universidade nove de julho programa de pÓs ......monoartrite induzida com zymosan. em ambos os...
TRANSCRIPT
-
1
UNIVERSIDADE NOVE DE JULHO
PROGRAMA DE PÓS GRADUAÇÃO EM CIÊNCIAS DA REABILITAÇÃO
MARCELO DE PAULA A. SILVA
Efeito do treinamento físico combinado com laser de baixa potência em monoartrite
experimental
São Paulo
2015
-
2
MARCELO DE PAULA A. SILVA
Efeito do treinamento físico combinado com laser de baixa potência em monoartrite
experimental
Orientação:
Profa. Dra. Stella Regina Zamuner, PhD.
Coorientação:
Profa. Dra. Kátia De Angelis, PhD.
São Paulo
2015
TESE apresentada a Universidade Nove de Julho –
UNINOVE como requisito para obtenção do título de
Doutorado em Ciências da Reabilitação.
-
3
Silva, Marcelo de Paula A.
Efeito do treinamento físico combinado com laser de baixa potência em
monoartrite experimental. / Marcelo de Paula A. Silva. 2015.
105 f.
Tese (doutorado) – Universidade Nove de Julho - UNINOVE, São
Paulo, 2015.
Orientador (a): Profa. Dra. Stella Regina Zamuner.
1. Monoartrite. 2. Laser de baixa potência e treinamento físico. I. Zamuner, Stella Regina. II. Titulo
CDU 615.8
-
4
-
5
A sabedoria é a coisa principal; adquire pois a
sabedoria, emprega tudo o que possuis na
aquisição de entendimento. Provérbios 4(todo):7
https://www.bibliaonline.com.br/acf/pv/4/7+#v7
-
6
DEDICATORIA
Á Jesus Cristo. Á meu Pai Gilberto Alves da Silva; minha Mãe Julia Barbosa Queiroz da Silva;
minhas irmãs Jeanne Paola, Nicole Kerolin, Carolie Keterin e meu irmão Gil Polarah. Meus
sobrinhos Yam Di Luca, Luan e minha sobrinha Maria Elloyse.
-
7
AGRADECIMENTO
A minha orientadora Profa. Dra. STELLA REGINA ZAMUNER que admiro a
cada dia mais por sua disponibilidade em me ensinar, trocar saberes, sonhos e projetos. Eterna
gratidão por ter me escolhido!
A minha coorientadora Profa. Dra. KATIA DE ANGELIS por sua excelência na
velocidade e acumulo de conhecimentos. Especial agradecimento pela confiança,
ensinamentos e auxílio acadêmico.
A minha coorientadora Profa. Dra. IRIS CALLADO SANCHES pelo gratificante
aprendizado, expertises, dicas e apoio enriquecendo incomensuravelmente minha trajetória.
Aos que compuseram as bancas de qualificação e defesa norteando minha caminhada
com suas arguições, incentivando-me a reflexões contínuas: Prof. Dr. José Antonio, Prof. Dr.
Rodolfo de Paula, Prof. Dr. Paulo de Tarso, Profa. Dra. Ivani Credidio Trombetta, Profa. Dra.
Maria Claudia Irigoyen, Profa. Dra. Maricilia Silva Costa, Prof. Dr. Danilo Sales Bocalini e
Profa. Dra. Cristina Maria Fernandes.
Ao Reitor Prof. Eduardo Storópoli da Universidade Nove de Julho e respectivos
colaboradores empenhados na qualidade do crescimento acadêmico á manutenção da
infraestrutura da instituição.
Aos Professores Doutores do programa de pós graduação em Ciências da Reabilitação,
que em suas aulas professaram conhecimentos que me auxiliam em meu caminhar profissional:
Profa. Dra. Cláudia Santos Oliveira, Profa. Dra. Daniela Aparecida Biasotto Gonzalez, Prof. Dr.
Dirceu Costa, Profa. Dra. Fernanda de Cordoba Lanza, Prof. Dr. João Carlos Ferrari Corrêa,
-
8
Profa. Dra. Kristianne Porta Santos Fernandes, Profa. Dra. Luciana Maria Malosá Sampaio, Prof.
Dr. Luis Vicente Franco de Oliveira, Profa. Dra. Raquel Agnelli Mesquita Ferrari, Profa. Dra.
Regiane Albertini de Carvalho, Profa. Dra. Simone Dal Corso, Prof. Dr. Andrey Jorge Serra e
Prof. Dr. Humberto Dellê. Assim como, a todos que oportunamente escutei e dialoguei.
Aos parceiros de pesquisa e troca de experiências: Profa. Dra. Christiane Malfitano,
Profa. Dra. Janaina Brito, Profa. Dra. Martha Manchini, Profa. Ms. Nathalia Bernardes, Profa.
Ms. Daniele Dias, Prof. Ms Filipe Conti, Prof. Ms. Guilherme Lemos, Profa. Ms. Renata Kely
da Palma, Profa. Ms. Morgana, Prof. Ms. Fernando Alves, Profa. Ms. Sarah Freitas e Profa.
Camila. Laboratório de Fisiologia Translacional – UNINOVE.
Aos parceiros de bancada e congressos: Profa. Dra. Nikele Andrade, Profa. Ms. Eliadna
Silva, Profa. Ms. Camila Silva, Prof. Ms. Agnelo Alves, Profa. Ms. Luciana Silva, Prof. Ms.
Adriano Santos e aos que me auxiliaram no Laboratório de Pesquisa – UNINOVE.
Aos companheiros de iniciação cientifica: Erico Gemignani, Daniel Munhoz, Larissa
Hirata, Stefano Fonseca e Welbert Oliveira, agradeço a vocês...
Aos parceiros de encontros, que em gestos singulares me auxiliaram: Prof. Marcio José
Figueira Chaves (valeu por tudo), Ligia Barbosa, Kamila Santos Cerrados, Camila Camarão,
Juliana Ribeiro, Luciana Dandara, Samara Vezzaro, Angela Santos, Rodrigo Barros, Karem
Viegas, Simone Moraes, Carla Duarte... Vossas ajudas me foram muito importante.
A CAPES - Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior e ao O
Programa de Suporte à Pós-Graduação de Instituições de Ensino Particulares/ PROSUP, pelo
suporte financeiro e estímulo a buscar o conhecimento.
Á DEUS! Por Ele, para Ele tudo o que sou e serei. Sempre!
Efeito do treinamento físico combinado com laser de baixa potência em monoartrite
experimental
-
9
A monoartrite (MA) é causada por inflamação monoarticular e representa considerável
problema de saúde pública em todo o mundo. Para avaliação desta patologia utilizou-se dois
protocolos (P1 e P2). Em P1 foi avaliado o efeito da terapia a laser de baixa potência (LBP) no
influxo de células inflamatórias, a liberação de mediadores inflamatórios, as metaloproteinases
(MMPs) e o processo de reparação intra-articular. Em P2 ocorreu associação do treinamento
físico (TF) e o LBP para avaliarmos alterações sistêmicas utilizando a variabilidade da
frequência cardíaca (VFC) e as alterações articulares locais em modelo experimental de
monoartrite induzida com zymosan. Em ambos os protocolos utilizou-se Ratos Wistar machos
(220-280 g) que receberam injeção intra-articular de zymosan (1 mg / 50 mL de uma solução
salina estéril) no joelho direito. Em P1 os ratos foram irradiados imediatamente, 1 h, e 2 h após
a administração de zymosan com LBP (660 nm, 10 mW, 2,5 J / cm2, 10 s). No grupo de controlo
positivo, os animais foram injetados com a dexametasona (fármacos anti-inflamatórios) 1 h
antes da administração de zymosan. Em P2 os ratos foram adaptados à esteira (10 min/d, 5 d e
0,3 km/h), 48 h após ocorreu a administração de zymosan seguindo da continuidade do TF
moderado e LBP (660 nm, 5 mW, 2,5 J/cm2, 20s, 0.04 cm2, 0.1 w/cm2) duas vezes por semana,
sempre antes do TF, durante 4 semanas.Os resultados demonstraram que em P1 o tratamento
com o LBP inibiu significativamente o influxo de leucócitos, a libertação de IL-1 e IL-6 e
também a atividade de metaloproteinase 2 e 9. Em P2 ocorreu a mensuração da pressão arterial
(PA) e variabilidade da frequência cardíaca (VFC). Ratos treinados apresentaram menor peso
corporal, aumento da velocidade máxima de corrida e menor frequência cardíaca em
comparação com os grupos sedentários. Além disso, ratos submetidos a TF mostraram um
aumento de Intervalo de Pulso (IP) e diminuição na Banda de Baixa Frequência (BF) e
Variância da Pressão Arterial Sistólica (VAR PAS) em relação ao grupo sedentário com MA.
Os ratos submetidos a TF associado ao LBP também mostraram uma diminuição na (BF) e na
Razão da Banda de Alta Frequência / Baixa Frequência (AF / BF), um aumento de VAR PAS,
Variância RR (VAR-RR) e AF em relação ao grupo sedentário com MA. Além destes ocorreu
melhora na capacidade funcional e uma diminuição de influxo leucocitário na cavidade
articular. A análise histológica mostrou uma histoarquitetura preservada da membrana sinovial
e uma redução do deposito de colágeno nos grupos com TF e associação TF e LBP em
comparação ao grupo sedentário com MA. TF e LBP causaram uma redução na liberação de
IL-1β no líquido sinovial e membrana sinovial. Além disso, a IL-10 foi aumentada no grupo
com associação de TF e LBP. A terapia com LBP foi eficaz na redução do processo inflamatório
e inibe a ativação de proteases (gelatinase), sugerindo menor degradação do tecido de colágeno
no modelo experimental. Além disso, na MA ocorre um desequilíbrio do sistema simpático, o
que sugere um envolvimento autonômico precoce. Um programa de TF moderado associado ao
LBP pode exercer efeitos benéficos no balanço autonômico cardiovascular e melhora da
capacidade funcional. Quanto aos efeitos deletérios nos joelhos dos ratos. Associação de TF e
LBP foi eficaz na proteção da articulação em modelo experimental de monoartrite, levando a
uma melhora na regulação de citocinas inflamatórias e melhor organização histoarquitetura
intra articular.
