Made available in DSpace on 2014-06-11T19:27:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-02Bitstream added on 2014-06-13T19:14:30Z : No. of bitstreams: 1 rino_as_me_botfmvz.pdf: 1140459 bytes, checksum: 5597016d559daa886389f4ed9c58c88b (MD5) / Universidade Estadual Paulista (UNESP) / As fibras musculares esqueléticas são entidades versáteis, capazes de alterar seus fenótipos de acordo com a demanda energética. Para avaliar os efeitos do exercício de enduro no músculo esquelético, foram utilizados neste estudo 36 equinos da raça Puro Sangue Árabe divididos em dois grupos: treinados e não treinados para enduro. Foram obtidos fragmentos musculares da região profunda do músculo glúteo médio e processados para técnicas histoquímicas e imunoistoquímica, utilizando-se anticorpos específicos contra isoformas de cadeia pesada de miosina (MyHC) lenta e rápida. As fibras identificadas pela histoquímica e imunoistoquímica foram analisadas uma a uma. Determinou-se a porcentagem dos diferentes tipos de fibras musculares, e avaliou-se que, a porcentagem das fibras do tipo IIA foi significativamente mais alta do que a porcentagem das fibras dos tipos I e IIX no grupo não treinado (P<0,05). Em contra partida, a porcentagem das fibras do tipo I foi significativamente mais alta do que a porcentagem das fibras dos tipo IIA e IIX no grupo treinado (P<0,05). Também foram analisados, a média da área de corte transversal e o menor diâmetro da fibra e observou-se que, a média de corte transversal das fibras do tipo IIX foi significativamente maior comparada à média de corte de transversal das fibras dos tipos I e IIA em ambos os grupos. Em conclusão pode-se afirmar que, o exercício de enduro em equinos aumenta a população das fibras de contração lenta, em detrimento da população de fibras de contração rápida / The skeletal muscle fibers are versatile entities capable to change their phonotype according to energy demand. To evaluate the effects of exercise over equine skeletal muscle in this study, 36 Arabian horses divided in 2 groups trained and untrained for endurance were used. The samples was obtained from de deep region of the gluteus medius muscle and processed for histochemistry and immunohistochemistry using specific monoclonal antibodies to selected fast and slow isoforms myosin heavy chain (MyHC). The histochemical and immunohistochemical categorization of the muscle fiber types were compared fiber by fiber. It was analyzed the percentage of different fiber types where, in the untrained group, the percentage of the fiber type IIA was higher significantly than the percentage of fiber types I and IIX (P<0.05). Unlike, the group trained showed a higher proportion of fiber type I than the fiber type IIX (P<0.05). The cross section area and lesser fiber diameter were also analyzed, and showed that, CSA of fiber type IIX was higher significantly than fibers I and IIA (P<0.05) in both groups. In conclusion, endurance exercise in equines increase the population of slow-twitch fibers in detriment of fast-twitch fibers

Orientador: Rogério Martins Amorim / Banca: Marcos Jun Watanabe / Banca: Maria Verônica de Souza / Resumo: As fibras musculares esqueléticas são entidades versáteis, capazes de alterar seus fenótipos de acordo com a demanda energética. Para avaliar os efeitos do exercício de enduro no músculo esquelético, foram utilizados neste estudo 36 equinos da raça Puro Sangue Árabe divididos em dois grupos: treinados e não treinados para enduro. Foram obtidos fragmentos musculares da região profunda do músculo glúteo médio e processados para técnicas histoquímicas e imunoistoquímica, utilizando-se anticorpos específicos contra isoformas de cadeia pesada de miosina (MyHC) lenta e rápida. As fibras identificadas pela histoquímica e imunoistoquímica foram analisadas uma a uma. Determinou-se a porcentagem dos diferentes tipos de fibras musculares, e avaliou-se que, a porcentagem das fibras do tipo IIA foi significativamente mais alta do que a porcentagem das