Use of magnetic resonance imaging for noninvasive characterization and follow-up of an experimental injury to normal mouse muscles.

A magnetic resonance imaging protocol was tested in a cardiotoxin-induced myonecrosis of hindlimb muscles of three normal mice to assess the usefulness of data provided by longitudinal follow-up of a few individuals. Magnetic resonance imaging examinations were performed sequentially at 4 T between days 1 and 11 post-injury. Axial T1-weighted images after injection of a paramagnetic contrast agent were used to determine the volume of lesions from regions of increased signal intensity. T2 measurements were performed from a single-slice ten-echo acquisition centered upon the largest section of lesion. Early after injury, a very large T2 increase was observed. As recovery proceeded, T2 values progressively decreased toward normal values. Similarly, the volumes of lesions decreased to virtually zero by days 10-11. The evolution of these indices followed the same time scheme observed in histological studies. The use of a volume probe allowed accurate measurement of T2 values, and the acquisition of volumetric data. Such magnetic resonance imaging follow-up should help gather valuable information using few animals.

Differential modulation of alpha, beta and gamma enolase isoforms in regenerating mouse skeletal muscle.

Nothing is known about the expression of the glycolytic enzyme enolase in skeletal muscle alterations such as myofiber degeneration and regeneration. Enolase is a dimeric enzyme which exhibits cell type specific isoforms. The embryonic form, alphaalpha, remains expressed in most adult tissues, whereas a transition towards specific isoforms occurs during ontogenesis in two cell types with high energy requirements: alphagamma and gammagamma in neurons, alphabeta and betabeta in striated muscle cells. During murine myogenesis, beta enolase transcripts are detected early in the forming muscles, and the beta gene is further upregulated at specific stages of muscle development. The alpha and beta subunits exhibit characteristic developmental microheterogeneity patterns. High levels of beta enolase subunits characterize the glycolytic fast-twitch fibers of adult muscles. We have investigated the expression of enolase subunits in a mouse experimental model of muscle regeneration. Following a single intramuscular injection of the necrotic agent cardiotoxin, we observed a rapid decrease in the level of the major muscle enolase subunit beta, accounting for the drop in total enolase activity that correlated with the degeneration of myofibers. Concomitant with the regeneration of new fibers, beta subunit levels began to increase, reaching normal values by 30 days after injury. Changes in the embryonic and ubiquitous subunit, alpha, mimicked those occurring during development by two aspects: modifications in electrophoretic variants and redistribution between soluble and insoluble compartments of muscle extracts. Imunocytochemical analyses of alpha and beta enolase subunits first revealed a homogeneous labeling within myofibers. Striations characteristic of normal adult muscle tissue were visible again by day 14 after injury. A perinuclear alpha and beta immunoreactivity was often observed in regenerating myofibers but its functional significance remains to be elucidated. Double labeling experiments with anti-gamma enolase and FITC-alpha bungarotoxin allowed us to follow the neuromuscular junction remodeling that occurs during muscle regeneration despite the absence of nerve injury.

Expression of matrix metalloproteinases 2 and 9 in regenerating skeletal muscle: a study in experimentally injured and mdx muscles.

