ARS VETERINARIA, Jaboticabal, SP, Vol. 20, nº 1, 001-008, 2004.
ISSN 0102-6380
MORPHOMETRIC STUDY OF HEPATIC
MEGAKARYOCYTOPOIESIS IN NEW ZEALAND WHITE RABBITS
DURING INTRAUTERINE AND POSTNATAL DEVELOPMENT
(ESTUDO MORFOMÉTRICO DA MEGACARIOCITOPOIESE HEPÁTICA EM
COELHOS DA RAÇA NOVA ZELÂNDIA BRANCO DURANTE O DESENVOLVIMENTO
INTRA-UTERINO E PÓS-NATAL)
(ESTUDIO MORFOMÉTRICO DE LA MAGACARIOCITOPOYESIS HEPÁTICA EN
CONEJOS DE LA RAZA BLANCO DE NUEVA ZELANDA DURANTE EL
DESENVOLVIMIENTO INTRAUTERINO Y POS NATAL)
M. R. PACHECO*1, N. FERREIRA2, V. R.MELO3, L.S.O. NAKAGHI1,
S. M. BARALDI-ARTONI4, L.N. GANECO5, A.C.F. CARVALHO6
SUMMARY
We studied the ratios of cells of the megakaryocytic line to other tissue constituents in the liver of New Zealand
White rabbits in the intrauterine phase and in the immediate postnatal period. Six females were sacrificed, two of them on
the 15th day of pregnancy for embryo liver collection, and two each on the 22nd and 29th day of pregnancy for fetus liver
collection. Six other females were allowed to complete gestation and the liver of newborn rabbits was obtained on the 10th,
21st and 32nd day after birth. By calculating percentage values of the tissue constituents in the liver by the morphometric
technique of CHALKLEY, counting 300 points per animal, we observed that cells of the megakaryocytic line presented the
greatest mean percentual values on the 29th day of pregnancy and the smallest percentual values on the 21st and 32nd day
of postnatal life.
KEY-WORDS: Morphometry. Megakaryocytopoiesis. Rabbits
RESUMO
Estudou-se a proporção de células da linhagem megacariocítica em relação aos demais constituintes tissulares do
fígado de coelhos da raça Nova Zelândia Branco na fase intra-uterina e no período pós-natal. Seis fêmeas foram sacrificadas,
duas delas no 15º dia de prenhez para a coleta do fígado dos embriões e duas no 22º e 29º dia de prenhez para a coleta do
fígado dos fetos. Seis outras fêmeas mantiveram a prenhez até o final e o fígado dos coelhos recém-nascidos foi colhido
no 10º, 21º e 32º dia após o nascimento. Para o cálculo dos valores percentuais dos componentes tissulares do fígado pela
técnica morfométrica da CHALKLEY, contaram-se 300 pontos por animal e observou-se que as células da linhagem
megacariocítica apresentaram os maiores valores percentuais médios no 29º dia de prenhez e os menores no 21º e 32º dia
do período pós-natal.
*1
Professor Assistente Doutor da FCAV/Unesp – Campus de Jaboticabal/SP. Rod. Prof. Paulo Donato Castellani, km 5. CEP.
14884-900. End. Eletrôn.: [email protected]
2
Professor Titular da FMVZ - USP, São Paulo/SP.
3
Professor Doutor da FMRP - USP, Ribeirão Preto/SP.
4
Professor Adjunto da FCAV/Unesp - Campus de Jaboticabal/SP.
5
Doutorando da CAUNESP da FCAV/Unesp – Campus de Jaboticabal/SP.
6
Doutorando da FCAV/Unesp – Campus de Jaboticabal/SP.
1
M. R. PACHECO, N. FERREIRA, V. R. MELO, L. S. O. NAKAGHI, S. M. BARALDI-ARTONI, L. N. GANECO, A. C. F. CARVALHO. Morphometric study of hepatic
megakaryocytopoiesis in New Zealand white rabbits during intrauterine and postnatal development./ Estudo morfométrico da megacariocitopoiese hepática em coelhos da
raça Nova Zelândia branco durante o desenvolvimento intra-uterino e pós-natal./ Estudio morfométrico de la magacariocitoyesis hepática en conejos de la raza blanco
de Nueva Zelanda durante el desenvolvimiento intrauterino y pos natal Ars Veterinaria, Jaboticabal, SP, Vol. 20, nº 1, 001-008, 2004.
