Muscle regeneration

Summary Against the background of the importance of growth hormone (GH) for normal muscle growth, a study was performed to investigate whether lack of GH after hypophysectomy affects the cell proliferation and the local production of insulin-like growth factor-I (IGF-I) in the early stages of muscle regeneration in adult rats. The level of IGF-I in the serum of hypophysectomised rats was reduced to about 30% of that of controls. The incorporation of [methyl-³H]thymidine into the regenerating muscle showed a peak 6 days after the operation and then gradually declined to the end of the period of study 30 days after initiation of regeneration by ischemic necrosis. The DNA content rose to a maximum level after 6–8 days, and remained high after 30 days. There was no major difference in the incorporation of [³H]thymidine in regenerating muscle of hypophysectomised and control rats, but the DNA concentration in the regenerating muscles of hypophysectomised rats was significantly reduced after 30 days. There was a corresponding reduction in the number of nuclei per muscle fibre, indicating that hypophysectomy has a small effect on the cell proliferation during the early stages of muscle regeneration. Immunohistochemical demonstration of IGF-I in the regenerating muscle revealed the transient presence of immunoreactive material in satellite cells and myotubes after 6 to 8 days of regeneration but no immunoreactivity after 30 days. No obvious difference was observed between hypophysectomised and control rats, indicating that the endogenous production of IGF-I in regenerating skeletal muscle can occur independently of GH.Supported by the Swedish Medical Research Council (Project no. 7122 and 7120)     

Role of the basement membrane in the regeneration of skeletal muscle

In many experimental models of skeletal muscle damage and in human muscle disease, empty basement membrane tubes remain following the destruction of muscle fibres. In the present study we test the hypothesis that the empty basement membrane tubes play an essential role in the orientation of regenerating muscle fibres. Two groups of 15 Wistar rats were used. In one group, aqueous barium chloride (BaCl2) solution was injected into the right quadriceps muscle; in the other group, freshly prepared 2% trypsin solution was similarly injected. The different stages of muscle cell necrosis and regeneration were observed by histology, by immunofluorescence using an anti-basement membrane antibody, and by transmission (TEM) and scanning electron microscopy (SEM) in animals killed 1-77 days following injection. Although there was muscle fibre necrosis at sites of BaCl2 injection, empty basement membrane tubes were well preserved. Myoblasts grew along the empty basement membrane tubes and by 77 days, the regenerated muscle fibres at the site of the injection were well oriented. Trypsin not only destroyed muscle fibres but also destroyed the basement membrane tubes; in the early stages of regeneration the myoblasts were disorientated but by 77 days, regeneration was comparable to that seen in the barium chloride injected muscle. The results of this study suggest that preservation of empty basement membrane tubes is not essential for the orientation of regenerating myoblasts in skeletal muscle.     

Changes in muscle carnitine during regeneration

Carnitine in rat skeletal muscle was measured before, during, and after muscle regeneration. Early regenerating tibialis anterior muscle consequent to bupivacaine injection was found to have smaller amounts of total, free, and acyl carnitine per milligram wet weight, but returned toward normal values as muscle regeneration was completed. Accumulation of DL-[3H]carnitine per milligram wet weight in regenerating muscle was less than one-half that of control muscle at day 4 (P less than 0.005), but was not significantly different from the control value at day 7. Our results are consistent with the hypothesis that the reported decrease in muscle carnitine content in several different human neuromuscular diseases is in part a manifestation of muscle fiber regeneration.     

Experimental regeneration in canine muscular dystrophy—1. Immunocytochemical evaluation of dystrophin and β-spectrin expression

The expression of dystrophin and β-spectrin was examined from 1 to 56 days in regenerating muscle fibres in normal and dystrophic dogs, following necrosis induced by the venom of Notechis scutatis. Normal and dystrophic dog muscle regenerated at an equal rate and new myotubes were present in both at the periphery of necrotic fibres by 3 days. In normal dogs dystrophin was detected in the sarcoplasm of the regenerating fibres by 3 days and was localized to the plasma membrane by 4 days. The localization of dystrophin is independent of β-spectrin and was detected before β-spectrin, which was not observed until 5–6 days. Normal peripheral labelling of both was restored by 14 days in normal dogs. Normal β-spectrin labelling of regenerating dystrophic fibres was also restored by 14 days and is not dependent on the presence of dystrophin in dystrophic dogs. A proportion of regenerating fibres in normal and dystrophic dogs showed weak immunolabelling of β-spectrin prior to 14 days. This is a feature of immature muscle fibres. Antibodies to different domains of dystrophin bound to the periphery and sarcoplasm of regenerating fibres in dystrophic dogs, particularly during the first 7 days of regeneration, but the fluoresence was less intense than in normal dogs. Weak labelling with antibodies corresponding to the C-terminus of the rod domain of dystrophin persisted on dystrophic regenerating fibres up to 21 days. This may relate to developmental isoforms of dystrophin.     

