Protection of dystrophic muscle cells using Idebenone correlates with the interplay between calcium, oxidative stress and inflammation.

There is strong cross-talk between abnormal intracellular calcium concentration, high levels of reactive oxygen species (ROS) and an exacerbated inflammatory process in the dystrophic muscles of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). In this study, we investigated effects of Idebenone, a potent anti-oxidant, on oxidative stress markers, the anti-oxidant defence system, intracellular calcium concentrations and the inflammatory process in primary dystrophic muscle cells from mdx mice. Dystrophic muscle cells were treated with Idebenone (0.05 µM) for 24 h. The untreated mdx muscle cells were used as controls. The MTT assay showed that Idebenone did not have a cytotoxic effect on the dystrophic muscle cells. The Idebenone treatment was able to reduce the levels of oxidative stress markers, such as H2 O2 and 4-HNE, as well as decreasing intracellular calcium influx in the dystrophic muscle cells. Regarding Idebenone effects on the anti-oxidant defence system, an up-regulation of catalase levels, glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity was observed in the dystrophic muscle cells. In addition, the Idebenone treatment was also associated with reduction in inflammatory molecules, such as nuclear factor kappa-B (NF-κB) and tumour necrosis factor (TNF) in mdx muscle cells. These outcomes supported the use of Idebenone as a protective agent against oxidative stress and related signalling mechanisms involved in dystrophinopathies, such as DMD.

Coenzyme Q10 supplementation acts as antioxidant on dystrophic muscle cells.

Increased oxidative stress is a frequent feature in Duchenne muscular dystrophy (DMD). High reactive oxygen species (ROS) levels, associated with altered enzyme antioxidant activity, have been reported in dystrophic patients and mdx mice, an experimental model of DMD. In this study, we investigated the effects of coenzyme Q10 (CoQ10) on oxidative stress marker levels and calcium concentration in primary cultures of dystrophic muscle cells from mdx mice. Primary cultures of skeletal muscle cells from C57BL/10 and mdx mice were treated with coenzyme Q10 (5 µM) for 24 h. The untreated mdx and C57BL/10 muscle cells were used as controls. The MTT and live/dead cell assays showed that CoQ10 presented no cytotoxic effect on normal and dystrophic muscle cells. Intracellular calcium concentration, H2O2 production, 4-HNE, and SOD-2 levels were higher in mdx muscle cells. No significant difference in the catalase, GPx, and Gr levels was found between experimental groups. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. Our findings also suggest that the decrease of oxidative stress reduces the need for upregulation of antioxidant pathways, such as SOD and GSH.

Inside-out and standard vein grafts associated with platelet-rich plasma (PRP) in sciatic nerve repair. A histomorphometric study.

PURPOSE:: To evaluated the tubulization technique with standard and inside-out vein, filled or not with platelet-rich plasma (PRP), in sciatic nerve repair. METHODS:: Seventy male Wistar rats were randomly divided into five groups: IOVNF (Inside-Out Vein with No Filling); IOVPRP (Inside-Out Vein filled with PRP); SVNF (Standard Vein with No Filling); SVPRP (Standard Vein filled with PRP); Sham (Control). The left external jugular vein was used as graft in a 10 mm nervous gap. RESULTS:: In the morphological analysis of all groups, myelinated nerve fibers with evident myelin sheath, neoformation of the epineurium and perineurium, organization of intraneural fascicles and blood vessels were observed. In the morphometry of the distal stump fibers, SVPRP group had the highest means regarding fiber diameter (3.63±0.42 µm), axon diameter (2.37±0.31 µm) and myelin sheath area (11.70±0.84 µm2). IOVPRP group had the highest means regarding axon area (4.39±1.16 µm2) and myelin sheath thickness (0.80±0.19 µm). As for values of the fiber area, IOVNF group shows highest means (15.54±0.67 µm2), but are still lower than the values of the Sham group. CONCLUSION:: The graft filled with platelet-rich plasma, with use standard (SVPRP) or inside-out vein (IOVPRP), promoted the improvement in axonal regeneration on sciatic nerve injury.

