Skeletal muscle regeneration after crush injury in dystrophic mdx mice: an autoradiographic study.

The X-linked dystrophic animal model, the mdx mouse, shows an extraordinary capacity for sustained muscle regeneration compared with X-linked dystrophic golden retriever dogs and humans with Duchenne muscular dystrophy. To test the hypothesis that muscles of mdx mice might have inherently superior muscle regeneration to that in nondystrophic animals, muscle regeneration in response to a crush injury was examined in mdx mice and in the nondystrophic, parental strain C57Bl/10Sn. Autoradiographic techniques were used specifically to investigate the timing of muscle precursor replication after crush injury. Little difference in the regenerative capacity, either histologically or with respect to the timing of muscle precursor replication, was found between mdx mice and C57Bl/10Sn or other nondystrophic strains of mice.     

Analysis of dystrophin in fast- and slow-twitch skeletal muscles from mdx and dy2J mice at different ages

Muscles from mdx, control, and dy2J/dy2J mice at different ages were analyzed for dystrophin in an attempt to relate the chronology of the protein expression with the final phenotypes in regenerated, normal, and dystrophic muscle, respectively. Immunostaining and gold staining of electrophoresis gels were carried out in the investigation. At 5, 25, and 219 days of age, control muscles exhibited dystrophin bands in both the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles. Muscles from the mdx mice at comparable ages (8, 28, and 217 days) never exhibited bands for dystrophin, although titin, nebulin, myosin, and other protein bands were present at intensities comparable to those in control muscles. The dystrophin band was present in both the EDL and SOL from dy2J/dy2J dystrophic mice. As indicated by the present study, the dystrophin deficiency from mdx tissue is not transient. This suggests that dystrophin is not necessary for the success of mdx muscle regeneration.     

Absence of laminin α1 chain in the skeletal muscle of dystrophic dy/dy mice

In Duchenne muscular dystrophy (DMD) and laminin α2 defective congenital muscular dystrophies (CMD) there are reports of an induction of laminin α1 chain in regenerating muscle fibers. These studies are based on immunohistochemistry data with one monoclonal antibody alone. Based on these data we sought to establish if the laminin α1 chain is induced in the muscle of dy/dy mice. We found no evidence of induction of laminin α1 chain protein or mRNA in dystrophic dy/dy skeletal muscle fibers as determined by immunohistochemistry, Western blotting, Northern blotting, or PCR analysis. Our data point to the need for additional immunological reagents specific for human laminin-α1 to resolve whether the conflicting data on laminin-α1 distribution in human and mouse tissues is due to species differences or, alternatively, due to differences in reagent specificity. Our data might be important when designing therapy strategies for CMD. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1515–1524, 1997     

Muscle regeneration and mitochondrial calmitine increase in the dystrophic dy/dy mouse after intramuscular chlorpromazine injection

We studied the effect of chlorpromazine injection on the gastrocnemius muscles of C57BL/6J dy/dy dystrophic mice. Changes in mitochondrial calmitine concentrations and differences in microscopy studies, fibre typing and morphometry were compared in gastrocnemius muscles of dystrophic and control mice before and 2 and 21 days after injection. In both cases, calmitine reduction associated with muscle degeneration was observed 2 days after drug injection. Calmitine then increased, reaching a level at day 21 nearly identical to that of controls before injection. This increase was associated with muscle regeneration. These results clearly indicate that dystrophic mouse muscle can regenerate calmitine after drug-induced damage.     

A comparative freeze‐fracture study of plasma membrane of dystrophic skeletal muscles in dy/dy mice with merosin (laminin 2) deficiency and mdx mice with dystrophin deficiency