Palavras chaves: Monoartrite, laser de baixa Potência e Treinamento Físico.
Effect of physical training combined with low level laser therapy in an experimental
monoarthritis
-
10
Monoarthritis (MA) is caused by single-joint inflammation and represents a considerable public
health problem worldwide. To evaluate this pathology two protocols were used (P1 and P2). In
P1, the effects of low level laser therapy (LLLT) in the influx of inflammatory cells, the release
of inflammatory mediators, metalloproteinases (MMPs) and the process of intra-articular
repair, were evaluated. In P2 the combination of exercise training (ET) and the LLLT were used
to evaluate systemic changes using the heart rate variability (HRV) and local joint changes in
experimental model of monoarthritis induced by zymosan. In both protocols male Wistar rats
(220-280 g) was used. Rats received intra-articular injection of zymosan (1 mg / 50 mL of
sterile saline) into the right knee. P1 rats were irradiated immediately, 1 hour and 2 hr after
zymosan administration with LLLT (660 nm, 10 mW, 2,5 J / cm2, 10 s). In the positive control
group, rats were injected with Dexamethasone (antiinflammatory agents) 1 hr before
zymosan.administration. P2 rats were adapted to the treadmill (10 min / d 0.3 5 km / h) after 48
h the zymosan was administrated and the moderate ET and LLLT (660 nm, 5 mW, 2,5 J/cm2,
20s, 0.04 cm2, 0.1 w/cm2) was applied. The LLLT was applied twice a week, always before TF,
during 4 weeks. Our results demonstrated that in P1 LLLT treatment significantly inhibited the
influx of leukocytes, release of IL-1 and IL-6 and also metalloproteinase activity 2 to 9. In P2
the measurement of arterial pressure (AP) and heart rate variability (HRV) was measured.
Trained rats had lower body weight, increased maximum speed racing and lower heart rate
compared to the sedentary groups. Furthermore, ET rats showed an increase in pulse interval
(PI) and decrease in the low frequency band (LF) and the systolic arterial pressure variance
(VAR SAP) compared to sedentary group MA. ET associated with LLLT also showed a
decrease in (LF) and Reason of Band High Frequency / Low Frequency (HF/LF), increase VAR
SAP, variance RR (VAR-RR) e HF. compared to the sedentary group with MA. In addition,
there was an improvement in functional capacity and a decrease of leukocyte influx in the joint
cavity. Histological analysis showed a histoarchitecture preserved the synovial membrane and
a reduction in collagen deposit in the groups with ET and ET associated and LLLT compared
to the sedentary group with MA. ET and LLLT caused a reduction in the release of IL-1β in
synovial fluid and synovial membrane. Furthermore, IL-10 was increased in the group
association of ET and LLLT. LLLT was effective in reducing inflammation and inhibits
activation of proteases (gelatinase), suggesting less degradation of collagen tissue in an
experimental model. In MA occurs an imbalance of the sympathetic system, suggesting an early
autonomic involvement. A moderate ET program associated with LLLT may have beneficial
effects on the cardiovascular autonomic balance and improved functional capacity. With regard
to deleterious effects in the rat knee, ET and LLLT association was effective in protecting the
joint in an experimental model of monoarthritis, leading to an improvement in the regulation of
inflammatory cytokines and better intra articular histoarchitecture organization.
Keywords: monoarthritis, low level laser and Exercise Training.
S U M Á R I O
-
11
Lista de Tabelas ............................................................................................................ xii
Lista de Quadros ......................................................................................................... xii
Lista de Figuras
...............................................................................................................
xiii
Lista de Abreviaturas
......................................................................................................
xiv
1. Contextualização
.........................................................................................................
16
1.1. Monoartrite
...............................................................................................................
17
1.2. Zymosan e Inflamação ........................................................................................ 18
1.3. Treinamento Físico ............................................................................................. 19
1.4. Variabilidade da Frequência Cardíaca .................................................................... 20
1.5. Laser de Baixa Potência
...........................................................................................
22
2. Objetivos ............................................................................................................. 26
3. Resultados .......................................................................................................... 28
4. Artigo 1 (Publicado) ........................................................................................... 28
5. Artigo 2 .............................................................................................................. 48
6. Artigo 3 .............................................................................................................. 63
7. Considerações finais .......................................................................................... 95
REFERÊNCIAS
ANEXOS (Ata de Aprovação, Comitê de Ética, Currículo resumido, Artigo 1)
-
12
LISTA DE TABELAS
Artigo 2
Table 1. Body weight and Maximal Exercise Test (MET) ……………………………. 64
Table 2. Hemodynamic Assessments
................................................................................
65
Table 3. Autonomic variables at after 35 days of induction arthritis
……………………
66
Artigo 3
Table 1. Maximal Exercise Test (MET)
………………………………………………….
87
LISTA DE QUADROS
Quadro 1. Índices estatísticos, no domínio do tempo da VFC. ..........................................
20
Quadro 2. Fórmulas matemáticas para cálculos de parâmetros do LBP. ..........................
22
-
13
LISTA DE FIGURAS
Artigo 1
Figure 1 - LLLT effect on leukocyte influx into the joint cavity induced by zymosan
….
44
Figure 2 - Effect of LLLT on the release of IL-1β and IL-6 into the joint cavity
………..
45
Figure 3 - Histological demonstration of the collagen fibers of the synovium
…………..
46
Figure 4 - Effect of LLLT on MMP-2 and 9 activities on joint lavage
…………………..
47
Artigo 2
Figure 1 - Time course line showing the treatment protocol the experimental
procedures
63
Artigo 3
Figure 1 - Time course line showing the 4 weeks of the experimental procedures …….
88
Figure 2 - Effect of ET and LLLT on leukocyte influx into the joint cavity in
monoarthritis rats ……………………………………………………………………….
89
Figure 3 - Histological demonstration of the synovium ………………………………..
90
Figure 4 - Histological demonstration of the fiber collagen ……………………………
91
Figure 5 - Collagen formation in longitudinal histological sections of rat knee ……... 92
xii
-
14
Figure 6 - Effect of ET and LLLT on the release of IL-1β in joint cavity of
monoarthritis rats
………………………………………………………………………...
93
Figure 7 - Effect of ET and LLLT on the release of IL-10 in joint cavity of
monoaarthritis rats
………………………………………………………………………..
94
LISTA DE ABREVIATURAS
A Area
ACR American College of Rheumathology
ACSM American College of Sports Medicine
AF Banda de Alta frequência
ANOVA Análise de variância
AR Artrite Reumatóide
ATP Adenosine trifosfato
BF Banda de Baixa frequência
Bpm Batidas por minute
Ca Cálcio
CD Compact disco
CDC Centers for Disease Controland Prevention
Cm Centímetros
COBEA Colégio Brasileiro de Experimentação
Animal
COX-2 Ciclo – oxigenase – 2
DCV Doença Cárdio Vascular
De Densidade de energia
Dp Densidade de potência
EGFr Fator de Crescimento de Epiderme
EPAC Exercise and Physical Activity Conference
Hz Hertz
i.p. Intraperitoneal
InGaAlP Indio – Galio – Alumínio - Fósforo
IL Interleucina
INF- δ Interferon gama
J Joule
xiii
-
15
K Potássio
Kg Kilo grama
Laser Light Amplification by Stimulated Emission
of Radiation
LBP Laser de baixa potência
M Média
MEC Matriz Extracelular
MFC Media da Frequência Cardíaca
Mg Miligrama
MmHg Milímetro de Mercúrio
MMP Metaloproteinases
Mn Morfonucleadas
Mw Mili watts
Na Sódio
NF KB Fator Nuclear de Cadeia Leve Kappa
potenciador de células ativadas B
Nm Nanômetro
NO Oxído Nítrico
NSAIDs Drogas Anti-inflamatórias não Esteroides
PAD Pressão Arterial Diastólica
PAS Pressão Arterial Sistólica
PBS Tampão Fosfato Salina
PGE 2 Prostaglandina E
Pmn Polimorfonucleadas
R Erro padrão estimado
ROS Espécies Reativas de Oxigênio
RR Rate Rate
S Salina
SNA Sitema Nervoso Autônomo
T Tempo
TEM Teste de Esforço Máximo
TGF- β Fator de Crescimento Beta
TLRs Toll-like Receptores
TNF- α Fator de Necrose Tumoral Alfa
USA United State America
VFC Variabilidade da Frequência Cardíaca
VO2 Volume de Oxigênio
VPAS Variância da Pressão Arterial
W Watts
C Controle
MA Monoartrite
MAT Monoartrite + treinamento físico
MATL Monoartrite + treinamento físico + laser de
baixa potência
ZY Zymosan
Λ
Lambda
xiv
-
16
1. CONTEXTUALIZAÇÃO
A monoartrite é uma patologia com caráter inflamatório que acomete uma única
articulação. Está associada a fatores genéticos, obesidade, estresse biomecânico e trauma. Seu
diagnóstico se divide em inflamatório e não inflamatório. Afeta milhões de pessoas ao redor do
mundo [1, 2], situação que requer desenvolvimento de estudos e ações terapêuticas eficazes.
A monoartrite experimental se torna uma fonte de pesquisas, que contribui para a
avaliação e intervenção. Estudos mimetizam a inflamação articular utilizando o zymosan (ZY),
um polissacarídeo obtido da parede celular de levedura [3, 4], o qual induz influxo leucocitário
com presença de células morfomononucleadas (Mn) e polimorfonucleadas (PMN), citoquinas,
interleucinas e ação de metaloproteinases [5], situação similar em pacientes.
Na ação terapêutica ao tratamento da inflamação articular, o laser em baixa potência
(LBP), sugere ações anti-inflamatórias e condroprotetoras [6], além de não ser invasivo.
Sistemáticas pesquisas com LBP mostram atenuação à dor crônica em articulação
comprometida [7] através de analgesia, aumento da microcirculação [8], regeneração de
xv
-
17
colágeno, proliferação de fibroblastos [9], de células osteoprogenitoras, osteoblastos e
regeneração óssea [10].
