The gastrocnemius muscle was removed from the hindlimb of female rodents, minced into 1mm3 fragments and autotransplanted. Experimental groups had the sural or tibial nerves laid in the mince as follows: both nerves (BN), sural only (SN), tibial only (TN) or no nerve (NN). Animals were sacrificed 45 days postsurgery and the regenerate and contralateral muscles studied for fiber composition and aerobic capacity. Regenerate mass and aerobic capacity were significantly less than control and followed the order BN > TN > SN > NN. The percentage of Types I, IIA and IIB fibers were also different between treatment groups. The data suggests (1) either a more rapid development of axons associated with IIA fibers following nerve transection or greater reinnervation capacity, and/or (2) a shift in the relative number of these axons in the regenerating nerve.

Successful antiretroviral (ARV) treatment is associated with suppression of HIV viral load and the reduction of clinical disease progression. Despite marked improvements in ARV medication, side effects from long-term treatment, such as loss of muscle mass do occur. The mechanism by which ARVs affect muscle mass is unclear, however, published in vitro data suggests a negative effect on myoblast fusion during differentiation. The objective of this study was therefore to determine the effect of ARVs on processes required for successful myogenesis; these included proliferation, migration during wound repair, and differentiation. C2C12 mouse skeletal myoblasts and human primary culture skeletal (HSk) myoblasts were incubated with Zidovudine (nucleoside reverse transcriptase inhibitor-NRTI), Tenofovir (nucleotide reverse transcriptase inhibitor-NtRTI) or Ritonavir (protease inhibitor-PI) at a concentration range of 0.01 μM to 10 μM. Proliferation was determined using crystal violet and migration was analyzed using a 2D wound healing assay. The commitment of myoblasts into the myogenic lineage was assessed via the expression of the transcription factor Pax7. Differentiation was measured by assessing the fusion index of multinucleated myotubes. C2C12 myoblast proliferation was observed to increase significantly in response to Tenofovir (1 μM and 10 μM). In HSk cells however, proliferation was observed to decrease significantly in response to Tenofovir (1 μM). Zidovudine had no consistent effect on C2C12 proliferation at any dose tested, but caused a decrease in HSk myoblast proliferation (0.01 μM and 0.1 μM); however this was statistically non-significant. A small dose-dependent increase in C2C12 and HSk cell number, although not significant, was seen in response to Ritonavir. Wound closure results revealed both dose-dependent and time-dependent effects of Tenofovir and Zidovudine on human myoblast migration, with significant decreases in the rate of wound closure (4-7 hours) noted at 0.1 μM and 0.01 μM doses respectively. Zidovudine had no significant effect on migration while Ritonavir (0.01 μM) was observed to significantly increase percentage wound closure of human myoblasts, suggesting an increased ability to migrate during wound repair. Differentiation results indicated a decrease in myoblast fusion in response to all three ARVs. However only Ritonavir was shown to negatively affect myosin heavy chain expression. Further research into the exact mechanism of decreased fusion is required. To our knowledge, this study is the first to suggest that selected ARVs may significantly influence myoblast regeneration capabilities by modulating myoblast proliferation, migration, differentiation and fusion, and thereby decrease their myogenic capability. Extended human myoblast studies on differentiation could confirm this hypothesis. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

Regeneration of the gastrocnemius muscle in 25 white rats was reinvestigated using reimplantation of minced muscle fragments in order to compare the fiber profiles of the regenerate and normal contralateral gastrocnemius. Fiber profiles were prepared from both the regenerate and the normal muscle 45 days postoperatively using myosin ATPase histochemistry.Counts made of type 1, 2A, 2B, and 2C fibers showed a significant (P<(0.05) change in the profile of the regenerated muscle compared to the contralateral muscle.The results indicated that although there was a change in the fiber profile at 45 days, the regenerated muscle appeared to follow a path of developmental recapitulation. Changes in the fiber appeared to be related to reinnervation of the regenerate.Ball State UniversityMuncie, IN 47306

