350-450g) sexually experienced were previous treated with vehicle (control, n = 8) or DEX
(1 mg/kg, n = 10) 2 hours before of exposed to receptive females (220-280g) by 40 minutes. Later the tapes were analyzed and
quantified the following parameters: mount latency (ML), intromission latency (IL), number of incomplete mounts (IM), number
of intromissions (NI), ejaculatory latency (EL); latency to first mount after ejaculation (LFMA), latency to first intromission after
ejaculation (LFIA), number of incomplete mounts after ejaculation (IMA), number of intromissions after ejaculation (NIA), total
number of ejaculations (NE). In experiment 2, male rats (290-350g), sexually inexperienced, were treated with vehicle (control, n
= 9) or DEX (1mg/kg, n = 11) and, 2 hours after, were placed in an apparatus where there was a receptive female (sexual
incentive) and a sexually experienced male (social incentive), by 20 minutes. After analyzing the videos were quantified the
following parameters: time in seconds spent in the female incentive zone (FIZ) and male incentive zone (MIZ), frequency of
visits to each incentive zone. Then we calculated the score of preference. The behaviors of the male rats were started in the dark
cycle, from 20:00 pm. The experiments were conducted with the approval of the Ethics Committee of the Federal University of
Alfenas (protocol 306/2010). Results With respect to experiment 1, we observed that administration of DEX reduces the NI (14.8
± 1.7 vs. Control: 24.1 ± 3.6 intromission, p = 0.02), reduction in EL (671 ± 130 vs. Control: 1155 ± 151 seconds, p = 0.02),
reduced LFMA (252 ± 13.5 vs. Control: 327 ± 19.2 seconds, p = 0.004), reduction in LFIA (253 ± 13 vs. Control: 333 ± 19.9
seconds, p = 0.003) and increase in NE (3.8 ± 0.4 vs. Control: 2.3 ± 0.2 ejaculations, p = 0.01). Regarding the ML and IL, which
were not affected by DEX administration since these parameters are influenced by sexual experience and it was common to both
groups. Considering the experiment 2, we observed that administration of DEX increased the FIZ (738.5 ± 42.88 vs. Control:
584.4 ± 45.74 seconds, p < 0.05), reduced the MIZ (219.1 ± 26.58 vs. Control: 333.6 ± 48.18 seconds; p < 0.05), increased the
score of preference for female (0.77 ± 0.03 vs. control: 0.64 ± 0.04, p < 0.05). The other parameters showed no significant
difference.
Conclusions:
The behaviors changes caused by the acute administration of DEX suggest a better performance and sexual motivation in male
rats.
Keywords: Dexamethasone, Glucocorticoids, Hormonal control of behavior, Male rats, Sexual behavior
Financial Support: Fapemig; CNPq; CAPES; FINEP
Resumo:21-147
FUNCTIONAL CORRELATES OF ABERRANT BUNDLES IN CALLOSAL DYSGENESIS
Lazarev, V. V. 1; Monteiro, M. C. 1,2,3; Oliveira-souza, R. 2; Deazevedo, L. C. 1; Moll, J. 2; Lent, R. 3;
Tovar-moll, F. 2,3
1
Instituto Fernandes Figueira, IFF
2
Instituto D'Or de Pesquisa e Ensino , IDOR
3
Universidade Federal do Rio de Janeiro, UFRJ
Objectives:
Callosal dysgenesis (CD) is characterized by a developmental failure of formation of the major commissural bundle. Previous
studies had demonstrated structural evidence for abnormal white-matter pathways (Probst and Sigmoid bundles) in this
pathology. However, evidence for a functional role for the Sigmoid bundle is still lacking. Here we combined diffusion tensor
imaging (DTI) with quantitative electroencephalography (EEG) in order to address, in vivo, the functional correlates of these
pathways and the relationship between structural and functional neuroplasticity in human CD.
Methods and Results:
Volumetric anatomical (3DT1) and DTI (2,5mm isotropic voxel) images were acquired (3T, Achieva Philips) in five patients
with CD and fifteen healthy volunteers. Neurophysiological assessment was performed by 16-electrode EEG registration (Bio-
logic, USA) and analysis of the Coefficients of Coherence (CChr) (Brainsys - Neurometrics, Russia) in five EEG frequency
bands. The Intelligence Quotient Test was obtained from all the subjects. Group comparison was performed using the t-test. The
patients had lower intelligence quotients within normal range. In the patients, conventional images showed typical anatomical
characteristics of CD, including partial dysgenesis (n=2), agenesis (n=2) or hypoplasia (n=1) of the corpus callosum (CC). In
addition, DTI and tractography confirmed the presence of two abnormal white-matter tracts: (i) the Probst bundles (PB)
connecting intrahemispheric cortical regions in all the patients and, (ii) the Sigmoid bundles, connecting the frontal lobe with the
contralateral occipito-parietal cortex (n=3, except for agenesis). General analysis of CChr showed an increased intrahemispheric
and decreased interhemispheric coherence in patients as compared to controls. In the patients with partial CD or CC hypoplasia, a
relatively higher interhemispheric coherence in the frontal areas was observed. These three patients also presented higher
coherence connectivity between the right frontal and left parietal areas and, in certain frequency bands, bilateral increase in some
other long-distance contralateral coherent connections of the fronto-polar and frontal regions with the occipital, parietal and
central ones.
Conclusions:
The results corroborate previous anatomical findings in CD, showing the presence of abnormal bundles formed during the
development. The neurophysiological results correlate with observed structural changes, since an increased intrahemispheric
connectivity may be related to the presence of the Probst bundle. In addition, the anatomical connectivity of the Sigmoid bundle
may underlie the higher interhemispheric coherence observed between contralateral anterior and posterior areas in partial CD and
CC hypoplasia. The changes in electrophysiological connectivity observed in CD might be the functional counterpart of the
abnormal white-matter connections detected in these patients.
Keywords: callosal dysgenesis, diffusion tensor imaging, EEG Coherence
Financial Support: CAPES, CNPq, FAPERJ
Resumo:21-148
EFFECTS OF 8-PHENYLTHEOPHYLLINE ON CATALEPSY BEHAVIOR INDUCED BY HALOPERIDOL, IN
MICE
Araújo, R. S. ; Ribeiro, T. S. ; Souza, A. S.
Fundação Universidade Federal de Mato Grosso do Sul , UFMS
Objectives:
Adenosine plays an important hole on basal nuclei functions; however dopamine and adenosine interactions on these nuclei are
not completely understood. The aim of this study was assess the effects of an A1 selective adenosine antagonist, 8phenylteophylline, on catalepsy behavior induced by the D2 dopamine antagonist receptor, haloperidol, in mice.
Methods and Results:
Swiss male mice, 25-35g weight, were initially injected by intraperitoneal route with saline or haloperidol and after 30 minutes
injected by intraventricular route with saline or 8-phenylteophylline (5 and 10nM), performing a total of 6 experimental groups:
(n=8/group): i) saline + saline; ii) saline + 8-phenylteophylline (5nM); iii) saline + 8-phenylteophylline (10nM); iv) haloperidol
(2 mg/kg) + saline; v) haloperidol (2mg/kg) + 8-phenylteophylline (5nM); e vi) haloperidol (2mg/kg) + 8-phenylteophylline
(10nM). After drug administration (5, 35 and 65 minutes) the animals were submitted to catalepsy test measuring the latency
(sec.) on the bar test. The catalepsy data were log transformed (log10 + 1) and analyzed by two-way ANOVA of repeated
measures followed by Duncan post hoc test. The catalepsy time in each experimental group 5, 35 e 65 minutes after drug
administration were: i) saline + saline: 5min=1.13±0.58s; 35min=1.38±0.56s; 65min=0.75±0.25s; ii) saline + 8-phenylteophylline
(5nM): 5min=0.63±0.26s; 35min=1.13±0.30s; 65min=1.75±0.45s; iii) saline + 8-phenylteophylline (10nM): 5min=0.25±0.16s;
35min=0.38±0.26s; 65min=0.00±0.00s; iv) haloperidol (2mg/kg) + saline: 5min=35.75±3.64s; 35min=115.63±30.88s;
65min=177.50±32.00s; v) haloperidol (2mg/kg) + 8-phenylteophylline (5nM): 5min=53.13±20.05s; 35min=29.13±9.88s;
65min=35.63±15.13s; and vi) haloperidol (2mg/kg) + 8-phenylteophylline (10nM): 5min=104.50±36.10s; 35min=68.00±36.85s;
65min=41.63±20.37s. There was a significant effect of experimental group, no effect of time and significant interaction between
group X time (Two-way ANOVA of repeated measures; group: p<0.05).
Conclusions:
Antagonism of dopamine receptors by haloperidol produced catalepsy on the animals. This effect was partially reverted by 8phenylteophylline an A1 adenosine antagonist receptor in both doses used (5 and 10nM). This effect was observed only 35 e 65
minutes after drugs administration.
Keywords: BEHAVIOR, CATALEPSY, HALOPERIDOL, 8-PHENYLTHEOPHYLLINE , MICE
Financial Support: UFMS, CNPq.
Resumo:21-149
MEDIAN NERVE REPRESENTATION IN RAT S1 AND IMMEDIATE PLASTICITY AFTER ACUTE FOREPAW
DEAFFERENTATION.
Oliveira, J. T. ; Bittencourt-navarrete, R. E. ; Martinez, A. M. B. ; Franca, J. G.
Universidade Federal do Rio de Janeiro, UFRJ
Objectives:
Transection of peripheral nerves interrupts sensory inputs to the central nervous system, resulting in cortical plasticity. In this
study we investigated the representation of the median nerve in rat primary somatosensory cortex (S1) after acute section of all
other brachial plexus nerves.
Methods and Results:
Three adult Wistar rats had their right brachial plexus transected, except for the median nerve. A contralateral craniotomy was
performed followed by electrophysiological mapping of the representation of the forepaw and adjacent body parts in S1. The
median nerve was then transected and a remapping of the same cortical region was performed. After electrophysiological
mapping small DY crystals were placed at 3 or 4 cortical sites as fiducial markers. The animals were transcardially perfused with
a fixative solution. The cortex was flattened and processed for cytochrome oxidase histochemistry. Superimposition of barrel
fields with the functional maps revealed that all the forepaw barrel subfield (FBS) was responsive to forepaw stimulation carried
by the median nerve alone. Most sites responded to light touch stimuli in the forepaw, fewer sites responded to deep touch or
movement stimulation. After median nerve transection, we observed an expansion of the representation of vibrissae, hindpaw,
and inferior lip into regions previously responsive to the forepaw in the FBS.
Conclusions:
We conclude that the median nerve can relay responses to the whole FBS and that part of this barrel subfield can become acutely
responsive to stimulation of other body parts once the forepaw is completely deafferented.
Keywords: brachial plexus, cortical mapping, median nerve, plasticity, somatosensory cortex
Financial Support: CAPES, Cnpq and Faperj
Resumo:21-150
EFFECT OF CENTRAL INJECTION OF HIDROGEN PEROXIDE ON 2% SUCROSE AND FOOD INTAKE.
Zanella, R. C. ; Menani, J. V. ; Colombari, E. ; Colombari, D. S. A.
Fisiologia e Patologia/ Faculdade de Odontologia Araraquara , UNESP
Objectives:
Reactive oxygen species (ROS) produced endogenously may act in the brain modulating autonomic and behavioral responses.
Previously we have demonstrated that intracerebroventricular (icv) injection of a ROS (hydrogen peroxide, H2O2) reduced the
dipsogenic responses to icv injection of angiotensin II and carbachol (cholinergic agonist) or to intragastric load of hypertonic
saline. In order to test whether central H2O2 effects would be specific to water intake, we investigated the effects of icv injection
of H2O2 on 2% sucrose and food intake.
Methods and Results:
Male Holtzman rats (280-320 g) with stainless steel guide-cannulas implanted into the lateral ventricle (LV) were used. In the
first protocol (n = 5), animals were trained to ingest 2% sucrose for 2 h daily over 7 days. Phosphate buffered saline (PBS; 1 µl)
or H2O2 (5 µmol/1 µl) was injected into the LV 1 minute before starting the measurement of 2% sucrose intake. In the second
protocol (n = 11), PBS (1 µl) or H2O2 (5 µmol/1 µl) was injected into the LV 1 minute before starting the measurement of food
intake by 24 h food-deprived rats. The treatment with icv H2O2 reduced 2% sucrose intake (0.7 ± 0.4 ml, vs. PBS: 5.1 ± 1.6 ml/2
h, p < 0.05). The treatment with icv H2O2 did not reduce food intake (10.3 ± 0.6 vs. PBS: 10.7 ± 0.9 g/2 h, p >0.05), but reduced
meal-associated water intake (8.3 ± 0.8 vs. PBS: 12.8 ± 1.2 ml/2 h, p < 0.05).
Conclusions:
The results suggest an inhibitory role for H2O2 acting centrally on fluid intake (water and sucrose), without changing food
intake, which excludes a non-specific effect of H2O2 acting centrally on all ingestive behaviors.
Keywords: CENTRAL HIDROGEN PEROXIDE, SUCROSE INTAKE, FOOD INTAKE
Financial Support: CNPq, FAPESP.