Outra terapia não invasiva é o treinamento físico com evidências em contribuir ao
remodelamento do tecido cartilaginoso, desde que não seja de alta intensidade, pois isto é fator
importante para a prevenção e ou promoção da artrite [11, 12]. O papel do treinamento físico e
sua intensidade nos constituintes da articulação são complexos e influenciam de diferentes
formas as vias de sinalização na expressão gênica dos constituintes intra articular [13].
Diminuir a terapêutica farmacológica e cirurgias [14, 15], aumentar as possibilidades de
terapias não invasivas é indicação do Colégio Americano de Reumatologia, que recomenda
exercícios de alongamentos, resistidos e aeróbicos [16]. Há estudos experimentais
demonstrando que o LBP contribui para o tratamento não invasivo de doenças reumatólogicas
[17].
1.1. MONOARTRITE
Na MA doença debilitante 50% dos casos são auto limitante. A MA mostra alteração do
perfil inflamatório envolvendo uma única articulação. Pode ser classificada em aguda, sub-
aguda e crônica, dependendo do tempo e da evolução dos processos fisiológicos [18]. A
avaliação clínica atenta-se aos sintomas de dor, edema, redução de movimento (principalmente
pela manhã), fragilidade e inflamação articular. É considerada uma das principais doenças de
incapacidade funcional na população idosa [19].
As doenças reumáticas são caracterizadas por inflamações localizadas e sistêmicas de
etiologia ainda com muitas lacunas devendo ter abordagem multidisciplinar [20].
As ações multifatoriais da inflamação local na articulação e interações com os tecidos
adjacentes ao progredirem causam efeitos deletérios aos tecidos [21].
-
18
A expectativa e qualidade de vida da população, que sofre de inflamação articular são
dependentes de fatores genéticos e acesso a terapias multiprofissionais, sendo três vezes mais
frequentes na mulher do que no homem. 30 a 50 % das mortes em pacientes com doenças
reumáticas crônicas de longo prazo são decorrentes de complicações cardiovasculares ou
doença cardiovascular (DCV) [22].
Neste sentido em 1945 FLETCHER e LEWIS-FANNIG, encontraram hipertensão em
45% de pacientes com inflamação articular, auxiliando a correlacionar desordens músculo
esqueléticas, reumatologia e DCV [23].
1.2. ZYMOSAN E INFLAMAÇÃO
β-glucanos são polissacarídeos presentes em fungo a exemplo o Saccharomyces
cerevisiae. Sendo o Zymosan, que é principalmente composto de β-glucanos, um preparado
insolúvel encontrado nas paredes celulares daquele fungo. Interiorizado ao organismo os β-
glucanos liga-se a Dectina-1 e aos toll-like receptores (TLRs), componentes da função imune
inata orgânica, estes intermedeiam ativação de espécies reativas de oxigênio (ROS), do NF-kB
e posterior secreção de citocinas inflamatórias [24].
O Zymosan é utilizado em modelos inflamatórios exercendo ação sobre células
endoteliais, osteoclastos, linfócitos, neutrófilos e macrófagos. Caracterizando influxo de células
[25].
A inflamação induzida por Zymosan desencadeia uma série de eventos tais como
ativação de citocinas, quimiocinas, angiogênese, sinovite, e degradação da cartilagem [3].
Citocinas pró inflamatórias como o Fator de Necrose Tumoral alfa (TNF- α), o
Interferon gama (INF- δ) e as Interleucinas (IL) IL1, IL2, IL3, IL6, IL8, IL12 e 1L18, bem
como as citocinas antiinflamatórias como Fator de crescimento beta (TGF- β) e as Interleucinas
-
19
(IL) IL4 e IL10 (ou antagonistas das citocinas) agem por controlar o processo [26, 27]. Outra
conseqüência do processo inflamatório é na matriz extracelular (MEC), que esta em constante
remodelamento e com a presença de inflamação ocorre alterações na produção e
remodelamento das fibras de colágeno [28, 29].
1.3. TREINAMENTO FISICO
A monoartrite, inflamação articular é desencadeada por diversos fatores sendo alguns
podendo receber influências diretas do treinamento físico, como intensidade alta e fatores
endógenos e exógenos [30].
Não obstante o sedentarismo contribui para efeitos deletérios músculo esquelético como
a hipotrofia, assim também como a imobilização articular leva a distrofia [31] que acarretam a
degradação articular.
Evidências com diferentes abordagens consideram o treinamento físico personalizado
aos que sofrem de processos inflamatórios articulares, coadjuvante na modulação da dor [32]
outras abordam o estilo de vida somado a treinamento físico moderado e recreação, como
prevenção a artroplastia de joelho [33].
As recomendações ao tratamento de pacientes acometidos de inflamação articular
incluem terapias multimodais, indicando combinação de ações terapêuticas aliadas à educação
dos pacientes, treinamento físico, dieta e estilo de vida saudável [34]. Neste sentido, o Centers
for Disease Controland Prevention (CDC- USA) e o American College of Sports Medicine
(ACSM), tem associado à diminuição da dor, habilidade funcional e ao tratamento de
inflamação articular a prática regular de treinamento físico com intensidade moderada de
esforço percebido e ou monitorada em unidades metabólica [35]. Em 2002 Exercise and
-
20
Physical Activity Conference (EPAC) recomendou ás pessoas acometidas de inflamação
articular o treinamento físico moderado de 3 vezes por semana, somando 30 minutos ao dia.
Quanto ao tipo de treinamento físico recomenda-se o alongamento, resistido e o
aeróbico, fortalecido por consenso de evidências científicas apontadas pelo American College
of Rheumathology (ACR) [36], recomendações estas que promovem, também, alterações
cardiovasculares que contribuem a qualidade de vida, contudo há indagações a respeito de
considerar o impacto articular causado durante o treinamento físico aeróbico mesmo moderado.
Na MA diagnósticos diferenciais incluem progressão dos efeitos deletérios a poliartrite.
Nas doenças reumatologicas o perfil inflamatórios em longo prazo tendem a comprometer o
sistema cardiovasular, logo desordens musculoesqueléticas e genéticas decursam ao surgimento
de doenças cárdicas incluindo pericardite [37, 38, 39].
1.4. VARIABILIDADE DA FREQUÊNCIA CARDÍACA
Ferramentas de observação da Variabilidade da Frequência Cardíaca (VFC) apontam a
capacidade dos sistemas cardiovascular e sistema nervoso autônomo (SNA) em responder a
estímulos fisiológicos múltiplos e ambientais como o estresse mental, alterações
hemodinâmicas e metabólicas, sono, ortotastismo, respiração e treinamento físico, bem como
em compensar desordens induzidas por patologias [40, 41].
Em linhas gerais a VFC descreve as oscilações das batidas do coração (intervalos R-R)
no decorrer do tempo, podendo aferir as influencias do SNA sobre o nódulo sinusal,
Cronologicamente em 1965 a VFC teve uma maior atenção a partir das observações de
HON e LEE durante o sofrimento fetal, algum tempo depois, no ano de 1977, WOLF et al.
mostraram que uma menor VFC aumenta o risco de morte após o infarto agudo do miocárdio
(IAM) e em 1987, KLEIGER et al. confirmaram que a VFC é um importante e independente
-
21
preditor de mortalidade após IAM. Logo, alta VFC significa adaptabilidade, caracterizando
mecanismos SNA eficientes. Uma crescente atenção a VFC na historia, que atualmente persiste.
De posse destas informações, pode-se avaliar qualitativamente a VFC e inferir uma serie
de diagnósticos. Grupos de estudiosos [40] copilaram diversos dados unificando-os e com isto
desenvolveu-se programas computacionais e recomendações técnicas em saúde, que auxiliam
a uma avaliação quantitativa com acurácia específica e abrangente, as quais culminaram com
estratégias de mensuração da VFC, contribuindo a pratica clínica de diagnósticos em saúde,
bem como na pesquisa científica.
Método Domínio do Tempo calcula a dispersão das oscilações da média da frequência
cardíaca por um período prolongado, (Quad. 1). Método Domínio da Frequência avalia a
densidade espectral das oscilações cardíacas através da observação de bandas de frequência,
sendo as mais utilizadas as Banda de Alta Frequência (AF / 0,2 a 0,4 Hz) e a Banda de Baixa
Frequência (BF / 0,02 a 0,07 Hz).
Quadro 1. Índices estatísticos, no domínio do tempo da VFC.
Sigla Descrição
SDNN*
Desvio padrão de todos os intervalos de pulsos normais gravados em um
intervalo de tempo, expresso em ms;
SDANN
Representa o desvio padrão das médias dos intervalos de pulsos normais, a
cada 5 minutos, em um intervalo de tempo, expresso em ms;
SDNNi
É a média do desvio padrão dos intervalos de pulsos normais a cada 5
minutos, expresso em ms;
RMSSD
É a raiz quadrada da média do quadrado das diferenças entre intervalos de
pulsos normais adjacentes, em um intervalo de tempo, expresso em ms;
pNN50
Representa a porcentagem dos intervalos de pulsos adjacentes com
diferença de duração maior que 50 ms.
-
22
* (NN) Intervalo Normal – Normal, ou ciclos cardíacos de cada registro do complexo QRS o
qual representa a despolarização ventricular, resultante da despolarização do nó sinusal.
Existem também os métodos não lineares que estão presente em todos os seres vivos. Métodos
de avaliação da VFC, que se destacam, dentre vários, são a teoria do caos e o mapa de retorno
tridimensional, porém a necessidade de validações [42].
1.5. LASER DE BAIXA POTÊNCIA
O LASER, Light Amplification by Stimulated Emission of Radiation ou amplificação
da luz por emissão estimulada de radiação originalmente descrito em 1960, por THEODORE
MAIMAN, sobe a forma de um laser de rubi que era utilizado, inicialmente, em alta potencia
por conta da intensidade. Sua aplicação na saúde se fazia e faz como ativação de agentes
fotodinâmicos, em cirurgias (bisturi a laser) e terapias ablativas.
