O efeito do intervalo da estimulação elétrica no músculo desnervado de rato / The effect of time interval between electrical stimulation on the denervated rat muscle

OBJETIVO: Comparar o efeito da estimulação elétrica (EE) aplicada diariamente e em dias alternados na densidade de área do tecido conjuntivo (TC) e na área de secção transversa (AST) das fibras do músculo desnervado. MATERIAIS E MÉTODOS: Trinta e cinco ratos foram divididos em grupos controle (C), desnervado (D), desnervado + eletroestimulado diariamente (EED) e desnervado + eletroestimulado em dias alternados (EEA). A aplicação da EE no músculo gastrocnêmio teve início 24 horas após lesão nervosa do tipo axoniotmese, sendo a mesma aplicada durante 20 e 30 dias. Cortes transversais foram corados com HE para mensurações da AST e densidade de área de TC. Análise estatística: teste Shapiro Wilk, seguido pela análise de variância (ANOVA) F (one-way) e teste de Tukey (5 por cento). RESULTADOS: Na análise da densidade de área do TC, observou-se que somente o Grupo EED apresentou valores similares ao Grupo C nos dois períodos analisados. No Grupo 20 dias, não houve diferença na AST quando comparados os grupos submetidos à EE com o Grupo D (p> 0,05), e após 30 dias todos os grupos experimentais alcançaram valores similares ao Grupo C. CONCLUSÕES: A EE não foi eficiente para minimizar a atrofia das fibras musculares. Entretanto, o TC foi responsivo à EE, sendo a aplicação diária mais benéfica ao músculo do que a aplicação em dias alternados, sugerindo que o intervalo de aplicação da EE em músculo desnervado é variável importante para as adaptações do TC.

OBJECTIVE: To compare the effect of electrical stimulation (ES) applied daily and on alternate days, on the area density of the connective tissue (CT) and on the cross-sectional area (CSA) of the denervated muscle fibers. METHODS: Thirty-five rats were divided into the following groups: control (C), denervated (D), denervated + daily electrical stimulation (D+DES) and denervated + alternate-day electrical stimulation (D+ES). The application of ES on the gastrocnemius was started 24 hours after nerve damage of axonotmesis type and was applied for 20 and 30 days. Cross-sections were stained with hematoxylin-eosin to measure the CSA and area density of CT. The statistical analysis consisted of the Shapiro Wilk test followed by analysis of variance (ANOVA) F (one-way) and the Tukey test (p< 0.05). RESULTS: Analysis of the area density of CT showed that only the D+DES Group presented values similar to those of the C Group, for the two analysis periods. There was no difference in CSA in the 20-day Group between the ES Groups and the D Group (p> 0.05). After 30 days, all the experimental groups reached CSA values similar to the C Group. CONCLUSIONS: The ES was inefficient for minimizing the muscle fiber atrophy. However, the CT was responsive to ES, and daily applications were more beneficial for the muscle than were alternate-day applications, thus suggesting that the interval for applying ES to denervated muscle is an important variable for CT adaptation.

The onset and duration of mobilization affect the regeneration in the rat muscle.

UNLABELLED: The effects of different mobilization protocols for muscle regeneration after myotoxin injury was compared in the rat tibialis anterior (TA) muscle. Adult Wistar rats were divided into control (C); mobilized (M); injury (I); injury + late mobilization (LM) and injury + early mobilization (EM) groups. Muscle injury was induced by intramuscular lidocaine injection. The exercised animals were mobilized for 5 and 8 days during 15 and 45 minutes/session. The swimming started 1 hour or 3 days after injury. All animals were killed 8 days after the injury, together with the control group, when the TA muscles were weighted and excised. Cross sections were obtained by cryostat and submitted to Toluidine Blue stain. Qualitative morphological characterization of muscle regeneration and quantitative analysis of muscle fiber and non-muscle fiber area density were performed. The I and late mobilization groups showed decreased muscle mass when compared to all other groups. All injured animals showed signs of muscle fiber damage, although signs of early regenerated muscle fibers were more evident in injury + mobilization groups. Only the EM groups submitted to 45 minutes of exercise had increased muscle fiber and decreased non-muscle fiber area density values when compared to I group (p<0.05). CONCLUSION: the regeneration process is related to the onset of exercise, since animals submitted to early mobilization showed improved regeneration when comparted to LM groups. Besides, the length of session is also important for accelerating the regeneration process, as it was observed that 45 minutes was better than 15 minutes duration.