Resumo:21-151
OXIDATIVE STRESS IN THE BRAIN OF 21 MONTHS OLD RATS AFTER AGUTE ADMINISTRATION OF AN
AQUEOUS EXTRACT OF THE MEDICINAL MUSHROOM AGARICUS BRASILIENSIS (A. BLAZEI)
Sá-nakanishi, A. B. de ; Soares, A. A. ; Comar, J. F. ; Natali, M. R. M. ; Peralta, R. M. ; Bracht, A.
Departamento de Bioquímica, Universidade Estadual de Maringá, UEM
Objectives:
Reactive oxygen species (ROS) are involved in the mechanism of several neurodegenerative processes related to ageing. The
mushroom Agaricus brasiliensis (formerly known as A. blazei), popularly known as cogumelo do sol, has been amply utilized in
popular medicine as a potential therapeutic agent for treatment of several diseases. It is generally accepted that A. brasiliensis has
antioxidant properties, which are attributed to its contents in vitamins A and C, &beta-carotenes and several phenolic compounds.
If the mushroom also exerts antioxidant effects in the ageing brain, however, is still an open question.
Methods and Results:
Male Wistar rats, (with 21 months) were treated, by gavage, during 21 days with a lyophillized aqueous extract of A. blazei (200
mg kg-1 day-1). Control rats (same age, aged rats) were treated with saline during the same period. After fasting for 24 hours, the
rats were decapitated, the brain removed and rapidly freeze-clamped in liquid nitrogen. The tissue was homogenized in 100 mM
phosphate buffer (pH 7.4) and used for determining the following oxidative state markers: substances reactive to thiobarbituric
acid (TBARS), reduced glutathione (GSH), reduced protein sulphidryl groups (reduced thiols) and the activities of the enzymes
catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR). Brains of rats that were 3 months old were also
processed. These rats comprise the adult controls. The results revealed that the TBARS levels increased with ageing: 1.59±0.09
(n=5) and 2.07±0.17 (n=5) nmol.(mg protein)-1 (p = 0.04) in adults and aged rats, respectively. However, the A. brasiliensis
treatment significantly reduced TBARS of aged rats to 1.55±0.04 (n=4) (p=0.04 versus aged controls). The GSH and thiol levels
were not changed upon ageing: 2.61±0.15 (n=5) and 2.54±0.04 (n=9) &muGSH.(mg protein)-1 (p=0.62), in adults and aged rats
respectively; 76.91±3.3 (n=5) and 76.64±2.12 (n=6) nmols thiols.(mg protein)-1 (p=0.94) in adults and aged rats respectively. A.
brasiliensis treatment, however, increased the thiol levels in the aged animals (83.17±1.56 ng (mg protein)-1; n=5; p=0.04).
Ageing did not change the activity of the antioxidant enzymes: CAT, 12.90±0.57 (n=5) and 12.89±0.74 (n=7; p=0.99)
&mumol.min-1.mg-1; SOD, 1.86±0.12 (n=4) and 1.70±0.07 (n=8; p=0.28) units.mg-1; GR, 16.09±0.67 (n=5) and 14.51±.1.05
nmol.min-1.mg-1 (n=6; p=0.26). A. brasiliensis treatment, however, increased the activity of CAT and SOD in aged rats:
17.78±0.86 (n=5; p=0.001) &mumol.min-1.mg-1 and 2.04±0.07 (n=6; p=0.002) U.mg-1, respectively.
Conclusions:
It can be concluded that treatment with the aqueous extract of Agaricus brasiliensis protects the brain against the oxidative stress
that occurs during ageing. Possibly, this protection minimizes the occurrence of those disorders associated to ageing that involve
the participation of free radicals.
Keywords: Ageing, Medicinal mushroom, Oxidative stress, Rat brain, Reactive oxygen species
Financial Support: CNPq
Resumo:21-152
DEPRESSIVE-LIKE AND ANXIOGENIC EFFECTS IN ADULT MICE EXPOSED TO ORGANOPHOSPHATE
PESTICIDES DURING NEONATAL LIFE
Lima C. S. 1; Dutra-tavares A. C. 1; Nunes F. 1; Filgueiras, C. C. 1; Manhães, A. C. 1; Meyer, A. 2; Abreuvillaça Y. 1
1
Departamento de Ciências Fisiológicas/ IBRAG, UERJ
2
Instituto de Estudos em Saúde Coletiva, UFRJ
Objectives:
Aim:Epidemiologic studies suggest that organophosphates (OPs) are etiologically linked to mood disorders and cognitive deficits
and that certain subpopulations, particularly children, may be especially vulnerable. Here, we investigated whether early exposure
to chlorpyrifos (CHLOR) and methamidophos (METH) at doses well below the threshold for systemic toxicity and that cause
only 20% of acetylcholinesterase (ACHE) inhibition elicit behavioral disorders at adulthood.
Methods and Results:
Methods: From postnatal day 3 to 9 (PN3-PN9), Swiss mice received daily s.c. injections of CHLOR (3mg/kg), METH (1mg/kg)
or DMSO vehicle (CONT). These doses were selected because we have previously shown that they cause small and similar
inhibitions of AChE in brain. At adulthood (PN60) mice were submitted to a battery of tests: the forced swimming test (N= 46),
in which the animal is placed in a cylindrical container with water to swim for 10 min and the tail suspension test (N= 55), in
which the animal is hanged by the tail for 6 min. In both tests, the immobility time is quantified and used as a measure of
depressive-like behavior. Anxiety levels were evaluated by the elevated plus maze test (N= 40) which consists of a suspended
maze with two open arms and two arms enclosed by walls. The time that animals remain in open and closed arms is measured.
Learning/memory was evaluated by the step down passive avoidance (N= 53), in which the animals learn that stepping down
from a platform is followed by a foot shock and on subsequent exposures to the task they stay longer on the safe platform.
Locomotor activity was evaluated by the open field test (N= 51), in which animals are placed in a square box and the traveled
distance in cm is quantified. Results were evaluated by analyses of variance (ANOVAs) on all factors: treatment and sex.
Treatment effects and interactions were followed by post-hoc analyses using Fisher‘s Protected Least Significant Difference tests
(FPLSD). Results: Animals exposed to methamidophos presented an increase in immobility time in the forced swimming test
when compared to control animals (METH vs CONT: 36.8±9.5 vs 8.36±4.6; P=0.006), indicating a depressive-like behavior. In
the tail suspension test there were no significant differences between groups. In the elevated plus maze, animals exposed to
chlorpyrifos presented a decrease of total time in the open arms (CHLOR vs CONT, 2.92±2.4 vs 26.94±10.3; P=0.012) and in the
percentage of time in the open arms (CHLOR vs CONT, 2.21±1.67 vs 8.14±2.39; P=0.013), indicating an anxiogenic effect.
There were no significant differences in locomotor activity (open field) and in learning/memory (passive avoidance).
Conclusions:
Conclusion: Our results demonstrate that exposure to chlorpyrifos and methamidophos during development elicit behavioral
alterations at adulthood. These results corroborate previous studies that suggest that OP exposure elicit mood disorders and
reinforce the importance of studying long-term effects. Finally, since the doses of methamidophos and chlorpyrifos used in the
present study elicit similar levels of ACHE inhibition (≈20%), distinct behavioral outcomes associated either to methamidophos
or to chlorpyrifos exposure suggest that ACHE inhibition alone does not fully explain our results.
Keywords: anxiety, behavior, chlorpyrifos, depression, methamidophos
Financial Support: CAPES and CNPq, and grants from FAPERJ and MSSM (Award Number
D43TW000640)
Resumo:21-153
EFFECTS OF 8-PHENYLTHEOPHYLLINE ON MOTOR BEHAVIOR IMPAIRMENT INDUCED BY
HALOPERIDOL, IN MICE
Ribeiro, T. S. ; Araújo, R. S. ; Schiaveto-de-souza. A.
Dpt. of Morphophysiology, Universidade Federal de MS, UFMS
Objectives:
Adenosine plays an important hole on basal nuclei functions; however dopamine and adenosine interactions on these nuclei are
not completely understood. The aim of this study was assess the effects of an A1 selective adenosine antagonist, 8phenylteophylline, on motor behavior impairment induced by the D2 dopamine antagonist receptor, haloperidol, in mice.
Methods and Results:
: Swiss male mice, 25-35g weight, were initially injected by intraperitoneal route with saline or haloperidol and after 30 minutes
injected by intraventricular route with saline or 8-phenylteophylline (5 and 10nM), performing a total of 6 experimental groups:
(n=8/group): i) saline + saline; ii) saline + 8-phenylteophylline (5nM); iii) saline + 8-phenylteophylline (10nM); iv) haloperidol
(2 mg/kg) + saline; v) haloperidol (2mg/kg) + 8-phenylteophylline (5nM); e vi) haloperidol (2mg/kg) + 8-phenylteophylline
(10nM). After drug administration (60 minutes) the animals were submitted to open field test measuring horizontal (quadrants
crossed - HE) and vertical (rearing - VE) exploration. Gait analysis was performed in a runway platform measuring
anteroposterior (APD) and laterolateral deviation (LLD) between fore hind footprint and by stride length (SL) (mm). Comparison
among experimental groups was performed bu one-way ANOVA followed by Duncan post hoc test. The behavior results to each
experimental group 60 minutes after drug administration were: i) saline + saline: HE=78.38±8.01; VE=29.13±3.48;
APD=7.62±1.85; LLD=4.58±0.75; SL=2.79±0.19; ii) saline + 8-phenylteophylline (5nM): HE=109.63±6.94; VE=24.00±3.26;
APD=4.48±0.78; LLD=4.00±0.42; SL=2.92±0.07; iii) saline + 8-phenylteophylline (10nM): HE=94.75±17.73; VE=11.88±3.16;
APD=7.06±1.28; LLD=4.76±0.66; SL=2.69±0.06; iv) haloperidol (2mg/kg) + saline: HE=18.50±4.12; VE=4.63±1.51;
APD=7.68±0.69; LLD=5.45±0.44; SL=2.48±0.09; v) haloperidol (2mg/kg) + 8-phenylteophylline (5nM): HE=25.38±5.14;
VE=4.38±1.24; APD=7.51±1.45; LLD=5.01±0.64; SL=2.69±0.13; and vi) haloperidol (2mg/kg) + 8-phenylteophylline (10nM):
HE=13.75±6.61; VE=1.13±0.88; APD=6.23±1.31; LLD=5.73±1.16; SL=2.82±0.38. Groups that received haloperidol (iv, v and
vi) crossed less quadrants than other groups in spite of 8-phenylteophylline application (Duncan, p0.05)
Conclusions:
Antagonism of dopamine receptors by haloperidol produced impairment on animal motor behavior, specifically on horizontal and
vertical exploration, that was not reverted by 8-phenylteophylline an A1 adenosine antagonist receptor in both doses used (5 and
10nM). Apparently, animal gait was not affected by dopamine antagonism.
Keywords: BEHAVIOR, HALOPERIDOL, 8-PHENYLTHEOPHYLLINE, IMPAIRMENT, MOTOR
Financial Support: UFMS and CNPq
Resumo:21-154
EFFECT OF CHRONIC INTAKE OF DIETS RICH IN HYDROGENATED VEGETABLE FAT ON THE
PERFORMANCE OF RATS IN THE ELEVATED PLUS MAZE.
Ferreira, A. K. ; Lins, P. G. ; Ladislau, H. F. L. ; Rocha-de-melo, A. P. ; Borba, J. M. C. ; Pereira-da-silva,
M. S.
Depto. de Nutrição- Lab. de Fisiologia da Nutrição - LAFINNT, UFPE
Objectives:
Aim: Observe the effect of chronic intake of a diet high in hydrogenated vegetable fat on the performance of rats in the elevated
plus maze (EPM).
Methods and Results:
Methods and Results: Female Wistar rats were fed during gestation and lactation with diets containing 7% soybean oil (control,
C) or 14% of hydrogenated vegetable fat (experimental; E). After weaning (21 days) the male pups of the control (C, n = 18) and
experimental (E, n = 11) group were fed the same diets of their respective mothers. The animals were weighed at 1 st, 7th, 14th, 21st
and 30th day of life. At 35 days of age, each animal of both groups was put in the center of the maze with the head orientated to
one of the closed arms for 5 minutes. The session was filmed by a video camera, installed on the roof of the experimental room,
for subsequent analysis of the following behavioral categories: number of entries into the open and closed arms; total time spent
in the open (OA) and closed arms (CA) and exploration of the segments of the open arms. Pups of the E group showed a lower
weight gain at the 1st (E= 6 – 1; C= 6,82 - 0,852, median–interquartile) and at the 7th (E= 14 – 2,75; C= 15,83 – 4,9925) lactation
day when compared to the group C group. There was no significant difference in the number of entries in the OA and in the CA
between the groups (OA: E= 7,90 ± 0,89 and C= 7,61 ± 0,80; CA: E= 10,36 ± 1,08 and C= 10,5 ± 0,80; Mean ± SEM). The same
was observed about the time spent (sec) in the arms of the maze (OA: E= 73 ± 12,94 and C= 110 ± 15; CA: E= 127 ± 17,48 and
C= 153 ± 12; Mean ± SEM ). On the other hand, the frequency of visits into the different segments of the OA was increase for the
segment 2 of the E group when compared to the C group ( E= 4,0-2,75 and C= 2,5-1,0; median-interquartile). No significant
differences were observed for segment 1 which correspondes to the nearest third of the central area and the segment 3 which
corresponds to the extremity of the arm.