The dystrophin-related protein, utrophin, is expressed on the sarcolemma of regenerating human skeletal muscle fibres in dystrophies and inflammatory myopathies.

Utrophin is the 400 kDa protein product of an autosomal homologue (DMDL) of the dystrophin gene. In normal skeletal muscle, utrophin is expressed in vascular smooth muscle, endothelium and nerves but not in mature muscle fibres except at the neuromuscular junction. We have examined the expression of utrophin in a wide range of human skeletal muscle diseases using monoclonal antibodies against three C-terminal epitopes. Utrophin is consistently expressed in all basophilic, regenerating fibres irrespective of the underlying disease or expression of dystrophin. It is also found in regenerating fibres from a normal volunteer. In Duchenne and Becker dystrophies, as well as in dermatomyositis, sarcolemmal staining for utrophin is also seen in larger fibres which are not obviously regenerating. These studies do not support the idea that utrophin occupies membrane attachment sites only when dystrophin is absent or reduced, but would be consistent with utrophin expression as part of an activated foetal programme during regeneration.     

Expression of utrophin (dystrophin–related protein) during regeneration and maturation of skeletal muscle in canine X–linked muscular dystrophy

The regulation of utrophin, the autosomal homologue of dystrophin, has been studied in the canine X–linked model of Duchenne muscular dystrophy. Dystrophic muscle has been shown to exhibit abnormal sarcolemmal expression of utrophin, in addition to the normal expression at the neuromuscular junction, in peripheral nerves, vascular tissues and regenerating fibres. To establish whether this abnormal presence of utrophin in dystrophic muscle is a consequence of continued expression following regeneration, or is attributable to a disease related up–regulation, the expression of utrophin was compared immunocytochemically with that of dystrophin, β–spectrin and neonatal myosin in regenerating normal and dystrophic canine muscle, following necrosis induced by the injection of venom from the snake Notechis scutatis. In normal regenerating muscle, sarcolemmal utrophin and dystrophin were detected concomitantly from 2–3 d post–injection, prior to the expression of β–spectrin. Down–regulation of utrophin was apparent in some fibres from 7 d, and it was no longer present on the extra–junctional sarcolemma by 14 d. Neonatal myosin was still present in all fibres at this stage, but dystrophin and β–spectrin had been fully restored. In dystrophic regenerating muscle, downregulation of utrophin occurred from 7 d, although it persisted on some fibres until 28 d, longer than in normal muscle. At 42 d, however, utrophin in dystrophic muscle was only detected in a population of small fibres thought to represent a second cycle of regeneration, with no immunolabelling of mature fibres. The results show that most utrophin is down–regulated in regenerating dystrophic fibres, prior to neonatal myosin, thus abnormal sarcolemmal expression of utrophin in dystrophic muscle is unlikely to be a continuation of the maturational process. Persistence of both utrophin and neonatal myosin, however, suggest a delay in the maturation of dystrophic muscle. In addition, a second cycle of degeneration and regeneration in dystrophic muscle does not occur whilst utrophin is still present, suggesting it may have a protective role against fibre damage and necrosis.     

The fate of desmin and titin during the degeneration and regeneration of the soleus muscle of the rat

Summary We studied the fate of desmin and titin in rat skeletal muscle during a cycle of degeneration and regeneration induced in vivo by the inoculation of a snake venom. Cryosections of muscle were labelled using antibodies to the two proteins, and examined at fixed time points after venom injection. Early pathological changes in the muscle, such as hypercontraction, preceded the loss of desmin. Immunolabelling using anti-desmin antibodies showed that desmin bridges were still intact when adjacent myofibrils were no longer aligned. The results suggested that although the hydrolysis of desmin is not necessary for the hypercontraction of muscle fibres, it probably contributes to complete fibre breakdown. Titin, or at least the part which lies close to the M-line, remained intact longer than desmin, but was also hydrolysed prior to complete disintegration of the fibres. Both desmin and titin were re-expressed in the regenerating myotubes by 2 days after venom inoculation, and became well organised even before the myofibrils became aligned. We conclude that desmin and titin are involved in both establishing and maintaining the structural integrity of the muscle fibres.Supported by the Muscular Dystrophy Group of Great Britain, the Wellcome Trust and the MRC     