PALAVRAS-CHAVE: Morfometria. Megacariocitopoiese. Coelhos.
RESUMEN
Fue estudiada la proporción de células del linaje megacariocítico, en relación con los demás constituyentes tisulares,
en el hígado de conejos de la raza Blanco de Nueva Zelanda, en la fase intrauterina y en el período pos natal. Seis hembras
fueron sacrificadas, dos en el 15o día de gestación, para la colecta del hígado de los embriones, y dos en los días 22o y 29o,
para la colecta del hígado de los fetos. Otras seis hembras llevaron a término la gestación y el hígado de los conejos recién
nacidos fue colectado en los días 10o, 21o y 32o después del nacimiento. Para el cálculo de los valores porcentuales de los
componentes tisulares del hígado fue usada la técnica morfométrica de CHALKLEY. Fueron contados 300 puntos por
animal y se observó que las células del linaje megacariocítico presentaron los mayores valores porcentuales medios en el
día 29o de gestación y los menores en los días 21o y 32o del período pos natal.
PALABRAS CLAVE: Morfometría. Megacariocitopoyesis. Conejos.
INTRODUCTION
In mammals, an early event after traumatic injury is
the transitory contraction of the damaged blood vessels,
but this has little effect on hemostasis, i.e., on the
stanching of blood (BERNE & LEVY, 1990). Although, 1
to 2 seconds after the occurrence of an endothelial lesion
numerous platelets, essential for hemostasis, adhere to
the damaged surface forming a dense aggregate. This is
the beginning of the hemostatic plug, which is completed
with the formation of a blood clot. Platelets contribute
substances that accelerate clotting which are essential
for clot retraction after clot formation. Furthermore,
platelets release substances with vasoconstrictive
properties, especially 5-hydroxytryptamine (5-HT or
serotonin). There are indicative that platelets may be
deposited on the capillary endothelium, thus contributing
perhaps to the integrity of the latter. Platelet depression
or thrombocytopenia causes a hemorrhagic disorder,
which manifests as persistent bleeding after cuts or
wounds and as the appearance of petechiae, ecchymoses
and blood leakage from capillary beds (MOUNTCASTLE,
1978).
In view of the importance of platelets, cells that
originate from megakaryocytes by a process of exocytosis
in which they detach from the cell surface (BANKS, 1992),
for blood clotting, we undertook the present study to
determine the proportion of cells of the megakaryocytic
line in relation to the remaining tissue constituents of the
liver in New Zealand White rabbit embryos, fetuses and
newborns, and to determine the time of peak and decline
of hepatic megakaryocytopoiesis. We thought it would
be interesting to investigate, in addition to its relevant
2
metabolic function, the participation of the liver in
megakaryocytopoiesis, platelet genesis and hemostasis
during the various phases cited above, and due to the
scarcity of literature on the morphometry of hepatic
megakaryocytopoiesis and on account of the intense
utilization of rabbits as laboratory animals.
MATERIALS AND METHODS
Twelve female New Zealand White rabbits were
mated to males of the same breed. After pregnancy was
verified by abdominal palpation, the animals were kept in
individual metabolic cages fitted with fixed feeders and
automatic water spounts, with food (production ration)
and water ad libitum throughout the experimental period.
Hepatic megakaryocytopoiesis was studied during
the intrauterine phase and during the postnatal period.
Six rabbits were sacrificed on the 15th, 22nd and 29th day of
pregnancy, two per period, and the liver of 15 day embryos
and 22 and 29 day fetuses was collected. Six other rabbits
were allowed to complete pregnancy and, after parturition,
the same study was conducted on 10-, 21- and 32-day-old
newborn animals. The liver of embryos, fetuses and
newborns was fixed in Bouin’s solution for 24 hours and
routinely processed for paraffin embedding. Semiserial 7μm sections were obtained with a microtome, stained with
Masson trichrome by the method of BEHMER et al. (1976)
and observed under the light microscope for
morphometric analysis. To determine the proportions of
cells of the megakaryocytic line and of the remaining tissue
components of the liver, 300 points per animal were counted
by the technique of CHALKLEY (1943). The values
M. R. PACHECO, N. FERREIRA, V. R. MELO, L. S. O. NAKAGHI, S. M. BARALDI-ARTONI, L. N. GANECO, A. C. F. CARVALHO. Morphometric study of hepatic
megakaryocytopoiesis in New Zealand white rabbits during intrauterine and postnatal development./ Estudo morfométrico da megacariocitopoiese hepática em coelhos da
raça Nova Zelândia branco durante o desenvolvimento intra-uterino e pós-natal./ Estudio morfométrico de la magacariocitoyesis hepática en conejos de la raza blanco
de Nueva Zelanda durante el desenvolvimiento intrauterino y pos natal Ars Veterinaria, Jaboticabal, SP, Vol. 20, nº 1, 001-008, 2004.
