UNIVERSIDADE NOVE DE JULHO
PROGRAMA DE PÓS-GRADUAÇÃO EM BIOFOTÔNICA APLICADA ÀS
CIÊNCIAS DA SAÚDE
Efeitos do Laser de Baixa Intensidade (830 nm) na Inflamação Pulmonar
Aguda em um Modelo de Síndrome do Desconforto Respiratório Agudo
(SDRA) Intra e Extrapulmonar Induzida por LPS
Aluno: Manoel Carneiro de Oliveira Junior
Orientador: Prof. Dr. Rodolfo de Paula Vieira
São Paulo, 30 de Setembro de 2013
1
Manoel Carneiro de Oliveira Junior
Efeitos do Laser de Baixa Intensidade (830 nm) na Inflamação Pulmonar
Aguda em um Modelo de Síndrome do Desconforto Respiratório Agudo
(SDRA) Intra e Extrapulmonar Induzida por LPS
Dissertação
apresentada
á
Universidade Nove de Julho,
para obtenção do título de
Mestre em Biofotônica Aplicada
às Ciências da Saúde.
Orientador: Prof. Dr. Rodolfo de Paula Vieira
São Paulo, 30 de Setembro de 2013
2
FICHA CATALOGRÁFICA
3
4
DEDICATÓRIA
Dedico a todos meus familiares, por ter
suportado meus maus humores ao longo de minha pesquisa.
A minha esposa Thais pelas horas despendidas,
aos meus pequenos Gabriel e Gustavo que são a
razão da minha vida e tudo isto é pra vocês.
Amo Vocês.
Aos meus pais, irmãos e familiares por me
mostrarem o caminho certo.
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AGRADECIMENTOS
Agradeço principalmente ao meu orientador Prof. Dr. Rodolfo pelos
ensinamentos passados ao longo deste curso e por ter me orientado com muita
paciência.
A Universidade Nove de Julho pela bolsa de Mestrado, a Diretoria de pós
graduação em Biofotônica, a Profa. Regiane Albertini e Prof. Rodrigo Martins
pela oportunidade de ingresso no Mestrado.
A Profa. Dra Ana Paula, por ter me ensinado muitos procedimentos e pelos
conhecimentos passados, cuja paciência, dedicação e experiência me
ajudaram muito.
Agradeço as amigas: Vanessa Roza da Silva, Flávia Regina Greiffo, ao nosso
grupo de pesquisa: Ricardo, Paulo, Adilson, Nicole e Ana Roberta, aos colegas
de curso por estarem sempre ao meu lado nas horas de aperto e nas horas
que não sabíamos o que fazer. As minhas amigas Ana Paula Souza e Elis
Cabral Victor pela paciência de terem me escutado nos momentos que pensei
em desistir e a todos os Profs. que passaram na minha vida acadêmica
passando seus conhecimentos com afinco.
As amigas Nilsa Regina Damaceno e Francine M. Almeida (FMUSP) pela ajuda
em nosso projeto, meu muito obrigado.
As técnicas do laboratório Ângela e Luciana pela paciência e pelos
ensinamentos das técnicas que me foram úteis para desenvolver este projeto,
meus agradecimentos. E principalmente a Deus por me dar forças e paciência
para completar este objetivo de vida.
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Resumo
A síndrome do desconforto respiratório agudo (SDRA) é uma síndrome
que apresenta altas taxas de mortalidade, que pode ser resultante tanto de
insultos pulmonares como extrapulmonares. A síndrome é caracterizada pela
insuficiência respiratória proveniente da resposta inflamatória que cursa com
alteração de permeabilidade alvéolo-capilar, edema e hipoxemia refratária aos
altos fluxos de oxigênio. Um dos mais importantes mecanismos que
determinam a severidade desta injúria é a magnitude da lesão da barreira
epitélio alveolar. A possibilidade de reparação do epitélio em um estágio
precoce é o maior determinante da recuperação. Muitas das modalidades
terapêuticas baseiam-se na tentativa de diminuição da inflamação pulmonar
para minimizar a lesão inicial, a qual se deve em grande parte ao processo
inflamatório mediado pela ativação local e sistêmica por citocinas como TNF-α
e IL-1β. Um número crescente de estudos relata que o laser de baixa
intensidade apresenta efeitos antiinflamatórios em modelos de SDRA induzida
por LPS e isquemia e reperfusão da artéria pulmonar. No entanto, até o
momento, apenas lasers no espectro vermelho (650 – 655 nm) foram
estudados. Portanto, o presente estudo tem como objetivo investigar o papel do
laser de baixa intensidade (LBI), na faixa do infravermelho (830nm), 3J/cm2,
35mw, 80 segundos por ponto (03 pontos por aplicação), na inflamação
pulmonar, usando um modelo de SDRA de origem pulmonar (LPS
intratraqueal) e também extrapulmonar (LPS intraperitoneal). A aplicação do
laser foi realizada diretamente em contato com a pele, em três pontos do tórax
(correspondente ao final da traquéia - ponto 01, pulmão direito - ponto 02 e do
pulmão esquerdo ponto 03), por três vezes, 01 hora após a administração de
LPS. Camundongos BALB/c (n = 40) machos foram distribuídos em Controle (n
= 08; não administrado com LPS), IT 10 (n = 07; LPS intratraqueal; 10
µg/camundongo), IT + Laser (n = 09; LPS intratraqueal; 10µg/camundongo +
Laser), IP (n= 07; LPS intraperitoneal; 100µg/ camundongo), IP + Laser (n = 09;
LPS intraperitoneal; 100 µg/camundongo + Laser). Os animais foram
eutanaziados vinte e quatro horas após a administração de LPS. Foi avaliada a
contagem de células totais e diferenciais no lavado bronco alveolar (LBA), os
7
níveis de citocinas (IL-1β, IL-6, IL 10, KC e TNF-α), a densidade de neutrófilos
no parênquima pulmonar.
Os resultados demonstraram que o LBI significativamente reduziu o
número de células totais e de neutrófilos no Lavado Bronco Alveolar (LBA), o
número de neutrófilos no parênquima pulmonar, e os níveis de citocinas próinflamatórias no
LBA
tanto
no
modelo
de
SDRA
pulmonar
quanto
extrapulmonar. Portanto, concluímos que o laser infravermelho 830nm é eficaz
para reduzir a inflamação pulmonar, em ambos os modelos de SDRA
intrapulmonar e extrapulmonar induzida por LPS.
Palavra
Chave:
neutrófilos.
SDRA,
laserterapia,
inflamação
pulmonar,
citocinas,
8
Abstract
Acute respiratory distress syndrome (ARDS) is a syndrome that presents
high mortality rates, and the results of both insults pulmonary or extrapulmonary (pneumonia or septic shock) are high, and is a disease
characterized by respiratory insufficiency from the inflammatory response that
leads to alteration of alveolar-capillary permeability, pulmonary edema and
hypoxemia refractory to high flow oxygen. One of the most important
mechanisms that determined the severity of this injury is the magnitude of the
injury of alveolar epithelial barrier. The possibility of repairing epithelial at an
early stage is the major determinant of recovery. Many of therapeutic modalities
based on the attempt to decrease lung inflammation to minimize the initial injury
and much of the inflammatory process occurs through activation of local and
systemic cytokines such as TNF-α and IL-1β. A growing number of studies
report that Low Level Laser Therapy (LLLT) have anti-inflammatory effects in
models of LPS-induced pulmonary ARDS, however, so far, only the red
spectrum lasers were studied. Therefore, this study aimed to investigate the
role of infra red laser (830nm), 3J/cm2, 35mw, 80 seconds per point (03 points
per
application),
in
pulmonary
inflammation,
lung
using
LPS
model
(intratracheal) and also extrapulmonary (intraperitoneal) inducing ARDS. The
laser application was performed directly in contact with the skin in the chest
three points (corresponding to the end of the trachea - Section 01 right lung point 02 and left lung - point 03), three times, beginning 01 hour after LPS
administration. BALB / c mice (n = 40) were divided into control (n = 08; not
administered LPS), IT (n = 07; intratracheal administered LPS (10 µg / mouse),
IT + LLLT (n = 09; intratracheal LPS administered (10 µg / mouse) + LLLT), IP
(n = 07; LPS administered intraperitoneal (100 µg / mouse), IP + LLLT (n = 09;
administered intraperitoneal LPS (100 µg / mouse) + LLLT). Twenty-four hours
after administration of LPS and Laser, animals were euthanized and the lungs
removed for studies of pulmonary inflammation: Total cell count and differential,
bronchoalveolar lavage (BAL), cytokines (IL-1beta, IL-6, IL-10, KC and TNF-α),
BAL levels were also analyzed quantitatively the number of neutrophils in the
lung parenchyma in lung tissue using histomorphometry techniques. Results
showed that LLLT significantly reduced pulmonary and extra-pulmonary LPS
9
induced in both configurations Experimental of ARDS, as evidenced by a
reduction in the number of total cells and neutrophils in BAL, reduced levels of
IL-1β, IL-6, KC, and TNF-α in BAL fluid as well as the number of neutrophils in
the lung parenchyma. Therefore, we conclude that the 830nm infrared laser is
effective in reducing pulmonary inflammation in both models pulmonary or
extrapulmonary LPS-induced experimental ARDS.
Keyword:
neutrophils.