Matrix metalloproteinases (MMPs) cooperatively degrade all components of the extracellular matrix (ECM). Remodeling of ECM during skeletal muscle degeneration and regeneration suggests a tight regulation of matrix-degrading activity during muscle regeneration. In this study, we investigated the expression of MMP-2 and MMP-9, in normal muscles and their regulation during regeneration process. We further investigated their secretion by C2C12 myogenic cell line. Two models of muscle degeneration-regeneration were used: (1) normal muscles in which necrosis was experimentally induced by cardiotoxin injection; (2) mdx muscles which exhibit recurrent signs of focal myofiber necrosis followed by successful regeneration. MMPs were studied by zymography; their free activity was quantified using 3H-labeled gelatin substrate and mRNA expression was followed by Northern hybridization. Muscle degeneration-regeneration was analyzed by conventional morphological methods and in situ hybridization was performed on muscle sections to identify the cells expressing these MMPs. Results show that MMP-2, but not MMP-9 expression, is constitutive in normal muscles. Upon injury, the active form of MMP-2 is transiently increased, whereas MMP-9 is induced within 24 h and remains present for several days. Quantitative assays of free gelatinolytic activity show a progressive and steady increase that culminates at 7 days postinjury and slowly returns to normal levels. In adult mdx mice, both pro and active forms of MMP-2 and MMP-9 are expressed. Northern blot results support these findings. Zymography of C2C12-conditioned medium shows that myogenic cells produce MMP-2. By in situ hybridization we localized MMP-9 mRNA in inflammatory cells and putative activated satellite cells in injured muscles. Our data allow the correlation of the differential expression of pro and/or active forms of MMP-2 and MMP-9 with different stages of the degeneration-regeneration process: MMP-9 expression is related to the inflammatory response and probably to the activation of satellite cells, whereas MMP-2 activation is concomitant with the regeneration of new myofibers.

Matrix metalloproteinases MMP-2 and MMP-9 in denervated muscle and injured nerve.

Nerve crush or axotomy results in a transient or longterm denervation accompanied by remodelling in nerve, muscle and neuromuscular junctions. These changes include an increased turnover of several extracellular matrix molecules and proliferation of Schwann cells in injured nerves. Given the role of matrix degrading metalloproteinases MMP-2 and MMP-9 (gelatinases-type IV collagenases) in extracellular matrix remodelling, we investigated their regulation and activation in denervated muscles and injured nerves in mice. For this, immunofluorescence using MMP-2 and MMP-9 antibodies was carried concomitantly with gelatin zymography and quantification of gelatinase activity using [3H]-gelatin substrate. Results show that in normal mouse muscles MMP-2 and MMP-9 are localized at the neuromuscular junctions, in Schwann cells and the perineurium of the intramuscular nerves. In denervated mouse muscles, MMP-2 immunolabelling persists at the neuromuscular junctions but decreases in the nerves whereas MMP-9 immunolabelling persists at the neuromuscular junctions but is enhanced in degenerated intramuscular nerves. Denervated muscles did not show any significant change of gelatinolytic activity or expression pattern, while injured nerves exhibited a transient increase of MMP-9 and activation of MMP-2. In conclusion, this study demonstrates that MMP-2 and MMP-9 are expressed at mouse neuromuscular junctions and that their localization and expression pattern appear not to be modified by denervation. Their modulation in injured nerves suggests they are involved in axonal degeneration and regeneration.

Expression of utrophin and its mRNA in denervated mdx mouse muscle.

Utrophin is a large cytoskeletal protein which shows high homology to dystrophin. In contrast to the sarcolemmal distribution of dystrophin, utrophin accumulates at the postsynaptic membrane of the neuromuscular junction. Because of its localization within this compartment of muscle fibers, expression of utrophin may be significantly influenced by the presence of the motor nerve. We tested this hypothesis by denervating muscles of mdx mouse and monitoring levels of utrophin and its mRNA by immunofluorescence, immunoblotting and RT-PCR. A significant increase in the number of utrophin positive fibers was observed by immunofluorescence 3 to 21 days after sectioning of the sciatic nerve. Quantitative analyses of utrophin and its transcripts in hindlimb muscles denervated for two weeks showed only a moderate increase in the levels of both utrophin (approximately 2-fold) and its transcript (approximately 60 to 90%). The present data suggest that although utrophin is a component of the postsynaptic membrane, its neural regulation is distinct from that of the acetylcholine receptor.

Absence of extraocular muscle pathology in Duchenne's muscular dystrophy: role for calcium homeostasis in extraocular muscle sparing.

