UNIVERSIDADE NOVE DE JULHO

Programa de Ps-Graduao em Biofotnica Aplicada s Cincias

da Sade

Romildo Torres da Silva

EFEITO DO LASER DE BAIXA POTNCIA (660NM) NA TENDINITE

INDUZIDA POR COLAGENASE EM TENDO CALCNEO DE RATOS

So Paulo, SP 2013

ROMILDO TORRES DA SILVA

EFEITO DO LASER DE BAIXA POTNCIA (660NM) NA TENDINITE

INDUZIDA POR COLAGENASE EM TENDO CALCNEO DE RATOS

Dissertao apresentada ao Programa de Ps-Graduao em Biofotnica Aplicada s Cincias da Sade da Universidade Nove de Julho, para obteno do Ttulo de Mestre em Cincias. rea de Concentrao: Sade Orientador: Prof. Dr. Rodrigo Labat Marcos

So Paulo, SP 2013

Torres-silva, R. Efeito do laser de baixa potncia (660nm) na tendinite induzida

por colagenase em tendo calcneo de ratos. / Romildo Torres da Silva 2013.

52 f.

Dissertao (mestrado) Universidade Nove de Julho - UNINOVE, So Paulo,

2013.

Orientador (a): Prof. Dr. Rodrigo Labat Marcos.

1. Tendinite. 2. Inflamao aguda. 3. Tendo calcneo. 4. Colagenase. I. Marcos, Rodrigues Labat. II. Titulo

CDU 615.831

Dedico este trabalho:

minha esposa, Ana Maria

Aos meus filhos Thiago, Reinaldo e Juliana

As minhas netas Milena e Alice.

AGRADECIMENTOS

A Deus, o nosso Grande Arquiteto do Universo, sem ele nada seria

possvel.

A minha famlia, em especial ao meu grande amor minha esposa Ana

Maria.

Ao meu orientador Prof. Dr. Rodrigo Labat Marcos, pela orientao

nos trabalhos desenvolvidos, dedicao apoio e amizade.

Ao Prof. Dr. Rodrigo lvaro Brando Lopes Martins, pela confiana,

oportunidade e pela amizade.

A todos os amigos de sala, em especial a Ana, Manolo e Elis.

A todos os Professores Doutores que contriburam direta e

indiretamente para a realizao do trabalho.

MUITO OBRIGADO!!!!!!!!!

O mundo de um cego definido pelo limite de seu tato. O de um

homem ignorante, pelos limites do seu conhecimento. J o mundo de um

grande homem definido pelo tamanho do seu conhecimento.

(Autor desconhecido)

RESUMO

TORRES-SILVA, R. Efeito do laser de baixa potncia (660nm) na tendinite

induzida por colagenase em tendo calcneo de ratos. 52f . Dissertao

(Mestrado em Biofotnica Aplicada a Cincias da Sade) Universidade UNINOVE,

So Paulo, 2013.

Tendinopatias so distrbios comuns com uma variedade de tratamentos e terapias

e a Laserterapia aparece como uma alternativa no tratamento destas doenas. Ns

investigamos os efeitos da irradiao laser no modelo de tendinite induzida por

colagenase, em tendes calcneo de ratos, verificando sua ao em importantes

marcadores inflamatrios. Mtodos: Foram utilizados ratos machos (Wistar) divididos

em grupos: Controle (C), Tendinite No Tratado (NT), Tratamento com Diclofenado

de Sdio (D) ou Laser (1J) e (3J). A tendinite foi induzida por colagenase

(100g/tendo) sobre o tendo calcneo que foi removido para anlises. Foi

verificado a expresso gnica de COX-2, TNF-, IL-6 e IL-10 (RT-PCR). Resultados

e discusso: A laserterapia (660nm, 100mW, 3J), utilizada no tratamento da tendinite

induzida por colagenase em tendo calcneo de ratos, foi eficaz na reduo de

importantes marcadores inflamatrios, tornando-se uma ferramenta promissora para

o tratamento de leses tendneas.

Palavras-chave: Tendinite. Inflamao Aguda. Tendo calcneo. Colagenase.

Laser de baixa potncia.

ABSTRACT

TORRES-SILVA, R. Effect of the low level laser therapy (660nm) in the induced

tendinitis by collagenase in aquilles tendon of rats. 52pg. Master in Applied

Biophotonics the Health Sciences - UNINOVE, So Paulo , 2013.

Tendinopathy is a common disease with a variety of treatments and therapies. Laser

therapy appears as a alternative treatment. We research the effects of the laser

irradiation in tendinitis model induced by collagenase injection on Achilles tendon

rats, verified its action in important inflammatory markers. Methods: The male rats

(Wistar) were used and divided in groups: Saline (C), Tendinitis (NT) and tendinitis

treated with Sodium diclofenac (D) or Laser (1J) and (3J). The tendinitis was induced

by collagenase (100g/tendon) on the Achilles tendon which was removed for

analyzes. The gene expression for COX-2; TNF-; il-6 and IL-10 (RT-PCR) was

measured. Results and discussion: The Laser therapy (660nm, 100mW, 3J) used

in the treatment of the tendinitis induced by collagenase in Achilles tendon in rats,

was effective in the reduction of important inflammatory markers, becoming a

promising tool for the treatment of tendon diseases.

Word-key: Tendinitis. Acute inflammation. Achilles tendon. Collagenase. Low

Intensity laser therapy.

SUMRIO

1- INTRODUO..............................................................................................11

1.1- Tendo...............................................................................................12

1.2- Arquitetura interna dos tendes..........................................................13

1.3- Tecido colgeno.................................................................................14

1.4- Processo Inflamatrio .......................................................................14

1.5- Inflamao do Tendo........................................................................15

1.5.1 Cicloxigenase 2 (COX-2)...................................................................16

1.5.2 Interleucinas IL-6 e IL-10...................................................................17

1.5.3 Fator de necrose tumoral TNF-........................................................18

1.6- Tratamentos......................................................................................18

1.7- Laserterapia.......................................................................................19

2- OBJETIVOS..................................................................................................22

3- MATERIAL E MTODOS.............................................................................23

3.1 Animais...............................................................................................23

3.2 Grupos Experimentais........................................................................23

3.3 Induo da tendinite............................................................................24

3.4 Aplicao da Terapia com Laser de Baixa Potncia (LBP)..............25

3.5 Anlises de expresso gnica por RT-PCR.....................................25

3.6 Anlise estatstica...............................................................................26

4- RESULTADOS................................................................................................27

4.1 Artigo submetido para publicao.......................................................27

5- CONSIDERAES FINAIS.............................................................................43

6- REFERNCIAS..............44

7- ANEXO 1 (Aprovao do comit de tica e pesquisa)....................................52