Curto período de imobilização provoca alterações morfométricas e mecânicas no músculo de rato / Short-term immobilization causes morphometric and mechanical alterations in rat muscles

OBJETIVO: Analisar as características morfométricas e mecânicas dos músculos sóleo e gastrocnêmio após imobilização na posição de encurtamento. MÉTODO: 20 ratos Wistar (250 ± 20g) foram distribuídos igualmente em grupos imobilizado e controle. A imobilização foi realizada no membro posterior esquerdo por meio de órtese de resina acrílica, com a articulação do tornozelo em flexão plantar máxima. Após 7 dias da imobilização, a massa muscular, número e comprimento de sarcômeros em série, área das fibras musculares, densidade de área de tecido conjuntivo intramuscular e força máxima de ruptura do tríceps sural foram avaliados. Os dados foram analisados pela ANOVA e teste de Tukey (p< 0,05). RESULTADOS: O músculo sóleo imobilizado apresentou alterações em todas as variáveis morfométricas analisadas, enquanto que, no músculo gastrocnêmio, algumas adaptações não foram observadas. Na análise do ensaio de tração, o grupo imobilizado apresentou redução de 20 por cento na força máxima de ruptura muscular. CONCLUSÃO: Os resultados deste estudo revelaram que curto período de imobilização promove alterações nos parâmetros morfométricos das fibras musculares, com repercussões na mecânica muscular. Tais resultados permitem sugerir a necessidade da reabilitação em músculos submetidos à imobilização, mesmo a curto prazo, para que a mesma possibilite o retorno precoce das características musculares normais.

OBJECTIVE: to analyze the morphometric and mechanical characteristics of the soleus and gastrocnemius muscles after immobilization in a shortened position. METHODS: 20 Wistar rats (250 ± 20g) were divided equally into immobilized and control groups. The left hind limb was immobilized by means of an acrylic resin orthosis, with the ankle joint at maximum plantar flexion. After seven days of immobilization, the muscle mass, number and length of sarcomeres in series, muscle fiber cross-sectional area, density of the intramuscular connective tissue area and tensile strength of the triceps surae muscle were evaluated. The data were analyzed by the ANOVA and Tukey tests (p< 0.05). RESULTS: The immobilized soleus muscle presented changes in all the morphometric variables analyzed, while some of these changes were not observed in the gastrocnemius muscle. Analysis of the traction test showed that the immobilized group presented a 20 percent decrease in the maximum tensile muscle strength. CONCLUSION: The results from this study showed that short-term immobilization causes changes to the morphometric parameters of the muscle fibers, with repercussions on muscle mechanics. These results suggest the need for rehabilitation of muscles subjected to immobilization, even if only for a short period, in order to achieve early recovery of normal muscle characteristics.

Regeneração do músculo tibial anterior em diferentes períodos após lesão por estimulação elétrica neuromuscular / Regeneration of the tibialis anterior muscle at different times following injury induced by neuromuscular electrical stimulation