fibras dos tipos I e IIX no grupo não treinado (P<0,05). Em contra partida, a porcentagem das fibras do tipo I foi significativamente mais alta do que a porcentagem das fibras dos tipo IIA e IIX no grupo treinado (P<0,05). Também foram analisados, a média da área de corte transversal e o menor diâmetro da fibra e observou-se que, a média de corte transversal das fibras do tipo IIX foi significativamente maior comparada à média de corte de transversal das fibras dos tipos I e IIA em ambos os grupos. Em conclusão pode-se afirmar que, o exercício de enduro em equinos aumenta a população das fibras de contração lenta, em detrimento da população de fibras de contração rápida / Abstract: The skeletal muscle fibers are versatile entities capable to change their phonotype according to energy demand. To evaluate the effects of exercise over equine skeletal muscle in this study, 36 Arabian horses divided in 2 groups trained and untrained for endurance were used. The samples was obtained from de deep region of the gluteus medius muscle and processed for histochemistry and immunohistochemistry using specific monoclonal antibodies to selected fast and slow isoforms myosin heavy chain (MyHC). The histochemical and immunohistochemical categorization of the muscle fiber types were compared fiber by fiber. It was analyzed the percentage of different fiber types where, in the untrained group, the percentage of the fiber type IIA was higher significantly than the percentage of fiber types I and IIX (P<0.05). Unlike, the group trained showed a higher proportion of fiber type I than the fiber type IIX (P<0.05). The cross section area and lesser fiber diameter were also analyzed, and showed that, CSA of fiber type IIX was higher significantly than fibers I and IIA (P<0.05) in both groups. In conclusion, endurance exercise in equines increase the population of slow-twitch fibers in detriment of fast-twitch fibers / Mestre

No description available.

The SH3 and cysteine rich domain 3 (Stac3) gene is expressed specifically in skeletal muscle and essential for skeletal muscle contraction and postnatal life in mice. In this dissertation project, I conducted two studies to further understand the role of STAC3 in skeletal muscle development, growth, and contraction. In the first study, I compared the contractile responses of hindlimb muscles of Stac3 knockout and control mice to electrical stimulation, high [K+]-induced membrane depolarization, and caffeine and 4-chloro-m-cresol (4-CMC) activation of ryanodine receptor (RyR). Frequent electrostimulation-, high [K+]-, 4-CMC- and caffeine-induced maximal tensions in Stac3-deleted muscles were approximately 20%, 29%, 58% and 55% of those in control muscles, respectively. 4-CMC- and caffeine-induced increases in intracellular calcium were not different between Stac3-deleted and control myotubes. Myosin-ATPase and NADH-tetrazolium reductase staining as well as gene expression analyses revealed that Stac3-deleted hindlimb muscles contained more slow type-like fibers than control muscles. These data together confirm a role of STAC3 in EC coupling but also suggest that defective EC coupling is only partially responsible for the significantly reduced contractility in Stac3-deleted hindlimb muscles. In the second study, I determined the potential role of STAC3 in postnatal skeletal muscle growth, fiber composition, and contraction by disrupting Stac3 gene expression in postnatal mice through the Flp-FRT and tamoxifen-inducible Cre-loxP systems. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei without affecting the total myofiber number. Postnatal Stac3 deletion decreased limb muscle strength. Postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced maximal force output in limb muscles. Similarly, postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum. These results demonstrate that STAC3 is important to myofiber hypertrophy, myofiber type composition, contraction, and EC coupling in postnatal skeletal muscle. / Ph. D.