The intramembranous particle (IMP), orthogonal array (OA) and orthogonal array subunit particle (OASP) densities of skeletal muscle plasma membranes of merosin deficient dy/dy mice and their control mice at 7, 14 and 28 days after birth were analysed by freeze‐fracture electron microscopy. Similar studies were performed on dystrophin‐deficient mdx mice with mild muscle weakness at 28 days after birth for the comparison with those of dy/dy mice with severe muscle weakness at the same age. In the pre‐clinical stage of dy/dy mice at 14 days after birth, the membranes showed a significantly decreased density of OAs ( P <0.01 by Wilcoxon rank‐sum test) as compared with control mice, while those in the clinical stage of dy/dy mice at 28 days after birth showed normal IMP density but a marked depletion of OA density ( P <0.01). Moreover, at 28 days after birth, the reduction of OAs in the plasma membranes of dy/dy mice was more marked than that of mdx mice ( P <0.05 by Wilcoxon rank‐sum test). These results provided us with the information that the OA density was affected more severely with merosin deficiency than with dystrophin deficiency, and again supported our previously proposed concept that the clinical severity in muscular dystrophies correlated with the OA density.     

Gamma irradiation can reduce muscle damage in mdx dystrophic mice

We report the effects of a single gamma irradiation delivered to the soleus muscle of one limb of normal and mdx mice at the age of 16–20 days. At 45, 75 and 90 days of age transverse cryostat sections from the mid-belly of the muscles were used for microscopic examination. In normal mice the growth of fibres was appreciably reduced by irradiation without fibre loss. In the irradiated soleus of mdx mice the number of the regenerated centrally nucleated fibres was very small and the total number of fibres was remarkably reduced. The number of the peripherally nucleated fibres, presumably surviving since the birth of the animal, was almost consistently larger than in the contralateral non-irradiated limb. The cross-sectional area of the irradiated fibres was smaller. It is well known that proliferation and fusion of satellite cells are required both for regeneration after fibre damage and for the normal postnatal growth of muscle fibres: irradiation appears to reduce regeneration and growth. It is suggested that irradiation reduces damage by reducing fusion associated with growth. Our hypothesis indirectly indicates a significant link between dystrophin deficiency and fibre necrosis and accounts well for many features of mouse dystrophy under natural and experimental conditions. Received: 2 October 1997 / Revised: 26 January 1998, 23 April 1998 / Accepted: 2 June 1998     

Age-related differences in regeneration of dystrophic (mdx) and normal muscle in the mouse

The mdx mouse, a genetic homologue of human Duchenne muscular dystrophy (DMD), has been attributed with a greater regenerative capacity of its skeletal muscles. Here, we have tested the hypothesis that muscles of mdx mice regenerate better than those of nondystrophic animals. We studied muscle regeneration resulting from a denervation-devascularization injury (DD) of extensor digitorum longus muscle (EDL) at 3 weeks and 2 months in mdx and wild-type (C57BL/10) mice. Histological and morphometrical studies of muscle regeneration were made from 3 to 180 days later. When DD was performed in 3-week-old C57BL/10 mice, the percentages of nonperipheral nuclei in regenerated fibers decreased progressively over 3 months. This decrease did not occur in animals where DDs were performed at 2months, suggesting that two different populations of muscle precursor cells are mobilized in muscle regenration in mice at these two ages. Moreover, mdx EDL muscle regenerated similarly to the controls for up to 60 postoperative days, as shown by distribution of mean diameters and percentage of nonperipheral nuclei of muscle fibers. After 60 postoperative days, necrosis/regeneration characteristics of mdx muscles recurred, suggesting that mdx-regenerated muscle fibers remain susceptible to degeneration.© 1995 John Wiley & Sons, Inc.     

Blockade of TNF in vivo using cV1q antibody reduces contractile dysfunction of skeletal muscle in response to eccentric exercise in dystrophic mdx and normal mice

This study evaluated the contribution of the pro-inflammatory cytokine, tumour necrosis factor (TNF) to the severity of exercise-induced muscle damage and subsequent myofibre necrosis in mdx mice. Adult mdx and non-dystrophic C57 mice were treated with the mouse-specific TNF antibody cV1q before undergoing a damaging eccentric contraction protocol performed in vivo on a custom built mouse dynamometer. Muscle damage was quantified by (i) contractile dysfunction (initial torque deficit) immediately after the protocol, (ii) subsequent myofibre necrosis 48 h later. Blockade of TNF using cV1q significantly reduced contractile dysfunction in mdx and C57 mice compared with mice injected with the negative control antibody (cVaM) and un-treated mice. Furthermore, cV1q treatment significantly reduced myofibre necrosis in mdx mice. This in vivo evidence that cV1q reduces the TNF-mediated adverse response to exercise-induced muscle damage supports the use of targeted anti-TNF treatments to reduce the severity of the functional deficit and dystropathology in DMD.     