ARDS,
laser
therapy,
pulmonary
inflammation,
cytokines,
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Sumário
1. INTRODUÇÃO ................................................................................................... 13
1.1. Síndrome do Desconforto Respiratório Agudo (SDRA) ......................................... 13
1.2. Processo Inflamatório e Citocinas.............................................................................. 14
1.3. Reparação ..................................................................................................................... 15
1.4. Fatores envolvidos na coagulação .......................................................................... 166
1.5. Laser de Baixa Intensidade ......................................................................................... 16
1.5.1. Laser de Baixa Intensidade nas Doenças Pulmonares................................. 177
2. OBJETIVOS .................................................................................................... 178
3. MATERIAIS E MÉTODOS ................................................................................... 188
3.1. Animais ......................................................................................................................... 188
3.2. Grupos Experimentais................................................................................................ 188
3.3. Modelos Experimentais de Indução da SDRA ....................................................... 188
3.4. Análise da Inflamação Pulmonar.............................................................................. 189
3.5. Protocolo Experimental .............................................................................................. 199
3.5.1 Modelo de indução da SDRA Intratraqueal ...................................................... 199
3.5.2. Modelo de Indução da SDRA Extrapulmonar ................................................. 199
3.6. Aplicação com LBI ........................................................................................................ 20
3.7. Coleta de Sangue ......................................................................................................... 20
3.8. Lavado Broncoalveolar (LBA) ..................................................................................... 21
3.9. Análise dos Níveis de Citocinas ................................................................................. 21
3.10. Proteínas Totais ........................................................................................................ 201
3.11. Histologia - Análise da Densidade de Neutrófilos no Parênquima Pulmonar . 211
3.12. Análises Estatísticas......................................................................................212
4. RESULTADOS ................................................................................................... 22
4.1.Artigo submetido para revista Respiratory Physiology and Neurobiology .......... 23
5. CONSIDERAÇÕES FINAIS ................................................................................... 46
6. REFERÊNCIAS BIBLIOGRÁFICAS ...................................................................... ....48
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ANEXOS.......................................................................................................................54
Anexo I - Aprovação do Comitê de Ética em Uso de Animais (CEUA) da Uninove......54
Anexo II - Paper em segunda revisão...........................................................................57
Anexo III - Paper publicado...........................................................................................80
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LISTA DE ABREVIATURAS
SDRA – Síndrome do Desconforto Respiratório Agudo
IL – Interleucinas
LPS – Lipopolissacarídeo
TLBI – Terapia Laser de Baixa Intensidade
LBI – Laser de Baixa Intensidade
IT – Intratraqueal
IP – Intraperitoneal
LBA – Lavado Bronco Alveolar
PBS – Phosphate Buffered Saline – Tampão Fosfato Salino
mW – Miliwatt
Nm – Nanômetro
µg – Micrograma
µl - Microlitro
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1. Introdução
1.1. Síndrome do Desconforto Respiratório Agudo (SDRA)
As doenças pulmonares intersticiais compreendem uma variedade de
afecções que possuem em comum o acometimento do interstício pulmonar, por
distorção, fibrose ou destruição, sendo na maioria das vezes visualizada
radiologicamente como um infiltrado intersticial (1). SDRA é o termo utilizado
para designar a insuficiência respiratória proveniente da resposta inflamatória
que cursa com alteração da permeabilidade alvéolo-capilar, edema pulmonar e
hipoxemia refratária aos altos fluxos de oxigênio (2, 3, 4).
As anormalidades patológicas do pulmão, na SDRA, originam-se de uma
grave lesão da unidade alvéolo-capilar, seguida pelo extravasamento do líquido
intravascular, gerando edema. À medida que o processo evolui, o edema é
substituído pela necrose celular, hiperplasia epitelial, inflamação e fibrose,
caracterizando uma lesão alveolar difusa (5). A SDRA pode ser dividida em três
fases, sendo cada fase variável de acordo com o tempo e a evolução clínica da
doença: a “fase exsudativa”, de edema e hemorragia, a “fase proliferativa”, de
organização e reparação, e a “fase de fibrose”.
A fase exsudativa estende-se, geralmente, durante a primeira semana
após o início da insuficiência respiratória. A fase proliferativa é o estágio de
organização dos exsudatos intra-alveolares e intersticiais, observados na fase
aguda. Na fase fibrótica, o pulmão é totalmente remodelado por tecido rico em
fibras de colágeno. Além do colágeno, há um aumento de outras proteínas de
matriz extracelular, como a elastina, proteoglicanos e lamininas. A fibrose
compromete assim todo o sistema alvéolo-capilar, envolvida nas trocas
gasosas, levando à hipoxemia grave refratária e hipertensão arterial pulmonar,
responsáveis pela fase terminal da SDRA (5).
Muitos estudos (7) mostram que a prevalência de SDRA intrapulmonar é
maior quando comparada com a extrapulmonar, entretanto, existem estudos (8)
que demonstram uma igualdade na prevalência dos dois tipos.
O índice de mortalidade entre os insultos pulmonares e extrapulmonares
varia consideravelmente, porém alguns estudos demonstram um aumento da
mortalidade no grupo de etiologia direta (9), enquanto outros demonstram
relações entre aumento de mortalidade com insulto indireto (8).
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1.2. Processo Inflamatório e Citocinas
A barreira alveolar normal é composta de três diferentes estruturas: o
endotélio capilar, o espaço intersticial, incluindo a membrana basal e a matriz
extracelular, e o epitélio alveolar. O epitélio alveolar consiste de células
alveolares tipo I e II. A superfície de células tipo I engloba cerca de 90 % da
área
alveolar.
As
células
cubóides
alveolares
tipo
II
são
células
multifuncionais. Elas produzem surfactante, são importantes para ativar
clearence do líquido alveolar e representam as células progenitoras que
regeneram o epitélio alveolar após a injúria (2).
Em estudos histológicos de tecidos pulmonares provenientes de
pacientes com SDRA, a primeira lesão aparece como edema intersticial
seguido por lesão severa do epitélio alveolar. O epitélio alveolar usualmente
exibe extensiva necrose das células alveolares tipo I deixando uma erosão,
porém, mantendo a membrana basal recoberta com membranas hialinas. A
célula epitelial tipo I é altamente vulnerável a lesões, entretanto, a tipo II é a
célula mais resistente e pode funcionar como progenitora celular para
regeneração do epitélio após a lesão.
A interleucina 8 (IL-8) é um forte fator quimiotático para neutrófilos e é
encontrado nos pulmões em altas concentrações em pacientes com SDRA.
Os níveis de IL-8 nos pulmões também servem como fator prognóstico para o
desenvolvimento da SDRA, uma vez que foi demonstrado que pacientes com
níveis aumentados de IL-8 significativamente desenvolvem mais SDRA do
que pacientes com níveis mais baixos (11).
Estudos experimentais demonstram que em modelos de sepsis, a
cascata de citocinas consiste em TNF-α, IL-1β, IL-6, IL-1ra, sTNF-R e IL-10
(12). As duas primeiras citocinas da cascata são TNF-α e a IL-1β, sendo
produzidas localmente. Essas citocinas são usualmente referidas como próinflamatórias e tanto o TNF-α como a IL-1β, estimulam a produção de IL-6 e
ambas (IL-1β e IL-6) podem apresentar papel tanto pró-inflamatório como
antiinflamatório. O TNF-α tem sido reportado como um importante modulador
na injúria pulmonar aguda (13). Estudos experimentais têm sugerido que os
níveis plasmáticos de TNF-α aumentam durante a injúria pulmonar e,
15
bloquear seus efeitos biológicos, através de anticorpos, induz a uma
diminuição da severidade da lesão (12). Além disso, injeções intravenosas de
TNF-α induzem à injúria pulmonar aguda com seqüestro de neutrófilos e
aumento da permeabilidade microvascular (3,13).
Após o início da lesão inicial, a produção de citocinas pelo parênquima
pulmonar aumenta, as quais são liberadas pelos macrófagos e ativadas via
p38 MAPK (14).
1.3. Reparação
A perda da integridade do epitélio-alveolar gera conseqüências
funcionais e patológicas severas, como por exemplo: Um influxo de proteínas
e edema para o espaço aéreo com deposição de membrana hialina na
membrana basal lesada, hiperplasia da célula alveolar tipo II típica da fase
proliferativa da SDRA, células alveolares tipo II migrando e iniciando a
proliferação ao longo do septo alveolar na tentativa de recobrir a membrana
basal lesada e restabelecer a continuidade do epitélio alveolar. Dentro da
parede alveolar, fibroblastos proliferam e migram para a membrana basal
através do exsudato fibroso intra-alveolar. Se o exsudato fibroso pode
resolver esse processo lesivo, a restauração da arquitetura normal do pulmão
pode ser alcançada. Entretanto, se a célula alveolar tipo II migrar sobre a
superfície da organização tecidual granular, ocorre uma transformação de
exsudato intra-alveolar para tecido intersticial, e a fibrose intersticial do
pulmão pode se desenvolver (2), gerando uma reparação não eficiente, mas
sim fibrótica.
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1.4. Fatores envolvidos na coagulação
Trabalhos demonstram que a inflamação sistêmica está associada com
a ativação da coagulação e o sistema fibrinolítico. (15). O maior iniciador da
cascata de coagulação é o Tissue Factor (TF), sendo o receptor e cofator
para o fator de coagulação VII no plasma. A síntese de TF é induzida por
mediadores inflamatórios como a IL-6, IL-8, e MCP-1 (15).