Duchenne muscular dystrophy (DMD) is characterized by clinical weakness and progressive necrosis of striated muscle as a consequence of dystrophin deficiency. While all skeletal muscle groups are thought to be affected, enigmatically, the extraocular muscles (EOM) appear clinically unaffected. Here we show that dystrophin deficiency does not result in myonecrosis or pathologically elevated levels of intracellular calcium ([Ca2+]i) in EOM. At variance with a previous report, we find no evidence for dystrophin-related protein/utrophin up-regulation in EOM. In vitro experiments demonstrate that extraocular muscles are inherently more resistant to necrosis caused by pharmacologically elevated [Ca2+]i levels when compared with pectoral musculature. We believe that EOM are spared in DMD because of their intrinsic ability to maintain calcium homeostasis better than other striated muscle groups. Our results indicate that modulating levels of [Ca2+]i in muscle may be of potential therapeutic use in DMD.

M-cadherin localization in developing adult and regenerating mouse skeletal muscle: possible involvement in secondary myogenesis.

In this work, we investigated the distribution of the Ca(2+)-dependent cell adhesion molecule, M-cadherin, in mouse limb muscle during normal development and regeneration. Using two unrelated anti-M-cadherin peptide antibodies, we found scarce M-cadherin immunostaining during primary myogenesis (E12-E14) with no accumulation at areas of cell-cell contact. In contrast, the staining sharply increased in intensity at E16, remained high during secondary myogenesis (E16-P0) but disappeared soon after birth. During secondary myogenesis, M-cadherin was specifically accumulated at the characteristic sites of insertion of secondary myotubes in neighbouring primary myotubes. M-cadherin was also accumulated at the areas of contact between fusing secondary myoblasts and myotubes in vitro. In the adult normal and regenerating muscle, we did not detect M-cadherin accumulations at the surface of myofibres. All together, these observations suggest that M-cadherin is specifically involved in secondary myogenesis.

Expression of a recombinant dystrophin in mdx mice using adenovirus vector.

Genetic deficiencies may be compensated by delivery of the appropriate gene to the affected tissue(s) by somatic gene transfer. In this study, recombinant adenoviruses (defective for replication) carrying a cDNA coding for a truncated dystrophin or 'minidystrophin' (Ad.dys), associated to adenoviruses carrying a beta-galactosidase reporter gene (Ad.beta gal), were administered locally to evaluate the biochemical correction of the genetic defect in mdx mice mutants. Both genes were placed under the control of muscle specific regulatory elements. Two weeks after a single intramuscular injection of Ad.dys, injected muscles showed a significant increase in the percentage of dystrophin positive fibres when compared to muscles either untreated or injected with Ad.beta gal only. Intramuscular injection of the adenoviral expression vectors elicited a local deleterious effect on muscle morphology, rarefaction of myofibres at the site of injection, calcifications and fibrosis were much more marked in comparison to control muscles injected with vehicle. beta-galactosidase was exclusively expressed within myofibres in a segmental fashion. Regional co-localization of beta-galactosidase and dystrophin expression gives further support to the demonstration of adenoviral induced expression of the recombinant genes.

[Muscular reconstruction by myogenic cell graft]. / Reconstruction musculaire par greffe de cellules myogéniques.

We have developed an experimental model to test the ability of cultured satellite cells to form new muscle fibers when grafted in an irreversibly injured muscle. Association of X-irradiation to autotransplantation reduced extensor digitorum longus muscles of adult rats to a cystic structure formed by a peripheral rim of surviving muscle fibers surrounding a central space devoid of myofibers. Grafting autologous satellite cells, multiplied and labelled in vitro, into this central space gave rise to new myotubes which developed and matured to form fully differentiated myofibers. Mecanophysiological recordings confirmed the improvement of functional parameters particularly a 4 fold increase of twitch and tetanic tensions in grafted muscles. Furthermore, we examined the role of old basal lamina in architectural organisation of the reconstituted muscle by electron microscopy and immunofluorescence using antibodies to laminin, fibronectin, type IV collagen and heparan sulfate proteoglycan. We observed the persistence of immunoreactivity to all components; anti-laminin antibodies for example, evidenced a clear fascicular organization of ghost basal lamina which are progressively repopulated by the grafted cells. We finally investigated in vivo proliferation of the grafted cells by an autoradiographic study of H3-thymidine incorporation in the regenerated fibers and demonstrate that grafted cells proliferate for, at least, 1 week after cell grafting.

