Luis Henrique Rapucci Moraes

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.

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.

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

Abstract 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.

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.

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.

Histological assessment of mdx mice treated with antioxidant supplementation

Resumen pt: Os Autores utilizaram procedimentos histopatologicos para revelar o efeito da suplementacao com os antioxidantes nacetilcisteina e deferoxamina em fibras...