Normalmente, o TF é expresso em células em contato direto com o
sangue, mas podem ser expressos em células intravasculares principalmente
monócitos e células endoteliais quando estimulados especialmente por
estímulos inflamatórios incluindo o LPS. Quando aumentado, o TF pode ser o
responsável pelas manifestações trombóticas em vários estados inflamatórios,
como ocorre na SDRA (15).
1.5. Laser de Baixa Intensidade
Laser é um dispositivo que emite luz através de um processo de
ampliação óptica baseado na emissão estimulada de radiação eletromagnética.
O termo laser originou-se da sigla light amplification by stimulated emission of
radiation ou, luz amplificada por estimulação emitida por radiação. Lasers
diferem de outras fontes de luz por possuir luz coerente (formada por ondas de
mesma freqüência e direção), colimada (onde as ondas eletromagnéticas
andam na mesma direção) e ser monocromático (possuir uma única cor) (43).
Por suas propriedades especiais, o laser é hoje utilizado nas mais diversas
aplicações: médicas (cirurgias), na Fisioterapia com o efeito antiinflamatório,
regenerador e analgésico, na indústria (cortar metais, medir distâncias),
pesquisa científica (pinças ópticas, hidráulica, física atômica, óptica quântica,
resfriamento
de
nuvens
atômicas,
informação
quântica),
comerciais
(comunicação por fibras ópticas, leitores de códigos de barras), no campo
bélico (miras lasers) e mesmo todos os dias em nossas casas (aparelhos
leitores de CD, DVD e Blu-Ray, laser pointer usado em apresentações com
projetores).
17
É produzido por materiais como o cristal de rubi dopado com safira, mistura de
gases no caso do hélio e neônio, dispositivos de estado sólido como Laser
Díodo, moléculas orgânicas como os lasers de corante (44).
No campo da pesquisa a energia laser tem sido investigada como
alternativa de tratamento nos processos de regeneração dos tecidos biológicos
há aproximadamente 20 anos. Vários trabalhos desenvolveram-se perante a
evidente necessidade de se reduzir o tempo de reparação dos tecidos,
principalmente em doenças consideradas incapacitantes e têm sido relatados
efeitos positivos da terapia laser de baixa intensidade (TLBI) no reparo de
lesões de tecidos como: músculos (17); nervos periféricos (18); pele (19) e
ossos (20,21), entre outros tipos de tecidos. (22)
1.5.1. Laser de Baixa Intensidade nas Doenças Pulmonares
Existem grandes evidências na literatura sobre os efeitos do LBI nas
doenças pulmonares (23, 24, 25, 26), alguns estudos demonstram que o laser
em combinação com outras modalidades terapêuticas apresentam significativa
melhora de pacientes com bronquite crônica, promovendo a função de
drenagem dos brônquios, facilitando a normalização do estado imunitário do
paciente, e contribuindo para a otimização dos processos de peroxidação
lipídica (27). Já outros estudos demonstraram que o laser (660nm, 30 mw), na
inflamação pulmonar aguda induzida tanto pela isquemia e reperfusão do
intestino quando pela administração de LPS inibe significativamente a
inflamação pulmonar e a liberação de citocinas pró-inflamatórias, além de
estimular a liberação da IL-10 (28). Aimbire F. ET AL relata em seus estudos
(29) que a ação do laser Ga-Al-As (685 nm) na inflamação pulmonar induzida
por LPS, reduziu as respostas inflamatórias da hiper-reatividade traqueal,
lavado bronco alveolar e a infiltração dos neutrófilos pulmonares devido a sua
interação seletiva de COX-2 com derivados de metabólitos.
2. Objetivos
Avaliar os efeitos do laser de baixa intensidade na faixa do infravermelho
(830 nm) na inflamação pulmonar aguda induzida pela administração de LPS,
tanto em modelo intrapulmonar quanto extrapulmonar de SDRA.
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3. Materiais e Métodos
3.1. Animais
Os animais foram obtidos do Biotério Central da Universidade Nove de
Julho
e
mantidos em
condições controladas de
umidade (50-60%),
luminosidade (12h claro/12h escuro) e temperatura (22°C - 25°C), água e
alimentação ad libitum. O experimento foi aprovado pelo Comitê de Ética da
Universidade Nove de Julho (CEUA) sob o n° AN0020_2013.
3.2. Grupos Experimentais
Foram utilizados 40 Camundongos BALB/c, machos, com 08 semanas
de idade, pesando aproximadamente 20 gramas, os quais foram distribuídos
aleatoriamente nos seguintes grupos experimentais: Controle (n = 08; não
administrado LPS), IT (n = 07; LPS intratraqueal 10µg/camundongo), IT + Laser
(n = 09; LPS intratraqueal 10µg/camundongo + Laser), IP (n = 07; LPS
intraperitoneal 100µg/camundongo), IP + Laser (n = 09; LPS intraperitoneal 100
µg/camundongo + Laser).
3.3. Modelos Experimentais de Indução a SDRA
O modelo experimental de indução a SDRA intrapulmonar e
extrapulmonar será o mesmo utilizado por Santos et. al., (30). Nesse trabalho,
os autores utilizaram a administração de Lipopolissacarídeo Escherichia coli
(LPS) nas doses de 10 µg (intratraqueal) e 100 µg (intraperitoneal) para o
desenvolvimento do modelo de SDRA intrapulmonar e extrapulmonar,
respectivamente. Neste estudo, os animais foram estudados 24 horas após a
administração do LPS.
3.4. Análise da Inflamação Pulmonar
Um dos mais importantes mecanismos que determina a severidade da
injúria pulmonar na SDRA é a magnitude da lesão da barreira epitelial. A
possibilidade de reparar o epitélio num estágio precoce é o maior determinante
da recuperação. Tratamentos específicos para acelerar o reparo do epitélio
alveolar ainda não existem. Muitas das modalidades terapêuticas testadas
atualmente baseiam-se na tentativa de diminuir a inflamação pulmonar para
19
minimizar a lesão inicial. Sabendo-se que grande parte do processo
inflamatório se dá pela ativação local e sistêmica de citocinas como TNF-α e IL1β.
3.5. Protocolo Experimental
3.5.1 Modelo de indução da SDRA Intratraqueal
Os animais do grupo IT (intratraqueal) e IT + L (intratraqueal + laser)
receberam LPS + solução salina 0,9% (10 µg/camundongo/ 50 µl) uma única
vez com o auxílio de uma micropipeta, sendo que os animais foram
anestesiados com Quetamina e Xilazina para este procedimento.
3.5.2. Modelo de Indução da SDRA Extrapulmonar
Os animais do grupo IP (intraperitoneal 100 µg/camundongo) e IP + L
(intraperitoneal 100 µg/camundongo + laser) receberam LPS através de
aplicação de uma única aplicação via intraperitoneal.
3.6. Aplicação do LBI
Uma hora após a administração de LPS os animais foram submetidos à
terapia com laser de baixa potência. Sendo utilizado laser com os seguintes
parâmetros:
Tipo de Laser
Infravermelho
Comprimento de onda
830 nm
Modo
Contínuo
Densidade
3J/ cm²
Potência
35 mW
Tempo de Irradiação por ponto
80 segundos
Os animais do grupo laser receberam 03 irradiações com intervalo de 1
hora entre cada irradiação diretamente em 03 pontos (conforme protocolo de
nosso grupo de estudo): 01 ponto traquéia, 01 ponto pulmão direito e 01 ponto
20
pulmão esquerdo, foram irradiados por 80 segundos cada ponto totalizando
240 segundos. Vinte quatro horas (24hrs) após a administração do LPS, os
animais foram anestesiados com Quetamina (100 x µg/kg) e Xilazina (10 x
µg/kg) (02 µ/g) e foram eutanaziados.
3.7. Coleta de Sangue
Após anestesia foi realizada uma incisão na região abdominal e através
da veia cava inferior coletado entre 0,5 ml e 1,0 ml de sangue. O sangue
coletado foi armazenado em eppendorf e utilizado 90 µl para contagem total de
células em Câmara de Neubauer, o restante foi centrifugado a 3000 RPM a 4°C
durante 10 min. O soro suspendido foi armazenado em tubo eppendorf a -70°C
para análise dos níveis de citocinas por ELISA.
3.8. Lavado Broncoalveolar (LBA)
Após coleta de sangue, foi realizada uma incisão na traquéia e os
animais foram canulados e os pulmões lavados com 03 x 0,5 ml de PBS. O
volume do lavado recuperado foi centrifugado a 1000 RPM a 4°C por 05
minutos. O sobrenadante armazenado a -70°C para posterior análise das
citocinas por meio de ELISA. O botão celular foi ressuspendido em 01 ml de
PBS e a determinação do número de células totais no LBA foi realizada por
meio de contagem na Câmara de Neubauer (31, 32, 33, 34). Alíquotas do
material ressuspendido foram utilizadas para preparação de lâminas de
cytospin as quais foram coradas com May-Grunwald-Giemsa (onde 300 células
foram contadas para a determinação da contagem diferencial) (31, 32, 33, 34).
