Elaine Minatel

Intrinsic laryngeal muscles are spared from myonecrosis in the mdx mouse model of Duchenne muscular dystrophy.

Intrinsic laryngeal muscles share many anatomical and physiological properties with extraocular muscles, which are unaffected in both Duchenne muscular dystrophy and mdx mice. We hypothesized that intrinsic laryngeal muscles are spared from myonecrosis in mdx mice and may serve as an additional tool to understand the mechanisms of muscle sparing in dystrophinopathy. Intrinsic laryngeal muscles and tibialis anterior (TA) muscle of adult and aged mdx and control C57Bl/10 mice were investigated. The percentage of central nucleated fibers, as a sign of muscle fibers that had undergone injury and regeneration, and myofiber labeling with Evans blue dye, as a marker of myofiber damage, were studied. Except for the cricothyroid muscle, none of the intrinsic laryngeal muscles from adult and old mdx mice showed signs of myofiber damage or Evans blue dye labeling, and all appeared to be normal. Central nucleation was readily visible in the TA of the same mdx mice. A significant increase in the percentage of central nucleated fibers was observed in adult cricothyroid muscle compared to the other intrinsic laryngeal muscles, which worsened with age. Thus, we have shown that the intrinsic laryngeal muscles are spared from the lack of dystrophin and may serve as a useful model to study the mechanisms of muscle sparing in dystrophinopathy. Muscle Nerve, 2006

Muscle regeneration in dystrophic mdx mice is enhanced by isosorbide dinitrate

Activation of muscle satellite cells, a fundamental step in the success of muscle regeneration is mediated by nitric oxide (NO). In this study, we investigated whether isosorbide dinitrate (ISD), an NO donor, could improve muscle regeneration in dystrophic mdx mice. The right tibialis anterior muscle of mdx and C57Bl/10 mice was injected with bupivacaine (0.3 ml, 33 mg/kg), a myotoxic agent, to induce muscle fiber regeneration. After bupivacaine injection, mice were treated with ISD (30 mg/kg; i.p.), verapamil (a non-NO donor vasodilator, 15 mg/kg, i.p.) or saline solution (vehicle, 0.3 ml, i.p.) for 20 days. Some bupivacaine-injected mice received no pharmacological treatment (control group). Muscle regeneration was evaluated by counting the total number of muscle fibers and measuring myofiber cross-sectional area. ISD significantly improved bupivacaine-induced muscle regeneration in mdx by increasing by 20% the total number of muscle fibers compared to the other groups. Spontaneous muscle regeneration, evaluated in the contralateral non-injected muscle, was not affected. ISD treatment did not affect myofiber cross-sectional area. Verapamil and saline had no effect on muscle regeneration. These results suggested that NO derived from ISD stimulated and/or recruited satellite cells. Pharmacological treatment with ISD could be clinically useful for improving muscle regeneration in Duchenne muscular dystrophy.

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.

Acetylcholine receptors and neuronal nitric oxide synthase distribution at the neuromuscular junction of regenerated muscle fibers

We investigated whether the changes in acetylcholine receptor (AChR) distribution and neuronal nitric oxide synthase (nNOS) expression reported for the skeletal muscle of mdx mice were a consequence of muscle fiber regeneration rather than of the absence of dystrophin. Degenerative‐regenerative changes in muscle fibers of the sternomastoid muscle of normal mice were induced by injecting lidocaine hydrochloride. Twenty‐one days later, AChRs were labeled with alpha‐bungarotoxin and nNOS with anti‐nNOS antibody, and observed under a confocal microscope. AChRs were distributed in continuous branches in normal fibers. Regenerated fibers showed disruption of AChRs distribution similar to that seen in muscle of mdx mice. This suggests that changes in AChRs distribution seen in mdx mice were probably a consequence of muscle fiber degeneration and regeneration, rather than a symptom of dystrophin deficiency. Conversely, there were no changes in nNOS distribution and expression in normal regenerated fibers, suggesting that the decrease in nNOS expression reported for mdx mice might be attributed to the absence of dystrophin.

Effects of fish oil containing eicosapentaenoic acid and docosahexaenoic acid on dystrophic mdx mice.

BACKGROUND & AIMS In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin leads to muscle degeneration and inflammation contributes to progression of the disease. In this study, we evaluated the effects of commercially available fish oil containing EPA and docosahexaenoic acid (DHA) on mdx. METHODS Mdx mice (14 days old) were treated with fish oil (FDC Vitamins; 0.002 g EPA and 0.001 g DHA) for 16 days by gavage. Control mdx mice received mineral oil (Nujol). Grip strength measurement was used for functional evaluation. The sternomastoid, diaphragm and biceps brachii muscles were removed and processed for histopathology and Western blot analysis. RESULTS Fish oil decreased creatine kinase and myonecrosis. In all muscles studied, the inflammatory area was significantly reduced after treatment (18.0 ± 3.0% inflammatory area in untreated mdx mice versus 4.0 ± 1% in treated mdx mice). Fish oil protected against the loss of muscle strength. Fish oil significantly reduced the levels of TNF-α and the levels of 4-HNE-protein adducts (30-34% reduction for both) in all muscles studied. CONCLUSIONS Commercially available fish oil may be potentially useful to ameliorate dystrophic progression of skeletal muscles, deserving further clinical trials in DMD patients.