Inside-out and standard vein grafts associated with platelet-rich plasma (PRP) in sciatic nerve repair. A histomorphometric study

Abstract Purpose: To evaluated the tubulization technique with standard and inside-out vein, filled or not with platelet-rich plasma (PRP), in sciatic nerve repair. Methods: Seventy male Wistar rats were randomly divided into five groups: IOVNF (Inside-Out Vein with No Filling); IOVPRP (Inside-Out Vein filled with PRP); SVNF (Standard Vein with No Filling); SVPRP (Standard Vein filled with PRP); Sham (Control). The left external jugular vein was used as graft in a 10 mm nervous gap. Results: In the morphological analysis of all groups, myelinated nerve fibers with evident myelin sheath, neoformation of the epineurium and perineurium, organization of intraneural fascicles and blood vessels were observed. In the morphometry of the distal stump fibers, SVPRP group had the highest means regarding fiber diameter (3.63±0.42 μm), axon diameter (2.37±0.31 μm) and myelin sheath area (11.70±0.84 μm2). IOVPRP group had the highest means regarding axon area (4.39±1.16 μm2) and myelin sheath thickness (0.80±0.19 μm). As for values of the fiber area, IOVNF group shows highest means (15.54±0.67 μm2), but are still lower than the values of the Sham group. Conclusion: The graft filled with platelet-rich plasma, with use standard (SVPRP) or inside-out vein (IOVPRP), promoted the improvement in axonal regeneration on sciatic nerve injury.

Beneficial cilostazol therapeutic effects in mdx dystrophic skeletal muscle.

This study evaluated the possible protective effects of cilostazol against myonecrosis in dystrophic diaphragm muscle in vivo, focusing on oxidative stress, the inflammatory response and angiogenesis. Young mdx mice, the experimental animal for Duchenne muscular dystrophy, received cilostazol for 14 days. A second group of mdx mice and a control group of C57BL/10 mice received a saline solution. In the mdx mice, cilostazol treatment was associated with reduced loss of muscle strength (-34.4%), decreased myonecrosis, reduced creatine kinase levels (-63.3%) and muscle fibres stained for immunoglobulin G in dystrophic diaphragm muscle (-81.1%), and a reduced inflammatory response, with a decreased inflammatory area (-22%), macrophage infiltration (-44.9%) and nuclear factor-κB (-24%) and tumour necrosis factor-α (-48%) content in dystrophic diaphragm muscle. Furthermore, cilostazol decreased oxidative stress and attenuated reactive oxygen species production (-74%) and lipid peroxidation (-17%) in dystrophic diaphragm muscle, and promoted the up-regulation of angiogenesis, increasing the number of microvessels (15%). In conclusion, the present results show that cilostazol has beneficial effects in dystrophic muscle. More research into the potential of cilostazol as a novel therapeutic agent for the treatment of dystrophinopathies is required.

Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity, Inflammation Response and Oxidative Stress.

The present study evaluated low-level laser therapy (LLLT) effects on some physiological pathways that may lead to muscle damage or regeneration capacity in dystrophin-deficient muscle cells of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). Primary cultures of mdx skeletal muscle cells were irradiated only one time with laser and analyzed after 24 and 48 hours. The LLLT parameter used was 830 nm wavelengths at 5 J/cm² fluence. The following groups were set up: Ctrl (untreated C57BL/10 primary muscle cells), mdx (untreated mdx primary muscle cells), mdx LA 24 (mdx primary muscle cells - LLLT irradiated and analyzed after 24 h), and mdx LA 48 (mdx primary muscle cells - LLLT irradiated and analyzed after 48 h). The mdx LA 24 and mdx LA 48 groups showed significant increase in cell proliferation, higher diameter in muscle cells and decreased MyoD levels compared to the mdx group. The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups. The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca2+]i. The mdx group showed significant increase in H2O2 production and 4-HNE levels compared to the Ctrl group and LLLT treatment reduced this increase. GSH levels and GPx, GR and SOD activities increased in the mdx group. Laser treatment reduced the GSH levels and GR and SOD activities in dystrophic muscle cells. The mdx group showed significant increase in the TNF-α and NF-κB levels, which in turn was reduced by the LLLT treatment. Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies.

Reduction of Oxidative Damage and Inflammatory Response in the Diaphragm Muscle of mdx Mice Using Iron Chelator Deferoxamine.