3.9. Análise dos Níveis de Citocinas
Os níveis de IL-1β, IL-6, IL-10, TNFα e KC no LBA e no soro, foram
avaliados utilizando kits comerciais de ELISA de acordo com as instruções do
fabricante (B & D Biosciences, Califórnia, EUA).
3.10. Proteínas Totais
Os níveis de proteínas totais no LBA foram avaliados através do kit BCA
da Thermo Scientific de acordo com as instruções do fabricante.
21
3.11. Histologia - Análise da Densidade de Neutrófilos no Parênquima
Pulmonar
Após a coleta do sangue e do LBA, os pulmões foram removidos em
bloco e fixados em formol 10% durante 24 horas e submetidos à rotina
histológica. As lâminas contendo os cortes dos pulmões em 05 µm foram
coradas com hematoxilina e eosina (HE). Com o intuito de avaliar os efeitos do
laser sobre a densidade de neutrófilos no parênquima pulmonar, foram
fotografados 15 campos aleatórios do parênquima pulmonar num aumento de
40x e então através da análise de imagem (utilizando-se o software Image Pro
Plus 4.0), foi avaliada a área de tecido e contado o número de células
polimorfonucleares (PMN) nessa área. Assim, o número de células PMN foi
expresso em número de células por mm² de área de tecido (35,36).
3.12. Análises Estatísticas
As análises foram avaliadas através do programa Graphpad Prism 5®
por one way ANOVA, poshoc test Newman Keuls para dados paramétricos.
22
4. Resultados
4.1. Artigo submetido para a revista Respiratory Physiology and
Neurobiology
Low level laser therapy reduces acute lung inflammation in a model of
pulmonary and extrapulmonary LPS-induced ARDS
Manoel Carneiro Oliveira-Junior1, Nilsa Regina Damaceno-Rodrigues2, Flávia Regina
Greiffo1, Francine Maria Almeida3, Vanessa Roza da Silva1, Regiane Albertini1,
Rodrigo Álvaro B Lopes-Martins1, Ernesto César P Leal-Junior1, Ana Paula Ligeiro de
Oliveira1, Rodolfo P Vieira1
1- Nove de Julho University. Rua Vergueiro 239/245, São Paulo – SP, CEP 01504000, Brazil.
2- University of Sao Paulo, School of Medicine, Department of Pathology (LIM 59). Av.
Doutor Arnaldo 455, São Paulo – SP, CEP 01246-000, Brazil.
3- University of Sao Paulo, School of Medicine, Department of Clinical Medicine (LIM
20). Av. Doutor Arnaldo 455, São Paulo – SP, CEP 01246-000, Brazil.
Running head: LLLT reduces lung inflammation.
Corresponding author
Rodolfo P Vieira, PhD
Rua Vergueiro 239/245, São Paulo – SP, CEP 01504-000, Brazil.
23
Phone/Fax +55 11 3385-9222 / 3385-9066
[email protected]
Abstract
Acute respiratory distress syndrome (ARDS) is a syndrome presenting high rates of
mortality, and may result from pulmonary or extrapulmonary insults. The present study
investigated the effects of 830nm laser, 3J/cm2, 35mW, 80 seconds per point (3 points
per application), on the pulmonary inflammation, using a pulmonary (orotracheal) and
also an extrapulmonary (intra-peritoneal) model of LPS-induced ARDS. The laser
application was performed in direct contact with skin in three points of the chest,
beginning 1 hour after LPS administration, for 3 times. BALB/c male mice were
distributed in Control (n=6; PBS), ARDS IT (n=7; LPS orotracheally administered
10ug/mouse), ARDS IP (n=7; LPS intra-peritoneally administered 100ug/mouse),
ARDS IT + Laser (n=9; LPS intra-tracheally administered 10ug/mouse), ARDS IP +
Laser (n=9; LPS intra-peritoneally administered 100ug/mouse). Twenty-four hours after
last LPS administration, mice were studied for pulmonary inflammation by total and
differential cell count in bronchoalveolar lavage (BAL), cytokines (IL-1beta, IL-6, KC
and TNF-alpha) levels in BAL fluid and also by quantitative analysis of neutrophils
number in the lung parenchyma. The results demonstrated that LLLT significantly
reduced pulmonary and extrapulmonary inflammation in LPS-induced ARDS in both
experimental settings, as demonstrated by reduced number of total cells (p<0.001) and
neutrophils (p<0.001) in BAL, reduced levels of IL-1beta, IL-6, KC and TNF-alpha in
BAL fluid and in serum (p<0.001), as well as the number of neutrophils in lung
parenchyma (p<0.001). Therefore, we conclude that infra-red 830nm laser is effective
to reduce pulmonary inflammation in both pulmonary and extrapulmonary model of
LPS-induced ARDS.
Key words: ARDS, LPS, LLLT, lung inflammation, cytokines, bronchoalveolar lavage.
24
1. Introduction
The acute respiratory distress syndrome (ARDS) is defined as respiratory
failure from inflammatory response that leads to alteration of alveolar-capillary
permeability, pulmonary edema and hypoxemia refractory to high oxygen flow [The
ARDS Definition Task Force., 2012; Matute-Bello et al., 2008]. Although several
causes of ARDS result in a uniform pathology, in the last stage, evidence suggests that
the pathophysiology may differ according to the type of primary insult. Thus, two forms
of ARDS have been described: ARDS with direct effects on lung epithelial cells; ARDS
reflecting lung involvement secondary to a systemic inflammatory response, being the
center of the injury, the pulmonary endothelial cell [The ARDS Definition Task
Force, 2012; Matute-Bello et al., 2008].
Many studies show that the prevalence of intrapulmonary ARDS is higher when
compared with extrapulmonary [Silva et al., 2009]. However [Eisner et al., 2001]
demonstrate an equal prevalence of both types, ant this issue remains controversial
[Eisner et al., 2001]. From pulmonary causes, pneumonia is the most direct cause of
injury, followed by aspiration of gastric contents and pulmonary trauma [Silva et al.,
2009]. The rate of death from pulmonary and extrapulmonary insults varies
considerably, however, [Suntharalingam et al., 2001], shows an increase in mortality in
the group of direct etiology, while [Eisner et al., 2001] found a direct relationship
between lung injury and increased mortality.
The scientific literature has reported anti-inflammatory effects of low-level laser
therapy (LLLT) in models of acute lung injury [De Lima et al., 2011, 2013].
Furthermore, a growing number of clinical studies are demonstrating the efficacy and
safety of LLLT for different pulmonary diseases, as asthma and chronic obstructive
pulmonary diseases (COPD) [Landyshev et al., 2002; Faradzheva et al. 2007;
Farkhutdinov et al. 2007; Kashanskaia et al., 2009]. For instance, some studies also
25
have demonstrated that application of LLLT for the treatment of patients with chronic
obstructive bronchitis accelerates the elimination of clinical symptoms, increases its
efficiency, promotes drainage function of the bronchi, facilitates standardization the
immune status of the patient, and contributes to the optimization of lipid peroxidation
[Farkhutdinov et al., 2007; Kashanskaia et al., 2009].
Therefore, the present study was designed aiming to fill a lack of information
regarding the effects of LLLT in a model of pulmonary and extrapulmonary LPSinduced ARDS in BALB/c mice.
2.
Materials and Methods
2.1.
Animals and Experimental Groups
Thirty-eight male BALB/c mice weighing between 25-30g were obtained from
the Animal Facility of the Nove de Julho University. All experimental procedures with
animals care followed the international recommendations for the use and care of
animals and were approved by the local ethical committee. All mice were housed in
bright rooms with controlled temperature (21°-23°C) and humidity (45%-65%) and 1212h light/dark cycle, with access to food and water ad libitum.
The animals were divided into 5 groups: Control (n=6), LPS orotracheal (n=7),
intra-peritoneal LPS (n=7), orotracheal LPS + laser (n=9), intra-LPS Laser peritoneal +
(n=9).
26
2.2 .
Pulmonary and Extrapulmonary Model of LPS-Induced ARDS
For the pulmonary model of LPS-induced ARDS, under anesthesia (ketamine
100mg/kg and xylazine 10mg/kg), using a 100ul micropipette, animals received LPS
(10ug/mouse) diluted in 50ul of PBS through an orotracheal instillation as previously
described [Vieira et al., 2011]. For the extrapulmonary model of LPS-induced ARDS,
animals received LPS (100ug/mouse) diluted in 50ul of PBS through an intra-peritoneal
injection.
2.3.
LLLT Protocol
One hour after LPS administration, LLLT treated groups received infrared laser
administration [continuous wave, 830nm, 3J/cm², 35MW, 80 seconds per point (3
points per application)], where point 1 was in the end part of trachea, point 2 in the right
lung and the point 3 in the left lung, in direct contact with skin. These 3 points
application totalized 240 seconds and an energy of 9J/cm². In total, LLLT groups
received the LLLT as described above for 3 times, in a 1 hour interval between each
application.
2.4.
Blood Collection, Processing and Analysis
Under anesthesia, the abdomen was open the 1 ml of blood was collected via
cava vein using a syringe without anti-coagulant and immediately centrifuged at 950 g,
4°C, during 7 minutes. The serum was collected and stored at -70°C for cytokines
measurement.
27
2.5.