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xii, 270 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Mammalian skeletal muscle can regenerate after injury and this response is primarily mediated by the satellite cell, a muscle stem cell. Following injury, satellite cells are activated to myoblasts, undergo rapid proliferation, migrate towards the injury site, and subsequently differentiate into myotubes in order to facilitate functional muscle repair. Fibrosis, caused by the secretion of structural extracellular matrix (ECM) proteins such as collagen I and fibronectin, by fibroblasts, impairs complete functional repair of the muscle. In this study, the role of the microenvironment during wound conditions was assessed by analysing the effect of specific extracellular matrix and growth factors on myoblast migration. The role of the Rho/ROCK pathway as a possible mechanism in mediating the effects seen was investigated. In order to analyse wound repair in an in vitro setting, we optimised and improved a wound healing model specifically designed for skeletal muscle repair. To this end we also developed a co-culture assay using primary myoblasts and fibroblasts isolated from the same animal. The studies showed that collagen I and fibronectin both increased myoblast migration in a dose-dependent manner. Decorin displayed opposing effects on cellular movement, significantly increasing collagen I-stimulated, but not fibronectin-stimulated, migration of myoblasts. ROCK inhibitor studies revealed a significant increase in migration on uncoated plates following inhibition with Y-27632 compared to untreated control. When cells were cultured on ECM components (Matrigel, collagen I, or fibronectin), the inhibitory effect of Y-27632 on migration was reduced. Analysis of ROCK and vinculin expression, and localization at the leading front, showed that ROCK inhibition resulted in loosely packed focal adhesion complexes (matrix dependent). A reduced adhesion to the ECM could explain the increased migration rates observed upon inhibition with Y-27632. We also investigated the role of TGF-β and decorin during wound repair, as TGF-β is a known pro-fibrotic agent. TGF-β treatment decreased wound closure rates; however, the addition of decorin with TGF-β significantly increased wound closure. The addition of ECM components, Matrigel and collagen I enhanced the effect seen in response to TGF-β and decorin; however, fibronectin negated this effect, with no increase in migration seen compared to the controls. In conclusion, the importance of extracellular matrix components in regulating myoblast migration and therefore skeletal muscle wound repair was demonstrated. We emphasize that, in order to gain a better understanding of skeletal muscle wound repair, the combination of ECM and growth factors released during wounding need to be utilised in assays which mimic the in vivo environment more closely. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Force production by a muscle is critical to maintaining proper function and overall health of a human or animal. Muscle adapts to increased loading with hypertrophy by activating a number of intracellular signaling cascades that regulate protein synthesis. The overall hypothesis is that force-dependent processes acutely activate growth-related signaling during active force generation. This project took two approaches. The first employed a general survey of muscles in which age-dependent changes in muscle activity differed. No conclusive activity-dependent signaling emerged however coordinated signaling among kinases broke down with age. The second approach utilized an in situ muscle preparation in which force production or metabolic costs were specifically controlled. Similar sub-maximal force levels generated by different methods found that force, per se, is not a primary modulator of growth-related signaling but that ERK phosphorylation is dependent on fiber-activation. Prolonging the duration of electrical stimulation applied to the nerve or increasing the frequency at which stimulations are applied was expected to increase the metabolic stress associated with contraction. Several growth-related kinases correlated with markers of metabolic stress, i.e. increased AMPK activity and decreased glycogen content, which were decoupled from force decline. This suggests energy depletion, specific to stimulation pattern, strongly influences the immediate response to high force contraction signaling. The overall conclusion is that signaling molecules previously implicated in force-dependent signaling lie much too downstream to relay strict force-dependent signaling.