obtained by this techniques were transformed to
percentages and the means and standard deviations were
then calculated.
RESULTS
The proportion of cells of the megakaryocytic line
in relation to the remaining tissue components of the liver
of embryos, fetuses and newborn New Zealand White
rabbits, determined by the morphometric technique of
CHALKLEY (1943), is reported as mean percent values
(Tabs. 1 and 2 and Figs.1.A and 2.A). The Tukey’s test
showed that these cells of the megakaryocytic line
increased intensely during the intrauterine phase, with a
maximum value on the 29th day of this phase (Fig.1.B),
followed by a progressive decrease in 10-21- and 32-day
old newborns (Fig.2.B). On the 15th day of intrauterine life,
the cells of the megakaryocytic line were less numerous
than blood cells, hepatocytes and vessels (sinusoid
capillaries), but were more numerous than centrolobular
veins, portal space and fibrous tunica (Glisson’s capsule).
On the 22nd day of intrauterine life, they were only more
numerous than the fibrous tunica. On the 10th day of
postnatal life, the behavior of these cells was similar to
that observed on the 22nd day of intrauterine life, with cell
numbers only exceeding the fibrous tunica. On the 21st
and 32nd day of postnatal life, these cells were less
numerous than all other tissue components of the liver,
clearly demonstrating the reduction of hepatic
megakaryocytopoiesis during this phase of life.
The F test demonstrated significative differences
at the 1% level of probability (p<0,01) among the mean
percent values obtained for every liver tissue components,
in all phases of this research.
DISCUSSION
The results of the present study concerning the
morphometric determination of the proportion of cells of
the megakaryocytic line in relation to the remaining tissue
components of the liver, and the time of maximum and
declining hepatic megakaryocytopoiesis in New Zealand
White rabbits show that this process progressed normally
as expected, characterizing this function of the liver in the
intrauterine phase, in young newborns and, rarely, with
adult rabbits. The data obtained in the present study agree
with those reported by SORENSON (1960) who, in an
electron microscopy study of hepatic hemocytopoiesis
in rabbits from the 12th to the 30th day of pregnancy,
observed that during the peak of hepatic hemocytopoiesis
the liver contained many more blood cells than
hepatocytes. Similarly, in the present study, using the
morphometric technique of CHALKLEY (1943), we
observed the highest mean values of blood cells
(56.0667%) and the lowest mean values of hepatocytes
(28.6000%) on the 22nd day of intrauterine life, characterizing
the high hematocytopoietic potential of the liver in this
phase, followed by a gradual reduction of these cells and
a progressive increase in hepatocytes. This investigator
also noted the presence of megakaryocytes on the 14th
day of intrauterine life, with an increased frequency at the
end of pregnancy but a decrease before birth.
Our results also agree with those reported by
HERTZBERG & ORLIC (1981) who, in an electron
microscopy study of New Zealand White rabbits,
observed a decline in hepatic hematocytopoietic activity
starting on the 24th day of pregnancy and detected only a
small amount of hemocytopoietic cells around the 5th day
of postnatal life. They also agree with the data reported
by BOCKMAN & GULATI (1989), who detected
megakaryocytes in the liver of rat fetuses from the 12th
day of pregnancy, with increasing numbers from the 13th
day to almost the end pregnancy, and a decrease at the
end of pregnancy, with only few of them being detected
on the 8th postpartum day. Also reflecting LEESON &
CUTTS (1972) reports, when studding, using light
microscopy, rabbit’s liver after birth development, between
days 1 and 6, and 8th, 10th, 15th , 70th and 112th days, evidenced
megakaryocytes in newborn with 5 days of life and, rarely,
in adult animals. The results observed in our studies, about
rabbit’s liver at 32 days of life, reflect these authors
evidences, when reporting liver’s hematocytopoietic
activity pause, in rabbits with 28 days of age, and imputing
liver, at this stage of life, an almost adult microscopic
architecture, finding, even though rarely, megakaryocytes.