Bronchoalveolar Lavage Fluid (BALF)
Aiming to access lung inflammation, the number of total and differential cells
count in BALF was performed. Briefly, under anesthesia, mice were submitted to
tracheotomy and canulated. Then, using a 1 ml syringe, a 3 x 0,5mL PBS washing was
applied and the recovery material was centrifuged at 800 g, at 4°C during 7 minutes.
The supernatant was stored at -70°C for cytokines analysis and the cell pellet was
ressuspended in 1 ml PBS. The number of total cells was counted using a
hematocytometer (Neubauer chamber) and the differential cells count were performed
through a cytospin preparation, stained with Diff Quick and 300 cells were counted
according to the hematological characteristic [Gonçalves et al., 2012; Ramos et al.,
2010].
2.6.
Inflammatory Mediators in BALF and in Serum
The levels of pro-inflammatory cytokines IL-1beta, IL-6, KC and TNF-alpha and
of anti-inflammatory cytokine IL-10 was evaluated in the BALF according to the
manufacturer’s instructions.
2.7.
Histomorphometric Study
To evaluate the effects of LLLT on parenchymal inflammation, one the
hallmarks of ARDS, the lungs were collected, fixed in 10% formalin and submitted to
histological routine. Briefly, 5 µm ticks lung slices were stained with hematoxylin and
eosin. Then, 15 aleatory fields of the lung parenchyma of each mouse were
photographed. By using the software Image Pro Plus 4.0, the air and tissue area of all
photomicrographs were determined. The number of polymorphonuclear (PMN) cells
(notably neutrophils) was counted in each photo according the morphological criteria by
an experienced research, blinded to the group’s description. Then, the number of PMN
cells per square millimeter of lung tissue was presented.
28
3. Results
3.1.
Inflammation in Bronchoalveolar Lavage Fluid (BALF) and in Lung
Tissue in the Pulmonary Model of ARDS
The figure 1 shows the inflammatory profile in BALF (total cells – panel 1A;
neutrophils – panel 1B) and the number of polymorphonuclear cells (notably
neutrophils – panel 1C) and the representative photomicrographs of control (panel 1D),
LPS IT (panel 1E) and LPS IT + laser (panel F) in the pulmonary (IT) model of ARDS.
The results shows that intra-tracheal administration of LPS significantly increased the
number of total cells (p<0.001) and neutrophils (p<0.001) in BALF when compared with
control group. On the other hand, LLLT significantly reduced the number of total cells
(p<0.001) and neutrophils (p<0.01) when compared with LPS group. LLLT also
significantly reduced the number of polymorphonuclear cells in the lung parenchyma
(p<0.001; panels 1C until 1F).
3.2.
Cytokines Levels in BALF in the Pulmonary Model of ARDS
The figure 2 shows the levels of IL-1beta, IL-6, KC, TNF-alpha and IL-10 in
BALF in a pulmonary model of ARDS (panels 2A to 2E, respectively). Panel 2A to 2D
shows that LLLT significantly reduced intra-tracheal LPS increased IL-1beta, IL-6, KC
and TNF-alpha (p<0.05). Panel 2E shows that no differences in the levels of IL-10 were
found when all groups were compared (p>0.05).
3.3.
Cytokines Levels in Serum in the Pulmonary Model of ARDS
The figure 3 shows the serum levels of IL-6 and TNF-alpha in a pulmonary
model of ARDS (panels 3A and 3B, respectively). In the panel 3A, the results show that
LLLT significantly reduced intra-tracheal LPS increased IL-6 levels (p<0.01). In panel
3B, the results show that LLLT significantly reduced intra-tracheal LPS increased TNFalpha levels (p<0.001).
29
3.4.
Inflammation in Bronchoalveolar Lavage Fluid (BALF) and in Lung
Tissue in the Extrapulmonary Model of ARDS
The figure 4 shows the inflammatory profile in BALF (total cells – panel 4A;
neutrophils – panel 4B) and the number of polymorphonuclear cells (notably
neutrophils – panel 4C) and the representative photomicrographs of control (panel 4D),
LPS IP (panel 4E) and LPS IP + laser (panel 4F) in the extrapulmonary (IP) model of
ARDS. The results shows that intra-peritoneal (IP) administration of LPS significantly
increased the number of total cells (p<0.001) and neutrophils (p<0.001) in BALF when
compared with control group. On the other hand, LLLT significantly reduced the
number of total cells (p<0.001) and neutrophils (p<0.001) when compared with LPS
group. LLLT also significantly reduced the number of polymorphonuclear cells in the
lung parenchyma (p<0.001; panels 4C until 4F).
3.5.
Cytokines Levels in BALF in the Extrapulmonary Model of ARDS
The figure 5 shows the levels of IL-1beta, IL-6, KC, TNF-alpha and IL-10 in
BALF in a pulmonary model of ARDS (panels 5A to 5E, respectively). Panel 5A shows
that intra-peritoneal LPS administration significantly increased the levels of IL-1beta
(p<0.001), while LLLT significantly its levels, compared with LPS group (p<0.01). Panel
5B and 5C shows that intra-peritoneal LPS administration significantly increased the
levels of IL-6 (p<0.001) and KC (p<0.001), respectively, while LLLT significantly its
levels, compared with LPS group (p<0.001). Panel 5D shows that while intra-peritoneal
LPS administration significantly increased the levels of TNF-alpha (p<0.01), LLLT
significantly reduced its levels (p<0.01). Similarly to intra-tracheal model of intrapulmonary ARDS, in the extrapulmonary model of ARDS (intra-peritoneal LPS
administration), no differences were observed in the levels of IL-10 (p>0.05).
30
3.6.
Cytokines Levels in Serum in the Pulmonary Model of ARDS
The figure 6 shows the serum levels of IL-6 and TNF-alpha in an
extrapulmonary model of ARDS (panels 6A and 6B, respectively). In the panel 6A, the
results show that LLLT significantly reduced intra-peritoneal LPS increased IL-6 levels
(p<0.001). In panel 6B, the results show that LLLT significantly reduced intra-peritoneal
LPS increased TNF-alpha levels (p<0.05).
4. Discussion
The present study showed for the first time the effects of LLLT (830nm)
reducing the acute pulmonary inflammation in a pulmonary and extrapulmonary model
of LPS-induced ARDS in BALB/c mice, revealing that LLLT (830nm) may inhibit acute
pulmonary inflammation independent of etiology of primary insult.
Acute respiratory distress syndrome (ARDS) presents high rates of morbidity
and mortality and the amount and the state (activation and apoptosis rate) of the
neutrophils may be correlated with the diseases severity and prognosis [Fialkow et al.,
2006]. In the present study, we found that both models (pulmonary and
extrapulmonary) of LPS-induced ARDS significantly increased the migration of
neutrophils to the lungs, accordingly to the previous studies [Matute-Bello et al., 2008;
Silva et al., 2009; Gonçalves et al., 2012; Ramos et al., 2010]. In the physiopathology
of ARDS, neutrophils contribute to the lung injury releasing several mediators, i.e. free
radicals,
proteases,
cytokines
and
chemokines
[Matute-Bello
et
al.,
2008].
Furthermore, the activation of neutrophils has been directly linked with ARDS’ severity
and mortality [Fialkow et al., 2006]. In this way, our results showed that LLLT was
effective to reduce the migration of neutrophils to the lungs, as demonstrated through
neutrophils counting in bronchoalveolar lavage and also by the quantitative analysis of
the neutrophils number in the lung parenchyma. These anti-inflammatory effects of
LLLT on neutrophils recruitment is particularly important, since that such effect was
31
observed in pulmonary and extrapulmonary model of LPS-induced ARDS, reinforcing
the beneficial effects of LLLT independent of the diseases etiology. This results are
also in agreement with previous studies that have demonstrated that LLLT was able to
reduce neutrophils migration in model of intestinal ischemia-reperfusion induce ARDS
[De Lima et al., 2011, 2013].
The modulation of neutrophilic inflammation in ARDS have been attributed to
release of several pro-inflammatory cytokines, for instance, IL-1beta, IL-6, IL-8 and
TNF-alpha [Matute-Bello et al., 2008]. Interleukin 1 beta (IL-1beta) is a potent proinflammatory cytokine and its increased levels in patients developing ARDS are related
with poor prognosis of disease [Meduri et al., 1995]. IL-1beta is thought to play a
central role in the beginning of inflammatory process and the neutrophils to be the main
source of IL-1beta release in during diverse inflammatory response [Cho et al., 2012].
IL-1beta also increases neutrophils survival, contributing for non-resolution of the
inflammation [Cho et al., 2012]. In the present study we found increased levels of IL1beta in both, pulmonary and extrapulmonary models of ARDS, in agreement with the
current literature [Matute-Bello et al., 2008; Cho et al., 2012]. The present study also
revealed that LLLT was capable to decrease the levels of IL-1beta in both models of
ARDS, pointing out the inhibitory effects of LLLT on the pro-inflammatory mediators
involved in the physiopathology of ARDS. Of note, a study has been found similar
results concerning the suppressive effects of LLLT on the levels of IL-1beta, however,
in a model of extra-pulmonary LPS-induced ARDS in rats [Aimbire et al., 2008].
Interleukin 6 (IL-6) is considered a pleiotropic cytokine, presenting a central role
in the physiopathology of ARDS, beyond to be correlated with poor prognostic for
disease [Meduri et al., 1995; Cho et al., 2012; Sharifov et al., 2013; Rojas et al., 2013].