Conclusions:
Conclusion: The chronic intake of a high fat diet based on the hydrogenated vegetable fat induced an increase of the weight gain
and seemed to promote a greater impulsivity / lower risk assessment in young rats.
Keywords: Chronic Intake, Elevated Plus Maze, Hydrogenated Vegetable Fat, Rats
Financial Support: PROSPESQ/UFPE
Resumo:21-155
SEROTONIN-INDUCED DIPSOGENIC RESPONSE IN PIGEONS (COLUMBA LIVIA): MEDIATION BY 5-HT 1/2
AND ALFA-1 RECEPTORS
dos Santos, T. S. 1; Melleu, F. F. 1; Souza, V. D. 1; Marino-neto, J. 1,2
1
DEPT. OF PHYSIOLOGICAL SCIENCES, CCB, UFSC
2
INSTITUTE OF BIOMEDICAL ENGINEERING, EEL-CTC, UFSC
Objectives:
Aim: In mammals, interactions between noradrenergic and serotonergic mechanisms have been shown to significantly modulate
several motivational states, including thirst and hunger, as well as the expression of sleep/waking signs. In birds, we have shown
that central injections of noradrenaline or serotonin (5-HT) can differentially affect drinking, feeding and sleep-like behaviors
(SLB). Intracerebroventricular (ICV) 5-HT injections in free-feeding/drinking (FF/D) pigeons (C. livia), besides hypophagy,
evoke intense drinking and SLB, which are negatively correlated to Fos-like activation in serotonergic brainstem neurons (Behav
Brain Res, 220:173, 2011). Central (ICV, intra-raphe) injections of 8-OH-DPAT (a 5-HT1A receptor agonist that reduces 5-HT
neuronal activity by autoreceptor activation) also elicit intense drinking and SLB, suggesting the existence of a tonic, 5-HT1Amediated, inhibitory influence of 5-HT circuits on thirst and sleep in this species.
Methods and Results:
Methods and Results: This study examined the effects of ICV injections of 5-HT (150 nmol) on drinking, feeding and SLB of
FF/D pigeons, pretreated ICV with metergoline (MET, 5-HT1/2 receptor antagonist; 15, 50,150 nmol), WAY-100635 (WAY, 5HT1A receptor antagonist; 0.1, 0.3, 1 nmol), MM77 (an antagonist equally potent at post-synaptic 5-HT1A receptors and at α1adrenoceptors; 23, 69 nmol) or prazosin (α1-adrenoceptors antagonist: 0.9, 2.7 nmol). MET injections completely blocked the
dipsogenic, hypophagic and hypnogenic effects of 5-HT treatments, and produced hyperphagic (but no drinking or sleeping)
responses when given alone, suggesting that all these effects are mediated by 5-HT1/2 receptors. WAY failed to affect the 5-HTinduced drinking, feeding or sleep responses, suggesting that its effect is not 5-HT1A receptor related. MM77 and prazosin dosedependently decreased 5-HT-induced drinking suggesting that dipsogenic responses are depends of that activation of α1
adrenoceptors. Sleep responses after 5-HT were potentiated by prazosin but were contradictorily affect by MM77, suggesting that
α1 adrenoceptor can participate of sleep modulation.
Conclusions:
Conclusion: These results showed the participation of 5-HT1/2, probably not 5-HT1A, receptors in the serotonin control of
drinking behavior in pigeons. And besides, as well as in mammals, indicate the important interation between serotonergic and
noradrenergic system in control of drinking behavior in bird, that can indicated relationships that are shared functional traits of
this circuits in amniotes.
Keywords: Drinking behavior, Serotonin, 5-HT1A receptor, Alfa-1 receptor, pigeon
Financial Support: Sources of research support: Capes, Cnpq, Fapesc
Resumo:21-156
AN INVERSE RELATIONSHIP BETWEEN ANNEXIN V AND TNF-ALPHA LEVELS IN CHRONIC STABLE
PATIENTS WITH SCHIZOPHRENIA
Pfaffenseller, B. 1,2,3; Francesconi, L. P. 2,3; Ceresér, K. M. 2,3; Mascarenhas, R. 2,3; Stertz, L. 1,2,3; Gubert, C.
M. 2,3; Lersch, C. 2,3; Gama, C. S. 2,3; Belmonte-de-abreu, P. 2,3
1
PPG Bioquímica/Universidade Federal do Rio Grande do Sul, UFRGS
2
Laboratório de Psiquiatria Molecular/HCPA, HCPA
3
INCT-Translacional em Medicina, INCT-TM
Objectives:
Apoptotic processes may alter the neuronal network and are associated with the pathogenesis of several neurodegenerative
diseases, such as schizophrenia. The high incidence of inflammatory processes is also known in schizophrenic patients. Annexins
belong to a family of proteins that bind both calcium and phospholipids and form calcium-dependent ion voltage channels within
the lipid bilayer, regulating fagocitosis, cellular signaling, apoptosis, leucocitic migration, proteins like phospholipase A2 and
several components of inflammation, such as cytokines. Among these, tumor necrosis factor (TNF-alpha) is a cytokine
stimulating systemic inflammation and acute phase reactions. The objective of this study was to evaluate annexin V and TNFalpha serum levels in patients diagnosed with schizophrenia and controls.
Methods and Results:
The study sample consisted of 38 patients diagnosed with schizophrenia from the Hospital de Clínicas de Porto Alegre matched
by age and sex. All patients and controls signed an informed consent form. Diagnosis was given by a trained psychiatrist and
according to the SCID-IV (Structured Clinical Interview for DSM-IV) and Opcrit 4.0. Disease severity was measured by BPRS
(Brief Psychiatric Rating Scale) and illness duration recorded in years. Serum annexin V and TNF-alpha levels were assessed by
enzyme-linked immunosorbent sandwich assay. There was a significant difference in annexin V and TNF-alpha levels between
patients and controls by Wilcoxon T test (p
Conclusions:
The high levels of annexin V in patients diagnosed with schizophrenia can be responsible for the diminished levels of TNF-alpha
due to its anti-inflammatory action. The identification of the role of inflammatory and anti-inflammatory mediators in
schizophrenia can lead to targeted interventions to reduce disease progression, apoptosis and handicap associated to the illness.
Keywords: ANNEXIN V, TNF-ALPHA, SCHIZOPHRENIA, APOPTOSIS, INFLAMMATION
Financial Support: CNPq, FIPE-HCPA
Resumo:21-157
M-TRIFLUOROMETHYL-DIPHENYL DISELENIDE PROTECTS AGAINST GA-INDUCED SEIZURES AND
REACTIVE OXYGEN SPECIES PRODUCTION IN PUP RATS.
Quines, C. B. ; Magni, D. V. ; Bruning, C. A. ; Nogueira, C. W.
Departamento de Química, UFSM
Objectives:
Glutaric acidemia type I (GA-I) is an inherited organic acid cerebral disorder caused by the impairment of mitochondrial enzyme
activity glutaryl-CoA dehydrogenase, which gives rise to an accumulation of glutaric acid (GA) in body fluids and brain tissue of
affected patients. Clinical manifestations of GA-I are predominantly neurological, including generalized convulsions. mTrifluoromethyl-diphenyl diselenide (CF3) is an organoselenium compound that has showed interesting pharmacological
properties, such as antioxidant and anticonvulsant activities. In this way, we evaluated whether administration of CF3 protects
against GA-induced seizures and reactive oxygen species (ROS) production.
Methods and Results:
Pup male Wistar rats were anesthetized and placed in stereotaxic apparatus for cannula insertion into the striatum (coordinates
relative to bregma: AP, 0 mm; ML, 3.0 mm; V, 3.0 mm from the dura). The experiments were performed 3 days after surgery
when animals did not show any sign of pain, infection or discomfort. Pup rats (21 days-old) were divided into 4 groups of 12
animals each. A control group administered with oil + saline, another group treated with CF3 + saline, a third group administered
with only oil + GA, and a fourth group administered with CF3 + GA. CF3 compound (50 mg/kg) or oil (vehicle) were infused by
intragastric gavage 30 min before the intrastriatal administration of GA (1 µL, 1.3 µmol/striatum) or saline. Immediately after the
intrastriatal injections we observed the latency and duration of seizures for 20 min. In the present investigation, we verify that
administration of CF3 increased significantly the latency for the first convulsive episode induced by intrastriatal injection of GA.
Besides, the treatment with CF3 decreased significantly the duration of seizures. In addition, the analysis of ROS demonstrated
significantly greater levels in the group administered only with GA when compared to the group administered with CF3 + GA.
Conclusions:
Previous administration of CF3 in pup rats from 21 days-old gave a protective effect against GA-induced seizures, demonstrated
by an increase of latency and a reduction of duration of convulsive episodes. Besides, the pre-administration of CF3 decreased
the GA-induced ROS levels. Although our results are promising, it is important to understand the mechanisms involved in the
protective effect of CF3 on GA-induced seizures for indicating a new anticonvulsant therapy for treatment of this organic
acidemia.
Keywords: Glutaric acid, m-Trifluoromethyl-diphenyl diselenide, seizures, reactive oxygen species, pup rats
Financial Support: CAPES/CNPq
Resumo:22-031
EFFECTS OF DIFFERENTS PROTOCOLS OF PHISICAL TRAINING ON FUNCTIONAL AND
MORPHOLOGYCAL ASPECTS OF RATS SUBMITED TO MEDIAN NERVE CRUSH INJURY
Neves, J. D. 1; Camelier, F. S. 1; Silva, S. A. D. 1; Arsego, N. R. 1; Marcuzzo, S. 1; Xavier, L. L. 2; Faccioniheuser, M. C. 1
1
Departamento de Ciências Morfológicas, UFRGS
2
Laboratório de Morfologia, PUCRS
Objectives:
The aim of the present study was to compare the effects of balance and coordination, repetition and the combination of these two
types of training protocols on functional recovery and morphometric outcome of the median nerve after crush injury.
Methods and Results:
46 male Wistar rats (3 months) were used. Procedures were approved by the Ethical Committee at the UFRGS. Animals were
randomly divided in five groups: sham sedentary (Sh-s, n = 8) - operated rats, without median nerve crush and unexercised;
lesioned sedentary (LSE, n = 9) - rats with median nerve crush and unexercised; repetition (LR, n = 9) - rats with median nerve
crush and repetition training; balance and coordination (LBC, n = 10) - rats with median nerve crush and balance and
coordination training; balance and coordination + repetition (LBCR, n = 10) - rats with median nerve crush and balance and
coordination + repetition training. For the surgical procedures, animals were anesthetized and nerve crush injury was performed
with 1 mm hemostatic forceps for 30s. Four days after the surgery, the animals from the LR, LBC and LBCR groups began
specific training for 6 weeks. During training, the animals underwent the behavioral assessment with the footfault test (FT). Fixed
samples of the distal portion of median nerve were prepared for electron microscopy and cut at 1 µm for assessing axonal density,
average myelinated fiber area, myelin sheath thickness, myelinated fiber diameter and axonal diameter. The results were analyzed
by one-way ANOVA for functional test and repeated measures ANOVA for morphometric analysis followed by post hoc
Duncan‘s with software Statystical. Footfault test revealed that the number of right forelimb errors was significantly decreased in
the Sh-s group 3 days postlesion, compared with all the lesioned groups: LSE (p
Conclusions:
The data showed evidence that balance and coordination training accelerate nerve regeneration after experimental traumatic
injury, spite footfault test not show differences between the treatments.
Keywords: crush injury, peripheral nerve regeneration, training protocol
Financial Support: CAPES and Cnpq
Resumo:22-032
A POSSIBLE ROLE FOR MMP-9 IN THE RAT RETINOTECTAL SYSTEM.
Oliveira-poleto, A. L. ; Cabral-miranda, F. ; Campello-costa, P. ; Serfaty, C. A. ; Oliveira-silva, P.
Department of Neurobiology, Institute of Biology, UFF
Objectives:
During postnatal development retinal projections must reach correctly their targets in the superior colliculus (SC) in order to
establish functional synapses. In this context, inappropriate axons are retracted and eliminated during the critical period of
synapses formation. These events guarantee the correct representation of the visual field in central visual nuclei. Matrix
metalloproteinases (MMPs) are a family of enzymes that degrade most of the extracellular matrix components, creating a
permissive environment for axonal growth and synaptic plasticity. MMPs activity is regulated by the tissue inhibitors of
metalloproteinases (TIMPs) and imbalance of MMPs/TIMPs during development impairs the formation of topography of neural
circuits. Previous data of our lab have shown that MMP-9 activity decreases during SC development and the inhibition of
gelatinases in newborn animals harms the refinement of neural circuitry in this structure. Our objective was to search for the
impact of blockade of MMP-9 activity, using SB-3CT (Calbiochem) in a concentrationof 648µM, which is recognized to be
specific to inhibit this molecule without interfere with others MMPs activity.
Methods and Results:
Newborn Lister Hooded rats were implanted with ELVAX containing SB-3CT and Horseradish Peroxidase was injected in
temporal retina of animals at PND6. Animals were submitted to a perfusion trough heart at PND7 with saline 0.9% followed by
aldehydes, afterwards the brains removed and left in sucrose solution for 24h. 40µ coronal sections were obtained in a cryostat
and stored in 0.1M phosphate buffer. Then sections were transferred to 0.1M acetate buffer and incubated with TMB and H2O2.