Mosaic expression of dystrophin in the cerebellum of heterozygote dystrophic (mdx) mice.

The monoclonal NCLDyauthor revealed the presence of dystrophin in the Purkinje cells of normal mice but not of mdx mice and a mosaic staining in Purkinje cells of heterozygote mdx mice. Dystrophin was present in the soma and the dendrites of the dystrophin positive Purkinje cells and was absent in both regions of the dystrophin negative Purkinje cells. However, the polyclonal antibody d10 produced a uniform labeling of all Purkinje cells not only in the normal mice but also in mdx and heterozygote mdx mice. This staining was attributed to a reaction of this antibody not only with dystrophin but also with a different isoform of dystrophin or with a dystrophin related protein present even in mdx mice.     

Terminal Schwann cell structure is altered in diaphragm of mdx mice

The diaphragm muscle of the mdx mouse is a model system of Duchenne muscular dystrophy, since it completely lacks dystrophin and shows severe fiber necrosis and loss of specific muscle force by 4-6 weeks of age. Changes in neuromuscular junction structure also become apparent around 4 weeks including postsynaptic acetylcholine receptor declustering, loss of postsynaptic junctional folds, abnormally complex presynaptic nerve terminals, and muscle fiber denervation. Normally, terminal Schwann cells (TSCs) cap both nerve terminals and acetylcholine receptors at the neuromuscular junction, and play a crucial role in regeneration of motor axons following muscle denervation by guiding axons to grow from innervated junctions to nearby denervated junctions. However, their role in restoring innervation in dystrophic muscle is unknown. We now show that TSCs fail to cap fully the neuromuscular junction in dystrophic muscle; TSCs extend processes, but the organization of these extensions is abnormal. TSC processes of dystrophic muscle do not form bridges from denervated fibers to nearby innervated endplates, but appear to be directed away from these endplates. Adequate signaling for TSC reactivity is present, since significant muscle fiber denervation and acetylcholine receptor declustering are present. Thus, significant structural denervation is present in the diaphragm of mdx mice and the ability of TSCs to form bridges between adjacent endplates to guide reinnervation of muscle fibers is impaired, possibly attenuating the ability of dystrophic muscle to recover from denervation and ultimately leading to muscle weakness.     

Chronic exercise accelerates the degeneration-regeneration cycle and downregulates insulin-like growth factor-1 in muscle of mdx mice

The aim of this study was to examine the effects of chronic running exercise on degenerative-regenerative processes in the hindlimb muscles of dystrophin-deficient mdx mice. The number of large-sized degenerative-regenerative groups (DRGs) was markedly decreased, whereas that of small-sized DRGs was unchanged by exercise. Expression of insulin-like growth factor-1 (Igf1), as well as a myogenic factor MyoD (Myod1), was downregulated in mdx muscles by exercise. The downregulation of Igf1 may well correlate with the decrease in the population of early regenerating fibers, which existed predominantly in DRGs, because IGF-1 was mainly localized in these fibers. Our data indicate that chronic exercise may accelerate the active cycle of degeneration-regeneration in mdx skeletal muscles. This means that mdx skeletal muscles can temporarily cope with work-induced injury by enhancing muscle regeneration and repair, but we speculate that an early decline of IGF-1 will accelerate age-dependent muscle wasting and weakness in the later stage of life in mdx mice.     