Em 1967 MESTERS et al reportou os efeitos do laser de baixa potência atérmico (LBP)
em experimentos com camundongos [43, 44] a este tipo de laser nos deteremos.
O laser em sua gênese é gerado, após excitação do átomo que produz radiação
eletromagnética. O feixe de luz do LBP é coerente (ondas do feixe em fase), monocromática
(comprimento de onda uniforme) e colimada (ondas do feixe paralelas); praticamente não existe
dispersão ou espalhamento deste feixe de luz, diferente de uma luz proveniente de uma
lâmpada.
De 1960 a 2013, cinco décadas se passaram e cada vez mais o laser ganha espaço na
área da saúde com ações em cicatrização de tecidos, condições inflamatórias, modulação de
processos regenerativos, anfigênese e proliferação de células tronco [43].
Na medicina moderna e suas múltiplas especialidades destaca-se a utilização do laser na
dermatologia, oftalmologia, cardiologia e neurologia. Assim, como também na odontologia,
fisioterapia, estética e demais áreas correlatas da saúde ou não, a exemplo a impressão gráfica,
-
23
leitura de CD e máquinas laser de gravação em insumos (canetas, plásticos especiais, acrílicos,
etc).
A fotomedicina ganha status através da utilização criativa da “bioestimulação a laser” e
ou “terapia laser de baixa potencia”, em tratamento de patologias. Situação com o referendo de
avaliações, intervenções e reflexões, que se somam em produções intelectuais [45].
Nestas, aspectos importantes são apontados como à indicação minuciosa de local de
aplicação e parâmetros utilizados [7, 46], procedimentos necessários a melhor reprodutibilidade
de métodos e resultados. A este contexto as formulas necessárias ao cálculo (Quad. 2).
Quadro 2. Formulas matemáticas para alguns parâmetros do LBP.
Aos aspectos acima mencionados, inclui pontos chaves de intervenção com o LBP,
sendo eles:
• O material em estado gasoso utilizado no Laser de emissão continua;
• Comprimento de onda, sendo seu símbolo o λ (lambda) e unidade de mensuração o nm
(nanômetro);
De (J/cm2) = P . t / A
De = densidade de energia (dose ou fluência) em (J/cm2)
J = joule
P = potencia (W)
t = tempo (seg)
A = area (cm2) tamanho do ponto ou largura do feixe
Dp = P / A Dp = densidade de potencia em (W/cm2)
P = potencia (W)
A = area (cm2) tamanho do ponto ou largura do feixe. (Quando a ponteira do
laser toca a área irradiada)
E = P / t E = Energia (J)
P = potencia (W)
t = tempo (seg)
A = π . r2
A = área (cm)
π = pi
r = raio
-
24
• O contato versus o não contato do equipamento na área de aplicação;
• Periodicidade no tratamento;
• O tipo de lesão e ou desordem musculoesquelética, bem como orgânica.
Aliado a este contexto estudos tem demonstrado uma ação positiva na cicatrização de
feridas em ambiente clínico, porém não há uma compreensão total dos mecanismos de ação, no
que se refere a dosimetria, efeitos microbiológicos e “janela terapêutica” [47].
Contudo cabe a necessidade de padronização dos parâmetros dosimétricos da
fototerapia, a exemplo, fluência de energia, energia total, irradiação, pois na literatura muitas
vezes estas nomenclaturas se misturam. Outras variáveis são o comprimento de onda, tamanho
do feixe, tempo e duração da aplicação, ponto (s) da aplicação e suas organizações. Em
específico no tratamento em reumatologia há décadas, o LBP é considerado moderadamente
favorável aos efeitos clínicos em doenças reumáticas. Sendo aplicação em baixa potencia (1 a
500 mW), comprimento de onda espectro vermelho ou próximo ao infravermelho (600 – 1.000
nm) e irradiação entre 0.001 e 5 W/cm2, contribui a regeneração tecidual, redução de
inflamação e alivio a dor, através dos mecanismos fotoquímicos, já seus efeitos térmicos são
irrelevantes [48]. Os efeitos do LBP decorrem primeiramente de mecanismos fotoquímicos,
sendo que os fótons da radiação eletromagnética são absorvidos por moléculas fotoacptoras ou
cromóforas. Secundariamente ocorre indução por fotofisiologia em processos celulares e
terciariamente ocorre uma cascata de sinalização e respostas biológica.
A mitocôndria é descrita na literatura com uma célula cromófora e tendo em sua cadeia
respiratória propriedade de afinidade a luz monocromática sendo assim considerado um nível
biológico primário da ação do LBP [49].
O LBP é promissor ao tratamento em reumatologia, a literatura aponta moderada
eficácia durante o tratamento clinico na analgesia e mobilidade articular [48] situação esta
-
25
reforçada por estudo histológico mostrando que na inflamação o LBP mostra ação positiva na
modulação da resposta inflamatória tanto inicial, quanto tardia [18].
Ainda, estudos desvelam uma ação do LBP na inflamação articular experimental em
inibir a formação do edema, analgesia e incapacidade articular [50] sugerindo uma ação anti-
inflamatória e clinicamente relevante.
O LBP apresenta diminuição na modulação de mediadores inflamatórios como o TNF-
α, ciclo-oxigenase (COX-2) e prostaglandina E (PGE2) e redução de edema [51] estes presentes
no processo inflamatório articular local e participante dos efeitos decorrente da inflação.
O modelo experimental com Zymosan induz a inflamação articular através do
acionamento do sistema imune inato é bastante utilizado em pesquisa correlacionando fármacos
e LBP, sendo desvelada eficácia ao tratamento, porém com a necessidade constante de
comparar as alterações dosimétricas e os efeitos biomoleculares [52, 51]. Pesquisas suscitam
uma constante preocupação com as recomendações ao tratamento com LBP aos que sofrem
com doenças reumáticas [53]. Outra situação ainda não esta consolidada na literatura são os
parâmetros, frequência do tratamento, fluência de energia e energia entregue no tratamento com
LBP.
2. OBJETIVOS
-
26
2.1. OBJETIVO GERAL
O objetivo geral deste estudo visa compreender e comparar os efeitos do laser de baixa
potência combinado com treinamento físico em artrite experimental.
OBJETIVO ESPECÍFICO
ARTIGO 1:
Avaliar o efeito da terapia a laser de baixa potência na artrite aguda induzida por
zimosan de joelho de rato, no que se refere:
Influxo de células inflamatórias;
Liberação de mediadores pró-inflamatórios;
Ação das metaloproteinases (MMPs);
Processo de reparo da cartilagem na cavidade articular.
ARTIGO 2:
Estudar o efeito do laser de baixa potência combinado com treinamento físico, na ação
sistêmica, após a indução da artrite de joelho, no que se refere:
A capacidade funcional;
Alteração de peso;
A Variabilidade da Frequência Cardíaca;
Efeitos Hemodinâmicos;
Efeitos Autonômicos.
ARTIGO 3
Estudar o efeito do laser de baixa potência combinado com treinamento físico, na ação
local, após indução da artrite de joelho, no que se refere:
A degradação da cavidade articular;
Ao recrutamento Leucocitário;
Á liberação de Citocinas IL-1b e IL-10.
-
27
Avaliação histológica da articulação do joelho.
RESULTADOS
-
28
ARTIGO 1
Protective Effect of Low Level Laser therapy (LLLT) on Acute Zymosan-Induced
Arthritis
Fernando Pereira Carlos1, Marcelo de Paula Alves da Silva1, Eliadna de Lemos Vasconcelos
Silva Melo1, Maricilia Silva Costa2, Stella Regina Zamuner*1
1 Universidade Nove de Julho, Rua Vergueiro, 234, São Paulo, SP, Brazil.
2 Institute of Research and Development, University of Vale do Paraíba, São José dos Campos,
SP, Brazil
Running title: LLLT IN ZYMOSAN-INDUCED ARTHRITIS
Corresponding author: Stella Regina Zamuner, PhD
Adress: Rua Vergueiro, 234, Bairro Liberdade, CEP 01504-000
Phone: 55-11 33859222
E-mail: [email protected] or [email protected]
ABSTRACT
mailto:[email protected]
-
29
The aim of this study was to evaluate the effect of low level laser therapy on acute zymosan-
induced arthritis, with respect to the laser action on inflammatory cells influx, release of pro-
inflammatory mediators, metalloproteinases (MMPs) activity into the joint cavity and the
cartilage repair process. Arthritis was induced in male Wistar rats (250–280 g) by intra-articular
injection of zymosan (1mg/50 mL of a sterile saline solution) into one rear knee joint. Animals
were irradiated immediately, 1 h, and 2 h after zymosan administration with a semiconductor
laser InGaAIP (660 nm, 10 mW, 2.5 J /cm2, 10 s). In the positive control group, animals were
injected with the anti-inflammatory drug dexamethasone 1 h prior to the zymosan
administration. Treatment with laser significantly inhibited leukocytes influx, the release of IL-
1 and IL-6 and also the activity of metalloproteinase-2 and 9, into the joint cavity. In conclusion,
laser therapy was effective in reducing inflammation to sites of injury and inhibit activation of
proteases (gelatinase) suggesting less degradation of collagen tissue in experimental model of
acute arthritis.
Keywords: inflammation, arthritis, LLLT, cytokines, MMP
-
30
INTRODUCTION
Arthritis is a musculoskeletal disorder that affects great part of the population [1] it affects
people of all ages, but the problem of degenerative diseases of bones and joints is very likely
to increase considerably as the population ages [2]. Clinical symptoms are characterized by
pain, reduced range of movement, tenderness, and inflammation.
The pathological processes involved in arthritis induce to a complete joint destruction [3].
One of the main factors that lead to joint destruction is the infiltration of inflammatory cells.