Partial correction of an inherited biochemical defect of skeletal muscle by grafts of normal muscle precursor cells

We have attempted to use allografts of normal muscle precursor cells (mpc) to insert donor nuclei, containing a normal genome, into growing or regenerating skeletal muscle fibres of mice with an inherited deficiency of the enzyme phosphorylase kinase (PhK). Analysis of the glucose-6-phosphate isomerase (GPI) isoenzymes of treated muscles showed that myonuclei of donor origin became incorporated into host muscle fibres in 8 of 9 regenerating autografts, but PhK activity was found only in the 3 grafts into which the largest numbers (1-3 x 10(6)) of mpc had been implanted. Following injection of normal mpc into growing PhK-deficient skeletal muscle, mosaic fibres containing myonuclei of donor origin were detected in only 11 of 192 muscles examined from 64 mice, but, of these 11 muscles, 5 contained PhK activity detectable by two separate assays in a further 4 muscles activity was detected by one or other assay.     

Evidence that 4S RNA is axonally transported in normal and regenerating rat sciatic nerves

Studies in regenerating goldfish optic nerves indicate that RNA may be axonally transported during optic nerve regeneration^{14, 18, 19}. The present study was performed to determine if the axonal migration of RNA could be demonstrated during regeneration of the rat sciatic nerve.Rats, which had only the left sciatic nerve crushed 10 days earlier, were injected bilaterally with [³H]uridine into the spinal cord at segmental levels L₅ and L₆, thus labeling ventral horn cells giving rise to the sciatic nerve. Six, 14 and 20 days later rats were sacrificed by cardiac perfusion of saline followed by 10% formaldehyde. Formaldehyde-precipitable radioactivity, identified as [³H]RNA, was 4–5 times greater in the regenerating sciatic nerve compared to the normal nerve and moved without impediment beyond the point of the crush into the regenerating portion of the nerve.The axonal migration of free unincorporated labeled RNA precursors was also demonstrated, raising the possibility that the distribution of [³H]RNA along the sciatic nerve might be entirely extra-axonal; i.e., free [³H]uridine is taken up by Schwann cells from the axon where it is incorporated into [³H]RNA. This interpretation of the data would also result in the appearance of a proximodistal distribution of RNA associated radioactivity. To determine whether any sciatic nerve [³H]RNA was due to axonal transport, rats which had only the left sciatic nerve crushed 10 days earlier were injected bilaterally with [³H]uridine into the spinal cord. Fourteen days after injection, rats were sacrificed and radioactivity present in the nerve was confirmed as RNA by SDS polyacrylamide gel electrophoresis. Radioactivity in the various RNA species 14 days after intraspinal injection showed the following distribution: 28 + 18S RNA — normal39.3%±2.1; regenerating45.4%±1.6; 4S RNA — normal43.0%±1.3; regenerating46.8%±2.7. Similar characterization of sciatic nerve RNA 1 or 3 days following the intravenous administration of [³H]uridine gave the following distribution: 28 + 18S RNA — normal72.4%±3.0; regenerating75.0%±3.6; 4S RNA — normal7.7%±1.3; regenerating10.7%±0.8.The intraspinal injection of [³H]uridine would label Schwann cell RNA and, in addition, any species of intra-axonal RNA, while intravenous injections would label Schwann cell RNA and not axonal RNA. If 4S RNA is in the axon, one would predict relatively more labeled 4S RNA following intraspinal injections than following intravenous injections. The data demonstrate an enrichment of 4S RNA in both normal and regenerating rat sciatic nerve following the intraspinal but not following the intravenous injection of labeled precursor. Therefore, we suggest that 4S RNA migrates axonally in both normal and regenerating sciatic nerves of rats.     

