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Persistent Muscle Fiber Regeneration in Long Term Denervation. Past, Present, Future.

Carraro U, Boncompagni S, Gobbo V, Rossini K, Zampieri S, Mosole S, Ravara B, Nori A, Stramare R, Ambrosio F, Piccione F, Masiero S, Vindigni V, Gargiulo P, Protasi F, Kern H, Pond A, Marcante A - Eur J Transl Myol (2015)

Bottom Line: Whether in mammals, humans included, this is a result of persistent de novo formation of muscle fibers is an open issue we would like to explore in this review.Although embryonic isoforms of acetylcholine receptors are known to be re-expressed and to spread from the end-plate to the sarcolemma of muscle fibers in early phases of muscle denervation, we suggest that the MHCemb positive muscle fibers we observe result from the activation, proliferation and fusion of satellite cells, the myogenic precursors present under the basal lamina of the muscle fibers.Some of the mandatory procedures, are ready to be translated from animal experiments to clinical studies to meet the needs of persons with long-term irreversible muscle denervation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurorehabilitation, Foundation San Camillo Hospital , I.R.C.C.S., Venice, Italy.

ABSTRACT
Despite the ravages of long term denervation there is structural and ultrastructural evidence for survival of muscle fibers in mammals, with some fibers surviving at least ten months in rodents and 3-6 years in humans. Further, in rodents there is evidence that muscle fibers may regenerate even after repeated damage in the absence of the nerve, and that this potential is maintained for several months after denervation. While in animal models permanently denervated muscle sooner or later loses the ability to contract, the muscles may maintain their size and ability to function if electrically stimulated soon after denervation. Whether in mammals, humans included, this is a result of persistent de novo formation of muscle fibers is an open issue we would like to explore in this review. During the past decade, we have studied muscle biopsies from the quadriceps muscle of Spinal Cord Injury (SCI) patients suffering with Conus and Cauda Equina syndrome, a condition that fully and irreversibly disconnects skeletal muscle fibers from their damaged innervating motor neurons. We have demonstrated that human denervated muscle fibers survive years of denervation and can be rescued from severe atrophy by home-based Functional Electrical Stimulation (h-bFES). Using immunohistochemistry with both non-stimulated and the h-bFES stimulated human muscle biopsies, we have observed the persistent presence of muscle fibers which are positive to labeling by an antibody which specifically recognizes the embryonic myosin heavy chain (MHCemb). Relative to the total number of fibers present, only a small percentage of these MHCemb positive fibers are detected, suggesting that they are regenerating muscle fibers and not pre-existing myofibers re-expressing embryonic isoforms. Although embryonic isoforms of acetylcholine receptors are known to be re-expressed and to spread from the end-plate to the sarcolemma of muscle fibers in early phases of muscle denervation, we suggest that the MHCemb positive muscle fibers we observe result from the activation, proliferation and fusion of satellite cells, the myogenic precursors present under the basal lamina of the muscle fibers. Using morphological features and molecular biomarkers, we show that severely atrophic muscle fibers, with a peculiar cluster reorganization of myonuclei, are present in rodent muscle seven-months after neurectomy and in human muscles 30-months after complete Conus-Cauda Equina Syndrome and that these are structurally distinct from early myotubes. Beyond reviewing evidence from rodent and human studies, we add some ultrastructural evidence of muscle fiber regeneration in long-term denervated human muscles and discuss the options to substantially increase the regenerative potential of severely denervated human muscles not having been treated with h-bFES. Some of the mandatory procedures, are ready to be translated from animal experiments to clinical studies to meet the needs of persons with long-term irreversible muscle denervation. An European Project, the trial Rise4EU (Rise for You, a personalized treatment for recovery of function of denervated muscle in long-term stable SCI) will hopefully follow.

No MeSH data available.


Related in: MedlinePlus

Semithin sections, toluidine blue stain. Note in A the gradient of size of muscle fibers and the presence of central nuclei in myofibers of denervated and regenerated rat soleus muscle 30 days after bilateral sciatectomy and unilateral marcaine-induced muscle damage. Spontaneous regeneration is also present in 30 days denervated contralateral muscle as suggested by the central nucleus in one myofiber (arrow in B). 40x.
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fig003: Semithin sections, toluidine blue stain. Note in A the gradient of size of muscle fibers and the presence of central nuclei in myofibers of denervated and regenerated rat soleus muscle 30 days after bilateral sciatectomy and unilateral marcaine-induced muscle damage. Spontaneous regeneration is also present in 30 days denervated contralateral muscle as suggested by the central nucleus in one myofiber (arrow in B). 40x.