Functional recovery induced by satellite cell grafts in irreversibly injured muscles.

Grafting autologous cultured satellite cells in irreversibly injured rat extensor digitorum longus EDL muscle leads to myofiber regeneration at the grafting site. In this study, we investigated whether cell grafts induced functional improvement and correlated mechanophysiological findings with histological observations. In cell grafted muscles, the number of myofibers did not differ significantly between 2 wk and 3 mo, whereas no regenerating myofibers were observed in ungrafted controls. During this period, the total number of myofibers in the cell grafted muscles represented 48.2-51.9% of that in normal muscles. The mean diameter of regenerated myofibers increased with time, reaching a maximum (32 microns) at the second mo and remained smaller than that of normal myofibers (47 microns). Muscle function was measured by mechanophysiological recordings of muscle response to supramaximal electrical stimulation of the nerve in situ. Cell grafted muscles exhibited a progressive improvement of all contractile parameters. After 3 mo, a 4-fold increase in absolute values of twitch and tetanic tension outputs was measured in cell grafted muscles when compared to ungrafted controls. However, these parameters remained much lower than in normal muscles (23.4% and 22.3% of control, respectively). This study showed that myogenic cell grafts replace degenerated myofibers and form functional myofibers. Functional improvement observed, between 2 wk and 3 mo after cell grafting, correlated with the development, differentiation, and maturation of the regenerated myofibers rather than with an increase in the number of regenerated myofibers.

Role of persisting basement membrane in the reorganization of myofibres originating from myogenic cell grafts in the rat.

Satellite cells grafted at the site of an irreversible muscle injury regenerate normal myofibres that become organized in fascicles. The role of the basement membrane in organization of the newly formed muscle fibres was investigated using polyclonal antibodies against laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan. In ungrafted muscles, original basement membranes were reactive to these antibodies at 7, 14 and 45 days after injury. Labelling of satellite cells with FITC-latex beads showed the labelled myoblasts and new myofibres within the remnants of old basement membranes at 7 days after cell implantation and thereafter. Electron microscopy of injured-ungrafted muscles showed persistence of electron dense material corresponding to thin layers of old basal laminae partially interrupted. After cell grafting, myotubes developed within these structures and were surrounded by redundant basal laminae. These results suggest that grafted cells are able to migrate inside the basement membranes which serve as scaffolding for their development.

Regeneration of skeletal muscle fibers from autologous satellite cells multiplied in vitro. An experimental model for testing cultured cell myogenicity.

An experimental model used to test in vivo myogenicity of autologous satellite cells multiplied in vitro is described. Free muscle autotransplantation served as the basis and was combined with x-irradiation. Administration of 1500, 2500, and 3500 rad doses 24 hours before or after ischemia showed that inhibition of spontaneous regeneration is dose dependent and more efficient when irradiation was applied before injury. A single dose of 2500 rad before injury resulted in the formation of a cystic structure ideal for cell implantation. FITC-latex beads and/or carbocyanine dyes were internalized by mononucleated satellite cells in vitro. Labeling did not affect survival or development of these cells. No sign of marker release or spreading from labeled to unlabeled cells was detectable unless by the fusion process. These labels were retained for several weeks. Grafting of labeled dense cellular suspensions into x-irradiated ischemic muscles indicated that satellite cells retain their myogenic characteristic and are able to reform fully differentiated muscle fibers.

Effects of growth hormone on skeletal muscle. II. Studies on regeneration and denervation in adult rats.