LISTA DE ABREVIAES

AP Fosfatase Alcalina

ATP Adenosina Trifosfato

-actina Anticorpo Policlonal

cDNA DNA Complementar

COX Ciclo-Oxigenase

DNAse Enzima catalizadora do cido Desoxirribonuclico

FP Fibrinognio Plasmtico

HILT - High Intensity Laser Treatment

IL Interleucina

JMT Juno Msculo-tendnea

JOT Juno steo-tendnea

LASER Light Amplification by Stimulated Emission of Radiation

LBP Laser de Baixa Potncia

LILT - Low Intensity Laser Treatment

MMP Metaloproteinase da Matriz

mW MiliWatt

nm Nanmetro

PMNs Polimorfonucleares

RNA cido Ribonuclico

RNAm cido Ribonuclico mensageiro

RT PCR reao de transcriptase reversa reao em cadeia da polimerase

TNF- Fator de Necrose Tumoral

LISTAS DE FIGURAS

Figura 1: Tendo Calcneo..............................................................................pag 12

Figura 2: Arquitetura interna dos tendes.........................................................pag 13

Figura 3: Esquema de grupos experimentais...................................................pag 23

Figura 4: Seqncia de Primers..................................................................pag 25-26

Figura 5: Tabela dos resultados......................................................................pag 44

11

1 INTRODUO

As doenas musculoesquelticas entre elas a tendinite, em virtude da sua

frequncia, do custo do tratamento, da reduo da funcionalidade laboral e do

desencadeamento de co-morbidades, necessitam de recursos financeiros

considerveis para o acompanhamento da sade dos indivduos vtimas destas

patologias, principalmente nos casos em que a incapacidade no trabalho persiste

alm do tempo mdio para o retorno s atividades laborativas (BERNARD B., 1997,

ARVIDSSON S. et al., 2008).

As tendinopatias so alteraes na sade do tendo, geralmente frequentes e

difceis de serem tratadas, incapacitando desde atletas profissionais e recreacionais

at pessoas comuns em seu local de trabalho. A tendinite tem um alto impacto na

qualidade de vida do indivduo que requer freqente atendimento mdico mesmo

nos casos simples, constituindo um desafio teraputico nos casos crnicos

(BARROS M. et al.; 2006; AITTOMAKI A. et al., 2006).

Sua ocorrncia alta, especialmente quando consideramos o tendo

calcneo, o patelar e o do cotovelo, onde a cronificao da doena comum

(LANGBERG e KONGS-GAARD, 2008), sendo objeto de preocupao entre as

classes de trabalhadores em diversas ocupaes, pela reduo da qualidade de vida

e pelo alto custo ao sistema de sade (LOISEL P., et al 2002). Quando a doena

cronifica, ocorre um aumento considervel dos recursos despendidos pelo

empregador, relacionados ao afastamento e a necessidade de relocao e

qualificao de outro para o exerccio desta funo (NGUYEN et al 2007).

Sua predominncia foi relatada entre 2% dos trabalhadores ativos e 55% dos

atletas (LIN et al, 2006). Nos Estados Unidos, foi observado um aumento de 39%

dos gastos com indenizaes e 62% com custos mdicos no perodo de 1991

2001. No Brasil, acidentes de trabalho e doenas ocupacionais somaram 509.062

dias de trabalho perdidos em 2000, responsvel por R$8.492.762,00 de gastos com

benefcios concedidos pelo INSS, apenas no estado da Bahia (SANTANA et al.,

2006).

Porm, a etiologia das tendinopatias ainda no foi totalmente explicada

(ALFREDSON e LORENTZON, 2000), mas sua ocorrncia elevada faz destas

doenas um grande problema scio-econmico onde as intervenes mdicas e

terapias para a reabilitao so ainda limitadas (LANGBERG e KONGS-GAARD,

12

2008). Sua preveno, a identificao das causas associadas e a promoo do

retorno seguro s atividades laborativas, portanto, tornaram-se tema de estudos no

campo da sade pblica (BARBE MF, 2006).

1.1 Tendo

Tendes saudveis apresentam colorao branca brilhante e textura

fibroelstica, mostrando grande resistncia a tenses mecnicas. Eles podem variar

muito quanto forma, e ainda quanto maneira como esto ligados ao osso. Alm

disso, a juno miotendnea (JMT) tambm mostra variaes anatmicas entre

diferentes msculos.

Figura 1. Tendo calcneo (Aquiles) de humano. (vista posterior). Fonte:

Wikipdia.org.

Tendes so estruturas anatmicas interpostas entre msculos e ossos

(figura 1), que transmitem a fora gerada no msculo para o osso, tornando possvel

o movimento articular (JZSA e KANNUS, 1997; KHAN et. al., 1999). Os elementos

bsicos dos tendes so fibras de colgeno e matriz extracelular rica em

proteoglicanas, formadas a partir de clulas denominadas tencitos. O colgeno

fornece a fora tnsil ao tendo, enquanto a matriz fornece suporte estrutural para

as fibras de colgeno e regula a transformao extracelular de pr-colgeno em

13

colgeno maduro (ASTROM, 1997). Os tenoblastos, clulas achatadas e distribudas

entre as fibras de colgeno sintetizam tanto a matriz extracelular, quanto o pr-

colgeno. O colgeno arranjado hierarquicamente em nveis de complexidade

crescente, comeando com protocolgeno, fibras, fascculos, bandas tercirias e

finalmente o tendo propriamente dito (JZSA e KANNUS, 1997; ASTROM, 1997;

KHAN et al., 1999).

1.2 Arquitetura interna dos tendes

O tendo completamente coberto pelo epitendo, uma camada fina de

tecido conjuntivo contendo suprimentos nervosos, vasculares e linfticos. O

epitendo se estende profundamente no tendo, entre as bandas tercirias e o

endotendo (figura 2). Mais superficialmente, o epitendo envolvido pelo

paratendo, um tecido conjuntivo areolar contendo basicamente fibrilas de colgeno

(KVIST et al., 1985), algumas fibrilas elsticas e uma linha interna de clulas

sinoviais (WILLIANS, 1986).

Figura 2. Organizao hierrquica da estrutura do tendo (das fibrilas de colgeno

at o tendo propriamente dito). Fonte: KASTELIC et al. (1978).

1.3 Tecido Colgeno

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As fibras formadas por colgeno representam dois dos trs principais tipos de

fibras do tecido conjuntivo, as fibras colgenas e as reticulares. As fibras elsticas

so formadas pela protena elastina.

O colgeno a protena mais abundante do organismo, representando cerca

de 30% do peso seco e pode ser classificado nos seguintes grupos: (1) colgenos

que formam longas fibrilas; (2) colgenos associados a fibrilas; (3) colgeno que

forma rede e (4) colgeno de ancoragem. O colgeno que forma os tendes, ossos,

dentina, derme e outros tecidos, est includo no 1o Grupo.

A sntese de colgeno foi primeiramente atribuda a uma variedade restrita de

tipos celulares como fibroblastos, osteoblastos e condroblastos. Atualmente, existem

evidncias convincentes de que outros tipos celulares so capazes de sintetizar o

colgeno (JUNQUEIRA e CARNEIRO, 2004).