Polymorphonuclear leukocytes (neutrophils) infiltration into inflamed tissues is a hallmark of
acute inflammation. These cells are predominant in the synovial exudates of a variety of
inflammatory arthropathies including rheumatoid arthritis [4]. Additionally, neutrophil influx
is always associated to the severity of the clinical picture in joint diseases (5). The mobilization
of these cells is mainly mediated by cytokines (IL-1β, IL-6), tumor necrosis factor-alpha (TNF-
), interferon- (INF-), platelet-derived growth factor (PDGF), transforming growth factor-
(TGF-) and nitric oxide (NO) [6-8]. Furthermore, elevated levels of pro-inflammatory
cytokines have direct implications for increased secretion of proteolytic enzymes (e.g.
matrixmetalloproteinases-MMPs) from stromal cells of the synovium and from chondrocytes
which, in turn, play a major role in eliciting cartilage damage [9].
Drug and non-drug treatments are used to relieve pain and/or swelling in patients with
arthritis. Non-steroidal anti-inflammatory agents (NSAIDs) and selective cyclooxygenase
(COX-2) inhibitors are commonly used as analgesic and anti-inflammatory agents in a number
of pathologies [10]. Although, the use of NSAIDs is limited due to the high incidence of
cardiovascular and gastrointestinal problems, it is widely used for inflammatory diseases such
as knee arthritis [11]. Likewise, TNF inhibitor therapy is often used to treat arthritis, but this
treatment is related with side effects on the systemic immune system [3]. These considerations
have prompted the search for alternative non-drug treatments for arthritis.
-
31
Low-level laser therapy (LLLT) has been used clinically and experimentally to treat a
wide variety of pathology conditions including musculoskeletal pathologies associated with
joint disease [12]. Even though LLLT has been used to treat several clinical conditions and has
also been studied in many animal models and in cell culture systems, its mechanisms are still
incompletely understood.
We recently demonstrated that LLLT, in two wavelengths (685 nm and 830 nm) was
effective in reducing edema formation, vascular permeability, and hyperalgesia in zymosan-
induced arthritis [11]. Therefore, the purpose of this study was to evaluate the mechanisms of
the effects of laser therapy in acute arthritis, induced by zymosan in the rat knee, with special
focus on inflammatory cells influx, the release of pro-inflammatory mediators and
metalloproteinase activity into the joint cavity.
MATERIAL AND METHODS
Laboratory animals
The experimental protocol was approved by our local ethics committee (protocol
number 0025/2011) that follows the guidelines of the Brazilian College of Animal
Experimentation. A total of 40 male Wistar rat were used for the study. Rats weighting 250-
280 g were housed in cages with free access to standard laboratory diet and drinking water.
Animals were kept in a 12:12-hour light-dark cycle at a temperature-controlled room (26ºC).
All experiments were designed to minimize animal suffering and to use the minimum number
of them associated with valid statistical evaluation.
Zymosan-induced acute inflammation in knee joint
Rats received an intra-articular (i.a.) injection of 1 mg zymosan (Sigma Chemical
Company, St Louis, MO, USA ), dissolved in 50 L of a sterile saline solution, into one rear
-
32
knee (stifle) joint. The procedure was done under anesthesia, using a mix of ketamine 80 mg/kg
(Hospira, Inc.; Lake Forest, IL, USA), xylazine 20 mg/kg (Lloyd, Inc.; Shenandoah, IA, USA)
intramuscularly.
Light sources, doses and treatment.
A low level laser InGaIP (aluminum gallium indium phosphide; MMOptics, Ltda, São
Carlos, SP. Brazil), operating continuous way in 660 nm wavelengths was used through the
whole experiment to irradiate the animals. The laser parameters were: 10 mW of power, 10 s
irradiation time and irradiated area of 0.04 cm2; which corresponds to a laser dose of 2.5 J/cm2.
The optical power of the laser was calibrated using a Newport Multifunction Optical Meter
(model 1835C). That laser dose, low enough to avoid any thermal effect, was chosen on the
basis of previous studies from our laboratory that had shown a beneficial effect of the low level
laser on the inflammatory process [11].
The rats were randomly divided into four groups with five animals per group. Saline
group: rats received an i.a. injection of saline; Zymosan group: rats received an i.a. injection of
zymosan (1mg/kg); Laser group: rats received an i.a. injection of zymosan and a laser treatment
in 660 nm; Dexamethasone group: rats received an i.a. injection of zymosan and was treated
with dexamethasone (0,4 mg/kg; i.p.) as an anti-inflammatory positive control (Sigma
Chemical Company), one hour before the zymosan injection. Animals of the laser group were
irradiated at times: 0, 1 and 2 h after induction of inflammation [11]. At the end of each protocol,
the animals in the respective groups were sacrificed in CO2 atmosphere.
Evaluation of leukocyte influx
The leukocytes recruited into the joint cavity were measured after induction of the
inflammatory reaction, as described above. After 6 hours of zymosan, saline or dexamethasone
-
33
injection, animals were sacrificed. Dissection was performed from the knees with the removal
of tibial-patello femoral ligament to expose the outer surface of the synovial membrane. Joint
lavage was collected from the cavity of the knee joint after two injections totaling 400 µL of
phosphate-buffered saline, pH 7,2 (PBS), containing 5 UI/mL heparin. Aliquots of the washes
were used to determine total cell counts in a Neubauer chamber after dilution in Turk solution
(0.2% crystal violet dye in 30% acetic acid). Differential leukocyte counts were performed on
stained Instant Prov.
Quantification of IL-1 and IL-6 concentrations
Sinovium washes were collected 6 h after i.p. injection of zymosan (1 mg/kg) or sterile saline,
as described above. After centrifugation, the supernatants were used for determination of IL-1
and IL-6 levels by a specific EIA. Briefly, 96-well plates were coated with 50 mL of the first
capture monoclonal antibody anti-IL-1 (2 mg/mL) or anti-IL-6 (2.5 mg/mL) and incubated for
18 h in the case of IL-1 or 2 h for IL-6 at 37°C. Following this period, 200 mL of blocking
buffer, containing 5% bovine serum albumin (BSA) in PBS/Tween 20, were added to the wells
and the plates were incubated for 2 h at 37 °C for IL-1 and overnight at 4 °C for IL-6. After
washing, 50 mL of either samples or standards were dispensed into each well and the plates
incubated for 2 h at 37 °C. Wells were washed, and bound IL-1 or IL-6 was detected by the
addition of the biotinylated monoclonal antibodies anti-IL-1 (5 mg/mL, 50 ml/well) or anti-IL-
6 (5 mg/mL, 50 mL/well), respectively. After incubation and washing, 50 mL of streptavidin–
peroxidase, in the case of IL-1, or avidin–phosphatase, in the case of IL-6, were added, followed
by incubation and addition of the substrate (50 mL/mL of s-phenylenediamine in the case of
IL-1 or 200 mL/mL r-nitrophenylphosphate in the case of IL-6). Absorbances at 450 nm were
recorded and concentrations of IL-1 and IL-6 were estimated from standard curves prepared
with recombinant IL-1 or IL-6.
-
34
Zymography
For the enzymatic assay, aliquots with 3 μl of supernatant from sinovium washes were
subjected to electrophoresis under non-reducing (100V a 4oC) polyacrylamide SDS gels (8%)
prepared with 1 mg/mL gelatin. After electrophoresis, gels were washed twice for 15 min each
with 2.5% Triton X-100 to eliminate SDS. Gels were then incubated overnight at 37ºC in
substrate buffer (50 mM Tris-HCl, pH 8.5, 5 mM CaCl2, and 0.02% NaN3). Gels were then
stained for 30 min with 0.05% Coomassie blue R-250 in acetic acid:methanol:water (1:4:5) and
distained in the same solution. All gels were prepared and run at the same time. The bands were
quantified by densitometry analyzed by public domain software Image J.
Histological procedures
At 6 hours after the induction of arthritis, the synovia was collected and submitted to
the classic histological method for embedment in paraffin: dehydration in increasing
concentrations of alcohol; clearing with xylol in order to allow the penetration of paraffin;
impregnation in paraffin baths and insertion in molds; cross-sectional cuts to a thickness of five
micrometers; and mounting. The material was stained with hematoxylin and eosin for the
determination of inflammatory cells in the injury site in each treatment and Picrosirius red stain
for the verification of collagen fibers.
To quantify the collagen fibers, the slices were observed in a polarized light microscope
Olympus coupled to an Olympus brand video camera. The images were digitized and
standardized and then analyzed through the Imag J software.
Statistical Analysis
Results are expressed as mean SEM. Differences among groups were analyzed by one-
way analysis of variance (ANOVA) followed by Tukey test. Values of probability lower than
-
35
5% ( p
-
36
zymosan (Zy) injection showing intense alteration in collagen fibers where we observe the
presence of areas with non-aligned fibers (arrow) and areas without collagen (star). LLLT
shows a better organization of the collagen fibers with similar appearance compared to
dexamethasone treatment. The percentage of collagen fibers in different groups was 76±6%,
31±10%, 68 ±16% and 71±11% in control, zymosan, LLLT and dexamethasone group,
respectively, demonstrating that the amount of collagen fibers in LLLT group is significantly
higher than the zymosan group (Fig. 3B).
Effect of LLLT on matrix metalloproteinase activity
LLLT effect on MMP-2 and MMP-9 activity was examined in the supernatant of
synovial lavage collected 6 and 12 hours after zymosan injection. The laser treatment
significantly decreased the release of MMP-9, both within 6 and 12 h after zymosan injection.
The supernatant collected at 12 h showed more intense band than those of supernatant collected
6 h after induction of inflammation (Fig. 4A and B). There was no statistical difference between
the laser and dexamethasone treatment used in this study. Figure 4C and 4D shows that the laser
treatment applied after the injection of zymosan significantly reduced MMP-2 activity,
similarly to dexamethasone, at 6 and 12 h after treatment.
DISCUSSION
Arthritic inflammation is a serious health problem that affects a large number of people
worldwide. LLLT was introduced as an alternative noninvasive treatment for arthritis about 20
years ago, but its effectiveness remains controversial. Moreover, the mechanisms involved in
the anti-inflammatory effect of LLLT are not yet established. Therefore, the knowledge of the
underlying mechanisms involved in the anti-inflammatory effect of laser treatment could lead
to the improvement of effective of a noninvasive therapy. In this study, we investigated the use
of LLLT on zymosan-induced arthritis, focusing on the acute phase.