Muscle transplantation and regeneration in the dystrophic hamster. Part 1. Histological studies

Minced tibialis anterior muscles have been transplanted between normal and dystrophic hamsters and examined histologically after periods ranging from a few hours to 376 days. Transplants which regenerated successfully established normal neuromuscular contact and tendon connections. The net weight of the transplant varied from 25–35% of that of the intact muscle.Degeneration and regeneration began first at the periphery and gradually progressed toward the centre of the transplant. The beginning of regeneration was marked by the appearance of basophilic cells under the basement membranes of the implanted muscle fibres. This was followed by the proliferation of myogenic cells and the formation of bi-, tri- and multi-nucleated myotubes. Cross-striations were first observed in regenerates examined at 10 days after transplantation. Most of the original implanted muscle fibres degenerated by 15 days and the regenerates consisted of myotubes, young muscle fibres, connective tissue, mononuclear cells and polymorphonuclear leucocytes.The mature regenerated fibres often showed splitting and contained central nuclei. All transplants contained randomly-orientated fibres. In auto- and heterotransplants of dystrophic muscle, fibres with a round and opaque appearance were observed. Otherwise there was no apparent difference in histology between normal and dystrophic regenerates.     

Calcineurin is a potent regulator for skeletal muscle regeneration by association with NFATc1 and GATA-2

The molecular signaling pathways involved in regeneration after muscle damage have not been identified. In the present study, we tested the hypothesis that calcineurin, a calcium-regulated phosphatase recently implicated in the signaling of fiber-type conversion and muscle hypertrophy, is required to induce skeletal muscle remodeling. The amount of calcineurin and dephosphorylated nuclear factor of activated T cells c1 (NFATc1) proteins was markedly increased in the regenerating muscle of rats. The amount of calcineurin co-precipitating with NFATc1 and GATA-2, and NFATc1 co-precipitating with GATA-2 gradually increased in the tibialis anterior muscle after bupivacaine injection. Calcineurin protein was present in the proliferating satellite cells labeled with BrdU in the damaged muscle after 4 days. In contrast, calcineurin was not detected in the quiescent nonactivating satellite cells expressing Myf-5. At 4 days post injection, many macrophages detected in the damaged and regenerating area did not possess calcineurin protein. Calcineurin protein was abundant in many myoblasts and myotubes that expressed MyoD and myogenin at 4 and 6 days post injection. In the intact muscle, no immunoreactivity of calcineurin or BrdU was detected in the cell membrane, cytosol or the extracellular connective tissue. In mice, intraperitoneal injection of cyclosporin A, a potent inhibitor of calcineurin, induced extensive inflammation, marked fiber atrophy, the appearance of immature myotubes, and calcification in the regenerating muscle compared with phosphate-buffered saline-administered mice. Thus, calcineurin may have an important role in muscle regeneration in association with NFATc1 and GATA-2.     

The regeneration of a hormone-sensitive muscle (levator ani) in the rat

In 1-month-old male rats the levator ani muscle was crushed and allowed to regenerate. The main questions were whether or not the course of regeneration is similar to that of limb muscles and whether or not the regenerating levator ani muscle is sensitive to the effects of testosterone. Regeneration was followed in three groups of rats: castrated, normal, and testosterone-treated. Histological analysis revealed no detectable differences between regeneration of the levator ani and other muscles. By 31 days both the gross weight and mean cross-sectional area of the muscle fibers in castrated rats were much less than in the other two groups. Testosterone-treated muscles were larger, but not significantly so, than normal regenerating muscles. Twitch and tetanic tensions were lowest in regenerating muscles from castrated rats and highest in those from hormone-treated rats. Both the contraction (time to peak) and half-relaxation times at 7 and 14 days were fastest in hormone-treated muscles and were very slow in castrated rats. By 30 days, the differences between normal and hormone-treated muscles had disappeared. It is concluded that the regenerating levator ani muscle is sensitive to the effects of testosterone.     