O colgeno um dos principais componentes da matriz extracelular,

responsvel pela resistncia trao atuando tambm na estabilidade do tecido

conjuntivo (MOALI C, 2012, WIDMER, C, et al 2012 ). Esta integridade e

estabilidade do tecido so reguladas principalmente atravs de um equilbrio entre

produo, organizao e degradao do colgeno (HULMES DJ 2002). O colgeno

fibrilar mais abundante no tendo primeiramente o colgeno do tipo I, seguido pelo

colgeno tipo II e III, sintetizados a partir do pr colgeno (HULMES, 2008).

1.4 Processo Inflamatrio

A inflamao uma resposta biolgica complexa do tecido estmulos

nocivos como, clulas danificadas, ou irritantes (FERRERO- MILIANI L. et al., 2007)

sendo um dos mecanismos de proteo do organismo com a funo de neutralizar e

remover este estmulos nocivos, promovendo o reparo. A inflamao aguda um

processo de curta durao, aparecendo geralmente dentro de alguns minutos ou

horas (COTRAN et al., 1998).

O mecanismo fisiopatolgico da inflamao explicado por um conjunto de

reaes locais e gerais do organismo, compondo uma srie de fenmenos

complexos que se associam e se complementam, formando uma reao em

cascata, que envolve clulas inflamatrias, como: neutrfilos, linfcitos e moncitos/

macrfagos (TEDGUI e MALLAT, 2001).

15

Durante a resposta inflamatria aguda, ao libertar mediadores inflamatrios

responsveis pelos sinais de inflamao, ocorre a vasodilatao e consequente

aumento do fluxo sanguneo, apresentando na rea inflamada vermelhido e

aumento do calor. O aumento da permeabilidade dos vasos sanguneos resulta em

uma exsudao do plasma e protenas de fluidos para os tecidos, se manifestando

como inchao. Alguns mediadores inflamatrios liberados durante este processo

podem alterar determinadas molculas dos vasos sanguneos permitindo a migrao

de leuccitos, principalmente de neutrfilos provocando um infiltrado inflamatrio. Os

neutrfilos migram por quimiotaxia at o local da leso (COTRAN et al., 1998).

1.5 Inflamao do Tendo

Historicamente, tendes so conhecidos como partes vulnerveis do corpo

humano. Hipcrates reportou que se o tendo de Aquiles (calcneo) fosse lesado,

poderia causar febre aguda, convulses, inconscincia e finalmente a morte.

Felizmente, leses no calcneo nos dias de hoje no esto associadas com morte,

mas algumas vezes podem causar condies crnicas dolorosas e de difcil

tratamento.

No processo inflamatrio crnico, ocorre o remodelamento da bainha

tendnea e do tecido sinovial, aumentando a quantidade de clulas inflamatrias.

Estas alteraes no tecido tendneo conduzem a um distrbio com reduo de sua

funcionalidade. Outra complicao est no fato que os tendes usam suas

extremidades para se ligarem em outras estruturas, como ossos e cartilagens e uma

vez alterado, este processo inflamatrio pode migrar para outros tecidos iniciando

um quadro pr-artrtico. Esta pode ser a explicao sobre como os tendes so

afetados pela inflamao crnica e como a tendinite pode influenciar no

aparecimento e evoluo de doenas inflamatrias nos ossos e cartilagens (HAYER

et al., 2007).

De maneira Geral, o tendo humano tem uma alta capacidade de suportar

tenses. Medies diretas em calcneos humanos registraram foras de at 9 kN

(correspondente a 12,5 vezes o peso corporal) durante uma corrida em velocidade

de 6 m/s. Entretanto, hoje sabemos que a fora mxima suportada desse tendo

16

uma varivel bastante individual, indicando tambm uma faixa ampla de

suscetibilidade a sobrecargas.

Existe um grande nmero de sugestes quanto aos fatores etiolgicos da

tendinite, mas so poucas as comprovaes cientficas. A causa determinante mais

comum o esforo exagerado de extenso sobre os tendes, ocorrendo distenso

de suas fibras, que por no suportarem a trao mecnica, podem apresentar

rupturas parciais desenvolvendo intensa e dolorosa reao inflamatria local

(ALFREDSON e LORENTZON, 2000).

Alm da sobrecarga mecnica conhecida como o principal fator causador das

tendinopatias, existem processos lesivos repentinos e o envelhecimento que

tambm contribuem para a degenerao do tendo que pode ocorrer na ausncia

(tendinose) ou na presena (tendinite) de resposta inflamatria (SUN et al., 2008).

1.5.1 Cicloxigenase 2 (COX-2)

Aps a leso do tendo, o organismo inicia uma srie de eventos para a

promoo do reparo tecidual que pode ter como resultado a alterao do tecido e

formao de cicatriz (LIN al, 2006). A cicatriz inicialmente fornece a continuidade

fsica do tecido, porm pode provocar a formao de tecidos adjacentes

indesejveis, impedindo o mecanismo de deslizamento do tendo (BEREDJIKLIAN

et al., 2003). J a inflamao intensa, com grande migrao leucocitria e formao

de citocinas pr-inflamatrias partir da Ciclo-oxigenase, promove a alterao do

tecido, pelo aumento de fibroblastos e mudana da matriz extracelular (LIECHTY et

al., 2000).

A prostaglandina H2 sintase, tambm conhecida como cicloxigenase uma

enzima integral da membrana celular, participando da cascata inflamatria.

Geralmente so constitutivas, porm a COX-2 pode ser induzida durante a resposta

inflamatria, influenciando ainda a formao de mediadores inflamatrios tais como

interleucinas, prostaglandinas e tromboxanos (KOMOTO J. et al 2006). Algumas

destas citocinas, entre elas a IL-1, IL-6 e IL-12 e ainda o TNF- so reprimidas

drasticamente no final do processo inflamatrio, durante o reparo tecidual (RANG

and DALE, 2012).

A expresso de COX-2 pode ser tambm aumentada aps a induo do

processo inflamatrio em tendes calcneos de ratos (MARCOS et al, 2011). Ao

17

contrrio, sua reduo foi observada aps terapias farmacolgicas e no

farmacolgicas, como na utilizao da laserterapia de baixa potncia (WANG et al,

2003; XAVIER et al, 2010; MARCOS et al, 2011).

1.5.2 Interleucinas IL-6 e IL-10

Uma maneira de reduzir o processo inflamatrio no tecido lesionado seria

controlar o processo de cicatrizao pela modulao do processo inflamatrio e dos

nveis de citocinas anti e pr-inflamatrias envolvidas no reparo (RICCHETTI et al.,

2008). Entre estas citocinas, as interleucinas teriam um papel importante na

modulao da inflamao do tendo (LIN et al., 2006).

A IL-6, uma citocina com papel central na inflamao aps processos

lesivos (SKUTEK M, et al., 2001). No processo inflamatrio agudo do tendo

observado um aumento expressivo de granulcitos e neutrfilos na bainha sinovial,

geralmente ativados pelo aumento de IL1 e IL6. A presena destas duas citocinas

esta diretamente relacionada a progresso da tendinite (HAYER et al., 2007). Outros

estudos sugeriram que as citocinas inflamatrias pode desempenhar um papel

durante a progresso das condies das tendinopatias, uma vez que tambm foram

detectados no processo da dor e ruptura do tendo ([FU SC, et al., 2002, NAKAMA

K, et al. , 2006).