-
37
The effects of LLLT were evaluated in cell migration into the rat knee joint. It is
noteworthy that most studies evaluate the cell influx in the synovia, whereas we did in joint
lavage. Moreover, the cells in the synovia of arthritis induced by zymosan are predominantly
lymphomononuclear while polymorphonuclear is the predominant cells in joint lavage [13]. To
verify that the laser was able to reduce the leukocytes influx into the joint cavity, this effect was
evaluate at 6 h after zymosan injection, period in which the peak of neutrophils influx into the
joint cavity occurred [13]. Our results clearly demonstrated that the laser radiation decreases
the migration of neutrophis in the sixth hour of inflammation, when applied immediately, 1st
and 2nd h after the induction of inflammatory arthritis, in the rat knee. Similar results were
described in the literature [14]. In another experimental model, it was observed a reduction of
leukocyte influx in the initial phase of carrageenan-induced pleurisy in rats after three
applications of LLLT 660 nm [15]. Similar data were found by Amano et al. [16] who treated
14 patients with knee arthritis and used the laser treatment. Also, we observed a better
organization of collagen structure in the laser-treated group. Thereby demonstrating the effect
of biostimulation with LLLT on membrane repair process.
The influx of inflammatory cells is an important factor that determines the course of
arthritis. Moreover, it is observed that the production of inflammatory cytokines such as IL-1β
and IL-6 are responsible for the disease progression and chronicity of the process. The literature
states that these cytokines can modulate the expression of metalloproteinase which is involved
in regulating the balance between cell and matrix, and when there is an unbalanced expression
of these enzymes the destruction of articular cartilage occurs. IL-1 is a dominant cytokine in
stimulating MMP expression by chondrocytes in experimental arthritis [17]. Also, several
authors [18, 19], reported that IL-6, involved in the pathophysiology of arthritis also enhances
production of osteoclasts. In the experimental model used in this study, the expression of IL-6
and IL-1 β had decreased after laser treatment, showing the same extent of reduction of
-
38
dexamethasone treatment. The reduction of this cytokine may suggest decreased activity of
MMP and/or osteoclasts production that causes cartilage and bone destruction. The same results
were found by Pallota et al., [14] showing a decreased in IL-1 and IL-6 after treatment with
LLLT.
A number of studies have demonstrated that MMPs are important mediators in
inflammatory and connective tissue diseases such as arthritis [20-22]. Of considerable
importance in arthritis are the gelatinase subfamily of MMPs, consisting of MMP-2 (gelatinase
A) and MMP-9 (gelatinase B). MMPs degrade various extracellular matrix proteins by
breaking them into their specific peptide bonds and are expressed in various cell types and
tissues, including vascular smooth muscle cells, endothelial cells, fibroblasts and inflammatory
cells [23]. Also, MMPs and cytokines (IL-1, IL-6 and TNF-α) are responsible for the
inflammatory signals that occur in the breakdown of cartilage [24]. In the present study LLLT
significantly decrease the activity of MMP 2 and 9 after zymosan injection, suggesting less
degradation of collagen tissue.
Conclusion
Collectively, the observations outlined above support the statement that the anti-
inflammatory effects of the LLLT tested in this study provide protective effects, countering
effectively the degradation of the joint cartilage network.
Acknowledgement - Financial support: Brazilian Council of Research—CNPq (Process No.
475083/2011-3).
-
39
REFERENCES:
1. Kidd BL (2002) Osteoarthritis and joint pain. Pain 123, (1-2):6
2. Castano AP, Dai T, Yaroslavsky I, Cohen R, Apruzzese WA, Smotrich MH,. Hamblin MR
(2007) Low level laser therapy for zymosan induced arthritis in rats: Importance of illumination
time. Lasers Surg. Med., 39:543-550
3. Yoshida S, Arakawa F, Higuchi F, Ishibashi Y, Goto M, Sugita Y, Nomura Y, Niino D,
Shimizu K, Aoki R, Hashikawa K, Kimura Y, Yasuda K, Tashiro K, Kuhara S, Nagata K,
Ohshima K. (2012) Gene expression analysis of rheumatoid arthritis synovial lining regions by
cDNA microarray combined with laser microdissection: up-regulation of inflammation-
associated STAT1, IRF1, CXCL9, CXCL10, and CCL5. Scand J Rheumatol. 41(3):170-179
4. Bezerra MM, Brain DS, GIRÃO CCV, GREENACRE S, KEEBLE J, ROCHA ACF (2007)
Neutrophils-derived peroxynitrite contributes to acute hyperalgesia and cell influx in zymosan
arthritis. Arch Pharmacol. 374:265-273
5. Harris Jr E (1991) Pathogenesis of rheumatoid arthritis: its relevance to therapy in the'90s.
Trans Am Clin Climatol Assoc. 102:260-268
6. Ed H (1990) Rheumatoid arthritis: pathophysiology and implications for therapy. N Engl J
Med. 322:1277-1289
7. Farrell AJ, Blake DR, Palmer R, Moncada S. (1992) Increased concentrations of nitrite in
synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases.
Ann rheum dis. 51:1219-1222
8. Cannon GW, Openshaw SJ, Hibbs Jr JB, Hoidal JR, Huecksteadt TP, Griffiths MM (1996)
Nitric oxide production during adjuvant induced and collagen induced arthritis. Arthritis
Rheum. 39:1677-1684
-
40
9. Nielsen RH, Christiansen C, Stolina M, Karsdal MA (2008) Oestrogen exhibits type II
collagen protective effects and attenuates collagen-induced arthritis in rats. Clin and Exp Imun,
152:21–27
10. Tascioglu F, Armagan O, Tabak Y, Corapci I, Oner C (2004) Low power laser treatment in
patients with knee osteoarthritis. Swiss Med WKLY. 134:254-258
11. Morais NCR, Barbosa AM, Vale ML, Villaverde AB, de Lima CJ, Cogo JC, Zamuner SR
(2010) Anti-inflammatory effect of low-level laser and light-emitting diode in zymosan-
induced arthritis. Photom Laser Surg. 28:227-232
12. Montes-Melina R, Mondroñero-Agreda MA, Romojaro-Rofroguéz AB, Gallego-Mendes
V, Prados-Cabiedas C, Marques-Lucas C, Péres-Ferreiro M, Martinez-Ruiz F (2009) Efficacy
of interferencial Low-Level laser therapy using two independent souces in the treatment of knee
pain. Photom Laser Surg. 27:467-471
13. Rocha FAC, Rocha JCS, Peixoto MEB, Jancar S, Cunha FQ, Ribeiro RA (2003) Efeito de
inibidores da sintase de óxido nítrico na dor inflamatória e influxo celular da artrite induzida
por zymosan em ratos; Effect of nitric oxide synthse in articular inflammatory pain and cellular
influx of zymosan-induced arthritis in rats. Rev bras reumatol. 43:206-217
14. Pallotta RC, Bjordal JM, Frigo L, Leal Junior ECP, Teixeira S, Marcos RL, Ramos L,
Messias FM, Lopes-Martins RAB (2012) Infrared (810-nm) low-level laser therapy on rat
experimental knee inflammation. Lasers Med. Sci. 27, 71-78
15. Boschi ES, Leite CE, Saciura VC, Caberlon E, Lunardelli A, Bitencourt S, Melo DA,
Oliveira JR (2008) Anti-Inflammatory effects of low-level laser therapy (660 nm) in the early
phase in carrageenan-induced pleurisy in rat. Lasers Surg Med. 40(7):500-508
16. Amano A, Miyagi K, Azuma T, Ishihara Y, Katsube S, Aoyama I, Saito I (1994)
Histological studies on the rheumatoid synovial membrane irradiated with a low energy laser.
Lasers Surg. Med. 15:290-294
-
41
17. van Lent PLEM, Hofkens W, Blom AB, Grevers L, Sloetjes A, Takahashi N, van Tits LJ,
Vogl T, Roth J, de Winther MP, van den Berg WB (2009) Scavenger Receptor Class A Type
I/II Determines Matrix Metalloproteinase–Mediated Cartilage Destruction and Chondrocyte
Death in Antigen-Induced Arthritis. Arthritis Rheum. 60:2954–2965
18. Sato K 2008 Th17 cells and rheumatoid arthritis--from the standpoint of osteoclast
differentiation. Allergol Int. 57:109-114
19. Schett G, Stach C, Zwerina J, Voll R, Manger B (2008) How antirheumatic drugs protect
joints from damage in rheumatoid arthritis. Arthritis Rheum. 58:2936-2948
20. Cunnane G, FitzGerald O, Hummel KM, Youssef PP, Gay RE, Gay S, Bresnihan B (2001)
Synovial tissue protease gene expression and joint erosions in early rheumatoid arthritis.
Arthritis Rheum. 44:1744–1753
21. Konttinen YT, Ainola M, Valleala H, Ma J, Ida H, Mandelin J, Kinne RW, Santavirta S,
Sorsa T, Lópes-Ortin C, Takagi M (1999) Analysis of 16 different matrix metalloproteinases
(MMP-1 to MMP-20) in the synovial membrane: different profiles in trauma and rheumatoid
arthritis. Ann Rheum Dis. 58:691–697
22. Xue M, March L, Sambrook PN, Jackson CJ (2007) Differential Regulation of Matrix
Metalloproteinase 2 and Matrix Metalloproteinase 9 by Activated Protein C: Relevance to
Inflammation in Rheumatoid Arthritis. Arthritis Rheum. 56:2864–2874
23. Raffetto JD, Ross RL, Khalil RA (2007) Matrix metalloproteinase 2-induced venous
dilation via hyperpolarization and activation of K+ channels: relevance to varicose vein
formation. J Vasc Surg. 45:373-380
24. Müller‐Ladner U, Kriegsmann J, Tschopp J, Gay RE, Gay S (1995) Demonstration of
granzyme A and perforin messenger RNA in the synovium of patients with rheumatoid arthritis.