CONTEXTUALIZAÇÃO: Lesões no musculoesquelético podem ser causadas pela própria contração muscular. OBJETIVO: Analisar em diferentes períodos a lesão do músculo tibial anterior (TA) induzida pela eletroestimulação. MATERIAL E MÉTODO: ratos Wistar macho (298,2 ± 16,0g) foram divididos nos grupos: eletroestimulado (EE) e analisado após 3 e 5 dias (n= 20) e controle (C), 3 e 5 dias (n = 14). O TA, mantido em alongamento, foi lesado por eletroestimulação neuromuscular (90 min, 30Hz, 1m/s, Ton/Toff 4s e 4mA). Após 3 e 5 dias, os animais foram sacrificados e os músculos retirados, sendo os cortes histológicos (10 μm) obtidos em criostato e corados com Azul de Toluidina. Os pesos corporal e muscular foram analisados estatisticamente pelo teste T-Student (p < 0,05). RESULTADO: Aumento do peso corporal final quando comparado com inicial em C3 e C5 (288,5 ± 18,3g x 308,5 ± 24,3g; 288,4 ± 15,0g x 305,5 ± 20,7g, respectivamente) e diminuição em EE3 e EE5 (305,0 ± 13,0g x 285,6 ± 13,2g; 306,1 ± 12,4g x 278,4 ± 20,9g, respectivamente). Peso muscular relativo do EE5 foi menor quando comparado com o C5 (0,20 ± 0,001 por cento x0,22 ± 0,01 por cento, respectivamente). Análise histológica mostrou variabilidade na extensão e nos sinais de fibras lesadas e/ou em regeneração e a região distal foi a mais lesada. Grupo EE3 apresentou predominância de infiltrado celular e hipercontração dos miofilamentos, e no grupo EE5 houve predominância de infiltrado celular, basofilia e fibrose. CONCLUSÃO: O período de 2 dias após eletroestimlação foi suficiente para observar diferença no processo de regeneração com maior susceptibilidade à lesão na região distal do músculo tibial anterior.

BACKGROUND: Skeletal muscle injuries may be caused by contraction of the muscle concerned. OBJECTIVE: To analyze the tibialis anterior muscle at different times following injury induced by electrical stimulation. METHOD: Male Wistar rats (298.2 ± 16.0g) were divided into two electrically stimulated groups evaluated after three and five days (n= 20) and two control groups, also evaluated after three and five days (n= 14). While stretched, the tibialis anterior muscle was injured by neuromuscular electrical stimulation (90 minutes, 30 Hz, 1 m/s, Ton/Toff 4 s and 4 mA). Three and five days afterwards, the animals were sacrificed and the muscles were removed. Histological sections were cut (10 μm) using a cryostat and were stained with toluidine blue. The body and muscle weights were statistically analyzed using Student's t test (p < 0.05). RESULTS: The final body weight was higher than the initial weight for the 3-day control group (288.5 ± 18.3g vs. 308.5 ± 24.3g) and 5-day control group (288.4 ± 15.0g vs. 305.5 ± 20.7g) and lower for the 3-day stimulated group (305.0 ± 13.0g vs. 285.6 ± 13.2g) and 5-day stimulated group (306.1 ± 12.4g vs. 278.4 ± 20.9g). The relative muscle weight in the 5-day stimulated group was lower than in the 5-day control group (0.20 ± 0.001 percent vs. 0.22 ± 0.01 percent, respectively). The histological analysis showed variance between the animals regarding the extent and signs of fiber damage and/or regeneration, and the distal region was the most injured. The 3-day stimulated group presented predominance of cell infiltrate and myofilament hypercontraction, while the 5-day stimulated group presented predominance of cell infiltrate, basophils and fibrosis. CONCLUSION: A period of two days following electrical stimulation was sufficient for showing a difference in the regeneration process. The distal region of the tibialis anterior muscle was more susceptible to injury.

Modelo de lesao no musculo tibial anterior de rato induzida pela estimulacao eletrica neuromuscular / Model for electrical stimulation-induced muscle damage