Analysis was made using a Zeiss microscope. Microscope analysis revealed that retinotectal projections in SC of animals treated
with SB-3CT appeared expanded and diffused in comparison with control rats, disrupting the cluster formation pattern observed
during development. This result led us to believe that the specific inhibition of MMP-9 activity in the first postnatal week might
delay or even abrogate the refinement of topography in rat‘s visual system.
Conclusions:
Our result suggests that MMP-9 activity have a key role in the refinement of retinotectal projections in SC, however further
analyses must be made to better elucidate the contributions of MMP-9 to neural plasticity.
Keywords: mmp-9, retinotectal, plasticity, development
Financial Support: Proppi/UFF & Faperj
Resumo:22-033
INTERFERON GAMMA INFLUENCES SYNAPSE ELIMINATION PROCESS WITHOUT AFFECTING THE
PERIPHERAL REGENERATION AFTER INJURY
Victório, S. C. S. ; Cartarozzi, L. P. ; Oliveira, A. L. R.
Depto. Anatomia/ UNICAMP, UNICAMP
Objectives:
Recent studies have indicated that the major histocompatibility complex (MHC I) expression by injured neurons and surrounding
glia plays an important role for neuronal repair, influencing the synaptic plasticity process. Additionally, interferon gamma
expression (IFNγ), a potent cytokine involved in MHC I expression, is upregulated after trauma. Taking into account that little is
known about the functional importance of IFNγ in the central nervous system (CNS) and peripheral nervous system (PNS) after
peripheral injury, the aim of the present work was to investigate the process of synaptic elimination in the spinal cord as well as
the motor recovery in IFNγ-/- mice, following sciatic nerve axotomy.
Methods and Results:
One week after transection and two weeks after crush lesion, the animals were sacrificed and had their lumbar spinal cord and
sciatic nerves processed for immunohistochemistry, Western blotting (WB) and electron microscopy. Newborn mice astrocyte
primary cultures were established in order to study the astrocytic respose in the absence of IFNγ expression. The lack of IFNγ
decreased the expression of MHC I (IFNγ-/-, 2.72±0.53; C57BL/6J, 5.21±0.48;ratio ipsi/contralateral; integrated density of pixels
x103; p0.05). The synaptophysin immunolabeling increased in the microenvironment surrounding large motoneurons of mutant
mice (IFNγ-/-, 4.41±0.6; C57BL/6J, 2.54±0.23; ratio ipsi/contralateral, integrated density of pixels; p0.05) and GFAP (IFNγ-/-,
2.27±0.24; C57BL/6J, 2.33±0.13; ratio ipsi/contralateral, integrated density of pixels; p>0.05) reactivity were equally increased
in both strains. In vitro, the astrocytes demonstrated similar GFAP levels, but the proliferation rate was higher in the wild type
mice. In the crushed nerves (distal stump), neurofilaments and p75NGFR immunolabeling were upregulated in mutant mice as
compared to the wild type, although it did not result in any improvement of locomotor recovery.
Conclusions:
The present results show that the lack of IFNγ affects the synaptic elimination process in the spinal cord. Such changes, however,
do not delay the peripheral nerve regeneration after nerve injury.
Keywords: interferon gamma, MHC class I, motoneuron, spinal cord
Financial Support: FAPESP
Resumo:22-034
STUDY OF SYNAPSES IN THE TEMPORAL CORTEX DURING AGING
Merlo, S. ; Nakayama, A. B. ; Brusco, J. ; Rossi, M. ; Carlotti Jr, C. G. ; Moreira, J. E.
FACULDADE DE MEDICINA DE RIBEIRÃO PRETO, FMRP USP
Objectives:
Analyze the temporal cortex of humans and rats: the number of excitatory, inhibitory, and electric synapses, the site of
postsynaptic contacts, the number of synaptic vesicles per terminal, and the expression of the proteins alpha-synuclein and
ubiquitin following the size and features of the lipofuscin granules.
Methods and Results:
Samples of temporal cortex of human subjects with different ages (20-28, 37-41 and 50-55 years, n=4/5) were collected from
patients with epilepsy who underwent temporal lobectomy. Samples from Wistar rats of 2, 6, 10 and 12 months were also
collected. Light, confocal, and electron microscopy, and western blots techniques were used as procedures. The data obtained on
lipofuscin granules were coincident with other studies that observed a higher area occupied by this structure in older mammals.
The granules seem to grow in volume, but not in number, with considerable increase of the electron lucid fraction (lipidic). There
was no difference in the alpha-synuclein and ubiquitin expressions between the experimental groups. The synaptic densities were
similar between the groups, as well as the postsynaptic contacts. Increase of the electron dense vesicles in inhibitory synapses,
appeared to be associated with the demand of catecholamines.
Conclusions:
Thal and Schlote (1994) also observed increase of the electron lucid portion and reduction of the electron dense portion with
aging. High levels of oxidative stress are related an increase of reactive oxygen species with aging (Yankner et al., 2008).
Already studies in mammals observed decrease in the number of synapses in older individuals. Huttenlocher (1979) examined the
synaptic density in layer 3 of the middle frontal gyrus, from infants to individuals 90 years of age. The author observed that the
synaptic density was constant in this region between the ages of 16 to 72 years, but declined markedly in individuals 74 to 90
years old. Therefore the results of the present work do not express totally the aging process, because the range of age used in
humans, and rats belong to a young age.
Keywords: AGING, TEMPORAL CORTEX, SYNAPSES, LIPOFUSCIN GRANULES, ELECTRON MICROSCOPY
Financial Support: FAEPA, CNPq, FAPESP
Resumo:22-035
DENDRITIC SPINE DENSITY IN HIPPOCAMPUS FOLLOWING NEONATAL HYPOXIA-ISCHEMIA IN RATS
SUBMITTED TO DAILY ENVIRONMENTAL ENRICHMENT
Deniz, B. F. 1; Rojas, J. J. 1; Miguel, P. M. 1; Diaz, R. 1; Hermel, E. E. S. 1; Achaval, M. E. 1; Netto, C. A. 1;
Pereira, L. O. 1
1
Departamento de Ciências Morfológicas, UFRGS
2
Departamento de Bioquímicas, UFRGS
Objectives:
Previous studies from our group showed the neuroprotective effects of environmental enrichment (EE) in rats submitted to the
neonatal hypoxia-ischemia (HI); however it didn‘t cause any change in lesion volume in the hippocampus. The aim of this study
was to investigate possible changes in dendritic spines density in the dorsal CA1 hippocampus region of rats submitted to HI and
exposed to EE.
Methods and Results:
Seven-day old Wistar male rats were submitted to HI procedure which consisted in permanent occlusion of the right common
carotid artery and posterior exposure to 90 minutes to hypoxic atmosphere. The pups were divided into four experimental groups,
with 5 animals per group: control maintained in standard environmental (CTSE); control exposed to environmental enrichment
(CTEE); submitted to hypoxia-ischemia and maintained in standard environmental (HISE); submitted to hypoxia-ischemia and
exposed to environmental enrichment (HIEE). At the 22 days old the EE started and continue during 9 weeks, being performed 6
days per week, 1 hour per day, during 9 weeks. Two days after the end of EE the animals were transcardially perfused with
fixative solution and their brains were submitted to Golgi method procedure. The morphological analysis was performed using a
camera lucida coupled to an optic microscope that allows simultaneous visualization and design of the sample. For each CA1
pyramidal cell the morphologic analysis included the dendritic spines number in 20 ìm of the selected most lateral tertiary
dendrites on the apical tree. From each rat, ten different dendrites were analyzed, five per hemisphere, one per neuron. For
statistical comparisons, the means of each rat were used and, after, a two-way analysis of variance (ANOVA) was conducted.
There was effect referent to the lesion (F (1,16) = 39.340, p < 0.01; F (1,16) = 55.104, p < 0.01) and environment (F (1,16) =
13.256, p < 0.01; F (1,16) = 20.680, p < 0.01) both in the left and right hemisphere, respectively. Hypoxic-ischemic maintained in
SE animals had lower dendritic spines densities when compared to both control groups (p
Conclusions:
These data of dendritic spines indicated a completely recovery in the left hemisphere and a partially recovery in the right
hemisphere after environmental stimulation in animals submitted to hypoxia-ischemia. Maybe these results could explain a
possible morphological effect to explain the functional recovery, previously published, consequent to environmental enrichment
in hypoxic-ischemic animals
Keywords: environmental enriched, hypoxia-ischemia, dendritic spine density
Financial Support: CAPES, CNPQ and FAPERGS.
Resumo:22-036
EFFECTS OF AXOTOMY ON SATELLITE GLIAL CELL ULTRASTRUCTURE IN FROG DORSAL ROOT
GANGLIA.
Rigon, F. ; Faccioni-heuser, M. C. ; Partata, W. A.
instituo de ciências biológicas da saúde, UFRGS
Objectives:
Peripheral nerve lesion lead to morphological changes in the cell bodies of the neurons of origin. Apoptosis and necrosis
contribute to neuronal death in this experimental condition. Signs of satellite glial cell (SGC) death also occur after peripheral
axotomy and the Tunel-positive cells are already present at day four, peaking at two months. Since the changes of SGC following
peripheral axotomy are not clearly understood, the present study was carried out to determine the effect of sciatic nerve
transection (SNT) on the ultrastructure of SGCs located in the dorsal root ganglia (DRG). Since the amphibian brain shares nearly
all the typical properties of the amniote brains, frogs were used as an experimental model in this study to offer a phylogenetic
basis of the effects of peripheral axonal injury.
Methods and Results:
Specimens of adult male frogs Rana catesbeiana weighing 100-200g, obtained from RANASUL (Imbé, RS), were divided in two
experimental groups (six animal/group): control (animals which did not undergo surgical manipulation) and SNT (the sciatic
nerve was exposed and transected approximately 5 mm distal to the sciatic notch). These animals were sacrificed 3 days after the
surgical procedure, since signs of mammal SGC death in this period were previously reported. After the sacrifice the DRGs of the
sciatic nerve ipsilateral to the lesion were dissected out and fixed in a 4% paraformaldehyde and 2.5% glutaraldehyde solution in
0.1M phosphate buffer (PB), pH 7.4, at room temperature. The ganglia were postfixed in osmium tetroxide in PB for one hour.
Afterwards, the sections were dehydrated in series of alcohol and acetone and embedded in araldite (Durcupan ACM, Fluka).
Semithin (900 nm) and ultrathin (70 nm) sections were obtained using a diamond knife (Drukker, Netherlands). The ultrathin
sections were mounted and stained with 2% uranyl acetate followed by lead citrate and examined by transmission electron
microscopy (JEM 1200 EX II). While in ganglionic neurons the earliest response to axotomy is the presence of irregularities in
the nucleus and perinuclear cytoplasm, no discontinuities were found in the cytoplasmic membrane and nucleolemma of the SGC
3 days after SNT. No changes in electron density and chromatin condensation were observed. There were more irregularities in
the SGC prolongation. These processes appeared more plicated than in the normal cell. Many free ribosomes, rough endoplasmic
reticulum and mitochondria were observed. No swelling was found in these organelles. They were scattered within the cytoplasm
and the perinuclear region showed increase in the amount of filaments. While a chromatolytic phenomenon was found in some
neurons, the SGCs did not show this characteristic in this time frame.
Conclusions:
These results showed that sciatic nerve transection did not induce SGC death at 3 days. Although frogs have a lower metabolic
rate in the nervous system than mammals, the fine structure morphology of the SGC was similar to that described for mammals.
Since it was suggested that SGCs support the mammalian neurons metabolically, the results obtained up to now clearly indicate
that SGCs are capable of actively synthesizing the proteins necessary to support the dorsal root neurons in the regenerative
process in the frog nervous system.
Keywords: axotomy, dorsal root ganglia, satellite glial cell
Financial Support: CNPq, FAPERGS
Resumo:22-037
ABSENCE OF TOLL LIKE RECEPTOR 2 EXPRESSION INFLUENCES THE INITIAL STEPS OF PERIPHERAL
NERVE REGENERATION AFTER AXOTOMY
Leite, G. A. ; Freria, C. M. ; Oliveira, A. L. R.
Department of Anatomy, Cell Biology and Physiology and Bioph, UNICAMP
Objectives:
Peripheral nerve regeneration after axotomy is a complex process that involves different signaling pathways. Following crush or
transection of a nerve, the Wallerian Degeneration (WD) process develops distally to the lesion site, mostly at the distal stump.
Such process is characterized by the fragmentation of myelin sheath, macrophage recruitment and Schwann cell proliferation.
Concurrently to the distal changes, retrograde phenomena takes place in the spinal cord, at the motoneuron cell body and
surrounding microenvironment, activating microglial cells and astrocytes in the central nervous system (CNS). Recent studies
have shown that glial cells, including Schwann cells, astrocytes and microglia express different toll like receptors (TLRs). The
TLRs are transmembrane recognition receptors, which identify pathogens and initiate a local immune response. Such receptors
might influence several events after injury and repair process within the nervous system. In this context, the present work aimed
at investigating the influence of knocking out the TLR2 on the peripheral nerve regeneration process after sciatic nerve axotomy.