Influence of non-neuronal cells on regeneration of the rat sciatic nerve

The ability of the rat sciatic nerve to regenerate into a previously frozen distal nerve segment was studied and compared to regeneration after a crush lesion. The regeneration rate in the frozen segment was 1.9 mm/day, which was approximately half of that observed after a crush lesion (3.3 mm/day). If an unfrozen nerve segment was left intact beyond the frozen section, the rate of regeneration increased to 3.2 mm/day. However, a fresh nerve segment sutured along the frozen segment did not significantly affect the rate of regeneration. Incorporation of [3H]thymidine in the regenerating nerve, analyzed after 1, 3 and 6 days, showed an increased labelling in the frozen segment. This increase spread from the proximal nerve segment into the frozen section. In nerves where a segment was left intact beyond the frozen section, [3H]thymidine incorporation was seen to enter the frozen section from both sides. The spreading of [3H]thymidine incorporation appeared to correlate with the rate of regeneration. However, the same pattern of incorporation could be observed in nerves where regeneration was detained by a transection. The results suggest that Schwann and/or other cells which invade the frozen nerve segment affect the rate of axonal elongation, and that the migration of these cells occurs independently of regenerating fibers.     

Localization of coxsackie virus and adenovirus receptor (CAR) in normal and regenerating human muscle

The primary receptor for Adenovirus and Coxsackie virus (CAR) serves as main port of entry of the adenovirus vector mediating gene transfer into skeletal muscle. Information about CAR expression in normal and diseased human skeletal muscle is lacking. C'- or N'-terminally directed polyclonal antibodies against CAR were generated and immunohistochemical analysis of CAR on morphologically normal and regenerating human skeletal muscle of children and adults was performed. In morphologically normal human muscle fibers, CAR immunoreactivity was limited to the neuromuscular junction. In regenerating muscle fibers, CAR was abundantly co-expressed with markers of regeneration. The function of CAR at the neuromuscular junction is currently unknown. Co-expression of CAR with markers of regeneration suggests that CAR is developmentally regulated, and may serve as a marker of skeletal muscle fiber regeneration.     

The response of optic tract glia during regeneration of the goldfish visual system. I. Biosynthetic activity within different glial populations after transection of retinal ganglion cell axons

We monitored biosynthetic activity of optic tract glia during regeneration of retinal ganglion cell axons in the goldfish and found that the greatest level of incorporated [3H]thymidine and [3H]leucine occurred in glia by 10-15 days after axotomy. During this period there was a marked increase in the number of oligodendroglia and multipotential glia near the site of injury with no change occurring in the astroglial population. Electron microscopic autoradiography showed that oligodendroglia and multipotential cells incorporated 5-7-fold more thymidine than did cells of intact control preparations. Though all glial cell types incorporated more [3H]leucine during axonal regeneration, oligodendroglia and multipotential cells together accounted for more than 90% of measured radioactivity. In order to characterize glial-stimulating events specific to axonal regeneration, we produced axonal degeneration in the optic tract by removal of the retina. Optic tract glia during axonal degeneration incorporated less amino acid when compared to glia associated with regenerating axons. The degenerating optic tract also had less 2',3'-cyclic nucleotide 3'-phosphohydrolase, an enzyme produced by oligodendroglia, than that found in the regenerating visual system. Our results suggest that in response to ganglion cell axotomy oligodendroglia and multipotential glia of the goldfish optic tract proliferate. Moreover, regenerating axons provide one type of stimulant for glial protein biosynthesis.     

Bcl-2 and bax immunohistochemistry in denervation-reinnervation and necrosis-regeneration of rat skeletal muscles

Bcl-2 and Bax immunohistochemistry was examined in the skeletal muscle of rats after cutting the sciatic nerve, as a model of denervation and reinnervation, and in the anterior tibialis muscle of rats after an intramuscular injection of metoclopramide, as a model of necrosis and regeneration of muscle fibers, to better understand the role of these proteins in muscle disorders. An increase in Bax immunoreactivity was seen in long-standing denervated and reinnervated muscle fibers. Bcl-2 and Bax immunoreactivity was limited to macrophages in necrotic muscle fibers at 24 h after the intramuscular injection of metoclopramide. However, increased Bax immunoreactivity was observed in regenerating muscle fibers by the fourth day after the injection. Muscle fiber nuclei with the morphological features of apoptosis were not observed in rat muscles after the intramuscular injection of metoclopramide or after the severing of the sciatic nerve. Furthermore, using the Tunel method, no stained nuclei were observed in the two groups of animals. Our observations in the experimental models of skeletal muscle denervation-reinnervation and necrosis-regeneration here described suggest that modification in the intensity of the Bax protein is not related to the process of cell death but rather that increased Bax expression is associated with muscle fiber regeneration.     