Oxidative stress and inflammatory processes strongly contribute to pathogenesis in Duchenne muscular dystrophy (DMD). Based on evidence that excess iron may increase oxidative stress and contribute to the inflammatory response, we investigated whether deferoxamine (DFX), a potent iron chelating agent, reduces oxidative stress and inflammation in the diaphragm (DIA) muscle of mdx mice (an experimental model of DMD). Fourteen-day-old mdx mice received daily intraperitoneal injections of DFX at a dose of 150 mg/kg body weight, diluted in saline, for 14 days. C57BL/10 and control mdx mice received daily intraperitoneal injections of saline only, for 14 days. Grip strength was evaluated as a functional measure, and blood samples were collected for biochemical assessment of muscle fiber degeneration. In addition, the DIA muscle was removed and processed for histopathology and Western blotting analysis. In mdx mice, DFX reduced muscle damage and loss of muscle strength. DFX treatment also resulted in a significant reduction of dystrophic inflammatory processes, as indicated by decreases in the inflammatory area and in NF-κB levels. DFX significantly decreased oxidative damage, as shown by lower levels of 4-hydroxynonenal and a reduction in dihydroethidium staining in the DIA muscle of mdx mice. The results of the present study suggest that DFX may be useful in therapeutic strategies to ameliorate dystrophic muscle pathology, possibly via mechanisms involving oxidative and inflammatory pathways.

Effect of N-acetylcysteine plus deferoxamine on oxidative stress and inflammation in dystrophic muscle cells.

OBJECTIVES: Oxidative stress and inflammatory process play an important role in the pathogenesis of Duchenne muscular dystrophy (DMD). We investigated whether deferoxamine (DFX) improves the antioxidant effects of N-acetylcysteine (NAC) on primary cultures of dystrophic muscle cells from mdx mice, the experimental model of DMD. METHODS: Primary cultures of skeletal muscle cells from mdx mice were treated with either NAC (10 mM), DFX (5 mM), or NAC plus DFX for 24 hours. The muscle cells of C57BL/10 mice were used as controls. RESULTS: Production of hydrogen peroxide (H2O2) and levels of 4-hydroxynonenal (4-HNE), tumor necrosis factor alpha (TNF-α), and nuclear factor kappa-B (NF-κB) were significantly higher in mdx muscle cells than in C57BL/10 muscle cells. Treatment with NAC, DFX, or NAC plus DFX significantly decreased H2O2 production (24, 58, and 72%, respectively), and levels of 4-HNE-protein adducts (62, 33, and 71%, respectively), TNF-α (32, 29, and 31%, respectively), and NF-κB (34, 38, and 52%, respectively) on dystrophic muscle cells. DISCUSSION: This study demonstrates that mdx muscle cells are able to produce key oxidative stress and inflammatory markers, without the interference of inflammatory cells, and shows that NAC plus DFX reduced the inflammatory and oxidative stress indicators, mainly H2O2 production and NF-κB levels by dystrophic fibers.

N-acetylcysteine treatment reduces TNF-α levels and myonecrosis in diaphragm muscle of mdx mice.

BACKGROUND & AIMS: Duchenne muscular dystrophy (DMD) is a genetic muscle disease caused by the absence of dystrophin. An established animal model of DMD is the mdx mouse, which is unable to express dystrophin. Inflammation, particularly the proinflammatory cytokine tumor necrosis factor alpha (TNF-α), strongly contributes to necrosis in the dystrophin-deficient fibers of the mdx mice and in DMD. In this study we investigated whether the antioxidant N-acetylcysteine (NAC) decreases TNF-α levels and protects the diaphragm muscle of mdx mice against necrosis. METHODS: Mdx mice (14 days old) received daily intraperitoneal injections of NAC for 14 days, followed by removal of the diaphragm muscle. Control mdx mice were injected with saline. RESULTS: NAC reduced TNF-α and 4-HNE-protein adducts levels, inflammation, creatine kinase levels, and myonecrosis in diaphragm muscle. CONCLUSIONS: NAC may be used as a complementary treatment for dystrophinopathies. However, clinical trials are needed to determine the appropriate dose for patients with Duchenne muscular dystrophy.

Histomorfometria da glândula submandibular de rato (Rattus novergicus) submetidos à dieta alcoólica – Parte II / Morphometric evaluation of submandibular gland of rat (Rattus Novergicus) submited to the alcohol diet – Part II

A literatura mostra que o alcoolismo é um dos maiores problemas médicos e sociais de quase todas as sociedadesneste século, e que ele produz alterações na cavidade oral, por isso pensou-se em realizar este trabalho para observar, histomorfométricamente, se esta substância produz alterações nas glândulas submandibulares de ratos. Para isso foram utilizados 15 ratos machos adultos, que foram divididos em três grupos de cinco animais cada um. Grupo Controle (GC), os animais receberam água e ração “ad libitum”; Grupo Alcoolizado (GA), os animais receberam uma mistura de água eálcool numa concentração de 25%; e Grupo Isocalórico (GI), onde os animais receberam uma solução de água e sacarose, com a mesma quantidade de calorias que os animais do grupo alcoolizado. Após 120 dias, de tratamento os animais foram eutanaziados e tiveram suas glândulas submandibulares removidas bilateralmente para análise histomorfométrica. Os resultados mostraram que nos animais do grupo GA ocorreu: menor crescimento na área dos ácinos seromucosos e seus núcleos; e maior crescimento nas áreas nos ductos granulares e estriados. Baseado nestes dados pode-se concluir que o álcool altera a morfologia das estruturas das glândulas submandibulares de ratos.