Methods and Results:
C57BL/6J (n=5) and C57BL/6J TLR2-/- (n=5) mice were subjected to unilateral sciatic nerve crush. Two weeks after surgery the
mice were sacrificed and perfused transcardially with fixative (10% formaldehyde in 0.1M PBS). Contralateral and lesioned
sciatic nerves were obtained, frozen and cryostate sectioned. Tissue samples from both groups were subjected to
immunohistochemistry with anti-neurofilaments, anti-laminin I, anti-collagen IV and anti-p75 NGFR antisera. Gait recovery after
sciatic nerve crush was performed up to five weeks post lesion in another set of control and knockout mice (n=5 for each group),
by using the CatWalk system (Noldus Information Technology). The results showed greater expression of p75NGFR,
neurofilaments and collagen IV in the control group as compared to knock out mice. However, regardless such protein expression
changes in the acute phase post lesion, no significant differences were observed in the functional analysis (four weeks postlesion: C57BL/6J 85,19% ± 12,30%, C57BL/6J TLR2-/- 64,52% ± 7,66%; five weeks post-lesion: C57BL/6J 72,25% ± 10,72%,
C57BL/6J TLR2-/- 71,00% ± 9,21%; average percentage of motor recovery ± standard error of mean).
Conclusions:
Altogether, the present results indicate that the absence of TLR2 negatively influences the initial response to injury, possibly by
postponing the nerve microenvironment reorganization. Nevertheless, as seen by the functional recovery, such initial delay does
not affect the axonal growth itself as well as the muscle reinnervation.
Keywords: Toll like receptor 2, Peripheral Nerve Regeneration, Wallerian Degeneration, Axotomy, Functional recovery
Financial Support: FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo.
Resumo:22-038
REELIN EXPRESSION DURING DEVELOPMENT OF RETINOCOLLICULAR PATHWAYS: MODULATION
THROUGH TRYPTOPHAN NUTRITIONAL RESTRICTION
Antonioli-santos, R. 1; Gonzalez, E. M. C. 1; Hedin-pereira, C. 2; Oliveira-silva, P. 1; Campello-costa, P. 1;
Serfaty, C. A. 1
1
Depto. de Neurobiologia/ Instituto de Biologia, UFF
2
Depto. de Anatomia/ Instituto de Ciências Biomédicas, UFRJ
Objectives:
During postnatal development, neural circuits are estabilished according to their interaction with the environment and also the
availability of nutrients. We studied the role of reelin – an extracellular matrix glycoprotein able to affect several steps in brain
development, from neuronal migration and positioning to dendritic maturation and synaptic plasticity – on the development of
retinocollicular connections in both normal Lister Hooded rats and rats with decreased levels of brain serotonin through a
nutritional restriction of tryptophan (Try-), an essential aminoacid acquired exclusively by food intake and the metabolic
precursor of serotonin.
Methods and Results:
As shown in our previous results, the distribution of uncrossed retinocollicular terminal fields was examined following
intraocular injections of horseradish peroxidase (HRP 30%) in aldehyde-fixed, free-floating sections (40μm) through the superior
colliculus at the tenth postnatal day (PND10). The neonatal tryptophan restriction alters the retinocollicular topographical
refinement, suggesting a delay in axonal elimination, associated to a decrease of neural plasticity (Exp. Neurol. 211:441, 2008).
In the present study, we performed western blot analysis to evaluate the content of reelin in the visual layers of the superior
colliculus. Reelin was differentially expressed in the collicular visual layers during postnatal development: low levels were found
at PND5 followed by a transient increase over the second postnatal week, reaching the highest expression at PND14. After the
third postnatal week, reelin expression showed a slight decrease and remained stable even after the end of the critical period
(PND28), when rats reach an adult like profile of retinotectal connections. Moreover, the immunofluorescence analysis showed
reelin-positive cells at PND10 with immunoreactivity predominantly located at perineuronal sites. Animals fed with a tryptophan
restricted diet (Try-) from the day of birth until PND10, showed an increase in reelin expression, as compared to control groups
fed with a regular rodent diet.
Conclusions:
The present results indicate the involvement of reelin in the specificity of retinocollicular connections during initial stages of
postnatal development. Also, reelin must be involved on the disruption of retinocollicular topography induced by the low levels
of tryptophan, possibly enabling the stabilization of inappropriate transitory synaptic contacts.
Keywords: reelin, retinocollicular pathway, tryptophan restriction, serotonin
Financial Support: CAPES, FAPERJ, CNPQ, PRONEX, PROPPI/UFF
Resumo:22-039
RELATIONS BETWEEN AGING AND NERVE CELLS IN RATS BRAIN - STEREOLOGICAL STUDY.
1
Correia, M. H. 1; Cruz, R. S. 1; Janeiro, V. 2; Melo, S. R. 1
Depto. de Ciências Morfológicas - Univ. Est. de Maringá-Pr, DCM - UEM
2
Depto. de Estatística - Univ. Est. de Maringá-Pr, DES - UEM
Objectives:
The neurons and glial cells represent the cells of nervous tissue that together provide the functions of the nervous system, the
relations with neurodegenerative diseases, aging and quantity of these cells has been estimated, however there is no consensus in
the literature. For these reasons, the main purpose of this study was to estimate the density (Vv) of neurons and glial cells in
normal rats brain at different ages.
Methods and Results:
This experiment used 20 male Wistar rats (Rattus norvegicus) at 10, 14, 18 and 24 months of age, five rats in each group. The
animal was perfused with Zamboni solution, and then the brain was removed, fixed in the same solution, processed in alcohol and
xylene series and embedded in paraffin, which was cut with six micrometers. For each animal three sections were processed with
the technique Picro Sirius Red, and fifteen fields were analyzed by stereological technique. We used video microscope coupled to
a video camera and monitor. Quantification of cells was achieved with the determination of neurons Vv (Vv [neu]) macroglia Vv
(Vv [mac]) and microglia Vv (Vv [mic]). Neurons were distinguished from glial cells based on shape and size of the nucleus and
were considered for the quantification the points from test system (M42) which touched in the nucleus. It was observed that Vv
[neu] in the 10m group was 4.76 in the 14m group was 3.81, in 18m group was 2.38 and was 2.86 in the 24m group. In the Vv
[mac] observed that the 10m group was 6.83, in the 14m group was 6.51, in the 18m group was 7.94 and was 6.98 in the 24m
group. And finally the Vv [mic] in the 10m group was 2.06, in the 14m group was 1.90, in 18m group was 2.38 and was 2.70 in
24m group. The analysis allowed us to infer that the Vv [neu], Vv [mac] and Vv [mic] in the analyzed groups was not statistically
significant, p> 0.05.
Conclusions:
This result shows that aging is not changing the number of neurons and glia. These results lead us to question what factors are
related to this stability in rats and whether this could be applied in human brain.
Keywords: Aging, Macroglia, Microglia, Neurons, Stereology
Financial Support: Fundação Araucária
Resumo:22-040
PNEUMOCOCCAL MENINGITIS: OLFACTORY ENSHEATHING CELL AS PUTATIVE SAFE HOST CELLS FOR
STREPTOCOCCUS PNEUMONIAE
Macedo-ramos, H. 1; Carvalho, L. A. 1; Teixeira, L. M. 2; Cavacante, L. A. 1; Baetas-da-cruz, W. 1,3
1
Laboratório de Neurobiologia do Desenvolvimento/IBCCFº/UFRJ, IBCCFº/UFRJ
2
Instituto de Microbiologia/UFRJ, IMPG/UFRJ
3
Campus Macaé/UFRJ, Campus Macaé/UFRJ
Objectives:
Pneumococcal Meningitis (PM) is an infectious disease caused by the gram-positive diplococcus Streptococcus pneumoniae (Sp).
PM can affect people of any age but is more commonly diagnosed in children in the first two years of life. It is usually believed
that PM induced by the intra-nasal route involves bacteremia before bacteria entry into the brain. The best demonstration of an
alternative route was that in which a strain of Sp that fails to survive in the blood stream was instilled into the nasal cavities and
recovered in the olfactory nerve and bulb as well as in the whole brain (Proc. Natl. Acad. Sci. U.S.A. 100:14363, 2003).
Olfactory ensheathing cells (OECs) comprise a special glial type derived from progenitors in the olfactory mucosa that ensheath
olfactory receptor axons. Axonal bundles extend toward the cribiform plate and enter the brain via the olfactory phila, thus,
providing a potential route for access of pathogens to the brain. In a recent paper, we have shown that OECs express MR and are
able to internalize Sp bacteria of the ATCC 49619 strain, by means of a mannan-blocked lectin, possibly MR itself (Neurosci.
Res. 69:308 2011). Based on the above, we decided to investigate possible variations in the expression levels of inducible nitric
oxide synthase (iNOS) in OECs cultures infected with Sp.
Methods and Results:
OEC cultures were infected by a suspension of living Sp (ATCC49619, American Type Culture Collection 49619) in a ratio of
100:1 bacteria:cells during 3 h, since formation of colonies after exposure, washing and recovery of bacteria from lysed host cells
was not significantly different after 1, 3 or 5h. To examine the levels of iNOS protein expression, we used a monoclonal antibody
against the iNOS (anti-iNOS) and bacteria were visualized with a polyclonal antibody (anti-pneumococcus) or Sytox green, an
optimal stain for bacterial DNA. Our results showed a significant decrease in the levels of iNOS expressed in OECs infected by
Sp when compared with (uninfected) controls.
Conclusions:
These results support our hypothesis of a prospectively important role of OECs as a host cell during the bacterial invasion of the
brain via the olfactory route.
Keywords: innate immunity, pattern recognition receptors (PRRs), inducible Nitric Oxide Synthase (iNOS), pathogen-associated
molecular patterns , pneumococcal meningitis
Financial Support: FAPERJ/CNPq
Resumo:22-041
REGION-SPECIFIC NEURONAL BINDING OF Aβ OLIGOMERS INJECTED CHRONICALLY INTO THE
CEREBRAL VENTRICLE OF ADULT MONKEYS.
Forny-germano L. 1; Lyra N. M. S. 1; Graça, D. F. 1; Brito-moreira J. 1; Bomfim, T. R. J. D. S. 1; Munoz, D.
P. 3; Ferreira S. T. 1; Houzel J. C. 1; de Felice. F. G. 1
1
LDN, Instituto de Bioquimica Médica, UFRJ, UFRJ
2
LFN, Instituto de Ciências Biomédicas, UFRJ, UFRJ
3
Queen's Centre for Neuroscience Studies, Queen's University, Queen's University
Objectives:
The histopatologial features found in post mortem Alzheimer‘s disease (AD) brains are plaques - extracellular deposits of Aβ
peptide- and neurofibrilary tangles - intraneuronal accumulations of hyperphosphorilated tau protein. However, neuropathological
studies failed to correlate plaque levels with clinical symptoms. The current hypothesis for AD is that soluble oligomers of the Aβ
peptide (known as Amyloid β-Derived Diffusible Ligands or ADDLs) accumulate in the brain and act as potent neurotoxins,
leading to the functional breakdown of specific synaptic circuits. Recent in vitro studies showed that, after binding to synaptic
sites, ADDLs can induce tau hyperphosphorylation and oxidative stress (Neurobiol. Aging 29:1334, 2008). Transgenic mice
expressing high levels of tau and APP (APPsw-tauvlw) shown that accumulation of oligomeric Aβ, but not of total amyloid
plaque, is an early event that faithfully predicts the selective pattern of regional neuronal vulnerability and closely correlates with
the extent of neuronal cell loss (J Neuropathol Exp Neurol 70; 360, 2011). Previously work from our group used chronic
injections of ADDLs into the lateral ventricule of rats to investigate the regionalization of ADDLs accumulation in rat brain Here,
we extend this model to a non-human primate, investigating whether biochemically characterized Aβ oligomers injected
chronically into the lateral ventricle of adult monkey may alter the levels of hyperphosphorilated tau protein, and bind selectivity
to neurons from specific brain regions.
Methods and Results:
An intraventricular canula was implanted into the lateral ventricle of three mid-aged (~9 years of age) cynomolgus monkeys
(Macaca fascicularis, weight 4.7-7.0Kg). Every 3 days for 21 days, monkeys received 100µg of freshly prepared ADDLs. One
animal was used as control. For immunohistochemistry, the fixed brains (3 ADDLs, 1 sham) were frozen-cut into 40µm sections
and processed for the detection of ADDLs, phospho-tau Ser396 or DAPI (nuclei). The ratio of ADDLs+/DAPI+ cells was
counted and compared across selected brain regions. Cellular levels of ptau were compared using NIH Image Java to measure
pixels per area. Our results showed that ADDLs could diffuse from the ventricle to the cerebral parenchyma and bind to cells
with typical neuronal morphology thoughout the brain. However, ADDLs binding was more frequent in regions involved in
higher cognitive functions. The mean fraction of ADDLs+ cells differed across regions: hippocampus: 3,4%; somatosensory
cortex: 2,5%; frontal cortex: 2,4%, thalamus: 2,3%, amigdalar complex: 1,7%; and striatum: 1,4%. No ADDLs labeling was
observed in the cerebellum. Preliminary results revealed a regionalized increase in ptau levels in ADDLs animals. Amygdala: 7
fold, frontal cortex: 2.5 fold and hippocampus: 1.5 fold. More regions are being quantified and additional analyses of the relation
between ptau levels and A oligomers will be developed.