The effects of human recombinant growth hormone (rhGH) on regenerating skeletal muscle after ischaemic necrosis and on denervated skeletal muscle were studied in normal adult rats. One group of rats was treated with 4 IE rhGH daily by subcutaneous injections, while control rats were injected with saline. The treatment with rhGH resulted in increased levels of insulin-like growth factor-I (IGF-I) in serum. Ischaemic necrosis was achieved in the extensor digitorum longus (EDL) muscle by cutting the supplying vessels and nerve fascicles at the entrance into the muscle. The wet weight and DNA: protein ration in the regenerating muscle were determined 2 and 4 weeks after the operation. The weight of the regenerating muscles in the rats treated with rhGH during the period of study was larger than in the control rats, while the DNA:protein ratio did not differ significantly between the groups. Denervation of the EDL and soleus muscles followed by subsequent reinnervation was obtained by freezing the sciatic nerve with a forceps chilled in liquid nitrogen. Rats treated with rhGH during the period of denervation and reinnervation, i.e. during the 4 weeks after the freezing of the sciatic nerve, revealed increased weight of both the reinnervated and normal muscles compared to corresponding muscles of control rats. Denervation of the EDL and soleus muscles without subsequent reinnervation was achieved by cutting the sciatic nerve at the level of the thigh. Four weeks after denervation the muscles showed atrophy, mainly affecting type 2 fibres in the EDL muscle and both type 1 and type 2 fibres in the soleus muscle.

[Muscular regeneration by autograft of in vitro multiplied satellite cells]. / Régénération musculaire par autogreffe de cellules satellites multipliées in vitro.

We used a free-grafting technique modified from that described by Carlson and Gutmann, to induce an ischemic injury of Extensor Digitorum Longus (EDL) muscle in adult rats. In these muscles, a peripheral rim, three to five muscle fibers thick, survive ischemia whereas the muscle fibers occupying the central part follow a sequential pattern of necrosis, degradation and regeneration. Association of X-irradiation to the experimental injury inhibits mitosis of myogenic cells, thus preventing, the regeneration of the injured fibers. At the 14th postoperative day, the muscle is reduced to a cystic structure. The center of this structure is occupied by a loose fibrinous and collagenic network with a few cellular elements. In vitro multiplied autologous satellite cells, labelled with fluorescent latex beads, were injected back into this preparation. Our results indicate that these cells have retained their myogenic characteristics and are able to reform fully differentiated muscle fibers.

Molybdic and tungstic heteropolyanions for "ionic fixation" of acetylcholine in cholinergic motor nerve terminals.

The treatment of neuromuscular junctions with phosphomolybdic acid (PMA) and silicotungstic acid (STA) heteropolyanions permits the visualization of electron dense precipitates in the synaptic vesicles of the cholinergic motor nerve terminals. At the light microscopic level, the uncolored molybdenum salt is visualized after reduction to molybdenum blue. The blue coloration is confined to the nerve terminals. Since PMA and STA are known as strong precipitating agents of quaternary ammonium compounds (cations) it is supposed that they have insolubilized in situ the acetylcholine (Ach) of the synaptic vesicles by means of a rapid ionic interaction. Furthermore, in spite of the strong acidity of PMA and STA solutions, the ultrastructure of the treated tissue is not significantly altered but on the contrary seems to be well preserved. The ionic insolubilization of Ach, added to the good preservation of the ultrastructure prompted us to use the term "ionic fixation".

Silicotungstic acid for cytochemical localization of water soluble substance(s) of cholinergic motor nerve terminal.

Silicotungstic acid (STA), an electron dense substance and a powerful precipitating agent of quaternary ammonium salts such as choline and acetylcholine, was employed on the frog motor end-plate in order to prove that STA reacts with diffusible substance(s) in nerve terminals. Thus, STA treatment and osmium tetroxide (OsO4) fixation were performed in three different ways. No reaction was detectable when STA treatment followed osmification, while simultaneous treatment with STA and OsO4 darkened both presynaptic and synaptic vesicle membranes. When STA was employed directly on fresh tissues which were subsequently fixed by OsO4, small black precipitates were observed in the synaptic vesicles and none on other synaptic structures. The possible reaction of STA with acetylcholine is discussed.