A IL-10 foi identificada inicialmente por MOSMANN et al. (1989), sendo

reconhecida por sua atividade antiimunolgica e antiinflamatria. Desde sua

descrio, a lista de clulas produtoras de IL-10 se expandiu rapidamente assim

como o nmero de clulas que so capazes de responder a essa citocina (MOSSER

et al., 2008). A IL-10 tambm conhecida por inibir a expresso de IL-6 e IL-8 e a

migrao de clulas inflamatrias no local da leso incluindo macrfagos e

moncitos (ALAM et al., 1994; RICCHETTI et al., 2008).

Durante o processo inflamatrio a interleucina IL-10 possui uma importante

funo biolgica de inibir a maturao e a diferenciao de clulas dendrtica em

moncitos e de inibir a produo de citocinas pr-inflamatrias exposio IL-10

que pode ainda inibir a inflamao, aumentando os receptores antagonistas de IL-1

pelos macrfagos (MOSSER et al., 2008).

18

1.5.3 Fator de necrose tumoral TNF-

O fator de necrose tumoral (TNF-) uma citocina pleitrpica, relacionada

tanto na sobrevivncia e proliferao celular como na morte celular no processo

apopttico. Esta citocina tambm distribuda e expressa em tencitos em

condies inflamatrias onde a apoptose e a proliferao celular acentuada

(HOSAKA et al., 2005).

O TNF- considerado uma citocina chave na origem de vrias doenas

osteomusculares, como Artrite Reumatide, Osteoartrite e Tendinite (HAYER et al.,

2007), porm, seu papel na doena do tendo ainda no foram bem determinada.

Sabe-se que em tendes de equinos, o TNF- pode apresentar aes anti-

apoptticas e apoptticas, controlando o nmero de clulas, sendo um fator

importante no progresso de degenerao do tecido tendneo (HOSAKA et al., 2005).

Os tendes so bastante afetados pela inflamao tambm por estarem

sujeitos a foras especficas e cargas mecnicas prolongadas (HAYER et al., 2007).

Nestas condies patolgicas, o tecido tendneo pode apresentar aumento dos

receptores de TNF- (R1 e R2) (HOSAKA et al., 2005).

Os nveis de TNF- so aumentados nos tecidos msculo-tendneos, em

situaes ps-traumticas (ELSAID et al., 2008). As cirurgias de tendo, cartilagem

e osso outro fator que pode dar incio a um processo inflamatrio. Aps uma leso,

sejam por um acidente ou por uma interveno mdica, os nveis de citocinas

inflamatrias como TNF- e IL so aumentados (AKESEN et al., 2009).

1.6 Tratamentos

Condies inflamatrias de tendes esto associadas a uma ampla variedade

de tratamentos propostos, algumas delas com poucas evidncias cientficas que

apoiem seu uso (KVIST, 1994; KHAN et al., 1999; ALFREDSON e LORENTZON,

2000; ANDRES et al., 2008; BJORDAL et al., 2008).

Os tratamentos da tendinite tm por finalidade a reduo da inflamao, que

se persistir, promove alteraes nos feixes das fibras de colgeno e aumento da

atividade de determinadas enzimas, prolongando-se assim as fases inflamatria e

proliferativa, favorecendo a deposio excessiva de tecido cicatricial, alterando

19

algumas caractersticas como a reduo da resistncia em comparao ao tendo

original (FERNANDES et al., 2003).

O tratamento mais comum o conservador, recomendado pela maioria dos

autores como estratgia inicial. Na maioria das vezes consiste em uma abordagem

multi-orientada, incluindo modelos de reabilitao combinada como repouso,

medicao a base de antiinflamatrios no esteroidais e corticides, crioterapia,

massagem, treinamento de fora.

A eficcia do tratamento aumenta com a determinao da fase em que o

processo inflamatrio no tendo se encontra. Na fase inicial do processo de

reparao, podem ser institudas prticas hidroterpicas frias, visando-se reduzir o

metabolismo na regio, e agentes antiinflamatrios esteroidais ou no-esteroidais

(ALVES e MIKAIL, 2006).

Os corticosterides so bem absorvidos em qualquer stio de administrao,

(tpica, oral, subcutnea, intramuscular, intralesional e intravenosa), porm podem

provocar efeitos deletrios no uso intralesional (POOL et al., 1980).

Os antiinflamatrios no esteroidais so as drogas utilizadas com mais

freqncia, como parte do tratamento inicial, principalmente na reduo da dor,

embora seja comum encontrarmos amostras de tendes durante a tendinopatia, com

ausncia de clulas inflamatrias que justifique esta preferncia (ALFREDSON et

al., 2003; ANDRES et al., 2008).

1.7 Laserterapia

Vrios trabalhos foram publicados para melhorar o entendimento dos efeitos

da terapia LASER no tratamento de doenas inflamatrias. Porm, apesar de todas

as investigaes realizadas e do importante uso clnico do laser de baixa potncia, o

conhecimento bsico de seus mecanismos de ao ainda muito deficiente.

O termo Laser um acrnimo para Light Amplification by emission of

radiation (amplificao da luz pela emisso estimulada da radiao). Embora Albert

Einstein originalmente tenha delineado os princpios subjacentes gerao deste

tipo de luz no incio deste sculo, foi somente em 1960 que Maiman produziu o

primeiro feixe de luz de laser e baseado neste prottipo surgiu vrios aparelhos. As

caractersticas que diferem a luz laser de uma lmpada so: monocromaticidade,

colimao e coerncia (SCHAWLOW,1995).

20

A coerncia uma das propriedades da luz laser, como citada acima, mas ao

penetrar no tecido, esta propriedade se perde nos primeiros extratos da pele. Isto

ocorre devido grande variedade de estruturas celulares que compe a pele

(Nomura et al, 1989). Segundo esses autores, apesar da perda da coerncia no

interior dos tecidos, a irradiao ainda absorvida pelas clulas, gerando alteraes

no seu metabolismo tanto em tecidos superficiais como profundos (SVAASAND,

1990).

Os lasers podem ser classificados em dois grandes grupos: os laseres

cirrgicos de alta intensidade (HILT High -Intensity Laser Treatment) e laseres no-

cirrgicos de baixa intensidade (LILT Low Intensity Laser Treatment).

Em geral, quase todas as aplicaes com HILT tomam por base os efeitos

fototrmicos e fotoablativos do laser no tecido, sendo usados para cortar, destruir,

soldar, remover tatuagens, entre outros efeitos. Em contraste, nas dcadas de 60 e

70 os pesquisadores voltaram-se para as aplicaes com LILT e essas se baseiam

nas interaes atrmicas da luz laser com o tecido, produzindo efeitos de

Biomodulao (KARU, 1998; BASFORD,1995).