The levels of IL-6 are increased in the lungs and also in the blood of humans and also
in animal’ models of ARDS [Meduri et al., 1995; Cho et al., 2012; Sharifov et al., 2013;
Rojas et al., 2013]. In the present study we found increased levels of IL-6 in
32
bronchoalveolar lavage fluid and in serum of mice in both pulmonary and
extrapulmonary model of LPS-induced ARDS. Of note, in both models, LLLT was able
to significantly reduce IL-6 levels in bronchoalveolar lavage fluid and also in serum, to
values very close to values of control group. These findings are extremely relevant,
since that increased levels of IL-6 are involved in the perpetuation of the inflammatory
state and also in pro-coagulant response in ARDS [Meduri et al., 1995; Fu et al., 2012].
Interleukin 8 (IL-8) and its functional homologue in mice (CXCL1/KC) present a
central role in the physiopathology of ARDS, primarily mediating the chemotaxis for
neutrophils [Meduri et al., 1995; Cho et al., 2012]. However, IL-8 and CXCL1/KC also
presents other important effects in the inflammatory process in ARDS, for instance,
increasing of neutrophils survival [Meduri et al., 1995; Cho et al., 2012; McGettrick et
al., 2006], and also are related with ARDS severity and mortality. In the present study
we found that in both models (pulmonary and extrapulmonary) of LPS-induced ARDS
the pulmonary levels of CXCL1/KC are significantly elevated. On the other hand, in the
present study, we also found that LLLT significantly reduced the pulmonary levels of
CXCL1/KC, event that may be involved in the anti-inflammatory effects of LLLT.
Tumor necrosis factor alpha (TNF-alpha) is a cytokine involved in neutrophils
adhesion and activation, and coagulation and edema formation, especially during
events of acute lung inflammation [Souza et al., 2002; Aimbire et al., 2006]. This
cytokine is accredited to be involved in IL-6 stimulation and release, playing a central
role in the inflammatory process in ARDS [Souza et al., 2002; Aimbire et al., 2006].
Also, increased levels of TNF-alpha are found in the lungs and also in the systemic
circulation of patients developing ARDS, reinforcing its role in the pathophysiology of
the disease [Meduri et al., 1995; Sharifov et al., 2013; Rojas et al., 2013]. In the
present study we found that the pulmonary and the extra-pulmonary model of LPSinduced ARDS coherently induced increases in the BALF and serum levels of TNFalpha. On the contrary, LLLT significantly reduced the TNF-alpha levels in both models
33
and also in both sites, in the lungs (in BALF) and also in the systemic circulation (in
serum). These inhibitory effects of LLLT are particularly important, considering the
potent pro-inflammatory effects and the central role of
TNF-alpha in the
pathophysiology of ARDS. Also, these results are in agreement with previous studies
that have demonstrated that LLLT significantly reduced the mRNA expression of TNFalpha in a model of immune-complex induce lung injury [Aimbire et al., 2006] and also
in an ex-vivo study using rat bronchi, where LLLT reduced bronchi hyper reactivity to
cholinergic agonist through a TNF-alpha dependent mechanism [Mafra et al., 2009].
Therefore, we conclude that LLLT present important anti-inflammatory effects
against the LPS-induced acute respiratory distress syndrome, independent of etiology
of disease.
34
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20. Ramos DS, Olivo CR, Lopes FDTQS, Toledo AC, Martins MA, Osório RAL,
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39
Figures and Figures Legends
Figure 1
Figure 1 – Inflammatory profile in BALF (total cells – panel A; neutrophils – panel B)
and the number of polymorphonuclear cells in the lung parenchyma (notably
neutryphils – panel C) and the representative photomicrographs of control (panel D),
LPS i.t. (panel E) and LPS i.t + laser (panel F) in the pulmonary (IT) model of ARDS. In
panel A, B and C, ***p<0.001; **p<0.01 and *p<0.05.
40
Figure 2
Figure 2 – Cytokines levels (IL-1beta, IL-6, KC, TNF-alpha and IL-10) in BALF in a
pulmonary (IT) model of ARDS. In panel A, B, C and D, *p<0.05.
41
Figure 3
Figure 3 – Cytokines levels (IL-6 and TNF-alpha) in serum in a pulmonary (IT) model
of ARDS (panels A and B, respectively). In panel A, **p<0.01 and in panel B,
***p<0.001.
42
Figure 4
Figure 4 – Inflammatory profile in BALF (total cells – panel A; neutrophils – panel B)
and the number of polymorphonuclear cells in the lung parenchyma (notably
neutryphils – panel C) and the representative photomicrographs of control (panel D),
LPS i.t. (panel E) and LPS i.t + laser (panel F) in the extra-pulmonary (IP) model of
ARDS. In panel A, B and C, ***p<0.001.
43
Figure 5
Figure 5 – Cytokines levels (IL-1beta, IL-6, KC, TNF-alpha and IL-10) in BALF in a
extrapulmonary (IP) model of ARDS. In panel A, B, C and D, ***p<0.001 and **p<0.01.
44
Figure 6
Figure 6 – Cytokines levels (IL-6 and TNF-alpha) in serum in an extrapulmonary (IP)
model of ARDS (panels A and B, respectively). In panel A, ***p<0.001 and in panel B,
*p<0.05.
45
5. Considerações Finais
O presente estudo mostrou pela primeira vez os efeitos da laserterapia
(830 nm) em uma comparação entre um modelo de SDRA intrapulmonar e
extrapulmonar em camundongos BALB/c, revelando que laser (830 nm) foi
eficaz na redução da inflamação pulmonar em ambos modelos experimentais.
A síndrome do desconforto respiratório agudo (SDRA) apresenta altas
taxas de mortalidade, morbidade, da quantidade e do estado (ativação e taxa
de apoptose) dos neutrófilos que podem ser correlacionados com o prognóstico
de doenças. No presente estudo, verificou-se que ambos os modelos
(intrapulmonar e extrapulmonar) de SDRA induzido por LPS aumentou
significativamente a migração dos neutrófilos para os pulmões, de acordo com
a literatura (37, 38, 39, 40). Além disso, nossos resultados mostraram também
que a laserterapia foi eficaz na redução da migração de neutrófilos para os
pulmões, tal como demonstrado por meio da contagem de neutrófilos no lavado
bronco alveolar e também no parênquima pulmonar.
Em consideração aos resultados, embora os apresentados aqui foram
obtidos a partir de um modelo experimental, nosso experimento demonstrou
que a inflamação pulmonar das vias aéreas foi reduzida com LBI através de
uma forma não invasiva, podemos propor que o laser de baixa intensidade
pode ser amplamente utilizado como uma terapia coadjuvante no tratamento
clínico de desordens pulmonares e como uma alternativa na tratamento da
inflamação pulmonar aguda, levando em consideração a reflexão, absorção e
penetração que podem influenciar diretamente o tecido a ser tratado, devido a
quantidade de luz penetrada, apesar do fato de que existe pouca informação
sobre como a luz pode modular o processo inflamatório pulmonar através de
alguns minutos de irradiação. Com isto em mente, é claro que uma
compreensão do mecanismo de ação da luz em lesão pulmonar aguda após
indução a SDRA seriam úteis para o desenvolvimento de tipos de tratamentos.
Concluímos que o presente estudo mostra a laser terapia como uma
excelente estratégia para o tratamento de SDRA, considerando que o laser
diminuiu as citocinas pró-inflamatórias e aumentou as citocinas inflamatórias
46
como demonstrado, possivelmente melhorando a função respiratória. No
entanto, mais estudos com o objetivo de entender os mecanismos celulares do
laser e os processos envolvidos nos efeitos antiinflamatórios devem ser
estudados.
47
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53
ANEXOS
Anexo I
Aprovação do Comitê de Ética em Uso de Animais (CEUA) da Uninove
54
55
56
Anexo II – Paper em segunda revisão
Exercise deactivates leukocytes in asthma
Rodolfo P Vieira1,2*, Ronaldo A Silva2, Manoel C Oliveira-Junior2, Flávia R Greiffo1, Ana
Paula Ligeiro-Oliveira1, Milton A Martins3, Celso R F Carvalho2
1- Nove de Julho University (UNINOVE), Sao Paulo, Brazil.
2- University of Sao Paulo, School of Medicine (LIM 34), Sao Paulo, Brazil.
3- University of Sao Paulo, School of Medicine (LIM 20), Sao Paulo, Brazil.
*Corresponding author
Rodolfo P Vieira, BSc, PhD.
Nove de Julho University (UNINOVE)
Laboratory of Pulmonary and Exercise Immunology (LIPEX)
Rua Vergueiro 239/245, Vergueiro
01504-000, São Paulo – SP, Brazil
Phone +55 11 3385-9222
Fax +55 11 3385-9222
Running title: Exercise reduces lung inflammation.