Freeze-fracture studies of myofiber plasma membrane in X chromosome-linked muscular dystrophy (mdx) mice

Summary The structure of the muscle plasma membrane of extensor digitorum longus muscles of X chromosome-linked muscular dystrophy (mdx) mice was studied by freeze-fracture technique at several time points after birth. The common denominator of the abnormalities was the decreased density of orthogonal arrays throughout all the time points examined. The results demonstrated that the ultrastructural features of the muscle plasma membrane alterations in mdx mice were similar to those in Duchenne dystrophy.Supported by grant (62A-2-20) from National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

Force and stiffness of old dystrophic (mdx) mouse skeletal muscles

It has recently been suggested, based on studies of tissue pathology, that the limb muscles of old mdx mice may be a good model for the muscular changes seen in human Duchenne muscular dystrophy. To test this hypothesis, we measured force and stiffness of soleus and extensor digitorum longus (EDL) muscles of old (20–21 months) mdx mice and age-matched controls. The mdx and control muscles generated similar twitch, tetanic, and eccentric forces. They were also equally stiff. The results show that the mechanics of aged mdx limb muscles differ greatly from Duchenne muscular dystrophy in humans, and disagree with the hypothesis. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:536–539, 1998.     

Genotyping mdx, mdx3cv, and mdx4cv mice by primer competition polymerase chain reaction

mdx, mdx3cv, and mdx4cv mice are among the most commonly used models for the study of Duchenne muscular dystrophy. Their disease is caused by point mutations in the dystrophin gene. Despite widespread use of these models, genotyping has not always been straightforward. Current methods require multiple polymerase chain reactions (PCRs), post-PCR manipulations, and/or special equipment/reagents. Herein we report a simple, robust PCR genotyping method based on primer competition. This approach could also be applied in genotyping other point-mutation models.     

The effects of altered metabolism (hypothyroidism) on muscle repair in the mdx dystrophic mouse

After dystrophic damage, the limb muscles of the mdx mouse recover very effectively compared to muscles in Duchenne muscular dystrophy (DMD) patients. Since thyroid hormone is required for muscle development and integrity, we examined whether a deficiency of the hormone, induced by 0.05% propylthiouracil (PTU) in drinking water over 8 weeks, would be deleterious to the myogenesis and muscle repair in control and mdx mice. Measured metabolic and growth parameters confirmed hypothyroidism in PTU-treated mice. Histological and morphometric techniques were used to study myogenesis and the repair of the tibialis anterior muscle (TA) after crush injury in mdx mice and their nondystrophic controls (C57B1/10ScSn). After 8 weeks, PTU-treated TA from mdx mice had larger crush sites and lower myotube density than TA in untreated mdx mice. In unoperated mdx TA, there was a larger proportionate area of active dystrophy and smaller fiber diameter in PTU-treated than in untreated mdx TA, which suggested that PTU increased the activity of dystrophy as well. In contrast, in control TA neither the regeneration of myotubes or fiber diameter were affected significantly by PTU. Therefore, these results suggest that mdx muscle regeneration is more affected by hypothyroidism than normal muscle repair. This may be due to the larger pool of muscle precursors in mdx than control muscle, and a possible impairment of precursor cell proliferation or fusion during myotube formation. © 1994 John Wiley & Sons, Inc.     

Spontaneous mechanical activity and evoked responses in isolated gastric preparations from normal and dystrophic (mdx) mice

This study examined whether alterations of the spontaneous and evoked mechanical activity are present in the stomach of the mdx mouse, the animal model for Duchenne muscular dystrophy. The gastric mechanical activity from whole-organ of normal and mdx mice was recorded in vitro as changes of intraluminal pressure. All gastric preparations developed spontaneous tone and phasic contractions, although the tone of the mdx preparations was significantly greater. Atropine reduced the tone of the two preparations by the same degree. Nomega-nitro-l-arginine methyl ester (l-NAME) significantly increased the tone and spontaneous contractions only in the stomach from normal animals, but did not affect on the mdx preparations. Effects ofl-NAME on tone and contractility were preserved in the presence of tetrodotoxin. In both types of tissues electrical field stimulation (EFS) induced a biphasic response: cholinergic contraction followed by slow relaxation. In nonadrenergic noncholinergic conditions, EFS induced a rapid relaxation followed by a slow component in both types of tissues. l-NAME abolished the rapid component, reduced the slow component and unmasked tachychinergic contractions. No significant difference was found in evoked responses. The enteric neurotransmission is preserved in mdx gastric preparations, although alterations in the ongoing production of nitric oxide are present.