The literature shows that alcoholism is the one of the most medical and a social problem of almost all society in this century, and that it produces alterations in the oral cavity. Therefore it was thought about carrying through this work to observe under morphological criteria, if this substance produces alterations in the rat submandibullary glands. For that purpose, 3 groups were used (5 rats in each group): control group (CG) received water and food “ad libitum”; alcoholic group (AG) received the solution containing water and 25% alcohol; and isocaloric group (IG) received a solution containing water and sucrose, with the same amount of calories as the animals in the alcoholic group. After 120 days of treatmentthe animals were sacrificed and had its submandibullary glands removed bilaterally for histomorphometric analysis. The results indicated that in the animals of group GA there was reduction in the area and nuclei of the seromucous acini. On the other hand, there was an enlargement in the area of the granular and striated ducts. Based in these data it can be concluded that the alcohol alters the morphology of the structures of the submandibullary glands of the rats.

Fat cell invasion in long-term denervated skeletal muscle.

There are several differences between red and white muscles submitted to different experimental conditions, especially following denervation: a) denervation atrophy is more pronounced in red than white muscles; b) the size of the fibers in the red muscles does not vary between different parts of the muscle before and after denervation, when compared to white muscles; c) the regional difference in the white muscles initially more pronounced after denervation than red muscle; d) red muscle fibers and fibers of the deep white muscle present degenerative changes such as disordered myofibrils and sarcolemmal folds after long-term denervation; e) myotube-like fibers with central nuclei occur in the red muscle more rapidly than white after denervation. Denervation of skeletal muscles causes, in addition to fibers atrophy, loss of fibers with subsequent regeneration, but the extent of fat cell percentage invasion is currently unknown. The present article describes a quantitative study on fat cell invasion percentage in red m. soleus and white m. extensor digitorum longus (EDL) rat muscles at 7 weeks for up to 32 weeks postdenervation. The results indicate that the percentage of fat cells increase after denervation and it is steeper than the age-related fat invasion in normal muscles. The fat percentage invasion is more pronounced in red compared with white muscle. All experimental groups present a statistically significant difference as regard fat cell percentage invasion.

Is there morphological difference between branchiomeric and somitic muscles submitted to alcohol consumption? An experimental study in rats (Rattus norvegicus)

O alcoolismo é considerado uma doenca que causa desordens físicas e também dependência. Mais de 18 milhões de pessoas nos Estados Unidos são alcoólatras e na Inglaterra, entre 1/3 à ½ delas apresentam algum tipo de desordem física. No geral a literatura está focada para as desordens que acometem os músculos do tronco. Esses músculos têm origem embriológica diferente dos músculos da mastigacão. O propósito desta pesquisa foi avaliar o efeito do álcool sobre os músculos da mastigacão (branquiméricos) no intuito de compará-lo com as alteracões que ocorrem nos músculos do tronco (miotômicos). Para isso 15 ratos machos Wistar, pesando ao redor de 250g foram utilizados. Os animais foram divididos em três grupos: Controle normal (N); Alcoolizado (A) e Isocalórico (I). Fragmentos dos músculos masseter, temporal e reto do abdome foram coletados e submetidos às reacões de m-ATPase (com pré-incubacões ácida e alcalina) e NADH-TR. As fibras puderam ser classificadas como FG, FOG e SO. Os resultados mostraram atrofia das fibras de contracão rápida (FG e FOG) nos músculos da mastigacão, embora esta atrofia, não tenha sido significante entre os grupos estudados. Por outro lado, atrofia significativa foi observada no músculo reto do abdome. Baseado nestes resultados pode-se concluir que o efeito do álcool sobre os músculos elevadores da mandíbula (m. masseter e m. temporal) é diferente se comparado aos observados em músculos somíticos (m. reto abdominal).

Is there morphological difference between branchiomeric and somitic muscles submitted to alcohol consumption? An experimental study in rats (Rattus norvegicus).