Desenvolver modelo alternativo de lesao muscular causada por eletroestimulacao (EE). Materiais e metodos: foram usados ratos Wistar (306+-12,4g) e o musculo analisado foi o tibial anterior(TA). O equipamento utilizado para EE foi o Dualpex 961 com os seguintes parametros: largura de pulso de 1m/s, intensidade de 3 ou 4 m/A. Ton/Toff de 4 s e frequencia de 30Hz durante 90 minutos, por meio de 2 eletrodos fixados no nervo fibular. Durante todo o periodo da EE, o TA encontrava-se imobilizado em flexao plantar maxima com pequena tala de madeira fixada por meio de fita crepe. Previamente a EE, o TA encontrava-se imobilizado em flexao plantar maxima com pequena tala de madeira fixada por meio de fita crepe. Previamente a EE, os animais foram anestesiados com hidrato de cloral (460 mg/kg - 1,4ml/300g, IP) e sacrificados apos 5 dias da inducao. Os musculos eletroestimulado e contralateral de cada animal foram pesados e divididos em tres regioes: proximal, media e distal. De cada regiao foram obtidas seccoes (10um) em criostato. Os cortes foram submetidos a coloracao de Azul de Toluidina e avaliados por meio de microscopio de luz. Resultados: Os musculos eletroestimulados apresentaram sinais caracteristicos de lesao muscular, sendo a regiao distal a mais comprometida. Foi tambem observada diminuicao do peso do musculo lesado quando comparado ao contralateral (0,20+-0,001 por cento, versus 0,25+-0,010 por cento; p<0,01, teste t Student, 5 por cento). Conclusao: O modelo proposto foi eficaz para producao de lesao. E de simples aplicacao e utiliza materiais de baixo custo e facil aquisicao, sendo uma alternativa aos modelos tradicionais para inducao de lesao muscular

Long-term regeneration of rat skeletal muscle after periodic contusions

Este estudo avaliou os aspectos morfologicos, as alteracoes na area e a incidencia dos tipos de fibras musculares na regeneracao a longo prazo do musculo tibialis anterior (TA) de ratos previamente submetidos a contusoes periodicas. Os animais sofreram 8 contusoes consecutivas: uma vez por semana, durante 8 semanas, e foram avaliados um (n=8) e quatro (n=9) meses apos a ultima contusao. As areas de secoes transversas foram avaliadas por intermedio da coloracao de Azul de Toluidina e reacoes da fosfatase acida, acetilcolinesterase e m-ATPase. Os musculos lesados apresentaram diminuicao de peso quando comparados com os musculos intactos (um mes: 0,77 +-0,15 g vs 0,91 +- 0,09 g, p=0,03; quatro meses: 0,79 +- 0,14 g vs 1,02 +- 0,07 g, p=0,0007, respectivamente) e mostraram grande incidencia de fibras fragmentadas e fibras com nucleos centralizados. Os musculos lesados apresentaram maior incidencia de fibras hibridas, qundo comparados com os musculos intactos ( um mes: 3,4 +- 2,1 por cento, vs 0,5 +- 0,3 por cento, p=0,006; quatro meses: 2,3 +- 1,6 por cento, vs 0,3 +- 0,3 por cento, p=0,007). Conclusoes: fibras musculares foram lesadas principalmente na porcao profunda do musculo TA, provavelmente devido ao fato de as mesmas terem sido comprimidas contra a tibia; contusoes periodicas nao alterarm a porcentagem de fibras musculares dos tipos I e II; e musculo TA periodicamente lesado levou quatro meses para apresentar area de fibra muscular similar ao musculo intacto

O musculo como um orgao de secrecao hormonal regulado pelo estimulo mecanico / The muscle as an organ of hormonal secretion regulated by mechanics stimulation

O objetivo dessa revisao e apresentar estudos recentes que identificaram a relacao entre o estimulo mecanico aplicado ao musculo e a expressao genica de fatores de crescimento pelo proprio musculo. Os fatores de crescimento, secretados pelos musculos esqueleticos, quando submetidos a um aumento de tensao muscular, sao similares ao fator de crescimento insulinico (IGF-I) e regulam a sinteses proteica e a hipertrofia muscular. Estudos recentes sugerem que um desses fatores, denominado `mechanical growth factor` (MGF), seja o sinal molecular para a hipertrofia muscular. Acredita-se que ele seja o principal fator de crescimento que controla o crescimento e a reparacao muscular esqueletica e cardiaca. Serao tambem abordadas algumas implicacoes entre o efeito desses homonios e a realizacao dos diferentes tipos de exercicios fisicos