Satellite cells are quiescent skeletal muscle specific stem cells that are activated in response to injury to aid in muscle repair and regeneration. The interaction of hepatocyte growth factor (HGF) with these cells is crucial for their activation. However, to date, research on the effect of HGF on skeletal muscle satellite cells has yielded conflicting data. Clarity is therefore required as to its effect on downstream myogenic processes. Furthermore, mouse and rat cell lines and primary culture have been widely used for in vitro studies to investigate the effect of HGF on skeletal muscle physiology and disease; very few studies have been carried out in primary cultured human skeletal myoblasts. As a result, we aimed to investigate and compare the effect of HGF (2, 10 and 50 ng/ml) on mouse C2C12 myoblast versus primary culture human skeletal myoblast (HSkM) proliferation, migration and differentiation. Proliferation was assessed via both cell counts and crystal violet assay, while migration was investigated using the scratch assay. Differentiation was determined via analysis of expression patterns of transcription factors implicated in myogenic commitment (i.e. Pax7, MyoD) as well expression of the structural protein Myosin Heavy Chain (MyHC). We demonstrate a dose-dependent effect of HGF on myoblast proliferation whereby an increase in proliferation was detected in response to 2 ng/ml HGF, whilst 10 ng/ml HGF resulted in a reduction in proliferation capacity of both C2C12 and HSkM myoblasts. Interestingly, the reduction in proliferation in response to 10 ng/ml HGF was accompanied by a down-regulation in Pax7 expression during differentiation of both mouse and human myoblasts. HGF also affected myoblast migration and differentiation in a dose-dependent manner that was inversely proportional to proliferation. HGF (10 ng/ml) stimulated an increase in myogenic commitment and terminal differentiation of C2C12 and HSkM myoblasts as reflected by the increased percentage MyoD positive cells, improved fusion and greater MyHC expression. C2C12 myoblast migration was also stimulated at this HGF concentration, but reduced in response to the lower HGF (2 ng/ml) dose. The decrease in proliferation following incubation with 10 ng/ml HGF, allows cells to exit proliferation into either a mode of migration or differentiation. Our data confirms the importance of HGF during myogenesis and highlights the sensitivity of satellite cells to changing HGF concentration. This has implications in the regulation of skeletal muscle wound repair. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Made available in DSpace on 2015-06-17T19:34:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-04. Added 1 bitstream(s) on 2015-06-18T12:46:48Z : No. of bitstreams: 1 000831507.pdf: 1599546 bytes, checksum: 662ec2cc8a79f99b1f64c6a60a89c9ff (MD5) / A neurorrafia látero-terminal sem lesão do nervo doador representou grande avanço na área de microcirurgia de nervos periféricos. O coto distal do nervo receptor é suturado na face lateral do nervo doador, sem prejudicá-lo. Desde 1994, o FK-506, uma droga imunossupressora, tem sido utilizado para impedir a rejeição do aloenxerto nos transplantes de órgãos e apresentou, em alguns estudos, como ação secundária, efeitos positivos sobre a regeneração nervosa periférica. O objetivo desse trabalho foi estudar se a administração de FK-506 traria benefícios na regeneração do nervo após neurorrafia látero-terminal. Foram utilizados 80 ratos Wistar, divididos em 4 grupos experimentais. Cada rato teve seu nervo fibular esquerdo seccionado e a extremidade distal suturada término-lateralmente ao nervo tibial. Os animais do grupo I, II e III foram submetidos à administração de FK-506 com dose de 1,0, 0,5 e 0,25 mg/kg/dia, respectivamente, durante dois meses, enquanto os animas do grupo Controle (GC) não receberam nenhuma droga. Após dois meses da cirurgia, os ratos foram submetidos ao teste da marcha, estudo eletrofisiológico e análise das fibras musculares e nervosas. O grupo II (GII) apresentou o músculo tibial cranial (MTC) com menor massa (p < 0,05) e maior amplitude (p = 0,019). Os resultados do número total de fibras nervosas foram maiores para GII (p < 0,001). Não houve diferença significativa entre os grupos para os índices funcionais. Com base em nosso modelo experimental, pudemos concluir que a administração de FK-506 na dose de 0,5 mg/Kg/dia diminuiu o ganho de massa corporal e massa do músculo tibial cranial