Disodium cromoglycate protects dystrophin-deficient muscle fibers from leakiness.

In dystrophin‐deficient fibers of mdx mice and in Duchenne dystrophy, the lack of dystrophin leads to sarcolemma breakdown and muscle degeneration. We verified that cromolyn, a mast‐cell stabilizer agent, stabilized dystrophic muscle fibers using Evans blue dye as a marker of sarcolemma leakiness. Mdx mice (n = 8; 14 days of age) received daily intraperitoneal injections of cromolyn (50 mg/kg body weight) for 15 days. Untreated mdx mice (n = 8) were injected with saline. Cryostat cross‐sections of the sternomastoid, tibialis anterior, and diaphragm muscles were stained with hematoxylin and eosin. Cromolyn dramatically reduced Evans blue dye–positive fibers in all muscles (P < 0.05; Students t‐test) and led to a significant increase in the percentage of fibers with peripheral nuclei. This study supports the protective effects of cromolyn in dystrophic muscles and further indicates its action against muscle fiber leakiness in muscles that are differently affected by the lack of dystrophin. Muscle Nerve, 2007

Acetylcholine receptor distribution and synapse elimination at the developing neuromuscular junction of mdx mice

The pattern of innervation of the vertebrate neuromuscular junction is established during early development, when junctions go from multiple to single innervation in the phenomenon of synapse elimination, suggesting that changes at the molecular level in the postsynaptic cell lead to the removal of nerve terminals. The mdx mouse is deficient in dystrophin and associated proteins that are part of the postsynaptic cytoskeleton. We used rhodamine‐α‐bungarotoxin and anti‐neurofilament IgG–FITC to stain acetylcholine receptors and nerve terminals of the sternomastoid muscle during postnatal development in mdx and control C57BL/10 mice. Using fluorescence confocal microscopy, we observed that, 7 days after birth, 86.7% of the endplates of mdx mice were monoinnervated (n = 200) compared with 41.4% in control mice (n = 200). By the end of the second postnatal week, all endplates were innervated singly (100% mdx and 94.7% controls, n = 200 per group). These results show that dystrophic fibers achieve single innervation earlier, perhaps because dystrophin or a normal cytoskeletal complex is implicated in this phenomenon. Muscle Nerve 28: 561–569, 2003

Nerve terminal contributes to acetylcholine receptor organization at the dystrophic neuromuscular junction of mdx mice.

Changes in the distribution of acetylcholine receptors have been reported to occur at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration rather than the absence of dystrophin. In the present study, we examined whether the nerve terminal determines the fate of acetylcholine receptor distribution in the dystrophic muscle fibers of mdx mice. The left sternomastoid muscle of young (1‐month‐old) and adult (6‐month‐old) mdx mice was injected with 60 μl lidocaine hydrochloride to induce muscle degeneration‐regeneration. Some mice had their sternomastoid muscle denervated at the time of lidocaine injection. After 10 days of muscle denervation, nerve terminals and acetylcholine receptors were labeled with 4‐Di‐2‐ASP and rhodamine‐α‐bungarotoxin, respectively, for confocal microscopy. In young mdx mice, 75% (n = 137 endplates) of the receptors were distributed in islands. The same was observed in 100% (n = 114 endplates) of the adult junctions. In denervated‐regenerated fibers of young mice, the receptors were distributed as branches in 89% of the endplates (n = 90). In denervated‐regenerated fibers of adult mice, the receptors were distributed in islands in 100% of the endplates (n = 100). These findings show that nerve‐dependent mechanisms are also involved in the changes in receptor distribution in young dystrophic muscles. In older dystrophic muscles, other factors may play a role in receptor distribution. Anat Rec 290:181–187, 2007.

Axonal sprouting in mdx mice and its relevance to cell and gene mediated therapies for Duchenne muscular dystrophy.

We investigated whether pre-terminal axons and motor terminals retained their ability to sprout in the murine X-linked muscular dystrophy (mdx). Immunofluorescence confocal microscopy observation of nerve terminals and acetylcholine receptors in mdx muscles with crushed and non-crushed nerves showed that most of the junctions had intraterminal sprouting and that the number of junctions with extraterminal sprouting increased after the nerve crush lesion. Since new dystrophin-positive muscle fibers generated by cell-mediated therapies need to be innervated to proceed with their maturation and dystrophin production, these results suggest that the use of inducing factors to increase the sprouting capacity of nerve terminals could be an additional tool in the success of cell-mediated therapies.

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.