Section: Immunology
57
Abstract
Leukocytes play a central role in asthma physiopathology. Aerobic training (AT)
reduces leukocytes recruitment to the airways, but the effects of AT on some aspects
of leukocytes activation in asthma are unknown. Therefore, the effects of 4 weeks of
AT on airway inflammation, pulmonary and systemic Th2 cytokines levels, leukocytes
expression of pro and anti-inflammatory, pro-fibrotic, oxidants and anti-oxidants
mediators in an experimental model of asthma was investigated. AT reduced the levels
of IL-4, IL-5, IL-13 in bronchoalveolar lavage fluid (BALF) (p<0.001), serum levels of IL5, while increased BALF and serum levels of IL-10 (p<0.001). In addition, AT reduced
leukocytes activation, showed through decreased expression of Th2 cytokines (IL-4, IL5, IL-13; p<0.001), chemokines (CCL5, CCL10; p<0.001), adhesion molecules (VCAM1, ICAM-1; p<0.05), reactive oxygen and nitrogen species (GP91phox and 3nitrotyrosine; p<0.001), inducible nitric oxide synthase (iNOS; p<0.001), nuclear factor
kB (NF-kB; p<0.001) while increased the expression of anti-inflammatory cytokine (IL10; p<0.001). AT also decreased the expression of growth factors (TGF-beta, IGF-1,
VEGF and EGFr; p<0.001). We conclude that AT reduces the activation of
peribronchial leukocytes in a mouse model of allergic asthma, resulting in decreased
airway inflammation and Th2 response.
Key words: asthma, exercise, immunology, allergy, cytokines.
58
Introduction
A growing number of studies point out the beneficial effects of regular practice
of aerobic training (AT) for the management of asthmatic individuals [2, 5, 6, 7, 9, 13,
19, 23, 24, 25, 26, 30, 31, 32, 36]. In summary, these studies demonstrate that AT
significantly improves asthma symptoms, including dyspnea and exercise-induced
bronchoconstriction (EIB), health-related quality of life, and also reduces corticosteroid
needing as well as reduces the levels of exhaled nitric oxide, suggesting a possible
anti-inflammatory effects of AT for the airways [7, 9, 23, 24, 25, 26, 30, 31]. More
recently, a study from Mendes et al (2011) demonstrated for the first time that AT
reduces eosinophilic inflammation in asthmatic patients, confirming the antiinflammatory effects of AT [24]. However, the mechanisms involved in the antiinflammatory effects of AT for asthma remains not fully elucidated.
In this way, a growing number of experimental studies have been performed
aiming to investigate the possible cellular and molecular mechanism underlying the
anti-inflammatory effect of AT in animals’ models of asthma, currently experimental
models of acute and chronic allergic airway inflammation [11, 12, 27, 28, 29, 34, 35,
37, 38, 39]. In general, these studies have demonstrated that AT reduces eosinophilic
and lymphocytic airway inflammation, Th2 cytokines production, nuclear factor kB (NFkB) activation, while increases the expression of anti-inflammatory cytokines IL-1ra and
IL-10 [11, 12, 27, 28, 29, 34, 35, 37, 38, 39]. From these studies, some initial evidences
of the cellular and molecular effects of AT in experimental models of acute and chronic
allergic airway inflammation were identified. For instance, Pastva et al., 2004 and 2005
demonstrated that part of the anti-inflammatory effects of AT could be attributed to
reduced NF-kB activation and glucocorticoid receptor expression in peribronchial
leuckocytes and also in airway epithelium [28, 29]. Following Pastva’s study, Vieira et
al., 2007 demonstrated that AT also induces the production of anti-inflammatory
59
cytokine IL-10 [37], a finding that was further confirmed by Silva et al., 2010, that
elegantly added the stimulatory effect of AT on IL-1ra expression [35].
However, the literature demonstrates that the leukocytes are responsible also
for the release of Th2 cytokines, growth factors and oxidants, which play a central role
in the inflammatory and remodeling process in asthma [3, 17, 18, 20, 21, 22, 33].
Therefore, the present study investigate the effects of AT on chronic allergic airway
inflammation, focusing on the effects of AT on peribronchial leukocytes activation (i.e.
expression of pro-inflammatory, anti-inflammatory, pro-fibrotic, oxidants and antioxidants and growth factors by leukocytes) involved in the inflammatory and
remodeling process in asthma.
Materials and Methods
This study was approved by the ethical committee of the School of Medicine of
the University of Sao Paulo. The “Guide for care and use of laboratory animals” was
followed (NIH publication 85-23, revised 1996). In addition, we state that the present
manuscript is in accordance to the IJSM’s ethical standard [10].
Animals and Experimental Groups
Thirty-two BALB/c male mice (20-25 g) were distributed in control (Control; n =
8), aerobic training (AT; n = 8), ovalbumin sensitized (OVA; n = 8) and ovalbumin
sensitized + aerobic training (OVA+AT; n = 8) groups.
We state that the immunohistochemical and the cytokines measurements in
bronchoalveolar lavage fluid (BALF) were performed in the samples of previous study
[37-39]. [
60
Treadmill Training and Test Protocol
Animals were adapted to treadmill training (15 min, 25% inclination and 0.2
km/h) during 3 days. In the following day, all animals were submitted to maximal
exercise test, as previously described [37, 38]. The physical test was repeated 30 days
after the beginning of AT. The results from physical test were presented in the previous
study [39]. The treadmill physical training was performed during 4 weeks, 5x/week, 60
minutes per session, at low intensity (corresponding to 50% of maximal exercise
capacity reached in the maximal exercise test). The exercise has started one day after
the first OVA or saline inhalation exposure [39].
Chronic Model of Allergic Asthma
Four intra-peritoneal (i.p.) injections of OVA (20ug per mouse) adsorbed with
aluminum hydroxide or saline solution for control groups (non-sensitized mice) were
performed on days 0, 14, 28 and 42. Twenty-one days after the first i.p. injection, mice
were challenged with aerosolized OVA (1%) or with a saline solution 3 times a week
until the 50th day [37, 38, 39].
Anesthesia and Animals’ Euthanasia
Seventy-two hours after the last inhalation day and exercise test, animals were
anesthetized by intramuscular injection of ketamine (50 mg/kg) and xylazine (40
mg/kg), and tracheostomized to collect bronchoalveolar lavage fluid (BALF). The blood
was collected through the abdominal vein for the cytokines quantification, followed by
euthanasia through exsanguinations.
Bronchoalveolar Lavage Fluid (BALF) Procedures
Lungs were gently washed with 1.5 ml of saline (administered as three 0.5ml
volumes) via the tracheal cannula. Total cell counts were performed using a
hematocytometer (Neubauer chamber) and the differential cell counts (300 cells per
61
lamina) were performed using cytospins preparations stained with May-GrunwaldGiemsa [27, 34, 37]. We clarify that the results of total and differential cell count was
already presented in the following previous study [39].
Cytokines Measurements
The levels of IL-4, IL-5, IL-10 and IL-13 were quantified in bronchoalveolar
lavage and in serum by ELISA using commercial kits (BD Elispot kit, CA, USA)
according to the manufacturer recommendation.
Lung Histology, Immunohistochemistry and Morphometic Analysis
Lungs were fixed in formalin and embedded in paraffin. Five-micrometer thick
sections were stained with hematoxylin and eosin for lung structure and inflammation
analysis [37]. Immunohistochemistry was performed with anti–IL-4, anti–IL-5, anti–IL10,
anti–IL-13, anti-CCL5, anti-CCL10, anti-VCAM-1, anti-ICAM-1, anti-GP91phox, anti-3nitrotyrosine, anti-NF-kB, anti-iNOS, anti-TGF-beta, anti-IGF-1, anti-VEGF and antiEGFr antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), using a biotin–
streptavidin–peroxidase method. With a 50-line, 100-point grid connected to the ocular
of the microscope, we assessed the peribronchial density of positive leukocytes for the
markers described above, using a point-counting technique [37]. Counting was
performed in 5 complete airways for each animal at 1,000x magnification. Results were
expressed as positive cells per square millimeter [37].
62
Statistical Analysis
Parametric and nonparametric data were expressed as means ± SD and as
medians ± 95% confidence interval (95% CI), respectively. Comparisons among
groups were performed by one-way analysis of variance followed by the StudentNewman-Keuls post hoc test (parametric data) or by one-way analysis of variance on
ranks followed by Dunn’s post-hoc test (nonparametric data); the significance level was
adjusted to 95% (p<0.05).
Results
BALF Levels of Pro-inflammatory Th2 and Anti-inflammatory Cytokines
Profile
The levels of pro-inflammatory Th2 cytokines (IL-4, IL-5, IL-13) and antiinflammatory cytokine (IL-10) in BALF are presented in Figure 1A–1D, respectively.
The results demonstrated that AT significantly reduced the levels of IL-4, IL-5 and IL-13
when compared with OVA group (p<0.01). The results also demonstrated that AT
significantly increased the levels of IL-10 in both non-sensitized (AT) and sensitized
(OVA+AT) groups (p<0.05).
Systemic Th2 (IL-5) and Anti-inflammatory (IL-10) Response
The levels of pro-inflammatory Th2 cytokine IL-5 and anti-inflammatory cytokine
IL-10 are presented in Figure 2A–2B, respectively. The results demonstrated that AT
significantly reduced the levels of IL-5 compared with OVA group (Figure 2A; p<0.01).
The results also demonstrated that AT significantly increased the levels of IL-10 in both
non-sensitized (AT) and sensitized (OVA+AT) groups (Figure 2B; p<0.05).