Arthritis Rheum. 38:477-484
-
42
Figure 1 - LLLT effect on leukocyte influx into the joint cavity induced by zymosan. Rats
were injected with zymosan (1 mg/kg) and treated with laser. Another group was pretreated
with dexamethasone (0,4 mg/kg). Rats were killed after six hour and inflammatory exudates
were withdrawn the joint cavity. Total leukocytes (A), polymorphonuclear (PMN) (B) and
mononuclear (MN) (C). LLLT was applied immediately, 1ª e 2ª hour after zymosan injection.
Results are presented as mean SEM (n=5). #p< 0,05 compared to saline and *p< 0,05
compared to zymosan group (ANOVA).
Figure 2 - Effect of LLLT on the release of IL-1β and IL-6 into the joint cavity. The animals
were injected with zymosan i.a. (1 mg/kg) or saline (control). The concentrations of IL-1β (A)
and IL-6 (B) were measured by ELISA in joint lavage fluid collected 6 h after injection of
zymosan. LLLT was applied immediately, 1ª e 2ª hour after zymosan injection. Results are
presented as mean SEM (n=5). #p< 0,05 compared to saline and *p< 0,05 compared to
zymosan group (ANOVA).
Figure 3 - Histological demonstration of the collagen fibers of the synovium.
Representative histological sections (picrossirius red under polarized light, 40x objective) of
the synovium. In (A) synovium of animals injected with saline presenting normal appearance
of collagen fiber, synovium of animals injected with zymosan showing disorganization of the
collagen fibers, synovium treated with laser and synovium treated with dexamethasone showing
a better organization of collagen fibers. In (B) quantification of collagen fibers in the synoviun.
Results are presented as mean SEM (n=5). #p< 0,05 compared to saline and *p< 0,05
compared to zymosan group (ANOVA). Objective 40x.
Figure 4 - Effect of LLLT on MMP-2 and 9 activities on joint lavage. Joint lavage was
collected 6 and 12 h after induction of inflammation and laser treatment as described in material
and methods. (A and C) Representative electrophoresis from three independent experiment. (B
-
43
and D) Densitometric analysis of joint samples intensity. # p
-
44
FIGURE 1
Total
0
1000
2000
3000
ASaline
Zy
Zy + LLLT
Zy + Dexa
* *
#
Cel
ls x
10
3/m
L
PMN
0
1000
2000
3000
B
**
#
Cel
ls x
10
3/m
L
MN
0
1000
2000
3000
C
6 h
* *
#
Cel
ls x
10
3/m
L
-
45
FIGURE 2
0
1
2
3
4
Saline Zy Zy +
LLLT
Zy +
Dexa
* *
#
A
ng
IL
-1
/0
.1 m
L
0.0
0.5
1.0
1.5
Saline Zy Zy +
LLLT
Zy +
Dexa
*
*
#
B
ng
/IL
-6/
0.1
mL
-
46
FIGURE 3
A
B
0
25
50
75
100
#
* *
Saline Zy Zy +
LLLT
Zy +
Dexa
Co
lla
gen
fib
ers
(%
)
B
-
47
FIGURE 4
0
1000
2000
3000
4000Zy
Zy +LLLT
Zy + Dexa
6 12
Time (h)
Saline
#
#
*
#
*
#
#
* #*
B MMP-9
Gela
tin
oly
tic a
cti
vit
y
(arb
itra
ry u
nit
s)
0
500
1000
1500
2000
Zy
Saline
Zy + LLLT
Zy + Dexa
D
6 12
Time (h)
#
#
*#
*
#
*#
*
MMP-2
Den
sito
metr
ic a
na
lysi
s
(Arb
itra
ry U
nit
s)
-
48
ARTIGO 2
Exercise training associated with low level laser therapy decreases Sympathetic
overactivity in experimental model of acute monarthritis in rat
Marcelo de Paula Silva MS 2, Iris Callado Sanches PhD 1, Felipe Fernandes Conti MS 1, Katia
De Angelis PhD 1, Stella Regina Zamuner PhD 2.
1 Laboratory of Translational Physiology – Universidade Nove de Julho
2 Laboratory of Cell Biology – Universdade Nove de Julho
Author address: Stella R. Zamuner, PhD
Post Graduation Program Sciences Rehabilitation UNIVERSIDADE NOVE DE JULHO –
UNINOVE/SP
Rua Vergueiro, 234, Bairro Liberdade, Cep:01504-000, São Paulo, Brasil.
Email: [email protected]
mailto:[email protected]
-
49
ABSTRACT
Background & objectives: It has been shown that the inflammatory process causes
autonomic changes in arthritis. The aim of this study was to evaluate the association of nom-
pharmacological therapies of exercise training (ET) and low-level laser therapy (LLLT) on the
inflammatory process and its influence on autonomic and cardiovascular regulation in the
experimental model of monoarthritis in rats.
Methods and Results: Thirty male Wistar rats were divided into: control (C);
monoarthritis (MA); MA + exercise training (MAT) and MAT + low level laser (MATL).
Monoarthritis was induced by intra-articular injection of zymosan (1 mg dissolved in 50 μl of
a sterile saline solution) into one rear knee joint. Moderate-intensity ET was performed on a
treadmill for 4 weeks and LLLT at 660 nm in a dose of 2.5 J/cm2 was applied twice a week.
Arterial pressure (AP) and heart rate variability (HRV) were measured. Trained rats presented
lower body weight, an increase in the maximum speed of running and lower heart rate compared
to the other groups. Furthermore, rats underwent to ET showed an increase of PI and a decrease
in LF and VAR SAP compared to MA group. Rats subjected to ET associated a LLLT also
showed a decrease in the LF and HF/LF and an increase of VAR SAP, VAR-RR and HF
compared to MA group.
Conclusion: Acute monoarthritis caused an imbalance of the sympathetic system, which
suggests an early autonomous involvement. A moderate exercise program associated with
LLLT can significantly exert beneficial effects on arthritic rats. These benefits were related to
the cardiovascular autonomic balance and improvement of functional capacity.
Keywords: monoarthritis, exercise training, low-level laser therapy, autonomic function.
-
50
INTRODUCTION
Cardiovascular dysfunction (CD) has been documented in rheumatoid arthritis (RA) as
an important predictor of mortality and survival [1], indeed, the mortality and morbidity of RA
patients is attributed more to its cardiovascular complications rather than the disease itself [2,
3, 4, 5]. In addition, literature shows that the cardiovascular risk in RA is related to the systemic
inflammatory burden as well as an increased prevalence of traditional risk factors [7, 8].
Currently therapeutic strategies for the treatment of arthritis include pharmacologic and
non-pharmacologic management and ultimately surgery [9]. The anti-inflammatory drugs
(corticosteroids and non-steroidal) are the most widely pharmacological treatment used in
arthritis. Although these anti-inflammatory drugs are commonly used to treat inflammation
associates with arthritis, it is often ineffective and may cause high incidence of adverse and
unwanted dangerous side effects in the gastrointestinal tract, which limit their use [10]. Non-
pharmacological treatments involve physiotherapy, aerobic and strength training exercises,
weight loss, wearing braces and orthoses and so on. Indeed, exercise training (ET) has been
recommended for managing of arthritis as a non-pharmacological therapy that has led to
improvements in function and arthritis symptoms [11, 12, 13, 14]. In this regard, studies have
shown that the regular practice of physical exercise attenuates the inflammatory response, joint
pain and swelling, thereby contributing to restoration of range of motion, muscle strength and
improves cardiovascular conditions [15, 16]. It is recommended that adults with arthritis do
moderate physical activity associated with muscle strengthening activities [17].
Another non-pharmacological treatment is low-level laser therapy (LLLT) that is
growing as an alternative to many medical conditions that require relief from pain and
inflammation [18]. Photobioestimulation with LLLT have been proposed to treat arthritis based
on the literature that shows a reduction of inflammatory cell in fluid of synovial washing and a
-
51
decrease in inflammatory citokines such as IL-1 and IL-6 and TNF-a [19, 9], angiogenesis
stimulation and reduction in the formation of fibrosis [20]. Also, we have shown that LLLT can
reduce hyperalgesia in a model of zymosan-induced arthritis [21].
Although arthritis predominantly affects the synovial joints it also leads to extra-
articular manifestations. In this regard, impressively increasing number of investigators have
reported on potential determinants of increased cardiovascular (CV) diseases in patients with
rheumatoid arthritis (RA) [1]. A recent review confirmed that the CV risk in RA is increased to
a similar magnitude to that seen in type 2 diabetes [7]. Also, it has been demonstrate that
elevations in circulating pro-inflammatory cytokines increases sympathetic activity [22, 23],
reduce cardiovagal baroreflex sensitivity [24] and reduce heart rate variability (HRV)-derived
indices of cardiac parasympathetic activity. Then, using a model of acute articular
inflammation, the monoarthritis (MA) induced by zymosan injection in the knee joint, we
investigated the effect of the inflammatory process and its influence on autonomic and
cardiovascular regulation in an acute arthritis. In order to evaluate autonomic and
cardiovascular alterations, we used direct measurements of blood pressure measurements for
30 min to the HRV for hemodynamic and autonomic functions analysised. We also tested the
hypothesis that exercise training combined with low level laser therapy causes changes in
hemodynamic and autonomic function.
MATERIAL AND METHODS
-
52
ANIMALS
The experiments were performed using thirty male Wistar rats (220–250 g) that were
kept in plastic cages with water and food ad libitum and maintained under a controlled
temperature on a 12-h light/dark cycle. The animals were randomized into four groups: control
sedentary (C, n=6); zymosan-induced monoarthritis (MA, n=8); MA + exercise training (MAT,
n=8) and MAT + low level laser therapy (MATL, n=8). All animal care was in accordance with
the guidelines of the Brazilian College for Animal Experimentation (COBEA) and was
approved by the ethics committee of the University, Protocol AN0017/2012.
ZYMOSAN-INDUCED MONOARTHRITIS
Rats received an intra-articular (ia.) injection of 1 mg zymosan [21] (Sigma Chemical
Company, St Louis, MO, USA), dissolved in 50 L of a sterile saline solution, into one rear
knee (stifle) joint. The procedure was done under general anesthesia, using a mix of ketamine
80 mg/kg (Hospira, Inc.; Lake Forest, IL, USA) / xylazine 20 mg/kg (Lloyd, Inc.; Shenandoah,
IA, USA) intramuscularly. Control animals received injections of sterile saline.