Postnatal skeletal muscle growth occurs through myonuclear accretion and high protein turnover rate. While fiber type composition of the muscle could affect protein turnover rate, less is known about how fiber type influences the regulation of protein synthesis and degradation signaling pathways. Thus, the hypothesis of this work was that variation in fiber type composition will differentially affect the regulation of signaling pathways related to protein turnover in skeletal muscle hypertrophy in growing pigs. Downregulated protein synthesis signaling and reduced expression of type II MyHC isoforms have been reported in skeletal muscles of low birth weight (LBWT) neonatal pigs. Therefore, we sought to determine whether these changes are sustained until weaning and would explain the reduction in LBWT pig growth compared to their normal birth weight (NBWT) sibling at weaning. Another objective was to determine whether the regulation of protein turnover signaling pathways are correlated to fiber type differences in skeletal muscles. Our data suggest that the longissimus dorsi (LD, glycolytic) muscle of LBWT pigs experienced compensatory growth while the soleus (oxidative) remained proportionally smaller. Growth of the LD was accompanied by upregulation of translation initiation. Additionally, there was no difference in expression of MyHC isoforms between NBWT and LBWT pigs. These data suggest the rapid growth of the LD of LBWT pigs may be attributed to an upregulation of protein synthesis signaling and occurred only in glycolytic muscles. A caveat in LBWT pig model is that the reduction in type II MyHC at birth is not the only factor that could influence muscle growth, and that other factors may have confounded our results. This is why we aimed to use β-adrenergic agonist as a means to induce a shift fiber type in muscles to a more glycolytic phenotype. Our objective was to determine the influence of the β-adrenergic agonist Ractopamine (RAC) induced slow-to-fast fiber type transformation on the regulation of protein synthesis and degradation pathways. Although supplementation improved translational capacity, enhanced S6K1 phosphorylation, and reduced the abundance of calcium-dependent proteases, RAC feeding had no effect on body or muscle weights. These results suggest that a fiber type transformation without other physiological influences does not alter protein turnover signaling in favor of hypertrophy in growing pigs. / Master of Science / Skeletal muscles grow by increasing the amount of protein contained within them. The amount of protein deposited is determined by the net balance between the rates at which proteins are synthesized and degraded. However, not all skeletal muscles grow at the same rate. One factor that is thought to influence protein synthesis and degradation rates is the types of muscle fibers that are present within a muscle. These fibers can display a range of contractile and metabolic characteristics, from slow-twitch oxidative fibers to fast-twitch glycolytic fibers. In the presented studies, we sought to determine whether changes in fiber type composition result in difference to the signaling pathways the regulate protein synthesis and degradation, ultimately leading to differences in the muscle growth of young pigs. We have previously shown reduced activation of the protein synthesis pathway in the skeletal muscle of low birth weight (LBWT) newborn pigs. These pigs also had lower expression of glycolytic fibers. In experiment 1, we aimed to compare the signaling pathways regulating protein synthesis and degradation in LBWT and normal birth weight (NBWT) pigs at weaning. We also sought to determine if the regulation of these signaling pathways changed between muscles with differing fiber type compositions. The glycolytic longissimus dorsi (LD) muscle of LBWT pigs grew rapidly between birth and weaning whereas the highly oxidative soleus did not. In addition, the LD of LBWT pigs had greater protein synthesis signaling and similar expression of muscle fibers compared with NBWT pigs, suggesting the improvement in protein synthesis signaling of LBWT pigs between birth and weaning may be related to a shift in fiber type. In experiment 2, we used a compound called ractopamine hydrochloride (RAC) to promote a slow-to-fast fiber type switch in the muscle of young pigs. With this study, we sought to determine the effect of this fiber type transformation, without the influence of birth weight, on the regulation of protein synthesis and degradation pathways. Although RAC-fed pigs showed some minor changes that could improve protein synthesis and decrease protein degradation, RAC feeding had no observable effect on body weight or muscle growth. These results suggest that a fiber type transformation alone is not enough to promote muscle growth in growing pigs.