We also emphasize that the highest mean values recorded
for vessels (sinsusoid capillaries) in the present study
were detected on the 15th day of intrauterine life (31.0667%)
and on the 10th day of postnatal life (11.9333%). We assume
that these were responsible for the induction of hepatic
hemocytopoiesis during the intrauterine phase and in
young newborns, in agreement with data reported by
MEDLOCK & HAAR (1983). These investigators, in an
ultrastructural study of the hemocytopoietic environment
of the liver of mouse fetuses between the 10th and 17th day
of intrauterine life, attributed to prehepatocytes and to
phagocytic cells (von Kupffer cells) lining regions of
hepatic sinusoids the probable function of synthesizing
and secreting substances, as well: erythropoietin and
interleukins (IL), respectively, which induces hepatic
hemocytopoiesis
and
consequently
megakaryocytopoiesis. BANKS (1992), confirmed that
3
M. R. PACHECO, N. FERREIRA, V. R. MELO, L. S. O. NAKAGHI, S. M. BARALDI-ARTONI, L. N. GANECO, A. C. F. CARVALHO. Morphometric study of hepatic
megakaryocytopoiesis in New Zealand white rabbits during intrauterine and postnatal development./ Estudo morfométrico da megacariocitopoiese hepática em coelhos da
raça Nova Zelândia branco durante o desenvolvimento intra-uterino e pós-natal./ Estudio morfométrico de la magacariocitoyesis hepática en conejos de la raza blanco
de Nueva Zelanda durante el desenvolvimiento intrauterino y pos natal Ars Veterinaria, Jaboticabal, SP, Vol. 20, nº 1, 001-008, 2004.
Tabela 1 - Comparative percent values of the liver tissue components of New Zealand White rabbit embryos on the 15th and
fetuses on the 22nd and 29th day of intrauterine life.
(1) The statistical analysis was carried out using data changed into ARC SEN “P/100
** Significative at the 1% level of probability (p<0,01)
A, B, C, D: The averages followed by the same letter do not differ from each other according to the Tukey’s test (p>0,05)
Tabela 2 - Comparative percent values of the liver tissue components of New Zealand White rabbit newborns on the 10th,
21st and 32nd day of life.
(1) The statistical analysis was carried out using data changed into ARC SEN “P/100
** Significative at the 1% level of probability (p<0,01)
A, B, C, D, E, F: The averages followed by the same letter do not differ from each other according to the Tukey’s test (p>0,05)
4
M. R. PACHECO, N. FERREIRA, V. R. MELO, L. S. O. NAKAGHI, S. M. BARALDI-ARTONI, L. N. GANECO, A. C. F. CARVALHO. Morphometric study of hepatic
megakaryocytopoiesis in New Zealand white rabbits during intrauterine and postnatal development./ Estudo morfométrico da megacariocitopoiese hepática em coelhos da
raça Nova Zelândia branco durante o desenvolvimento intra-uterino e pós-natal./ Estudio morfométrico de la magacariocitoyesis hepática en conejos de la raza blanco
de Nueva Zelanda durante el desenvolvimiento intrauterino y pos natal Ars Veterinaria, Jaboticabal, SP, Vol. 20, nº 1, 001-008, 2004.
B
b
Figura 1 - Histogram of the mean percent values of the liver tissue components of New Zealand White rabbit embryos on
the 15th and fetuses on the 22nd and 29th day of intrauterine life (A) and photomicrography of the morphological
liver aspects, on the 29th day of intrauterine life, evidencing: cell of the megakaryocytic line (a); blood cell (b);
hepatocyte (c) and sinusoid capillary (d) – Masson trichrome, 100X (B).