A terapia com laser de baixa potncia (LBP), incide sobre as reaes

atrmicas da luz com o tecido, ocasionando efeitos fotoqumicos (SCHAFFER et al.,

2000; HONMURA et al.,1993), ou seja, radiaes com baixa densidade de potncia

(DP) 0,01 w/cm2 1 w/cm2 e tambm baixa densidade de energia (DE), de 1

10J/cm2 (SCHINDL et al., 2000). Nesses limites se produz uma pequeno e no

significante aumento de temperatura, o qual no ultrapassa 1 grau Celsius

(KARU,1987).

O Laser de baixa potncia parece agir sobre organelas celulares

(mitocndrias e membranas), gerando aumento da sntese de ATP e modificando o

transporte inico. Dessa forma o laser, em curto prazo, acelera a gliclise e a

oxidao fosforilativa e em longo prazo a transcrio e a replicao do DNA (KARU,

1987).

Acredita-se que a ao do laser de baixa potncia sobre o tecido est

relacionada possibilidade de inibio do aparecimento de fatores quimiotticos nos

estgios iniciais da inflamao; de interferir com o efeito dos mediadores qumicos

induzidos pela inflamao (CAMPANA et al., 1998; CAMPANA et al. ,1999); inibir a

sntese das prostaglandinas (SATTAYUT et al., 1999; BJORDAL et al., 2006) alm

de inibir o esfncter pr-capilar, atravs de mediadores qumicos.

21

Estudos adicionais sobre o efeito antiinflamatrio do laser de baixa potncia

ainda se fazem bastante necessrios e seu mecanismo de ao deve ser

investigado a fim de compreender suas aes nos diferentes modelos experimentais

em que vem sendo utilizados.

Campana et al. (1998) quantificaram o nvel de fibrinognio plasmtico (FP)

em modelo de leso tecidual induzida por laparotomia e tratada com LBP e

diclofenaco de sdio. Nesse modelo experimental os resultados mostraram que o

nvel de FP que se encontrava aumentado nos tecidos lesados retornaram a nveis

normais aps o tratamento com LBP sendo semelhante ao tratamento com

diclofenaco de sdio. Esses resultados sugerem que a resposta inflamatria pode

ser normalizada ou reduzida pela ao fotoqumica do LBP, sugerindo uma ao

seletiva da COX-2, visto que a reduo do FP obtida com LBP foi semelhante ao

tratamento farmacolgico.

SATTAYUT et al. (1999), investigaram o efeito do laser As-Ga-Al (820nm)

com densidade de energia de 4J/cm2 e 19J/cm2 na produo de PGE2 em culturas de

clulas estimulada com IL-1 e observaram que a inibio da sntese de PGE2

ocorreu com 19J/cm2, sugerindo ser este o mecanismo analgsico para as dores

msculo - esquelticas.

O uso de lasers na prtica clnica objetivando o efeito antiinflamatrio em

diferentes doenas baseia-se em um nmero j razovel de publicaes de carter

cientfico.

Nos ltimos anos, inmeros estudos em diferentes situaes foram

realizados, fazendo com que a terapia laser j seja considerada como alternativa

teraputica para vrias doenas. (SALATE et al, 2005; FRIGO, 2009; CHOW, 2009;

CRESSONI ET AL, 2010; TUMILTY et al, 2012; ALFREDO et al, 2012; ALVES et al,

2013; BJORDAL et al, 2013; MARCOS et al, 2013; CASALECHI et al, 2013. partir

destes trabalhos, foi possvel entender parte do efeito da Terapia com Laser de

Baixa Potncia na reao inflamatria em cartilagem, na osteoartrite e tendinite, em

ratos e humanos. No entanto, importante ressaltar que pouco se conhece a

respeito do mecanismo de ao dos laseres no processo inflamatrio, onde os

estudos experimentais assumem grande relevncia.

22

2 OBJETIVOS

Estudar o efeito do laser de baixa potncia 660nm, 100mW na inflamao

aguda do tendo, investigando alteraes da expresso de enzimas e mediadores

inflamatrios como COX-2, IL-6, IL-10 e TNF-.

23

3 MATERIAL E MTODOS

3.1 Animais

Foram utilizados ratos Wistar machos pesando entre 200 e 250g (+/- 90 dias

de vida) com livre acesso a gua e rao, provenientes Biotrio do Instituto de

Cincias Biomdicas da Universidade de So Paulo.

Os animais foram mantidos em ambiente com temperatura controlada e ciclo

claro/escuro de 12 horas.

3.2 Grupos Experimentais

Os animais foram divididos em diferentes grupos conforme o esquema

abaixo:

Figura 3. Esquema dos grupos experimentais.

24

- Grupo 1: Controle (C) Tendes de animais que receberam injeo com soluo

salina 0,9% de NaCl (soro fisiolgico estril).

- Grupo 2: Tendinite no tratado (NT): Tendes de animais que receberam injeo

com soluo de colagenase.

- Grupo 3: Diclofenaco (D): Receberam injees de Colagenase e foram tratados

com Diclofenaco de Sdio (2,5mg/kg I.M., Voltaren, Novartis) imediatamente aps

a induo.

- Grupos 4: Irradiado (1J): Receberam injeo de colagenase e os tendes foram

irradiados, na regio da injeo, num nico ponto com Laser em 660nm na energia

de 1J e 100mW.

- Grupos 5: Irradiado (3J): Receberam injeo de colagenase e os tendes foram

irradiados, na regio da injeo, num nico ponto com Laser em 660nm na energia

de 3J e 100mW.

3.3 Induo da tendinite

Os animais foram anestesiados com injeo de cloridrato de ketamina

(90mg/Kg) e cloridrato de Xilazina (10mg/Kg). Depois de anestesiados, os animais

do grupo G2 ao G5 receberam 100 l de injeo transcutnea de colagenase (1

mg/ml; Sigma Chemical Co, Cat. C-6885) na regio posterior da pata direita do

Tendo calcneo. Animais do grupo controle receberam o mesmo volume de

soluo fisiolgica (NaCl 0,9%). A seguir, os animais retornaram para as caixas de

conteno separados em diferentes grupos experimentais. Aps 2 horas da induo

do processo inflamatrio pela injeo de colagenase, os animais foram

eutanasiados. A eutansia foi realizada atravs de overdose injetvel de

anestsico cloridrato de ketamina (180mg/Kg) e cloridrato de Xilazina (20mg/Kg). O

tecido tendneo foi removido para posteriores anlises.

25

3.4 Aplicao da Terapia com Laser de Baixa Potncia (LBP)

Os animais receberam o tratamento com laser de baixa potncia 660nm em

100mW de potncia (Thera Lase, DMC), 1 hora aps serem submetidos ao modelo

de inflamao aguda pela injeo de colagenase,

O tempo de Irradiao foi de 10 e 30 segundos, respectivamente para a

energia de 1J e 3J.

A aplicao do laser com um ponto por contato pontual, com dimetro de

0,028 cm.