A growing body of evidence shows that presynaptic nerve terminals throughout the nervous system are vulnerable to a range of traumatic, toxic and disease-related neurodegenerative stimuli. The aim of this study was to further characterise this vulnerability by examining the response of mouse α-motor nerve terminals at the neuromuscular junction (NMJ) to hypoxia-reperfusion injury. To address this aim, a novel model system was generated in which ex vivo skeletal muscle preparations could be maintained in an hypoxic environment, at an O2 concentration below in vivo normoxic values (<0.25% O2), for 2hr followed by 2hr reperfusion (2H-2R). Using this model system combined with quantitative assessment of immunohistological preparations as well as some ultrastructural observations, I present evidence to show that α-motor nerve terminals are rapidly and selectively vulnerable to hypoxia-reperfusion injury with no apparent perturbations to postsynaptic endplates or muscle fibres. I show that the severity of α-motor nerve terminal pathology is age and muscle type/location dependent: in 8-12wk old mice, nerve terminals in fast-twitch lumbrical muscles are more vulnerable than predominantly slow-twitch transversus abdominis and triangularis sterni. In 5-6 week old mice however, there is an age dependent increase in vulnerability of α-motor nerve terminals from the predominantly slow-twitch muscles while the fast-twitch lumbricals remained unaffected by age. The functional, morphological and ultrastructural pathology observed in α-motor nerve terminals following 2H-2R is indicative of selective and ongoing nerve terminal disassembly but, occurs via a mechanism distinct from Wallerian degeneration, as the neuroprotective slow Wallerian degeneration (Wlds) gene did not protect nerve terminals from these pathological changes. I also provide provisional evidence to show that 1A/II muscle spindle afferents and γ-motor nerve terminals are more resistant to hypoxia-reperfusion injury compared with α-motor nerve terminals. In addition to this, I also report preliminary finding that indicate that the oxygen storing protein, neuroglobin, maybe expressed at the mouse NMJ and report the difficulties of using mice that express yellow fluorescent protein (YFP) in their neurons for repeat/live imaging studies. Overall, these data show that the model of hypoxia-reperfusion injury developed in this study is robust and repeatable, that it induces rapid, quantitative changes in α-motor nerve terminals and that it can be used to further examine the mechanisms regulating nerve terminal vulnerability in response to hypoxia-reperfusion injuries. These findings have clinical implications for the use of surgical tourniquets and in the aetiology of many neurodegenerative diseases and neuropathic sequelae where mechanisms relating to hypoxia and hypoxia-reperfusion injury have been implicated.

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. The technical challenges of human skeletal muscle fiber type identification have evolved over the past three decades (8). The typical normal adult has roughly equal amounts of slow- and fast-twitch fibers, designated type I and II fibers. In addition, a variable portion of the type II fibers is mixed, containing both fast- and slow-twitch fiber markers, called type IIa fibers, whereas type II fibers that contain only the fast-twitch phenotype are designated type IIx in humans. Exercise training can cause modest shifts in fiber composition from one of these types to a contiguous type, with the relationship being type I to IIa to IIx or type IIx to IIa to I. The tail end of each myosin heavy chain is attached to the tail of another myosin heavy chain, and each of these forms a complex with two myosin light chains. Many heavy and light chain complexes are intertwined to form the thick filaments of each sarcomere. Thin filaments are composed of actin, troponin, and tropomyosin. The myosin heavy chains contain ATPase, which is essential for shortening of the contractile apparatus in the sarcomere, resulting in muscle-generated movement of a body part. The pH optimum of the ATPase has been classically the histochemical technique for identifying fast, slow, and mixed fibers. However, for more than a decade, monoclonal antibodies that correlated with the ATPase designation of fast, slow, and mixed fibers by bright-field or immunohistochemical methods have been used (2). The widely used fast and slow myosin monoclonal antibodies were obtained from mice immunized with only partially purified human skeletal muscle myosin antigens. More recently, antibodies that were raised against specific individual myosin heavy and light chain proteins became commercially available. The 15 human genes that code myosin heavy chains are designated MYH1, MYH2, MYH3, MYH4, MYH6, MYH7, MYH7B, MYH8, MYH9, MYH10, MYH11, MYH12, MYH13, MYH14, MYH15, and MYH16 (17). MYH9, MYH10, and MYH11 are expressed primarily in smooth muscle. At least eight separate genes that code myosin light chains, MYL1, MYL2, MYL3, MYL4, MYL5, MYL6, MYL6B, and MYLPF, have been identified, and at least three of these have a second isoform (3). Our initial investigation of the expression of myosin heavy and light chains using laser capture microdissection (LCM) to obtain specific fiber type samples from human vastus lateralis biopsies yielded some unexpected results. These observations led us to question which isoforms of myosin heavy and light chains are actually characteristic of “fast” or “slow” fibers in human skeletal muscle. We used immunoblots, mass spectroscopic (MS) proteomics, and next-generation sequencing of muscle homogenates and of LCM-generated samples of individual fiber types from normal control subjects and subjects with extremely different muscle fiber composition to approach this question by evaluating muscle specimens from subjects with diverse and extremely different fiber compositions. The hypothesis that drove these studies was that fibers of each type would have consistent myosin heavy and light chains that are characteristic of the fiber type. This is the first report that the abundance of different myosin heavy and light chains corresponds to different muscle fiber types.