erythropoietin is synthesized at the kidneys and liver and
interleukins produced by macrophages and T helper
lymphocytes. Beyond these substances, McDONALD et
al. (1975), included thrombopoietin, produced by kidneys,
as a megakaryocytopoiesis mediator. However, we
consider random the mean percentages obtained for
centrolobular veins, portal spaces and fibrous tunica
(Glisson’s capsule) in our study since these liver tissue
components are definitely not involved in
hemocytopoiesis or, more specifically, in hepatic
megakaryocytopoiesis. Thus, we believe that the above
reports agree with our results considering the intense
increase of cells of the megakaryocytic line in the
intrauterine phase which peaked on the 29th day, followed
by a progressive decrease in 10 - 21- and 32-day-old
newborns. Thus, we believe that the differentiation of
kidneys, liver, immunologic system, bone marrow,
connective tissue and skin in the fetal stage (29th day)
made a greater potential contribution to elaboration of
specific humoral substances as well erythropoietin,
thrombopoietin
and
interleukins
inducing
megakaryocytopoiesis. It is opportune to stand out,
according to classic literature reports (TIZARD, 1998),
that the majority of interleukins, also known as
hematopoietins, includes IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-9, IL-10, IL-11 and IL-13. Thinking so, it justify the
5
M. R. PACHECO, N. FERREIRA, V. R. MELO, L. S. O. NAKAGHI, S. M. BARALDI-ARTONI, L. N. GANECO, A. C. F. CARVALHO. Morphometric study of hepatic
megakaryocytopoiesis in New Zealand white rabbits during intrauterine and postnatal development./ Estudo morfométrico da megacariocitopoiese hepática em coelhos da
raça Nova Zelândia branco durante o desenvolvimento intra-uterino e pós-natal./ Estudio morfométrico de la magacariocitoyesis hepática en conejos de la raza blanco
de Nueva Zelanda durante el desenvolvimiento intrauterino y pos natal Ars Veterinaria, Jaboticabal, SP, Vol. 20, nº 1, 001-008, 2004.
A
B
Figure 2 - Histogram of the mean percent values of the liver tissue components of New Zealand White rabbit newborns
on the 10th, 21st and 32nd day of life (A) and photomicrography of the morphological liver aspects, on the 32nd
day of postnatal life, evidencing: cell of the megakaryocytic line (a); hepatocyte (b) and sinusoid capillary (c)
– Masson trichrome, 100X (B).
hypothesis that rabbit fetus, at 29th day of pregnancy,
have a high potential for producing interleukins,
illustrating that hematopoietin IL-6, is produced by
macrophages, T and B lymphocytes, stroma cells of the
bone marrow, fibroblasts and keratinocytes. With these
evidences it is appropriate to say that, in White New
Zealand rabbit fetus, with 29 days of pregnancy, exists
viability for chirurgical interference, intending scientific
researches, considering physiological mechanisms
already working, involved in thrombocytopoiesis and
sanguineous coagulation, following highest mean
6
percentual values of megakaryocytes found in the fetal
liver, at this stage of pregnancy. . Those affirmations are
in agreement with the literature papers as follows: INOUE
et al. (1993), who observed by electron microscopy in
megakaryocytes of rat bone marrow in vitro that the
interleukin-6 significatively increased the megakaryocytes
diameter. BURSTEIN et al. (1992), studying the effects of
interleukin-11 on megakaryocytes of murine and human
bone marrow, in vitro, noticed that this cytokine promotes
these cells maturation. INOUE et al. (1993), researching in
vitro megakaryocytes of rat bone marrow using electronic
M. R. PACHECO, N. FERREIRA, V. R. MELO, L. S. O. NAKAGHI, S. M. BARALDI-ARTONI, L. N. GANECO, A. C. F. CARVALHO. Morphometric study of hepatic
megakaryocytopoiesis in New Zealand white rabbits during intrauterine and postnatal development./ Estudo morfométrico da megacariocitopoiese hepática em coelhos da
raça Nova Zelândia branco durante o desenvolvimento intra-uterino e pós-natal./ Estudio morfométrico de la magacariocitoyesis hepática en conejos de la raza blanco
de Nueva Zelanda durante el desenvolvimiento intrauterino y pos natal Ars Veterinaria, Jaboticabal, SP, Vol. 20, nº 1, 001-008, 2004.
microscopy, verified that the interleukin-3, interleukin-6
and erythropoietin stimulated, with variable potencial, the
formation of cytoplasmic processes, which are
intermediary structures between megakaryocytes and
platelets in the sequence of maturation of these cellular
lineage. It was also observed that the interleukin-6
supports the megakaryocytes ploidy. BURSTEIN (1994),
investigating the effects of interleukin-6 on
megakaryocytes and on the platelet function in dogs,
noticed that these multifunctional cytokine is a potent
promoter of megakaryocytic maturation, as shown by
enhancing size, ploidt and platelet production. AN et al.