Axonal transport of glycerophospholipids in regenerating sciatic nerve of the rat during aging

The effect of age upon the axoplasmic transport of glycerophospholipids has been studied using as a model the regenerating sciatic nerve of young (2-month-old), young adult (6-month-old), middle-aged (16-month-old), and aged (20-month-old) male rats. The right sciatic nerve was crushed 0.5 mm down the incisura ischiadica. Four and nine days after the lesion, a mixture of [2-3H] glycerol and [methyl-14C] choline was bilaterally injected into the spinal cord, at a level of the L4-L5 vertebrae. The animals were killed 18 hr after the isotope injection. Proximal and distal portions of crushed nerve and of contralateral sham-operated ones were dissected and consecutive 5-mm segments were subjected to lipid extraction and analysis. The findings of the present study are summarized as follows: (1) The accumulation of labeled lipid material axonally transported four days after nerve injury was mainly located at the crush site in young, young adult, middle-aged, and aged rats. The accumulation of both 3H-glycerolipids and 14C-choline phospholipids in postcrush segments was markedly higher for young and young adult than for aged rats, four and nine days after crush; (2) the average rate of axonal regeneration, determined between days 4 and 9 following crush injury was 3.6 and 4.2 mm/day for 2-month-old and 6-month-old rats, respectively; it decreased to the value of 2.5 mm/day for 16-20-month-old rats.     

MUSCLE FIBRE SPLITTING AND REGENERATION IN DISEASED HUMAN MUSCLE

In soleus muscle of rat, necrosis of muscle fibres within a grossly intact endomysium was produced by 60–70°C Ringer's solution injected into the space around the muscle. After 2 weeks regenerated fibres appeared to be fragments of 'split' fibres. In split fibres of patients with myopathy all stages of myogenesis were seen in electron micrographs. Hence splitting of muscle fibres is thought to indicate regeneration rather than degeneration. Based on the morphology of the clefts through split fibres and of normal fetal myogenesis a hypothetical mechanism for splitting of muscle fibres is proposed. Myoblasts, myotubes and myofibres occur within a common basement membrane separated only by plasma membranes and a 20 nm wide gap. In light microscopy they appear as a single fibre. Many meshes of the endomysial framework remain empty and condense around regenerating fibres. Myotubes and young myofibres fuse laterally or become detached and appear by light microscopy to be fragments of a longitudinally split fibre. Though encircled by thick endomysial sheaths, connective tissue between them is sparse. The site of fusion may be marked by a strand of mitochondria or by internal nuclei. Incomplete fusion results in branched fibres or short longitudinal clefts or cavities. The prerequisite of this hypothesis is that regeneration is focal and takes place in a grossly intact endomysium. Repeated death of muscle fibres and focal regeneration in human myopathic muscle lead to dissociation of parenchyma and connective tissue. The mechanism is similar to the formation of pseudolobuli in cirrhosis of the liver.     

Laser microdissection-based expression analysis of key genes involved in muscle regeneration in mdx mice

We have used the mdx mice strain (C57BL/10ScSn-mdx) as an experimental subject for the study of reiterative skeletal muscle necrosis-regeneration with basement membrane preservation. In young mdx muscle, by means of Hematoxylin-Eosin staining, different types of degenerative-regenerative groups (DRG) can be recognized and assigned to a defined muscle regeneration phase. To evaluate the expression of known key-regulatory genes in muscle regeneration, we have applied Laser Capture Microdissection technique to obtain tissue from different DRGs encompassing the complete skeletal muscle regenerative process. The expression of MyoD, Myf-5 and Myogenin showed a rapid increase in the first two days post-necrosis, which were followed by MRF4 expression, when newly regenerating fibers started to appear (3-5days post-necrosis). MHCd mRNA levels, undetectable in mature non-injured fibers, increased progressively from the first day post-necrosis and reached its maximum level of expression in DRGs showing basophilic regenerating fibers. TGFbeta-1 mRNA expression showed a prompt and strong increase following fiber necrosis that persisted during the inflammatory phase, and progressively decreased when new regenerating fibers began to appear. In contrast, IGF-2 mRNA expression decreased during the first days post-necrosis but was followed by a progressive rise in its expression coinciding with the appearance of the newly formed myofibers, reaching the maximum expression levels in DRGs composed of medium caliber basophilic regenerating myofibers (5-7 days post-necrosis). mdx degenerative-regenerative group typing, in conjunction with laser microdissection-based gene expression analysis, opens up a new approach to the molecular study of skeletal muscle regeneration.