Conclusions:
Our results suggest that chronic intracerebroventricular injection of Aβ oligomers in adult monkeys is an useful model to study
the fate of such toxins in vivo. ADDLs might selectively target neurons in cognitive-related circuits and elevate ptau levels. Such
a regional specificity of the ADDLs attack may help to explain the specific cognitive impairments characteristic of the early
stages of AD.
Keywords: Ab oligomers, amyloid plaque, Alzheimer Disease, intracerebroventricular injection, tau hyperphosphorilation
Financial Support: CNPq, FAPERJ
Resumo:22-042
DENDRITIC ATROPHY IN HIPPOCAMPAL CA3 NEURONS IN PARALLEL TO CORTICOID AND
ANGIOTENSIN RECEPTORS REDUCTION IN ADULT RATS SUBMITTED TO IN UTERO PROTEIN
RESTRICTION
Lopes, A. D. S. 1; Torres, D. B. 1; A. J. Rodrigues3; J. J. Cerqueira3; J. M. Pego3; A. F. Godinho1; J. A. R.
Gontijo2; N. Sousa3; P. A. Boer1
1
Department of Morphology Biosciences Institute, UNESP
2
Department of Internal Medicine, UNICAMP
3
Life and Health Sciences Research Institute, ICVS
Objectives:
Aims: Corticoids have the ability to modulate hippocampal formation during the fetal period through their action in the
mineralocorticoid (MR) and glucocorticoid (GR) receptors. In this work, our objective was to investigate the effects of
intrauterine protein restriction in the adult hippocampal formation and expression/localization of GR and MR. Additionally, since
angiotensin receptor AT1 has been demonstrated to enhance learning and memory, we also studied its hipocampal expression
Methods and Results:
Methods: Pregnant rats were divided into two groups: a group was fed with normal diet (NP, 17% of protein n=9), and the other
received low protein diet (LP, 6% of protein n=9). Male body weight was measured in the day of birth. At 16 weeks of age, 4
animals from each experimental group were perfused with saline and brains processed for 3D neuronal reconstruction in GolgiCox stained slices. For each selected neuron, all branches of the dendritic tree were reconstructed at 600X magnification using a
motorized microscope attached to Neurolucida software. Fifteen neurons of regions CA1, CA3 and dentate gyrus (DG) were
studied for each animal. Total length of trees and number of dendritic branches were compared across groups using 1-way
analysis of variance. Other brains were processed for AT1, MR and GR immunohistochemistry (IH) (n=4) and for western
blotting (WB) (n=4).Results: LP offspring presented a significant reduction in birth weight (LP 5.34 ± 0.06 vs NP 6.23 ± 0.05,
n=95, p=0.0001). In adults, 3D analysis showed significant reductions in total length of CA3 basal, but not apical, dendrites
(length: LP 920,51 ± 307,54µm vs NP 1264,72 ± 445,95µm, p=0,01). On the contrary, CA1 and DG dendritic arborizations were
unaffected. By WB we verified that hipocampal expression of AT1 was 90% reduced in the LP group. IH revealed that GR
expression was decreased in CA1 and CA3 hippocampal regions; no differences were found in MR expression.
Conclusions:
Conclusions: Gestational protein restriction promoted significant length reduction of basal dendrites in hippocampal CA3 neurons
in parallel with reduced GR expression in CA1 and CA3 areas and a massive reduction of AT1. Since the hippocampal formation
is extremely vulnerable to both undernutrition and excessive corticosteroid concentration we suppose that elevated fetal exposure
to maternal corticoids may be related with the genesis of these alterations.
Keywords: FETAL PROGRAMMING, MEMORY, HIPOCAMPPUS, DENDRITIC STEREOLOGY, BEHAVIOUR
Financial Support: FAPESP and CAPES
Resumo:22-043
NUTRITIONAL MODULATION OF OMEGA-3 FATTY ACIDS ALTERS THE TOPOGRAPHICAL FINE TUNING
OF RETINOTECTAL PATHWAYS
Velasco, P. C. 1; Menezes, L. C. 1; Faria-melibeu, A. D. C. 1; Borba, J. M. C. 2; Costa, B. L. S. A. 2; Guedes,
R. C. A. 2; Campello-costa, P. 1; Serfaty, C. A. 1
1
Neurobiologia/Universidade Federal Fluminense, UFF
2
Nutrição/Universidade Federal de Pernambuco, UFPE
3
Fisiologia/Universidade Federal de Pernambuco, UFPE
Objectives:
The development and maturation of sensory systems depends on the correct pattern of connections which occurs during a critical
period when axonal elimination and synaptic plasticity are involved in the formation of topographical maps. Among the
mechanisms involved in synaptic stabilization, essential fatty acids (EFAs), which must be obtained through diet, appear as
precursors of signaling molecules involved in modulation of gene expression and also regulation of neurotransmitter production
and release. Omega-3 fatty acids, such as DHA are considered EFAs and are highly accumulated during fetal period and neonatal
development in the brain and retina. In the present work, we studied the effect of omega-3 fatty acids in stabilization of
connections in the visual system. We tested the effect of DHA nutritional restriction or supplementation in the structure of retinal
terminal fields in the rodent superior colliculus (SC). Moreover, we tested DHA deprivation on the expression of the subunits of
AMPA and NMDA glutamate receptors and also on the expression of the phosphorylated form of GAP-43 protein (pGAP43).
Methods and Results:
Female Lister Hooded rats were fed (30g/day/animal) for five weeks before mating with either a control (soy oil) or a restricted
(coconut oil) diet. Litters were then fed until postnatal day 13 (PND13), PND28 or PND42 when they received an intraocular
injection of HRP. Another group received a single retinal lesion at the temporal periphery at PND21. In the supplemented group,
animals received fish oil daily (3g/day/animal). Omega-3 restriction induced an increase in the optical density in the superficial
layers of the SC, as a result of axonal sprouting outside the main terminal zones. In the intermediate and ventral aspects of the
stratum griseum superficiale (SGS) we observed an increased optical density of retinotectal axons during critical period
(PND13), and also at PND28 and PND42. After a contralateral retinal lesion, omega-3 restricted group showed increased
sprouting of intact axons at the lesion site as compared with the control group. Alternatively, the supplemented group showed a
focalization in fiber density in the ventral region. Furthermore, omega-3 restriction decreased pGAP-43 expression and NR1 and
NR2A subunits of NMDA receptor as well at PND28. It also increased GluR1 and GluR2 subunits expression of AMPA receptor,
suggesting a disturbance in in synaptic stability.
Conclusions:
The data indicate that omega-3 fatty acid during development impairs the topographical map organization and induces abnormal
plasticity of retinotectal axons after the closure of the critical period. This effect was correlated to increased turnover/expression
in AMPA receptors and a decreased in NMDA subunit NR1 and NR2A. These results are consistent with a role of EFA as
stabilizing synaptic agents and are essential for topographical map organization.
Keywords: Retinotectal, omega-3 fatty acids, neural plasticity, nutritional, development
Financial Support: CAPES, CNPq, FAPERJ, PRONEX, PROPPi-UFF
Resumo:22-044
ULTRA-STRUCTURAL FEATURES IN THE POSTERODORSAL MEDIAL AMYGDALA OF MALE AND FEMALE
RATS.
Ikeda, E. T. 1; Brusco, J. 2; Rasia-filho, A. A. 3; Moreira, J. E. 1,2
1
Depart. Biologia Celular e Molecular e Bioagentes Patogênico, FMRP-USP
2
Depart. Neurociências e Ciências do Comportamento, FMRP-USP
3
Depart. Fisiologia , UFSCPA
Objectives:
The rat medial amygdala 〈MeA〉 is involved with the processing of olfactory information and the modulation of emotions and
social behaviors. The MeA posterodorsal subnucleus 〈MePD〉 is implicated in sexual behavior and has receptors for estrogens and
androgens. The number of dendritic spines is different between male and female rats along the estrus cycle on the MePD. The
aim of this work is to reveal the features of the left MePD synaptic structures on male and female rats along the phases of
diestrus, estrus, and proestrus.
Methods and Results:
Two Wistar rats of each group were anesthetized and their brain fixed by transcardiac perfusion of 2% paraformaldehyde, 2%
glutaraldehyde and 0.05% CaCl in cacodylate buffer. Brain slices were post-fixed in 1% OsO 4 , and 0.4 % uranyl acetate. The
samples were infiltrated in Embed 812 resin. Ultra-thin sections 〈60 nm〉 were mounted on copper single slot grids coated with
Pioloform, contrasted with uranyl acetate and lead citrate, observed and pictured under a Transmission Electron Microscope Zeiss
EM18. All the excitatory and inhibitory synapses found over dendrites, spines, soma or axons in the left MePD were
morphologically analyzed. The vesicles were classified as small round electron-lucid 〈SRV〉, small pleomorfic electron-lucid
〈SPV〉, large electron-dense 〈LEDV〉, and small electron-dense vesicles 〈SEDV〉. Clatrin-coated and priming vesicles were
counted as well. It was used the one-way ANOVA and post-hoc test of Tukey for the statistical analyze. In the four groups
studied there is a higher number of excitatory than inhibitory synapses, with more synapses on dendrites than on spines, somas
and axons. The number of clatrin-coated vesicles in the left MePD is higher on male 〈mean ± SD; 15.3 ± 11.6〉 than in female rats
in diestrus 〈3.7 ± 5.0〉, proestrus 〈8.6 ± 7.8〉, and estrus 〈8.9 ± 11.8; p < 0.01〉. Diestrus female have less clatrin-coated vesicles
than females in proestrus and estrus 〈p < 0.01〉. The number of priming vesicles per synapse area is higher on males 〈6.0 ± 5.9〉,
than in females in diestrus 〈3.1 ± 4.8〉, proestrus 〈3.3 ± 3.9〉 and estrus 〈4.0 ± 5.2〉. The total number of vesicles per terminal,
mainly SRV and LEDV, is higher on males 〈369.2 ± 137.8〉 than in female rats in diestrus 〈258 ± 140.5〉, proestrus 〈254 ±125.9〉
and estrus 〈277 ± 137.8; p < 0.01〉. Around 8% of synapses on dendritic spines, in the four groups studied, were
symmetrical⁄inhibitory; they are over thin and mushroom-like spines. Multisynaptic contacts are found in 6% of the total synapses
on spines. One excitatory besides an inhibitory terminal is found in 3% of the dendritic spines.
Conclusions:
This data suggest higher activity in the left male MePD than in female, as males have more clatrin-coated vesicles, vesicles in
priming and a higher number of vesicles in general. There are descriptions of inhibitory-like terminals and multisynaptic dendritic
spines in some brain regions, but this study is the first report of these terminals in the MePD. These results provide basic
morphological data for the MePD synapses under the influence of sex hormones as neurotransmitters.
Keywords: Medial amygdala, Electron microscopy, Synapses, Estrous cycle, Sexual dimorphism
Financial Support: FAEPA, CNPQ, FAPESP
Resumo:22-045
MODULATION OF GABA RELEASE BY GLUCOSE LEVELS IN THE CHICKEN RETINA
Maggesissi, R. S. 1,2; Mya-coreixas, V. S. 2; Carpi, R. S. 2; Gardino, P. F. 1; Calaza, K. C. 2
1
Universidade Federal do Rio de Janeiro, UFRJ
2
Universidade Federal Fluminense, UFF
Objectives:
Recurrent hypoglycemic episodes are considered a worrying condition for insulin-treated diabetic patients‘ health. Once diabetes
may cause vision loss, and glycemic oscillations may interfere with retinal function, this study aimed to analyze the effects of
glycemic fluctuations on morphological and neurochemical aspects of the retina.
Methods and Results:
Ex vivo chick retinas were kept under continuous perfusion with 95%O2/5%CO2 in physiological medium containing different
glucose concentrations (in mM): 0, 5.6 and 35 for 30 minutes. The consequences of hypoglycemia (0mM glucose) after 60
minutes were also analyzed. The samples were processed for Nissl staining, immunohistochemistry for ã-aminobutyric acid
(GABA) and lactate dehydrogenase (LDH) activity. Hypoglycemia promoted a time-dependent progressive loss of GABA
content from amacrine cells in the inner nuclear layer (INL) (mean±SEM, 61%±10, N=3) and ganglion cell layer (GCL) (73%±6,
N=3) and its processes. This effect was due to GABA release in the first 30 minutes of hypoglycemia, since it could be blocked
by type-1 GABA transporter inhibitor (NO-711). However, a longer exposure (60 minutes) of retinas to hypoglycemic conditions
induced swelling of the inner plexiform layer (two fold), increase in LDH release (indicating cell death) (191%±29, N=6) and
irreversible loss of GABA content from amacrine cells (19,6%±1,5, N=3). In contrast, hyperglycemia (35mM) during 30 minutes
augmented the number of GABA-positive horizontal cells (248%±21, N=3) and amacrine cells (124%±9, N=6).
Conclusions:
Retinal exposure to 0mM or 35mM glucose during 30 minutes induces opposite effects on GABA release both in horizontal and
amacrine cells. Furthermore, a longer period (60 minutes) of hypoglycemia induces retinal cell death.