e aumentou a regeneração das fibras nervosas, embora não tenha conseguido alterar a resposta funcional / The latero-terminal neurorrhaphy without injury to the donor nerve, represented great advances in microsurgery of peripheral nerves. The receptor nerve distal stump was sutured on the lateral side of the donor nerve, without harming it. Since 1994, FK-506, an immunosupressive drug, has been used to prevent allograft rejection in organ transplants and presented, in some studies, as a secondary action, positive effects on peripheral nerve regeneration. The aim of this work was to investigate whether the administration of FK-506 would benefit nerve regeneration after a latero-terminal neurorrhaphy. We used 80 Wistar rats, divided into four experimental groups. Each rat had its left fibular nerve sectioned and the distal stump sutured to the lateral of the tibial nerve. The animals of group I, II and III were subjected to the administration of FK-506 in an amount of 1.0, 0.5 to 0.25mg/K/day, respectively, for two months, while the aminals of the control group received no drug. Two months after surgery the rats were submitted to the walking test, eletrophysiological study and analysis of muscle and nerve fibers. The group II (GII) showed cranial tibial muscle (MTC) with lower mass (p < 0.05) and higher amplitude (p = 0.019). The results by the total number of nerve fibers was higher for GII (p < 0.001) . There was no significant difference between groups for functional indices. Based on our experimental model, we could conclude that the administration of 0.5 mg/Kg/dia of FK-506 decreased body mass gain and mass of MTC and increased regeneration of nerve fibers, although not able to change the functional response

Orientador: Fausto Viterbo / Banca: Alexandre L. R. Oliveira / Banca: Amilton Antunes Barreira / Banca: Carlos Nárcio Nóbrega de Jesus / Banca: José de Anchieta de Castro e Horta Junior / Resumo: A neurorrafia látero-terminal sem lesão do nervo doador representou grande avanço na área de microcirurgia de nervos periféricos. O coto distal do nervo receptor é suturado na face lateral do nervo doador, sem prejudicá-lo. Desde 1994, o FK-506, uma droga imunossupressora, tem sido utilizado para impedir a rejeição do aloenxerto nos transplantes de órgãos e apresentou, em alguns estudos, como ação secundária, efeitos positivos sobre a regeneração nervosa periférica. O objetivo desse trabalho foi estudar se a administração de FK-506 traria benefícios na regeneração do nervo após neurorrafia látero-terminal. Foram utilizados 80 ratos Wistar, divididos em 4 grupos experimentais. Cada rato teve seu nervo fibular esquerdo seccionado e a extremidade distal suturada término-lateralmente ao nervo tibial. Os animais do grupo I, II e III foram submetidos à administração de FK-506 com dose de 1,0, 0,5 e 0,25 mg/kg/dia, respectivamente, durante dois meses, enquanto os animas do grupo Controle (GC) não receberam nenhuma droga. Após dois meses da cirurgia, os ratos foram submetidos ao teste da marcha, estudo eletrofisiológico e análise das fibras musculares e nervosas. O grupo II (GII) apresentou o músculo tibial cranial (MTC) com menor massa (p < 0,05) e maior amplitude (p = 0,019). Os resultados do número total de fibras nervosas foram maiores para GII (p < 0,001). Não houve diferença significativa entre os grupos para os índices funcionais. Com base em nosso modelo experimental, pudemos concluir que a administração de FK-506 na dose de 0,5 mg/Kg/dia diminuiu o ganho de massa corporal e massa do músculo tibial cranial e aumentou a regeneração das fibras nervosas, embora não tenha conseguido alterar a resposta funcional / Abstract: The latero-terminal neurorrhaphy without injury to the donor nerve, represented great advances in microsurgery of peripheral nerves. The receptor nerve distal stump was sutured on the lateral side of the donor nerve, without harming it. Since 1994, FK-506, an immunosupressive drug, has been used to prevent allograft rejection in organ transplants and presented, in some studies, as a secondary action, positive effects on peripheral nerve regeneration. The aim of this work was to investigate whether the administration of FK-506 would benefit nerve regeneration after a latero-terminal neurorrhaphy. We used 80 Wistar rats, divided into four experimental groups. Each rat had its left fibular nerve sectioned and the distal stump sutured to the lateral of the tibial nerve. The animals of group I, II and III