EXERCISE TRAINING AND MAXIMAL EXERCISE TEST (MET)
Exercise training (ET) was performed on a motor treadmill at moderate intensity (~40
to 65% maximal exercise test) for 1 h a day, 5 days a week, for 4 weeks, with a gradual increase
in speed from 0.3 to 0.8 km/h. All animals were adapted to the procedure (10 min/day, 0.3
km/h) for 1 week before beginning the exercise training protocol. Exercise training groups were
-
53
subjected to a maximal exercise test, as described in detail in a previous publication [25, 26].
The tests were performed three times: 1) at the beginning of the experiment, 2) in the
intermediate protocol, and 3) in the final of the ET protocol. The purpose was to determine
maximal physical capacity and exercise training intensity. All groups inducing monoarthritis
(Zymosan) or saline in day 8. All groups was submitted to an initial MET in day 12. Only the
MAT and MATL group was submitted to an exercise training protocol before inducing
monoarthritis and MET protocol, while the C and MA groups did not exercise during period,
but MET realization for intermediate and final period. Subsequently, the MATL received
LLLT twice per week (#). Groups C, MA and MAT received therapy more with the power off
(Fig. 1).
LIGHT SOURCES, DOSES, AND TREATMENT
A low-level laser InGaIP (aluminum gallium indium phosphate; MM Optics Ltda; São
Carlos, São Paulo, Brazil), operating continuous ways in 660-nm wavelengths was used through
the whole experiment to irradiate the animals. The laser parameters were as follows: power
output 5 mW, energy density of 2.5 J/cm2, irradiation time 20 s, and beam area of 0.04 cm2;
which corresponds to a power density 0,1W/cm2. The optical power of the laser was calibrated
using a Newport Multifunction Optical Meter (model 1835-C). The laser dose, low enough to
avoid any thermal effect, was chosen on the basis of previous studies from our laboratory that
had shown a beneficial effect of the low-level laser on the inflammatory process [19, 21].
HEMODYNAMIC ASSESSMENTS
-
54
Thirty three days after zymosan-induced moarthritis, one catheter filled with 0.06 mL
of saline was implanted into the carotide artery of each anesthetized rat (80 mg/kg ketamine
and 12 mg/kg xylazine, i.p.). Twenty-four hours later, the arterial cannula was connected to a
strain gauge transducer (Blood Pressure XDCR; Kent Scientific, Torrington, CT, USA), and
the arterial pressure (AP) signals were recorded over a 30-minute period in conscious rats using
a microcomputer equipped with an analog-to-digital converter board (WinDaq, 2 kHz,
DATAQ, Springfield, H, USA). The recorded data were analyzed on a beat-by-beat basis to
quantify any changes in the mean AP (MAP) and heart rate (HR). The HRV of the autonomics
parameters in the time domain and frequency was assessed by computing the of short-term
recordings series [27].
STATISTICAL ANALYSIS
Statistical analysis was performed using ANOVA and Tukey test. The data was showed
in mean ± error standard. In all calculations we fixed the critical level of less than 0.05.
RESULTS
Body weight evaluations
There was no differences in body weight among groups at the beginning of the protocol
(~244 ± 6 g). At the end of the training period, MAT (344 ± 10 g) and MATL (345 ± 7 g) had
a lower body weight compared to C (362 ± 13 g) or MA (368 ± 15 g) animals (Tab. 1).
Maximal Exercice Test
Maximal exercise performance was evaluated by the response to the maximal exercise
test (MET). At the beginning of the experiment, the MET was similar among all groups (C,
0.75 ± 0.15; MA, 0.75 ± 0.15; MAT, 0.63 ± 0.09; MATL 0.71 ± 0.14; km\h). However, the
-
55
rats underwent to execice training showed an increase in the MET of running when compared
with C and MA groups after four weeks of exercicing training (MAT, 1.2 ± 0.09; and MATL,
1.3 ± 0.14; km\h) (Tab. 1).
Hemodynamic evaluations
Hemodynamic parameters can be observed in Table 2. No changes were observed in
systolic AP, diastolic AP or MAP among groups. However, rats underwent to exercice training
showed a significant lower resting HR compared to MA group (Tab. 2).
Cardiac Autonomic Modulation
The results of autonomic function evaluation are summarized in Table 3. Pulse interval
(IP) was reduced in MA group when compared with C group. In addition, absolute LF band,
VAR-SAP and LF-SAP were significant increased in MA group compared to C group.
Furthermore, these alterations were reflected in the LF/HF ratio, an autonomic balance index
that demonstrates an increase in the simpatic modulation. However, the RMSSD and HF
component were not changed by MA. Finally, the sensitivity of the baroreflex, which was
evaluated by alpha ratio, was also similar among groups. The animals subjected to ET showed
an increase of PI and a decrease in LF and VAR SAP compared to MA. The rats underwent to
an ET and a LLLT also showed a decrease in the LF and HF/LF and an increase of VAR SAP,
VAR-RR and HF compared to MA (Tab. 3).
DISCUSSION
This study was performed to investigate the effect of cardiovascular dysfunction (CD)
in rats with acute knee monoarthritis and the use of non-pharmacological approaches to reduce
the effects of arthritis. For this purpose, we evaluated the autonomic nervous system after four
-
56
weeks of induction of monoarthritis in rats (MA group). Our results demonstrated that while
MA rats showed unchanged blood pressure and heart rate, they indeed presented decreased PI
and LF/HF and a increased LF, VAR-SAP and LF-SAP compared to control rats indicating an
increase of sympathetic modulation as a result of inflammatory profile installed on knee.
Previous results from clinical studies suggest that decreased vagus nerve activity occurs in
subjects with acute inflammatory conditions [28, 29]. Few reports have assessed CD in patients
with early RA although they have shown that the autonomic nervous system dysfunction occurs
early in RA patients [30]. An important find is that, in our experimental model, the rats are
health, only the knee inflammation is occurring, inferring that inflammation process alone is
sufficient to elicit cardiovascular dysfunction.
ET has been identified as one of the most important behavioral strategies for
cardiovascular disease prevention, and just a slight increase in physical activity could benefit
sedentary individuals (like RA sufferers) [31]. In the present study, rats were submitted to a
four-week moderate intensity treadmill exercise as a treatment protocol. The result showed that
ET rats (MAT or MATL) had a lower body weight and an increase in the maximum speed of
running compared to sedentary groups (control or MA). Previous reports demonstrated that ET
could decrease the pain and improve function in the patients with RA and also slow the process
of the disease [32]. Furthermore, [33] showed that a training protocol for 4 weeks could
surprisingly almost treat arthritis symptoms of rats’ knee joint in 3 histological measures of
joint cartilage. Other studies also showed a beneficial effect in moderate exercise training on
arthritic joints of rats [34, 35]. So, it is possible that in our experimental model the ET reduces
pain and the degeneration of joint cartilage, which improves the capacity of the rats to running.
This study also demonstrates that ET has a positive effect on autonomic function of MA
rats, as measured by short-term heart rate and arterial pressure variability. Comparing the MAT
group to MA group, the MAT group showed better heart rate variability that was observed by
-
57
the decrease of LF and VAR-SAP, which is similar to results from former studies [3, 36].
Moreover, exercise training performed in the MAT group was able to normalize LF and VAR-
SAP.
Previous reports have shown that LLLT has beneficial effects in treating arthritis in
humans [37] as well as in animal model [19, 25, 38]. In this sense, a meta-analysis, based on
22 randomized controlled trials (668 people were in the laser therapy group and 565 people
were in the placebo laser group) showed the effectiveness of laser therapy by analyzing previous
clinical trials reported that LLLT might be a good alternative to NSAIDs drugs and the
association of LLLT with ET had a better effect than LLLT alone. [39]. In this study we showed
that the association of ET and LLLT increased the benefit of ET alone in the autonomic system.
ET performed in the MATL group was able to increase HF and VAR-RR and decreased LF/HF
compared to MAT group. In addition, MATL group maintained HF and VAR-SAP in the same
parameters as the MAT group. The fact that MATL group increased LF/HF demonstrated that
the association of ET and laser has a better effect in sympathovagal nerve modulation. This is
the first study to demonstrate that LLLT in trained monoarthritis rats increases LF/HF. In our
experimental model, ET combined with LLLT appears to have an advantageous effect on
autonomic function in monoarthritis rats as measured by short-term heart rate variability.
Especially, vagal modulation seems to be improved, and this can lead to a better cardiac health.
Regarding the findings of the present study, it can be concluded that acute joint
inflammation causes an imbalance of the sympathetic system, suggesting an early autonomic
involvement, which in the course of chronic rheumatic disease may affect cardiovascular
system. In addition, a moderate exercise program associated with LLLT can significantly exert
beneficial effects on monoarthritic rats. These benefits were related to the cardiovascular
autonomic balance and improved functional capacity.
-
58
Acknowledgement - Financial support: Brazilian Council of Research—CNPq/PROSUP.
REFERENCES
1 - SOLOMON A, et all.. Cardiovascular Disease Risk amongst African Black Patients with
Rheumatoid Arthritis:The Need for Population Specific Stratification. BioMed Research
International, 2014.
2 - PRIOR P, SYMMONS DP, SCOTT DL, BROWN R, HAWKINS CF. Cause of death in
rheumatoid arthritis. Br J Rheumatol. 1984.
3 - YADAV, R.K., GUPTA, R. and DEEPAK, K.K. A pilot study on short term heart rate
variability e its correlation with disease activity in Idian patients with rheumatoid arthritis.
Indian J. Med, 136, 2012.
-
59
4 - STRAUB, R., BAERWALD, C., WAHLE, M. and JANIG, W.Autonomic Dysfunction in
Rheumatic Diseases. Rheum Dis Clin N Am, 31, 2005.
5 - SEFEROVIC, P.M., RISTIC, A.D., MAKSIMOVIC, B., SI