Alcoholism is considered a physical dependence disorder. More than 18 million people are alcoholics in the USA and England and between 1/3 to ½ of them present some kind of physical disorder. In general the literature is focused on alcoholic trunk muscle disorders. These muscles have different embryological origins if compared to the masticatory muscles. The aim of this research was to evaluate the effects of alcohol on the masticatory muscles in order to compare them with the somitic muscles. For this purpose, 15 male Wistar rats weighing around 250g were used. The rats were divided into three groups: Normal control (N), Alcoholic (A) and Isocaloric (I). Slices of the masseter muscle, temporalis muscle and rectus abdominal muscle were harvested and submitted to histochemical reactions (m-ATPase: acid and alkaline pre incubation and NADH-TR). The myofibers were classified in SO, FOG and FG. The results showed atrophy of the fast fibers (FG and FOG) in the masticatory muscles but this atrophy was not statistically significant in this study (p< 0.05). On the other hand, significant atrophy occurred in the rectus abdominal muscle (p<0.05). Based on these data it can concluded that the effect of alcohol on the branchiomeric jaw elevator muscles (masseter and temporalis muscles) is different compared to the effect on somitic muscle (rectus abdominal muscle).

Morfometria dos tipos histoenzimológicos de fibras musculares do músculo reto abdominal de ratos (Rattus norvegicus) alcoolizados / Morphometry of histoenzymological types of muscular fibers of rectus abdominalis muscle of alcoholized rats (Rattus norvegicus)

Considerando que os usuários do álcool representam um boa parcela da população humana, que o uso prolongado do álcool em excesso causa multiplicidade de anormalidades, tanto bioquímicas, como morfológicas, resolveu-se estudar o efeito desta droga em uma mistura onde o álcool representa mais de 50 por cento das calorias usadas diariamente pelo rato, que apresenta uma atrofia seletiva das fibras do tipo II, semelhante à que ocorre no ser humano. Para isso foi retirada amostra do músculo reto abdominal de cinco animais de cada grupo Controle (C), Alcoolizado (A) e Isocalórico (I). As amostras foram submetidas às reações de m-ATPase (com pré-incubações ácida e alcalina) e NADH-TR, para classificar as fibras de acordo com os resultados obtidos nestas reações. Os resultados mostraram uma diferença significativa, tanto na freqüência das fibras do tipo FG, como nas do tipo FOG entre os grupos estudados. Quanto a área, os resultados apontaram uma diferença significativa apenas nas fibras do tipo FG, sendo menor nos animais do grupo A e maior nos do grupo C. Baseado nestes dados pode-se concluir que a alcoolização alterou o tamanho das fibras do tipo FG e a freqüência das fibras FG e FOG

Efeito do etanol sobre o músculo digástrico de ratos (Rattus norvegicus): uma avaliação histoenzimológica / Effect of ethanol in digastricus mucle in rats (Rattus norvegicus): an histoenzimologic evaluation

Estima-se que entre 1 milhão de adultos do hemisfério ocidental, 20.000 tenham anomalias nos músculos esqueléticos como conseqüência da ingestão de álcool. Estudos revelam que nestes casos ocorre uma fraqueza progressiva afetando predominantemente os músculos da coxa, ombro e quadris, e uma artrofia das fibras do tipo II ou de contração rápida. Na literatura consultada não foi observado relato do que acontece com os músculos da mastigação, por isso este estudo foi desenvolvido com o intuito de observar se as fibras de contração rápida do músculo digástrico apresentam alterações semelhantes às que ocorrem nos músculos do tronco e membros. Para tanto, amostras dos músculos digástrico e reto do abdome foram retiradas de cinco animais de cada grupo Controle (C), Alcoolizado (A) e Isocalórico (I). As amostras foram submetidas às reações de m-ATPase (com pré-incubações ácida e alcalina) e NADH-TR, para a classificação das fibras de acordo com os resultados obtidos nestas reações em FG (fast-twitch-glycolitic, FOG (fast-oxidative-glycolitic) e SO (slow-oxidative). Os resultados mostraram uma diferença significativa, na área das fibras dos tipos FG e FOG, entre os grupos estudados no músculo digástrico e na área das fibras do tipo FG no músculo reto do abdome; uma diferença sigificativa na freqüência das fibras dos tipos FG e FOG no músculo reto do abdome. Baseando-se nestes resultados, é possivel concluir que o etanol produz uma atrofia nas fibras da contração rápida dos músculos da mastigação, semelhante à dos músculos do tronco, mas com um coeficiente de atrofia diferente...