were subjected to the administration of FK-506 in an amount of 1.0, 0.5 to 0.25mg/K/day, respectively, for two months, while the aminals of the control group received no drug. Two months after surgery the rats were submitted to the walking test, eletrophysiological study and analysis of muscle and nerve fibers. The group II (GII) showed cranial tibial muscle (MTC) with lower mass (p < 0.05) and higher amplitude (p = 0.019). The results by the total number of nerve fibers was higher for GII (p < 0.001) . There was no significant difference between groups for functional indices. Based on our experimental model, we could conclude that the administration of 0.5 mg/Kg/dia of FK-506 decreased body mass gain and mass of MTC and increased regeneration of nerve fibers, although not able to change the functional response / Doutor

Orientador: Selma Maria Michelin Matheus / Banca: Maeli Dal Pai / Banca: Fausto Viterbo / Banca: Elaine Minatel / Banca: Carlos Eduardo Assumpção de Freitas / Resumo: A bupivacaina é um anestésico utilizado na prática médica e odontológica para bloqueio do nervo periférico e alívio da dor pré e pós-operatória devido à sua ação analgésica de longa duração. Suas principais limitações são a miotoxicidade, a neurotoxicidade e a inflamação. A laserterapia de baixa potência (LBP) tem sido utilizada para várias propostas terapêuticas, apresentando ação anti-inflamatória, regenerativa e analgésica. O objetivo do estudo foi avaliar o efeito do laser de Arseneto de Gálio (AsGa) sobre a morfologia das junções neuromusculares, fibras musculares e nervo associados ao músculo esternomastóideo de ratos após injeção de bupivacaina. 32 ratos Wistar machos adultos foram divididos em 2 grupos: Grupo Controle (C: n=16) e Grupo Laser (L: n= 16). Os grupos foram subdivididos seguindo os antímeros e substâncias injetadas: direito (bupivacaina 0,5%), esquerdo (Cloreto de sódio 0,9%). Após 24 horas houve aplicação de LBP (AsGa 904nm, 50mW, 4,8J) durante 5 dias consecutivos. A seguir, os animais foram eutanasiados, o sangue foi coletado para determinação da creatina kinase (CK); a porção superficial dos músculos esternomastóideos e os nervos associados foram dissecados, removidos e submetidos às seguintes análises: análise histopatológica e ultraestrutural; análises morfológica e morfométrica das JNMs (reação Esterase inespecífica), microscopia confocal de varredura a laser e análise ultraestrutural; e os nAChRs (alpha, beta e gama) e os níveis de TNF foram quanti... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bupivacaine is an anesthetic used in medical and dental practice as a peripheral nerve block and for relief of pre and postoperative pain due to its long duration analgesic action. Its principle limitations are myotoxicity, neurotoxicity, and inflammation. Low-level laser therapy (LBP) has been used for various therapeutic approaches, presenting an anti-inflammatory, regenerative, and analgesic action. The aim of the study was to evaluate the effects of the Arsenide Gallium laser (GaAs) on the morphology of neuromuscular junctions, muscle fibers, and the nerve associated with the sternomastoid muscle of rats after injection with bupivacaine. In total, 32 adult male Wistar rats were divided into 2 groups: Control group (C: n = 16) and Laser Group (L: n = 16). The groups were subdivided according to the antimeres and injected substances: right (0.5% bupivacaine), left (sodium chloride 0.9%). Twenty-four hours after the injection, LBP was applied (GaAs 904nm, 50mW, 4.8J) for 5 consecutive days. Subsequently, the animals were euthanized; blood was collected for determination of creatine kinase (CK); the surface portion of the sternomastoid muscles and associated nerves were dissected, removed, and submitted to the following tests: histopathological and ultrastructural analysis; morphological and morphometric analysis of the JNMs (nonspecific esterase reaction), confocal laser scanning microscopy and ultrastructural analysis; and the nAChRs (alpha, beta, and gamma) and TNF levels were quantified by Western blotting; in addition, nerve morphometry and quantification of muscle CK were performed. No alterations were observed in the antimere which received the sodium chloride, with or without laser application. The muscles receiving bupivacaine presented higher levels of inflammation, atrophy and necrosis, and muscle CK values; a greater number of central nuclei and... (Complete abstract electronic access below) / Doutor