There are gaps in the fundamental understanding of the expression of nitric oxide synthases (NOS) in the microvasculature. We examined co-localization of NOS1 (nNOS), NOS2 (iNOS) and NOS3 (eNOS) in the spinotrapezius muscle of young adult male Wistar-Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats according to fiber type using immunohistochemistry and brightfield microscopy. Data regarding fiber distribution, population and morphology data were collected. Alkaline phosphatase staining was used to determine capillary density and average number of capillaries around a fiber. Gel electrophoresis and Western blot techniques were used to compare myosin heavy chain (MHC) protein expression with fiber type population data and to determine NOS1-3 protein expression in whole muscle homogenate. This study should provide a more accurate understanding of differences in NOS expression between these two strains of rats.

As lesões tendíneas são uma das mais comuns que ocorrem nos esportes, sua freqüência é de 10 a 55% de todas as lesões ocorridas. A cabeça medial do músculo gastrocnêmio (GCm) é constituído por duas regiões bem distintas: uma região profunda \"vermelha\" e uma região superficial \"branca\". Dada essa característica de distribuição dos tipos de fibras, torna-se possível determinar se, no mesmo músculo, a tenotomia afeta as fibras de maneira diferente, dependendo do tipo de fibra predominante em cada região. O objetivo deste estudo foi avaliar o efeito da tenotomia e tenorrafia experimental na GCm. Foram usados 38 ratos Wistar machos pesando aproximadamente 300 g divididos em três grupos: Controle (C), Tenotomizado (T) e Tenorrafiado (R), avaliados aos 14 e 21 dias pós-cirurgia. Os animais foram mantidos sob as mesmas condições de alojamento, alimentação, temperatura umidade e luz. No grupo T, o tendão calcâneo do membro pélvico esquerdo foi dissecado e seccionado transversalmente no terço médio. No grupo R, este mesmo tendão foi imediatamente submetido à sutura de Kessler modificada após a tenotomia. Foram realizadas seções de 10 µm de espessura em criostato e, em seguida, estas secções foram submetidas às técnicas da hematoxilina e eosina, picro-sirius, NADH-tr e para análise em microscopia eletrônica de transmissão A significância estatística das diferenças inter-grupos foi determinada pela análise de variância, (ANOVA) e foi aceito p <0,05. Foram encontradas diferenças significativas entre os grupos C, T e R. Observou-se uma grande variação no tamanho e formato das fibras musculares e, ainda, uma desorientação e degeneração das miofibrilas e desorganização dos sarcômeros nos animais do grupo T. Ambos os grupos, T e R, apresentaram diminuição da área de secção transversa e comprimento da GCm. / Tendon injuries are one of the most commonly occurring in sports, its frequency is 10 to 55% of all injuries. The medial head of gastrocnemius muscle (GCm) consists of two clearly distinct regions: a deep region \"red\" and, a superficial region \"white\". Because of that characteristic distribution of fiber types, it becomes possible to determine if the same muscle tenotomy affects differently, depending on the predominant fiber type in each region. The aim of this study was to evaluate the effect of experimental tenotomy and tenorrhaphy on GCm. We used 38 male Wistar rats weighing approximately 300 g divided into three groups: control (C), tenotomized (T) and Tenorraphized (R). They were evaluated at 14 and 21 days after the surgery. Animals were kept under the same conditions of accommodation, feeding, temperature, humidity and light. In T group, calcaneal tendon of the left pelvic limb was dissected and sectioned in the middle third. In R group, the same tendon was immediately submmitted to the modified Kessler suture after tenotomy. Sections were performed in 10 µm thick in a cryostat and they were then stained according to hematoxylin and eosin, Picrosirius, NADH-tr methods and they were analyzed by transmission electron microscopy. The statistical significance of inter-group differences was determined by analysis of variance (ANOVA) and was accepted p<0.05. Significant differences were found between C, T and R groups. There was a wide variation in size and shape of muscle fibers and also a disorientation and degeneration of myofibrils and disorganization of sarcomeres in T group. Both T and R groups showed a decrease in cross-sectional area and length of the GCm.