3.5 Anlises de expresso gnica por RT-PCR

Os tendes removidos foram imediatamente armazenados -80 C para

anlise da expresso gnica dos compostos indicados na tabela abaixo, pela reao

em cadeia da polimerase reversa (PCR) em tempo real. O RNA total das amostras

foi extrado com o reagente Trizol (Gibco BRL, EUA), de acordo com instrues do

fabricante. Aps tratamento com DNAse, a sntese dos cDNAs foi feita pelo mtodo

da transcriptase reversa, empregando a enzima SuperScript (Invitrogen, 2g de RNA

total), na presena de mistura de primers randmicos e oligo dT. A anlise das

amostras em tempo real obedeceu um ciclo de desnaturao inicial (10min 95 C)

e quarenta ciclos de amplificao (30 seg de desnaturao 95 C e 1min de

anelamento e extenso 60 C).

As seqncias dos primers utilizados esto representadas na figura 4,

conforme Wang et al. (2004). Os resultados da expresso gnica foram

interpretados usando-se a frmula 2- Ct (Ct = nmero de ciclos necessrios para

atingir o limiar de fluorescncia acima do valor de fundo - background) que relaciona

a expresso do gene de interesse comparado quela do gene controle B-actina.

PRIMER SEQUNCIA

-ACTINA FOWARD: AAGATTTGGCACCACACTTTCTACA

REVERSE: CGGTGAGCAGCACAGGGT

COX-2 FOWARD: AGATCAGAAGCGAGGACCTG

REVERSE: CCATCCTGGAAAAGTCGAAG

26

IL-6

FOWARD: TGACCCAACCACAAATGC

REVERSE: CGAGCTCTGAAACAAAGGAT

IL-10 FOWARD: CATGGGTGTTGGGAAGAGAA

REVERSE: GCTTTCGAGACTGGAAGTGG

TNF- FOWARD: GGTGATCGGTCCCAACAAGGA

REVERSE: CACGCTGGCTCAGCCACT

Figura 4: Seqncia de Primers para anlise da expresso de mRNA, pela tcnica

de RT-PCR em tempo real.

3.6 Anlise estatstica

Os dados foram descritos em valores mdios seguidos dos respectivos

desvios padro. Todos os resultados foram submetidos anlise estatstica atravs

do teste de ANOVA com post-hoc de Tukey-Kramer para mltiplas comparaes.

Valores de p

27

4 RESULTADOS

4.1 Artigo submetido para publicao.

28

The Low Level Laser Therapy (LLLT) Operating in 660 nm Reduce Gene

Expression of Inflammatory Mediators in the Experimental Model of

Collagenase-induced Rat Tendinitis

Torres-Silva, R1; Lopes-Martins R.A.B.2, Bjordal J.M.3, Frigo L4, Rahouadj R.5, Arnold

G.6, Leal-Junior, E.C.P.1, Magdalou J.7, Pallota R.2, Marcos, R.L.1

1 - University of Nove de Julho, Biophotonics Applied to Health Sciences, So Paulo

01504-001, Brazil

2 - University of So Paulo (USP), Laboratory of Pharmacology and Experimental

Therapeutics, Institute of Biomedical Sciences (ICB), So Paulo 05508-900, Brazil

3 University of Bergen, Department of Global Health, Norway

4 Universidade Cruzeiro do Sul, Departamento de Cincias da Sade So Paulo

5 - Universit de Lorraine (UL), Laboratoire d'Energtique et de Mcanique

Thorique et Applique (LEMTA), UMR 7563 CNRS, F-54504 Vanduvre-ls-

Nancy, France

6 - Universit de Haute Alsace (UHA), Laboratoire Physique et Mcanique Textiles

(LPMT), F-68093 Mulhouse, France e Universit de Strasbourg (UdS), ICube, UMR

7357 CNRS, F-67411 Illkirch, France

7- Universit de Lorraine (UL), Ingnierie Molculaire, Physiopathologie Articulaire

(IMoPA), UMR 7365 CNRS, Biople, F-54505 Vanduvre-ls-Nancy, France

Correspondence Author

Rodrigo Labat Marcos

Programa de Ps-Graduao em Biofotnica Aplicada das Cincias da Sade

Universidade Nove de Julho

Rua Vergueiro 239-245

Email: rodrigolabat@yahoo.com.br

ABSTRACT

29

Tendinopathy is a common disease with a variety of treatments and therapies. Laser

therapy appears as an alternative treatment. Here we investigate the effects of laser

irradiation in an experimental model of tendinitis induced by collagenase injection on

rat calcaneous tendon, verifying its action in important inflammatory markers.

Methods: Male Wistar rats were used and divided in five groups: Control Saline (C),

non-treated tendinitis (NT) and tendinitis treated with sodium diclofenac (D) or Laser

(1J) and (3J). The tendinitis was induced by collagenase (100g/tendon) on the

Achilles tendon which was removed for analyzes. The gene expression for COX-2;

TNF-; IL-6; IL-10; MMP-3, 9, 13 (RT-PCR) was measured. Results and discussion:

The Laser irradiation (660nm, 100mW, 3J) used in the treatment of the tendinitis

induced by collagenase in Achilles tendon in rats, was effective in the reduction of

important inflammatory markers, becoming a promising tool for the treatment of

tendon diseases.

Keywords: Tendinitis. LLLT, Laser therapy, Tendinitis, inflammation, tendon, rats.

INTRODUCTION

Tendinopathies are changes in the tendon health, which are frequent and

difficult to treat, because of the high impact on quality of life of individuals that require

frequent medical care even in simple cases, being a therapeutic challenge in chronic

situations [1,2]. This fact results in a significant increase of resources expended by

the employer relating to the sick leave and the need for relocation and qualifying new

employees [3].

The high incidence tendon diseases make it a social problem which medical

therapies and interventions for rehabilitation are limited [4]. Therefore, its prevention,

the identification of associated causes and also the promotion of safe return for

working activities became the subject of studies in public health [5].

Tendons are composed by tenocytes which are responsible for the synthesis

of specific extracellular matrix and collagen, arranged in hierarchical levels, forming a

complex anatomical structure transmitting the force generated from the muscle to the

bone, making possible the joint movement [6,7].

The tendon tissue is covered by epitendon which provides a poor blood supply

when compared to other tissues. This may compromise its repair process after a

30

lesion due to the difficulty in removing metabolites or even the low bioavailability of

drugs commonly used in conventional therapy [8,9].

The most abundant collagen found in tendon tissue is the type I collagen

followed by type II and III. These proteins are organized in long fibrils conferring

resistance to the tissue. A balance between production and degradation of collagen

regulates the tissue integrity. Thus, changes in this balance can interfere directly in

the mechanical properties of the tendon [10,11,12].

There are a number of etiologic factors that can be attributed as tendinitis

inductors. The most common cause is the overexertion of tendons extension

promoting distension of collagen fibers leading to partial ruptures, developing an

intense and painful inflammatory reaction [13]. In this context, the most common

treatment of the inflammatory reaction in tendon tissue are the NSAIDs. The use of

NSAIDs still controversial in tendon diseases. However, an alternative to way to

reduce the inflammatory process in tendon tissue could be controlling the wound

healing process by modulating the inflammation and levels of anti- and pro-

inflammatory cytokines involved in tissue repair [14]. The cyclooxygenase II, for

example, is an integral enzyme that can be induced during this inflammatory process,

influencing the formation of important inflammatory cytokines, including the

interleukins (IL-1, IL-6, IL-10) and tumor necrosis factor (TNF - ). These cytokines

have a major role in the modulation of tendon inflammation [15]. IL-6, for example, is

a cytokine with a central role in inflammation after injurious processes [16]. In acute

inflammation of tendon is commonly observed a significant increase of both

granulocytes and neutrophil in synovial sheath, usually activated by increasing IL-1

and IL-6. The presence of these cytokines is directly related to progression of

tendinitis in a painful process after tendon rupture [17,18,19].