Keywords: GABA, Glucose, Retina
Financial Support: MCT/CNPq, INCT/INNT, CAPES, FAPERJ, PRONEX/MCT, PROPPi/UFF.
Resumo:22-046
NEUROBIOMECHANICAL OF THE RADIAL NERVE IN DIFFERENT POSITIONS OF THE UPPER LIMB.
Portes, B. B.
Centro Universitário Nossa Senhora do Patrocínio, CEUNSP
Objectives:
Peripheral nerves of upper limb present mobility in relation to their connective tissues along its anatomic path by the neural duct
and its interfaces that are the connection for the nerves to the soft tissue and bone. Pathophysiological changes like the formation
of fibrosis connective tissue may be responsible for changes in blood flow also lead to decrease the capacity of the sliding of the
nerve into these interfaces nerve during movements of the upper limbs, thereby increasing neural tension, common in patients
with overuse syndromes. Therefore, there are tests to cause neural tension to evaluate the presence of biomechanical restriction of
the nerve when subjected to the positions of the upper limb that stress it. Thus, the objective of this job is to evaluate the
neurobiomechanical of the radial nerve in different positions of the upper limb through topography and morphometric analysis.
Methods and Results:
In this study, we used seven bodies treated with formoldehyde in which they were done topographic evaluations and metric
length analysis of the radial nerve in the following positions taken from the right upper limb: P1- Anatomic Position; P2 Abduction of the shoulder (45°), internal rotation of the shoulder (45°), the extension of the elbow (0°) and wrist neutral (P2), P3
- shoulder abduction (45°), internal rotation of the shoulder (45°), extension of the elbow (0°), wrist flexion (60°) and ulnar
deviation of the wrist (20°). The morphometric data were submitted to the ANOVA Statistic Method (p
Conclusions:
The position of upper limb, which may cause greater tension in the radial nerve associated with the movements of abduction and
internal rotation of shoulder, elbow extension, flexion and ulnar deviation of the wrist, being more suitable therefore for the use
of manual technique of the neurobiomechanical evaluation of the radial nerve.
Keywords: Morfologia, Morfometria, Nervo Radial
Financial Support: There was not support for research.
Resumo:22-047
TOPOGRAPHIC ANALYSIS OF ULNAR NERVE AT DIFFERENT POSITIONS OF THE UPPER LIMB
Araújo, J. C. G. 1; Portes, B. B. 1; Iatecola, A. 1; Cunha, M. R. 1,2
1
Centro Universitário Nossa Senhora do Patrocínio, CEUNSP
2
Faculdade de Medicina de Jundiaí, FMJ
Objectives:
Aim:The neural compression syndromes peripheral upper limb are common in clinical medicine and the causes range from
trauma and occupational factors. As a result, the peripheral nerve loses its mobility along the path leading to anatomic change
sensory and motor. Technical evaluation manuals as neural tension at different positions of the upper limb as well as knowledge
of the topography of the affected nerve are essential for accurate diagnosis. Therefore, the purpose of this study was to evaluate
the topographic anatomy of the ulnar nerve in different positions of the upper limb adopted for clinical evaluation and palpation.
Methods and Results:
Methods and Results:A total of 7 formalinized cadavers were evaluated in which anatomical and measurement of the length of
the ulnar nerve as well as its distance from the middle third of the clavicle, scapula and coracoid process of the medial epicondyle
of the humerus. Measurements were performed in the following positions of the right arm: P1 - It´s an anatomical position; P2 The shoulder abduction , (90º), elbow flexion (90º) and wrist neutral, P3 - The shoulder abduction (90º), elbow flexion (90º) and
wrist extension (60º). The morphometric data were submitted to ANOVA (p
Conclusions:
Conclusion:The ulnar nerve has important topographical relations with bony prominences and neurovascular structures of the
upper limb with reference to the location of this nerve and understanding of associated diseases. Although no statistical difference
in the morphology between the positions adopted, we note that the position of the upper stretch increased the ulnar nerve, was
associated the movements of shoulder abduction, elbow flexion and wrist extension, so is the position most suitable for the use of
manual techniques for evaluation and mobilization of the ulnar nerve.
Keywords: Morfologia, Morfometria , Nervo Ulnar
Financial Support: Sources of research support:There was not support for research.
Resumo:23-139
BEHAVIORAL ANALYSIS OF RATS UNDER CHRONIC ANFEPRAMONE TREATMENT ASSOCIATED OR NOT
WITH EXERCISE TRAINING.
Salgueiro, J. S. 1; Borges, C. R. 1; Conceição, A. P. S. F. 1; Almeida, D. S. 1; Tieppo, C. A. 2; Lopes, C. 1,2
1
Neuroscience Nucleus/Methodist University of São Paulo, UMESP
2
Physiology /Faculty of Md. Sciences of Sta Casa de SP, FCMSCSP
Objectives:
Anfepramone, also known as diethylpropione(DEP)-(2[diethilamine]propiofenone) is one of the most used anorexigens in Brazil
for weigh control and obesity treatment. This drug acts on the central nervous system and it has the potenciality of induce
addiction and tolerance processes because of its induces mesolimbic plasticity (J. Pharmacol. Sci. Japan, 106 (1); 9-14; 2008).
This research investigate the behavioral alterations induced by the chronic treatment of female rats with anfepramone, mimicking
the main conditions of this drug use in Brazil.
Methods and Results:
Forty eight female Wistar rats, approximately 250 g and two months old were used in this research. Rats were randomized in four
groups of twelve animals, according to the 43 consecutive days received treatment: 1) anfepramone - 7 mg/kg i.p.; 2) control
anfepramone – same volume of saline solution i.p.; 3) anfepramone-exercise – same anfepramone treatment and 30 min of
swimming daily (water at 25oC); 4) control anfepramone-exercise – saline treatment associated with 30 min of swimming daily.
Behavioral tests included three trials at 15, 30 and 43 days of experiment at the open field and at elevated plus maze with aversive
stimulus (wind and sound noise at one same closed arm). It was evaluated some parameters of general motor activity, as reagings,
groomings, stand period, excrement count on the open field and the time rats spent on the closed arm with aversive stimulus or on
the open arm. On the open field, after 43 days, anfepramone increased the grooming behavior (1.00+/-0.28 vs 3.17+/-0.55, p
Conclusions:
Chronic treatment with anfepramone: 1) increased grooming behavioral alterations in rats which is associated with stereotyped
behavior in humans; 2) exercise training induces per se the same effect; 3) decreased aversive memory in rats, which is associated
with protective behaviors in humans, exercise training promotes this memory.
Keywords: Anfepramone, Neural Plasticity, Behavioral Analysis, Exercise training
Financial Support: Neuroscience Nucleus of Health School of Methodist University of São Paulo.
Resumo:23-140
5HT1A AND 5HT1B RECEPTORS IN ZEBRAFISH ANXIETY.
Puty, B. ; Maximino, C. ; Miranda, V. ; Herculano, A. M.
Universidade Federal do Pará, UFPA
Objectives:
Anxiety behaviors have been linked to the serotonergic system, especially with the 5HT1A and 5HT1B serotonin receptors. The
administration of SB-224289, an inverse agonist of the 5HT1B receptor, and WAY-100635, an antagonist of 5HT1A receptor,
allows us to evaluate the role of these receptors in a behavioral model of anxiety in zebrafish.
Methods and Results:
Adult zebrafish, with 3 months and weighting 300-500 mg, were treated acutely with SB-224289 (0.0, 2.5 or 5.0 mg/kg, i.p; n = 9
each) and WAY-100635 (0.0, 0.003 or 0.03 mg/kg, i.p.; n = 9 each). The animals were tested in the novel tank test. The
parameters analyzed were: 1- time spent on top (total time the animal spends at the top 1/3rd of the apparatus); 2- total
locomotion (number of 10 cm² squares crossed) ; 3- erratic swimming (number of high-speed zig-zag swim bouts); 4- freezing
(time that the animal spends without moving). Data was presented as mean±S.E.M (time on top, freezing) or median±IQR (total
movement, erratic swimming) and analyzed using parametric or non-parametric one-way ANOVAs. Both drugs produced an
inverted U-shaped effect on the time spent on the top of the tank (WAY-100635: control: 34.31 ± 14.68, n = 7; 0.003: 164.7 ±
36.93, n = 8; 0.03: 145 ± 27.98, n = 9; and SB-224289: control: 14.99 ± 5.481, n= 9; 2.5: 148.1 ± 39.24, n= 8 ; 5.0: 101.0 ± 18.07,
n= 8). SB-224289 showed a decrease in the number of erratic swimming (WAY-100635: control: 0 ± 1, n=8; 0.003: 1 ± 1, n=8;
0.03: 0 ± 0.5; and SB-224289: control: 9.459 ± 5.559, n=9; 2.5: 0 ± 0, n= 8; 5.0: 0 ± 0, n=9) . Both drugs had no effect on the
total movement (WAY-100635: control: 39.00 ± 86.25, n=8; 0.003: 81.50 ± 29,5, n=8; 0.03: 55 ± 15, n=9 and SB-224289:
control:141 ± 121, n=9; 2.5: 42 ± 58.75, n=8; 5.0: 175 ± 107,5, n=9).
Conclusions:
5-HT1A receptors are tonically stimulated by serotonin, leading to increased anxiety. 5-HT1B receptors are also associated with
anxiety-like behavior in zebrafish.
Keywords: receptors 5HT1A and 5HT1B, SB-224289 and WAY-100635, behavioral model of anxiety, in zebrafish
Financial Support: CNPq(Process 483336/2009-2).
Resumo:23-141
PARTICIPATION OF DOPAMINE D2-LIKE RECEPTORS IN THE HYPERLOCOMOTION EVOKED BY
NEUROPEPTIDE S IN MICE.
Souza, L. S. 1; Guerrini, R. 2; Calo´, G. 3; Gavioli, E. C. 1
1
Departamento de Biofísica e Farmacologia, UFRN
2
Department of Pharmaceutical Science , UNIFE
3
Section of Pharmacology and Neuroscience Center, UNIFE
Objectives:
Neuropeptide S (NPS) is a 20 amino-acid peptide recently identified in the brain and peripheral tissues of distinct species of
vertebrates. NPS is the endogenous ligand of a G protein-coupled receptor named NPS receptor (NPSR). The NPSR is widely
expressed in the mammalian brain, and higher levels were found in the limbic system, and in neurons from dopaminergic
pathways. Various central biological actions are evoked by NPS, such as hyperlocomotion, anxiolysis, antinociception, memory
improvement and anorexic effects. Recent findings suggest that there is an interaction between dopamine and NPS systems (J
Neurochem.,115(2):475-82, 2010; Peptides, 31(5):926-31, 2010). Thus, the present study aimed to investigate the involvement of
dopaminergic D2 receptors in the mediation of psychomotor stimulant effects of NPS in mice.
Methods and Results:
Female Swiss mice (28-33 g) were housed in groups of maximum 12 per cage (33x40x17 cm) with food and water ad libitum,
and under constant temperature (23±2ºC), and a 12-h light/dark cycle (lights on at 06:00 h). The spontaneous locomotor activity
was assessed in the open-field test which was made of formica (60x60x60 cm). The distance moved (m) during 30 min was
counted by using the software Any-Maze (Stoelting, USA). In order to evaluate the behavioral effects of NPS, animals were
subjected to an esterotaxic surgery to implant a cannula into the left lateral ventricle (icv; coordinates: AP -0.6; ML +1.1; DV -1.0
mm). The effects on locomotion of the icv injection of NPS (1 and 0.1 nmol/2 µl, 5 min prior to the test) and the intraperitoneal
treatment with haloperidol (0.3, 0.1 and 0.03 mg/kg, 15 min prior to the test) was assessed in this study. We observed that the
administration of NPS 1 nmol, but not 0.1 nmol, increased spontaneous locomotion in mice, particularly in the last 15 min of
observation (C=33.8±2.8, n=10; NPS 0.1:33.7±3.0, n=14; NPS 1=45.8±4.5*, n=8; *p
Conclusions:
These data suggest that dopamine D2-like receptors are mediating the hyperlocomotion induced by NPS in mice. Further studies
aiming to investigate the participation of dopaminergic receptors other than D2 are in progress by our research group.
Keywords: Dopamine, Locomotor activity, Mouse, Neuropeptide S
Financial Support: CNPq and UFRN/Propesq
Resumo:23-142
THE EXPOSURE OF YOUNG RATS TO NMDA ANTAGONIST OR OPIOID AGONIST PROMOTES
ALTERATIONS IN THE BEHAVIORAL RESPONSES.
Scarabelot, V. L. 1,2; Souza, A. C. 1,2; Medeiros, L. 1,2,3; Cioato, S. 1,2; D. 1,2; Fernandes, L. 4; Caumo, W. 1,2;
Torres, I. L. S. 1,2,3
1
Depto de Farmacologia Instituto de Ciências Básicas da Saúde, ICBS-UFRGS
2
Hospital de Clínicas de Porto Alegre, HCPA
3
Programa de Pós Graduação em Fisiologia - ICBS/UFRGS, PPGFISIO-ICBS/UFRGS
4
Faculdade de Fármacia - UNIVATES - Lajeado RS, UNIVATES
Objectives:
It is know that the immature nervous system is highly sensitive to the depressant effects of the anesthetic drugs and the exposure
of newborn and children to general anesthesia is a common practice on the modern medicine. For this, the objective of this study
was to evaluate the effects of acute intervention of pharmacological drugs (NMDA antagonist or opiod agonist) at P14 upon
behavioral responses evaluated at short-, medium- and long-term.