Regenerated rat skeletal muscle after periodic contusions

In the present study we evaluated the morphological aspect and changes in the area and incidence of muscle fiber types of long-term regenerated rat tibialis anterior (TA) muscle previously submitted to periodic contusions. Animals received eight consecutive traumas: one trauma per week, for eight weeks, and were evaluated one (N = 8) and four (N = 9) months after the last contusion. Serial cross-sections were evaluated by toluidine blue staining, acid phosphatase and myosin ATPase reactions. The weight of injured muscles was decreased compared to the contralateral intact one (one month: 0.77 + or - 0.15 vs 0.91 + or - 0.09 g, P = 0.03; four months: 0.79 + or - 0.14 vs 1.02 + or - 0.07 g, P = 0.0007, respectively) and showed abundant presence of split fibers and fibers with centralized nuclei, mainly in the deep portion. Damaged muscles presented a higher incidence of undifferentiated fibers when compared to the intact one (one month: 3.4 + or - 2.1 vs 0.5 + or - 0.3 percent, P = 0.006; four months: 2.3 + or - 1.6 vs 0.3 + or - 0.3 percent, P = 0.007, respectively). Injured TA evaluated one month later showed a decreased area of muscle fibers when compared to the intact one (P = 0.003). Thus, we conclude that: a) muscle fibers were damaged mainly in the deep portion, probably because they were compressed against the tibia; b) periodic contusions in the TA muscle did not change the percentage of type I and II muscle fibers; c) periodically injured TA muscles took four months to reach a muscle fiber area similar to that of the intact muscle

Regenerated rat skeletal muscle after periodic contusions.

In the present study we evaluated the morphological aspect and changes in the area and incidence of muscle fiber types of long-term regenerated rat tibialis anterior (TA) muscle previously submitted to periodic contusions. Animals received eight consecutive traumas: one trauma per week, for eight weeks, and were evaluated one (N = 8) and four (N = 9) months after the last contusion. Serial cross-sections were evaluated by toluidine blue staining, acid phosphatase and myosin ATPase reactions. The weight of injured muscles was decreased compared to the contralateral intact one (one month: 0.77 +/- 0.15 vs 0.91 +/- 0.09 g, P = 0.03; four months: 0.79 +/- 0.14 vs 1.02 +/- 0.07 g, P = 0.0007, respectively) and showed abundant presence of split fibers and fibers with centralized nuclei, mainly in the deep portion. Damaged muscles presented a higher incidence of undifferentiated fibers when compared to the intact one (one month: 3.4 +/- 2.1 vs 0.5 +/- 0.3%, P = 0.006; four months: 2.3 +/- 1.6 vs 0.3 +/- 0.3%, P = 0.007, respectively). Injured TA evaluated one month later showed a decreased area of muscle fibers when compared to the intact one (P = 0.003). Thus, we conclude that: a) muscle fibers were damaged mainly in the deep portion, probably because they were compressed against the tibia; b) periodic contusions in the TA muscle did not change the percentage of type I and II muscle fibers; c) periodically injured TA muscles took four months to reach a muscle fiber area similar to that of the intact muscle.

Effect of single and periodic contusion on the rat soleus muscle at different stages of regeneration.

This work analyzed the rat soleus muscle after single and recurrent contusions at different stages of regeneration. A noninvasive contusion was produced by a type of drop-mass equipment. The posterior region of the right hind limb received a trauma and both right and left soleus muscles were analyzed 1, 4, and 6 days after a single contusion (1x), and 6 and 30 days after periodic contusions (10x, one trauma per week for 10 weeks). Single contusion: there was no significant difference between right and left soleus muscle weight. All animals showed abundant signs of acute damage in the right soleus. AChE activity was identified in regeneration segments of the right soleus. Periodic contusions: there was an increase in the right soleus muscle weight (alpha = 5%) only in the animals evaluated 6 days after periodic contusions. The right soleus muscle showed a high incidence of chronic signs of damage, such as split fibers and a centralized nucleus, which predominated when compared with the acute signs. Right soleus muscles showed split fibers with AChE activity in both the proximal and middle regions. There was no difference in the incidence of muscle fiber types (I, II, and IIC) between right and left soleus muscles after periodic contusions. Skeletal muscle contusion is common in humans, especially in sport activities, where repetitive traumas are also frequent. The results of this work indicate that despite the regeneration process there is an important change in the morphological aspect of regenerated muscle fibers, which possibly affect muscle performance.