63
Peribronchial Leukocytes Expression of Th2 and Th1 Cytokines,
Chemokines and Adhesion Molecules
The expression of Th2 cytokines, Th1 cytokines, chemokines and adhesion
molecules are presented in Figure 3A–3D, respectively. The results demonstrated that
AT significantly reduced the expression of Th2 cytokines (IL-4, IL-5 and IL-13) by
leukocytes when compared with OVA group (Figure 3A; p<0.001). The expression of
Th1 cytokines (IL-2 and IFN-gamma) were not changed when compared all
experimental groups (Figure 3B; p>0.05). The results also demonstrated that AT
significantly reduced the expression of chemokines (CCL11 and CCL5) when
compared with OVA group (Figure 3C; p<0.01). In addition, AT also significantly
reduced the expression of adhesion molecules (VCAM-1 and ICAM-1) when compared
with OVA group (Figure 3D; p<0.01).
Expression of Oxygen and Nitrogen Reactive Species, Anti-inflammatory
Cytokine and NF-kB by Peribronchial Leukocytes
The expression of Gp91Phox and 3-nitrotyrosine (Figure 4A), iNOS (Figure 4B),
IL-10 (Figure 4C) and NF-kB (Figure 4D) are presented in Figure 4. The results
demonstrated that AT significantly reduced the expression of Gp91Phox and 3nitrotyrosine (Figure 4A; p<0.001). The results also demonstrated that AT significantly
reduced the iNOS expression (Figure 4B; p<0.001). On the other hand, AT in
sensitized mice significantly increased the expression of anti-inflammatory cytokine IL10 (Figure 4C; p<0.01). In addition, AT also significantly reduced the NF-kB expression
(Figure 4D; p<0.001).
64
Peribronchial Leukocytes Derived Growth Factors
Figure 5 A-D shows the expression of growth factors TGF-beta, IGF-1, VEGF
and EGFr, respectively. The results demonstrated that AT significantly reduced OVAinduced the expression of all growth factors investigated, as TGF-beta (p<0.01), IGF-1
(p<0.001), VEGF (p<0.001) and EGFr (p<0.01).
Discussion
The present study showed for the first time that AT inhibit the lung leukocytes
activation seen in an experimental model of allergic asthma, demonstrated through the
reduced expression of cytokines, chemokines, adhesion molecules, reactive oxygen
and nitrogen species, NF-kB and growth factors by peribronchial leukocytes, while
increases the expression of the anti-inflammatory cytokine IL-10.
Leukocytes play a central role in the pathophysiology of asthma [3, 17, 18, 20,
21, 22, 33]. Leukocytes, especially Th2 leukocytes are differentiated leukocytes
responsible for release of Th2 cytokines, i.e. IL-4, IL-5 and IL-13, which exert proinflammatory and pro-fibrotic effects on asthma [3, 17, 18, 20, 21, 22, 33]. In summary,
IL-4, IL-5 and IL-13 are involved in eosinophils, dendritic cells and T-lymphocytes
differentiation, proliferation and activation, exerting their effects both in the lungs as
systemically [3, 17, 18, 20, 21, 22, 33]. In the present study we found that AT
significantly reduced not only the expression of IL-4, IL-5 and IL-13 by leukocytes but
also the BALF levels of IL-4, IL-5 and IL-13, strongly suggesting that the reduced
expression of IL-4, IL-5 and IL-13 by lung leukocytes reflect leukocytes deactivation.
Furthermore, the results also demonstrated that AT significantly reduced the serum
levels of IL-5, showing that the effects of AT on allergic response is not limited to the
lungs, but also may involve a systemic component. However, the results found in the
present study may not be applied to circulating leukocytes, an issue that should be
investigated in further studies.
65
Beyond Th2 cytokines, chemokines, as CCL5 and CCL11 present an important
role in chronic allergic airway inflammation [4]. These chemokines regulates
eosinophils trafficking to the airways and are present at increased levels in the
asthmatic airways and also related with late onset asthmatic response [4]. In the
present study, we demonstrate that AT significantly reduced the expression of CCL5
and CCL11 by leukocytes, reinforcing the anti-inflammatory milieu induced by exercise.
However, many other mediators are involved in the eosinophilic trafficking to the
airways, as adhesion molecules. Adhesion molecules, i.e. VCAM-1 and ICAM-1 are
well studied molecules in inflammatory diseases and are found abundantly in the
airways of asthmatic patients and in animal models of asthma [8, 18, 40]. Increased
expression of these molecules is thought to exert a central role in the eosinophils
adhesion and transmigration during asthmatic inflammation [3]. Again, the present
study shown that AT significantly reduced the expression of ICAM-1 and VCAM-1 by
peribronchial leukocytes, accounting to the anti-inflammatory effects of AT.
Following unresolved chronic allergic airway inflammation, airway remodeling is
a key feature of asthma and is thought to be irreversible and the main component of
airway hyperresponsiveness and obstruction [21]. Airway remodeling is characterized
by hypertrophy and hyperplasia of airway epithelial cells and smooth muscle, mucus
hypersecretion and increased deposition of extra-cellular matrix proteins in airway walls
[21]. Different proteins families are involved in the remodeling process in asthma, as
growth factors (TGF-beta, IGF-1, VEGF and EGF), matrix metalloproteases (MMPs)
and tissue inhibitor of matrix metalloproteases (TIMPs) [1, 16]. Of note, growth factors
stimulate the synthesis of extra-cellular matrix proteins and are accredited to be the
main mediators involved in remodeling [1, 16]. In the present study we show for the first
time that AT inhibited the lung expression of TGF-beta, IGF-1, VEGF and EGFr in
OVA-sensitized animals. Therefore, these results explicitly show that AT may inhibit the
airway remodeling process.
66
As part of the anti-inflammatory and anti-fibrotic effects of AT in chronic allergic
airway inflammation, we have investigated the effects of AT on the expression of
reactive oxygen species (ROS), reactive nitrogen species (RNS), anti-inflammatory
cytokine IL-10 and nuclear transcription factor NF-kB. Regarding the role of ROS and
RNS in the pathogenesis of asthma, the literature clearly demonstrates that increased
ROS and RNS production modulates Th2 inflammatory and fibrotic response in asthma
[33, 41]. In agreement with the current literature, the present study demonstrated that
OVA sensitized animals presented increased expression of 3-nitrotyrosine and
Gp91phox [33, 41]. On the other hand, AT significantly reduced their expression,
confirming the inhibitory effects of AT on reactive oxygen and nitrogen species
synthesis by leukocytes, which may be involved in these anti-inflammatory and antifibrotic effects of AT in asthma. In addition, a growing number of studies demonstrates
that IL-10 present anti-inflammatory properties, by inhibiting the eosinoplilic
inflammation and Th2 cytokines release, notably IL-4, IL-5 and IL-13 [17, 22]. In this
way, the present results demonstrated that AT training significantly increased the
expression of IL-10 by leukocytes as well as increased the levels IL-10 in the lung and
also systemically. However, although we observe a strong stimulus from AT on IL-10
release by leukocytes, the exact molecular mechanisms of IL-10 mediating the antiinflammatory effects of AT in asthma remains to be further investigated. Finally, we
also investigated the effects of AT on NF-kB expression by peribronchial leukocytes.
Several studies show increased NF-kB expression in airways of asthmatic patients and
in animal models of asthma [8, 14, 15, 35, 38, 39]. These studies show that NF-kB
controls not only the expression of pro-inflammatory cytokines, but also the expression
of pro-fibrotic mediators [8, 14, 15, 35, 38, 39]. Our study has confirmed that
ovalbumin-induced chronic allergic lung inflammation is followed by increased
expression of NF-kB in leukocytes. However, again, the results of the present study
showed that AT significantly reduced NF-kB expression, possibly accounting as part of
67
mechanisms involved in the anti-inflammatory and anti-fibrotic effects of AT in a mouse
model of asthma.
In conclusion, the present study shows that aerobic training reduces chronic
allergic airway inflammation and remodeling in a mouse model of asthma and these
results seem to be partially mediated by deactivation of peribronchial leukocytes.
68
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Figure and Figure Legends
Figure 1
Figure 1 shows the levels of pro and anti-inflammatory cytokines in BALF. In figures 1
A, 1 B and 1 C, * p<0.01 when compared with all groups. In figure 1 D, * p<0.05 when
compared with Control group.
75
Figure 2
Figure 2 shows the levels of pro and anti-inflammatory cytokines in serum. In figure 2
A, * p<0.01 when compared with all groups. In figure 2 B, * p<0.05 when compared
with Control and OVA groups.
76
Figure 3
Figure 3 shows the expression of Th2 and Th1 cytokines, chemokines and adhesion
molecules by peribronchial leukocytes. In figure 3 A, * p<0.001 when compared with all
groups. In figure 3 B, no statistically differences were found comparing all groups. In
figures 3 C and 3 D, * p<0.01 when compared with all groups.
77
Figure 4
Figure 4 shows the expression of oxygen and nitrogen reactive species, antiinflammatory cytokine and NF-kB by peribronchial leukocytes. In figures 4 A, 4 B and 4
D, * p<0.001 when compared with all groups. In figure 4 C, * p<0.01 when compared
with all groups.
78
Figure 5
Figure 5 shows the expression of growth factors by peribronchial leukocytes. In figures
5 A and 5 D, * p<0.01 when compared with all groups. In figures 5 B and 5 C, *
p<0.001 when compared with all groups.
79
Anexo II – Paper publicado
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