As lesões tendíneas são uma das mais comuns que ocorrem nos esportes, sua freqüência é de 10 a 55% de todas as lesões ocorridas. A cabeça medial do músculo gastrocnêmio (GCm) é constituído por duas regiões bem distintas: uma região profunda \"vermelha\" e uma região superficial \"branca\". Dada essa característica de distribuição dos tipos de fibras, torna-se possível determinar se, no mesmo músculo, a tenotomia afeta as fibras de maneira diferente, dependendo do tipo de fibra predominante em cada região. O objetivo deste estudo foi avaliar o efeito da tenotomia e tenorrafia experimental na GCm. Foram usados 38 ratos Wistar machos pesando aproximadamente 300 g divididos em três grupos: Controle (C), Tenotomizado (T) e Tenorrafiado (R), avaliados aos 14 e 21 dias pós-cirurgia. Os animais foram mantidos sob as mesmas condições de alojamento, alimentação, temperatura umidade e luz. No grupo T, o tendão calcâneo do membro pélvico esquerdo foi dissecado e seccionado transversalmente no terço médio. No grupo R, este mesmo tendão foi imediatamente submetido à sutura de Kessler modificada após a tenotomia. Foram realizadas seções de 10 µm de espessura em criostato e, em seguida, estas secções foram submetidas às técnicas da hematoxilina e eosina, picro-sirius, NADH-tr e para análise em microscopia eletrônica de transmissão A significância estatística das diferenças inter-grupos foi determinada pela análise de variância, (ANOVA) e foi aceito p <0,05. Foram encontradas diferenças significativas entre os grupos C, T e R. Observou-se uma grande variação no tamanho e formato das fibras musculares e, ainda, uma desorientação e degeneração das miofibrilas e desorganização dos sarcômeros nos animais do grupo T. Ambos os grupos, T e R, apresentaram diminuição da área de secção transversa e comprimento da GCm. / Tendon injuries are one of the most commonly occurring in sports, its frequency is 10 to 55% of all injuries. The medial head of gastrocnemius muscle (GCm) consists of two clearly distinct regions: a deep region \"red\" and, a superficial region \"white\". Because of that characteristic distribution of fiber types, it becomes possible to determine if the same muscle tenotomy affects differently, depending on the predominant fiber type in each region. The aim of this study was to evaluate the effect of experimental tenotomy and tenorrhaphy on GCm. We used 38 male Wistar rats weighing approximately 300 g divided into three groups: control (C), tenotomized (T) and Tenorraphized (R). They were evaluated at 14 and 21 days after the surgery. Animals were kept under the same conditions of accommodation, feeding, temperature, humidity and light. In T group, calcaneal tendon of the left pelvic limb was dissected and sectioned in the middle third. In R group, the same tendon was immediately submmitted to the modified Kessler suture after tenotomy. Sections were performed in 10 µm thick in a cryostat and they were then stained according to hematoxylin and eosin, Picrosirius, NADH-tr methods and they were analyzed by transmission electron microscopy. The statistical significance of inter-group differences was determined by analysis of variance (ANOVA) and was accepted p<0.05. Significant differences were found between C, T and R groups. There was a wide variation in size and shape of muscle fibers and also a disorientation and degeneration of myofibrils and disorganization of sarcomeres in T group. Both T and R groups showed a decrease in cross-sectional area and length of the GCm.