Methods and Results:
Wistar male rats at fourteen day (P14) were divided into three groups: saline (SAL), S(+)-ketamine (KET; 20 mg/kg) and
fentanyl (FEN; 0.09 mg/kg) (n=6-14). The behavioral responses were evaluated at Open-Field (OF) and Elevated Plus-Maze
(EPM) tests, and they were performed at P14 (6h after drug administration), at P30 and at P60. The behaviors evaluated at OF:
number of line crossings, fecal boluses and rearing behaviors, latency to leave the first quadrant and time of grooming; and at
EPM: number of protected head-dipping movements (PHD) and non-protected head-dipping movements (NPHD); number of
entries in the open arms (EOA) and time spent on the open arms (TOA). Data were expressed as means ± standard error of the
mean (SEM). One-way ANOVA was performed to compare all groups, followed by the SNK. Student‘s t test for independent
samples was used to compare two groups (control group and FEN or KEN). Differences were considered statistically significant
if P < 0.05. The Institutional Research Committee approved all animal procedures (GPPG-HCPA: 100186), and measures were
taken to minimize pain and discomfort. At P14, when FEN e KET were compared with the SAL group, there was an increase in
inner crossings at OF (Student's t-test P < 0.05). At P30, the FEN group, at the EPM, showed a significant decrease in TOA and
EOA when compared to the SAL group (one way ANOVA/SNK, P < 0.05). At P60, the FEN group showed a significant increase
in NPHD, EOA and TOA when compared to other groups (ANOVA/SNK, P < 0.05) and to control (one way ANOVA/SNK, P <
0.05).
Conclusions:
These results indicate that fentanyl induced more changes in behavioral responses than S(+)-ketamine, especially in relation to
the anxiolytic state of rats.
Keywords: ketamine, fentanyl, young rats, open-field
Financial Support: FIPE/HCPA, CNPq, CAPES
Resumo:23-143
EFFECTS OF CANNABINOIDS IN THE PENTYLENETETRAZOLE MODEL OF CONVULSIVE SEIZURES IN
RATS
Vilela, L. R. ; Medeiros, D. ; Rezende, G. ; Oliveira, A. C. ; Moraes, M. F. ; Moreira, F. A.
Neurociências/Universidade Federal de Minas Gerais, UFMG
Objectives:
Epilepsy is a severe neurological disorder characterized by excessive or synchronous neuronal activity, which may lead to
convulsive seizures. The substances present in the plant Cannabis sativa, as well their synthetic counterparts (termed
cannabinoids), have been proposed as a treatment for this disorder. They act mainly through the cannabinoid (CB1) receptor in
the brain, which is also activated by endogenous substances, termed endocannabinoids. However, their effects in models
predictive of anti-convulsive activity have remained controversial. Thus, the aim of this study is to test these substances against
the generalized seizures induced by pentylenetetrazole (PTZ) in rats.
Methods and Results:
Methods: Male Wistar rats (6-8/group), weighing 280-310g, received intraperitoneal injections of vehicle or the cannabinoid
WIN 55212-2 (0.3-1.0-3 mg/kg). In an independent experiment they were treated with vehicle or the CB1 receptor antagonist
AM251 (0.3-1.0-3 mg/kg). Thirty minutes later they received intravenous infusion of PTZ, at a concentration of 10 mg/mL and a
rate of 0.5 mL/min. The ―threshold‖ of PTZ as well as the latency required to inducing minimum (partial myoclonus of limbs)
and maximum seizures (hyperextension of the forelimbs and hindlimbs with head flexion) were compared between groups
through analysis of variance followed by the Newman-Keuls test. Results: WIN 55212-2 (1 mg/kg) reduced both the latency (p <
0.001) and the threshold of PTZ (p < 0.01) required to induced minimal seizures, as compared to the vehicle control group.
Treatment with antagonist AM251, on the other hand (1 mg/kg), increased the threshold of PTZ (p < 0.05) required for minimum
seizure.
Conclusions:
Despite several pieces of evidence supporting anti-convulsive effects of cannabinoids, the present study points to a proconvulsive role of CB1 signaling in the PTZ model. Although the reasons for this finding are still unclear, further experiments are
under course to determine whether other interventions, such as blockade of endocannabinoid-hydrolysis, would actually attenuate
PTZ-induced seizures.
Keywords: cannabinoids , Epilepsy, convulsive seizures , endocannabinoids, pentylenetetrazole
Financial Support: Financial Support: FAPEMIG
Resumo:23-144
EARLY PRENATAL LPS EXPOSURE INCREASES SICKNESS BEHAVIOR AFTER AN IMMUNE CHALLENGE IN
ADULT RATS
Kirsten, T. B. ; Zager, A. ; Palermo-neto, J. ; Bernardi, M. M.
School of Veterinary Medicine, University of São Paulo, FMVZ-USP
Objectives:
Lipopolysaccharide (LPS) mimics infection with gram-negative bacteria and induces sickness behavior in many species generally
accompanied by a decrease in exploratory activity, social behavior, ingestive behavior, sexual behavior and induced anhedonia,
and poor learning and cognitive functions. Previous investigations by our group showed that prenatal LPS administration (100
µg/kg, i.p.) on gestational day 9.5 reduces striatal dopamine and metabolite levels (3,4-dihydroxyphenylacetic acid [DOPAC] and
homovanillic acid [HVA]) in adult male rats. However, behavioral parameters linked to motor system were not changed,
including open field general activity, haloperidol-induced catalepsy and apomorphine-induced stereotypy. The present study
investigated the prenatal LPS effects after an LPS challenge in adulthood, once adaptive plasticity processes which occur during
the animals‘ life can mask possible behavioral changes.
Methods and Results:
For this, dams of Wistar rats were submitted to the same conditions of treatment as our previous study (LPS on GD 9.5, 100
µg/kg, i.p.). The adult male pups were again treated, i.e., challenged with LPS (100 µg/kg, i.p.) on postnatal day 60-67, 90 min
before the experiments. Thus, there were four groups in the experiments (n = 7-9 for each group): SAL = prenatal saline
injection; LPS = prenatal LPS injection; SAL+LPS = prenatal saline and adulthood LPS injection; and LPS+LPS = prenatal and
adulthood LPS injection. This animals were evaluated for (1) open field general activity (traveled distance in cm and rearing
frequency); (2) spatial memory (T-maze spontaneous alternation); and (3) circulating proinflammatory cytokines interleukin-1
(IL-1) beta and tumor necrosis factor (TNF)-alpha levels. In relation to the other groups, results showed that LPS+LPS group
presented (1) decreased distance traveled (SAL = 3062.82±69.73, LPS = 3085.47±219.04, SAL+LPS = 2335.05±180.81 and
LPS+LPS=1416.44±214.75) and rearing frequency (SAL = 29.33±0.83, LPS = 30.00±1.21, SAL+LPS = 21.38±3.13 and
LPS+LPS=11.75±2.18); (2) impairment in spatial memory, i.e., hit score (SAL = 62.50% [1.0 (0.0 - 2.0)], LPS = 81.25% [2.0
(0.0 - 2.0)], SAL+LPS = 31.25% [0.0 (0.0 - 2.0)] and LPS+LPS 6.25% [0.0 (0.0 - 1.0)]); and (3) increased IL-1 beta levels
(SAL+LPS = 239.53±58.10 and LPS+LPS=511.73±103.45), but not TNF-alpha levels (SAL+LPS = 1021.10±310.68 and
LPS+LPS=972.03±163.38). In all the cases, the results were considered as significant if p < 0.05.
Conclusions:
Early prenatal LPS exposure increases sickness behavior after an immune challenge in adult rats, i.e., decreased exploratory
activity and spatial memory and increased IL-1 beta levels. Sickness behavior is a motivational state and present results might be
related with our previous results of striatal dopaminergic reduction. Taken together the data of previous and present study,
prenatal LPS modified brain plasticity, mainly of the dopaminergic system, which was masked during the animal development.
However, the immune challenge revealed these behavioral changes.
Keywords: Open field general activity test, Proinflammatory cytokines, Spatial memory T-maze
Financial Support: FAPESP
Resumo:23-145
BENEFICIAL EFFECTS OF OMEGA-3 FATTY ACIDS ON NATURALLY INDUCED ANXIETY-LIKE BEHAVIOR
BUT NOT ON MEMORY ACQUISITION IN RATS
Barcelos, R. C. S. ; Benvegnú, D. M. ; Boufleur, N. ; Emanuelli, T. ; Burger, M. E.
Universidade Federal de Santa Maria, UFSM
Objectives:
To evaluate the effects of the supplementation of the fish oil (FO) on anxiety-like behavior resulting from being in a new
environment in rats.
Methods and Results:
Methods and results: Male Wistar rats (250g±30) were fed with the same standard chow (SC) and supplemented for 28 days with
FO (0,1%) or soybean oil (SO- 0,1%), through of drinking water incorporation (polisorbate 80 1%). Gas chromatography (HP
6890) showed n-6/n-3 ratio (SC plus oils) of 1,31 and 10,47 for FO and SO, respectively. Rats orally supplemented with FO
(14mg/rat/day) showed lower locomotor (49,5%) and exploratory activities (48%), higher central locomotion (53%) and lower
number of fecal pellets (21%) than those treated with soybean oil (14mg/rat/day). By burying a smaller number of marbles, FOtreated rats showed reduced anxiety symptoms (56%), while their memory performance in a water-maze task was unchanged.
After 16h of fasting, FO reduced food intake (27%), confirming a less anxious behavior. Here we showed the effects of omega-3
on anxiety-like symptoms, which is related to development of stress of being in a new environment.
Conclusions:
We conclude that omega-3 can attenuate anxiety related to everyday life. Further studies should be conducted to assess the
influence of omega-3 together with anxiolytic drugs on anxiety disorders and depression.
Keywords: omega-3, fatty acids, anxiety, memory, rats
Financial Support: CNPq, CAPES, PRPGP, PROAP-UFSM
Resumo:23-146
HALOPERIDOL-LOADED POLYSORBATE-COATED POLYMERIC NANOCAPSULES SHOWED HIGHER
ANTIPSYCHOTIC EFFICACY IN RATS
Benvegnú, D. M. 1; Barcelos, R. C. S. 1; Boufleur, N. 1; Pase, C. S. 1; Reckziegel, P. 1; Roversi, K. 1;
Ourique, A. F. 2; Beck, R. C. R. 2; Burger, M. E. 1
1
Departamento de e Fisiologia e Farmacologia, UFSM
2
Faculdade de Ciências Farmacêuticas , UFRGS
Objectives:
Our objective was to prepare and evaluate the efficacy of haloperidol by its nanoencapsulation in polymeric nanoparticles.
Methods and Results:
Preparation and physicochemical characterization of nanocapsules suspension: Haloperidol-loaded nanocapsules (H-NC) were
prepared by interfacial deposition of preformed polymer (Int. J. Pharm. 55:r1, 1989) and a free suspension of haloperidol (0.25
mg/mL) was prepared in water using 5% (w/v) of polysorbate 80. Particle sizes, polydispersity indices and zeta potential were
measured by photon correlation spectroscopy using Zetasizer®. Drug content was assayed by HPLC, using as mobile phase
methanol/potassium phosphate monobasic pH 4.0 (60:40% v/v) and detected at 254 nm. Free drug was determined in the clear
supernatant following separation of nanocapsules by a combined ultrafiltration–centrifugation technique. Encapsulation
efficiency (%) was calculated by the difference between the total and free drug concentrations. All formulations appeared
macroscopically homogeneous similar to a milky bluish opalescent fluid (Tyndall effect). Haloperidol was efficiently
encapsulated (95 ± 1%). Nanocapsule suspension presented particles in the sub-micrometric range (between 200 and 300 nm),
low polydispersity (60.25), negative zeta potentials, acid pH values and drug content near 100%. In vivo experiments: Acute
pseudo-psychosis was induced and maintained with amphetamine injections (AMPH-8 mg/kg- ip) each 3 hours (0, 3, 6, 9 h) in
adult male Wistar rats (n=7). After 30 min of first AMPH administration, groups designated as free haloperidol (FH),
haloperidol-loaded nanocapsules (H-NC) and blank nanocapsules (B-NC) received a single injection of each respective
formulation (0.2 mg/Kg body weight ip). The AMPH and C groups received a single injection of polysorbate 80 suspension (5%
ip). Two observers quantified the stereotyped head behavior every 15 min according to scale scores (Psychopharmacol. 102:459,
1990). Kruskal–Wallis analysis revealed that AMPH treatment increased the head movements when compared to the control.
Rats treated with B-NC showed no reduction in head movements at any observation following each AMPH administration. On
the other hand, both H-NC and FH showed reduced behavioral scores at all observed times after the 1st and 2nd AMPH
administration, but H-NC demonstrated lower scores than FH. Following the 3rd dose, only the H-NC group showed decreased
movement scores.
Conclusions:
Our data demonstrated that the haloperidol was efficiently encapsulated and its therapeutic efficacy was improved, since it
presented a bet