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

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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

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FICHA CATALOGRÁFICA

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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

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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 pró-

inflamatórias 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: SDRA, laserterapia, inflamação pulmonar, citocinas,

neutrófilos.

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Abstract

Acute respiratory distress syndrome (ARDS) is a syndrome that presents

high mortality rates, and the results of both insults pulmonary or extra-

pulmonary (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

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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: ARDS, laser therapy, pulmonary inflammation, cytokines,

neutrophils.

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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 pró-

inflamatórias 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,

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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).

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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

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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 IL-

1β.

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 01504-

000, 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

rodrelena@yahoo.com.br

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 LPS-

induced 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 12-

12h 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 TNF-

alpha 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 intra-

pulmonary 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 pro-

inflammatory 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 IL-

1beta 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 LPS-

induced ARDS coherently induced increases in the BALF and serum levels of TNF-

alpha. 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 TNF-

alpha 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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Med. 184 (2), 215-223.

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 IL-

5, 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, IL-

5, IL-13; p<0.001), chemokines (CCL5, CCL10; p<0.001), adhesion molecules (VCAM-

1, ICAM-1; p<0.05), reactive oxygen and nitrogen species (GP91phox and 3-

nitrotyrosine; 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 (IL-

10; 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 anti-

inflammatory effects of AT [24]. However, the mechanisms involved in the anti-

inflammatory 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 (NF-

kB) 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 anti-

oxidants 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-Grunwald-

Giemsa [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-3-

nitrotyrosine, anti-NF-kB, anti-iNOS, anti-TGF-beta, anti-IGF-1, anti-VEGF and anti-

EGFr 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 Student-

Newman-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 anti-

inflammatory 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 3-

nitrotyrosine (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 IL-

10 (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 OVA-

induced 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 pro-

inflammatory 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 anti-

fibrotic 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 anti-

inflammatory 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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74

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, anti-

inflammatory 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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