In contrast, IL-10 is known for its anti-inflammatory activity. IL-10 inhibits both

the expression of the interleukins (IL-6; IL-8) and the migration of inflammatory cells

at the lesion site including macrophages and monocytes [14,20].

TNF- is a pleitropic cytokine related to cell survival and proliferation but also

to cell death in the apoptotic process expressed by tenocytes in inflammatory

conditions [21]. TNF- may be the key cytokine in the origin of several

musculoskeletal diseases such as Rheumatoid Arthritis, Osteoarthritis and tendinitis

[19], however, its role in tendon disease has not been well determined. It is known

31

that its expression is increased in tendons in traumatic situations or after surgery,

wherein there is a harmful process [21,22].

Thus, treatment of tendinitis aims to reduce inflammation, because its

persistence promotes alterations in collagen and an increase in specific activity of

certain enzymes fibers, extending the inflammatory and proliferative phases, favoring

the excess deposition of scar tissue, altering some characteristics as strength

reduction compared to the original tendon [23,24].

The most common treatment is still conservative and recommended by most

authors as the initial strategy. The NSAIDs are the drugs most commonly used as

part of initial treatment, especially in reducing pain [25,26]. However there are a few

scientific evidences to support its use [7,8,13,26,27].

In recent years, several studies have been performed in different situations,

leading laser therapy to be considered as a promising alternative therapy for a

numerous diseases, acting in the early stages of inflammation by inhibiting the onset

chemotactic factors through altering the presence of pro- and anti-inflammatory

mediators, modulating the inflammation [28,29,30,31].

From these studies, it was possible to understand part of the effect of therapy

using a Low Level Laser in inflammatory reaction of cartilage in osteoarthritis and

tendinitis of both rats and humans [9,24,26,28,32,33,34]

However, it is important to note that very few studies addresses the molecular

effects in gene expression over the lasers action in the inflammatory process,

specially in tendons.

This work aims to study the effect of low level laser 660nm, 100mW in acute

inflammation of tendon, investigating changes in the expression of enzymes and

inflammatory mediators such as COX-2, IL-6, IL-10 and TNF-.

MATERIALS AND METHODS:

All of the experimental procedures were submitted and approved by the

Ethical Committee of the University of Sao Paulo. Thirty male Wistar rats weighing

250 + 20 g were randomly divided and housed five per cage before the experimental

procedure. Food and water were provided ad libitum throughout the experiment. Rats

were anesthetized with xylazine and ketamine injection (90mg/Kg and 10mg/Kg,

respectively) before collagenase injection. All the necessary preoperative procedures

32

were performed in order to prevent discomfort and to avoid any infection. Skin was

surgically prepared and collagenase was injected in right leg (100 g/tendon)

percutaneously into the Achilles tendon, approximately 2 mm proximal to the

osteotendinous junction under anesthesia using a 30 G needle. The same volume of

PBS without collagenase was injected using the same procedure in a control group

(C). Thirty minutes after collagenase injection, one group (called D) was treated

using sodium diclofenac (Voltaren injectable Novartis 2.5 mg/kg) injected in the

gluteus muscle. Two other groups were treated by laser 1 h after collagenase

injection. A single LLLT was performed with an infrared laser unit (Thera Lase, DMC,

Brazil). The laser unit emitted a continuous optical radiation under a wavelength of

660 nm, with a power of 100 mW in a mean spot size area of 0.028 Cm2. Laser

irradiation was performed in skin contact at the site of collagenase injection with

doses of 1 J and 3 J, corresponding to irradiation times of 10 s and 30 s,

respectively. The laser energy doses were chosen according to previous studies [29].

The two groups were called L1J and L3J, for 1 and 3 J respectively. The last group

(called TEN) was not subjected to any treatment of tendinitis. Six animals of each

group were sacrificed with an overdose of xylazine and ketamine injection (270mg/Kg

and 30mg/Kg, respectively), 2 h after tendinitis induction for biochemical analysis.

After the removal of skin and connective tissue, Achilles tendons were dissected,

frozen in liquid nitrogen, and stored at 80 C for further analysis.

- RNA isolation and Real Time PCR analysis: At the Achilles tendons were dissected,

frozen in liquid nitrogen, and stored at 80C. Total RNA was isolated in the Trizol

reagent, according to the manufacturers instruction. DNase I was employed to digest

DNA to obtain RNA purification and the integrity of RNA was verified by agarose gel

electrophoresis. Total RNA (2 g) was used for first-strand cDNA synthesis (reverse

transcriptase [RT]) using SuperScript II. In addition, RNaseOUT was also added to

protect the RNA during this process. Three pooled RNA aliquots were routinely sham

reverse transcribed (i.e. RT omitted) to insure the absence of DNA contaminants.

Diluted RT samples (1:10) were submitted to real-time PCR amplification using

Platinum Sybr QPCR Supermix-UDG and specific oligonucleotides for COX-2

(forward: AGATCAGAAGCGAGGACCTG; reverse: CCATCCTGGAAAAGTCGAAG),

IL-6 (forward: TGACCCAACCACAAATGC; reverse: CGAGCTCTGAAACAAAGGAT),

IL-10 (forward: CATGGGTGTTGGGAAGAGAA; reverse:

GCTTTCGAGACTGGAAGTGG) and TNF- (forward:

33

GGTGATCGGTCCCAACAAGGA; reverse: CACGCTGGCTCAGCCACT). Beta-actin

was used as an internal control (forward: AAGATTTGGCACCACACTTTCTACA;

reverse: CGGTGAGCAGCACAGGGT). The conditions for PCR were as follows:

50C2 min; 95C2 min, followed by 30 cycles of 95C15 s; 60C1 min and

72C15 s. Ct values were recorded for each gene, and the results of genes of

interest were normalized to results obtained with the internal control gene. ddCT

were calculated and the results are expressed as fold increase. All oligonucleotides

and reagents utilized in this protocol were purchased from Invitrogen Co.

- Statistical analysis: Data are expressed as mean and standard error () of the

mean (SEM) and were analyzed using Student's t-test to evaluate the statistical

significance of the null hypothesis vs CTL and/or TEN. All data were statistically

evaluated by analysis of variance (ANOVA), followed by the Tukey test. Values with

P < 0.05 were considered to be statistically significant.

RESULTS

The figure 01 shows in the Panel A the COX-2 gene expression, 2 hours after

induction of tendinitis . We can observe that the tendinitis ( NT ) as well as the other

3 groups presented a significant increase in COX-2 gene expression when compared

to the control group after tendinitis induction (P < 0.001). However, none of the

groups were significantly inhibited by diclofenac or Laser treatments.