(1994), studying mouse megakaryocytes, evidenced that
the erythropoietin and interleukin-6 stimulated the
development of cytoplasmic processes on these cells,
considered proplatelet formation. TAJIKA et al. (1996),
researching trombocytopoiesis, in vivo and in vitro, by
means of electron and immunofluorescent mycroscopy in
megakaryocytes of mouses, which received interleukin-6
(10 mu-g/animal/day subcutaneously), verified bundle
formation of microtubules in the cytoplasm in about half
of these cells, in proportion to an increase in platelet
counts. It was concluded that the microtubule-bundle
formation is one maturational event in megakaryocyte
development and that interleukin-6 could accelerate this
event. ZUCKER-FRANKLIN & KAUSHANSKY (1996),
investigating the effect of thrombopoietin on
megakaryocytes culture isolated from the mouse bone
marrow, perceived by ultrastructure that the
thrombopoietin is able to drive the full maturation of the
megakaryocytes as evidenced by generation of granules,
demarcation membranes, and cytoplasmic fragmentation
into platelets. TANGE & MIYAZAKI (1996), studying the
synergistic effects of erythropoietin, interleukin-6 and
thrombopoietin on megakaryocytes culture isolated from
rat bone marrow, observed on both inverted phase
contrast microscopy and scanning electron microscopy
that a large number of proplatelet process clusters were
dose-dependently formed with the addition of these
substances. It was concluded that the erythropoietin and
the interleukin-6 were demonstrated to act synergistically
solely at low doses in the development of proplatelet
processes formation leading to platelet release DRAYER
et al. (2000), noticed increase in the number of
megakaryocyte progenitors, when stem cell cultures were
treated with interleukin-3 or interleukin-3 + interleukin-6 +
interleukin-11. These authors also reported that inclusion
of these cytokins to stem cell cultures can be a way to
accelerate platelets recuperation, in patients undergoing
treatment with high doses of chemotherapics MIYAZAKI
et al. (2000), revealed that interleukin -3 and thrombopoietin
incited, in vitro, megakaryocyte progenitors proliferation,
proceeding from umbilical cord and bone marrow, of
humans. However, the ability to produce mature
megakaryocytes, was superior at bone marrow
progenitors than at umbilical cord, in the same in vitro
culture method. KELLAR et al. (1988), notified that
thrombopoietic activity of rabbit plasma is attributed to a
humoral substance, thrombopoietin. They evidenced that
this substance stimulated the differentiation of
megakaryocyte precursors not identified to identified
megakaryocytes. They also attested that rabbit’s plasma
thrombopoietin incited in vivo platelets production and
in vitro megakaryocytopoiesis
Considering that the greatest mean percentual
value, for cells of the megakaryocytic line, was found in
White New Zealand rabbit fetus, with 29 days of
pregnancy, can be concluded that exists viability for
chirurgical interference, for scientific researches, in the
presence of physiological mechanisms, involved in
thrombocytopoiesis and sanguineous coagulation,
already working at this intrauterine stage, in this species.
Still considering the fact that the mean values detected
for these cells of the megakaryocytic line on the 10th day
of postnatal life were higher only with respect to the fibrous
tunica and were lower than all other liver tissue components
on the 21st and 32nd day of postnatal life, we also conclude
that the liver clearly demonstrates the reduction of
megakaryocytopoiesis during this phase of life.
ACKNOWLEDGMENTS
We wish to thank the professor José Carlos Barbosa
for his help and facilities used during the execution of this
research. This research was supported by a grant from
CAPES, São Paulo, Brazil.
ARTIGO RECEBIDO: JUNHO/2002
APROVADO: SETEMBRO/2003
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7
M. R. PACHECO, N. FERREIRA, V. R. MELO, L. S. O. NAKAGHI, S. M. BARALDI-ARTONI, L. N. GANECO, A. C. F. CARVALHO. Morphometric study of hepatic
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