In Panel B we can observe the IL6 gene expression at 2 hours after induction

of tendinitis. We could observe that there was an increase in gene expression of IL-6

in tendinitis untreated group (NT), (p

34

group. The group treated with sodium diclofenac (D) and the irradiated group (1J)

showed increased expression of the same gene, when compared with (NT).

The figure 01 panel D shows the TNF- gene expression, two hours after

tendinitis induction. We can observe that the (NT) group showed a significant

increase in TNF- gene expression when compared to the control group (C). It was

also observed that both groups diclofenac and Laser 1J failed to reduce the

expression of TNF-. Only the group treated with laser irradiation at the energy of 3J

was able to significantly reduce the TNF- expression when compared to NT group

(p

35

DISCUSSION

Low level laser therapy has been studied since the 60s being considered as a

therapeutic alternative with good perspectives for use specially in musculo-skeletal

and inflammatory disorders. In our study, we investigated the effects of low level

laser therapy on important inflammatory mediators, involved in acute inflammation of

the tendon.

Recent studies have focused on the discovery of new therapies able to

promote cytokines inhibition, such as IL-1, TNF- as well as metalloproteinases in

tendon diseases. Thus, tendinitis appears as the target of concern, both in the sports

medicine and labor.

Here we employed the nonsteroidal anti-inflammatory drug Sodium Diclofenac

as a pharmacological reference therapy to be compared to Laser irradiation in tendon

inflammation induced by collagenase. The experimental model used was the

tendinitis induced in Achilles tendon of rats by collagenase injection. This

experimental model of tendon inflammation is well known in the study of tendinitis in

which edema and acute destruction of the extracellular matrix are similar to those

found in natural tendon injury, being considered an excellent model that allows to

investigate molecular and histological changes [35].

The development of inflammation during the injury is a beneficial event that

aims to restore tissue homeostasis. In this process the formation of chemical

mediators with pro-inflammatory or anti-inflammatory action are produced from

cyclooxygenase pathways that also increase in inflammatory processes.

In tendinitis model, due to low vascularity or to transitions between tendinitis

and tendinosis periods, this tissue has not always inflammatory infiltrate. However,

COX-2 increase was observed in both cases which indicates in some extent, a tissue

degeneration as a result of the inflammatory process [36].

Previous study from our group was the first to determine the time course of

COX-2 gene expression, and the peak time after 2 hours of tendinitis induction [29].

From these data, we choose the period of 2 hours, the same used previously, to

36

study the effects of Laser irradiation, this time operating in 660nm, in the

experimental model of collagenase-induced rat tendinitis.

In the present study it was observed that COX-2 gene expression increased in

non-treated group (NT). In the same way, non-treated group presented significant

increases in the other pro-inflammatory citokines, IL-6 and TNF-.

The treated group with sodium diclofenac (D) also presented increase in COX-

2 and TNF- expression, similar to NT group and more IL-6 expression compare to

NT. On the other hand, the irradiated groups, especially 3J showed a slight decrease

in COX-2, IL-6 and TNF-, compared to NT.

Cyclooxygenase-2 increased can directly interfere in the formation of specific

inflammatory mediators, such as interleukins and tumor necrosis factor, by changing

the tissue repair process. TNF- is another factor that can modulate this process.

Their levels are generally increased in the presence of musculoskeletal diseases like

tendinitis and could be important in degenerative process of the tissue by inducing an

increase in inflammatory cells [19]. Thus, the increase of TNF- gene expression

after tendinitis induced may represent an increase in the inflammatory process

triggering other important inflammatory mediators, such as matrix metalloproteinases

[37].

Changes in tendon repair can lead to scar formation with different

characteristics of the original tissue. The scar initially provides the physical continuity

of the tissue, but the proliferation of adjacent tissue can be undesirable and hinder

the sliding mechanism tendon [14]. One way to reduce these changes would be

control the healing process, modulating the inflammation by cytokines levels,

involved in the repair, such as IL-6 , IL-10 and TNF- , observed in this work and

associated with disease progression. IL-10 for instance, sharing functional activities

such as suppression of proinflammatory events by inhibition of inflammatory

cytokines such as IL-1, IL-6, TNF- and also inhibit some MMPs that could hamper

tissue repair [15].

In this work, the expression of pro-inflammatory cytokines (IL-6 and TNF-)

were increased not only in the NT group, but also in diclofenac group (D) and even in

the irradiated group (1J). Only the irradiated group (3J) was able to reduce

37

expression of these pro-inflammatory cytokines. On the other hand, the expression of

antiinflammatory cytokines (IL-10) was increased in all groups where tendinitis was

induced, probably a natural action of the body to reduce inflammation, but in the

treated groups D, 1J and 3J, this increase was even higher.

Thus, the relationship between the amounts of IL-6 and IL-10 show a positive

anti-inflammatory response, mainly in the irradiated group 3J. These results together

suggest that in the non-treated group (NT) the inflammation remained high and

probably the highest level of tissue injury, on account of increase of TNF- to be

associated with increased of matrix metalloproteinase enzymes.

Previous results demonstrated that treatment with sodium diclofenac reduces

prostaglandin E2 synthesis and NK1 neuropeptides expression, receptors likely to

pain. However, our results showed that sodium diclofenac treatment failed to reduce

the expression of citokines (IL6 and TNF-). Diclofenac treatment failure may be

related to dose and administration in rats or specific tendon characteristics that is

poorly vascularized.

The sum of these factors is a strong indication that low level laser therapy in

660nm - 100mW and 3 Joules can be effective in reducing the acute inflammatory

process induced by collagenase in Achilles tendons of rats. However, further analysis

are needed to determine this laser modulation operating in 660 nm on matrix

metalloproteinases in tendon tissues during the inflammatory process. The

investigation of biochemical factors as protein expression of inflammatory mediators,

enzyme dosage and morphological and functional factors such as histological and

mechanical properties analyzes are important to understanding of this inflammatory

event and the action mechanism involved at cellular and structural changes in

tendinitis.

38

CONCLUSION

We can conclude that the model of tendinitis by collagenase-induced in rats

increased the expression of important inflammatory mediators. Treatment with

sodium diclofenac was not effective in reducing inflammatory mediators. The low

level laser therapy (660nm - 100mW) at 3 Joules of energy was effective in reducing

the inflammation in this model.

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43

5 CONSIDERAES FINAIS

O modelo de tendinite induzida por colagenase em ratos aumentou a

expresso de importantes mediadores inflamatrios relacionados tambm com a

degradao de colgeno. O tratamento com diclofenaco de sdio no foi eficaz na

reduo de mediadores inflamatrios. O laser de baixa potncia (660nm 110mW)

na energia de 3 Joules foi eficaz na reduo do processo inflamatrio neste modelo

de tendinite.

Figura 5: Tabela dos resultados; os valores do grupo NT foram comparados com o

grupo C, e os grupos tratados com D, 1J e 3J, com valores comparados com o grupo

NT.

44

6 REFERNCIAS

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