Mentions: Taken together, the above examples provide morphological evidence that very small myofibers may not only represent extreme examples of severely atrophied muscle fibers surviving long-term denervation (see below), but may also occur as the result of myofiber regeneration followed by atrophy in chronic denervation.6 Indeed, Mussini et al.9 have shown that marcaine damage of permanently denervated rat muscles produces unequivocal morphological features of muscle damage and regeneration and that repeated marcaine infusion of the aneural regenerated rat muscle was followed by a second round of muscle damage and regeneration, demonstrating that the regenerative process is accompained by survival and/or proliferation of myogenic cells with structural and functional characteristics of satellite cells (or, in other words, “stem cells”).9 Conclusive evidence of aneural satellite cell activation, proliferation, fusion and progression to small muscle fibers was obtained by gel electrophoresis which revealed the transient presence of the embryonic isoforms of the myosin light and heavy chains (MLCemb, MHCemb, respectively) in regenerating rat muscles,10 and by immunolabeling aneurally regenerating muscle cryosections (Fig. 3) with an antibody that recognizes epitopes of the MHCemb (Figs. 4 and 5).11,12 Note that in Fig. 4 the size of the regenerated muscle fibers increases up to 10 days (Fig. 4, A-C), at least, but that the immunostain starts to fade in several muscle fibers at this time of regeneration (Fig. 4, C). At 30 days of regeneration in aneural muscles (Fig. 3, A), indeed, the majority of the muscle fibers are negative for MHCemb protein and only some small myofibers are positive (Fig. 5, A). It is interesting to note that in rat muscle denervated for 30 days (Fig. 3, B) only one small round muscle fiber with a central nucleus is stained (Fig. 5, B) with the anti-MHCemb antibody that we are using for our studies in rodents and human muscles,13 while all other larger denervated muscle cells, with subsarcolemmal nuclei, are negative (Fig. 5, B). In our opinion, the anti-body we are using recognizes epitopes of the embryonic myosin heavy chains that are not re-expressed in adult muscle fibers during the first month of denervation, at least in the rat muscles.13 The same result was observed when the marcaine damage and the myogenic response occurred in rat muscles permanently denervated for 4 to 6 months.13 Further evidence of muscle fiber regeneration is the detection of myogenin and MRF4 gene expression in response to long-term muscle denervation,14,15 in spite of the down-regulation of activity-dependent genes in long-term denervated rat muscle.15 In conclusion, we are confident that expression of the MHCemb is the result of a cellular process of muscle regeneration.


Persistent Muscle Fiber Regeneration in Long Term Denervation. Past, Present, Future.

Carraro U, Boncompagni S, Gobbo V, Rossini K, Zampieri S, Mosole S, Ravara B, Nori A, Stramare R, Ambrosio F, Piccione F, Masiero S, Vindigni V, Gargiulo P, Protasi F, Kern H, Pond A, Marcante A - Eur J Transl Myol (2015)

Semithin sections, toluidine blue stain. Note in A the gradient of size of muscle fibers and the presence of central nuclei in myofibers of denervated and regenerated rat soleus muscle 30 days after bilateral sciatectomy and unilateral marcaine-induced muscle damage. Spontaneous regeneration is also present in 30 days denervated contralateral muscle as suggested by the central nucleus in one myofiber (arrow in B). 40x.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383182&req=5

fig003: Semithin sections, toluidine blue stain. Note in A the gradient of size of muscle fibers and the presence of central nuclei in myofibers of denervated and regenerated rat soleus muscle 30 days after bilateral sciatectomy and unilateral marcaine-induced muscle damage. Spontaneous regeneration is also present in 30 days denervated contralateral muscle as suggested by the central nucleus in one myofiber (arrow in B). 40x.
Mentions: Taken together, the above examples provide morphological evidence that very small myofibers may not only represent extreme examples of severely atrophied muscle fibers surviving long-term denervation (see below), but may also occur as the result of myofiber regeneration followed by atrophy in chronic denervation.6 Indeed, Mussini et al.9 have shown that marcaine damage of permanently denervated rat muscles produces unequivocal morphological features of muscle damage and regeneration and that repeated marcaine infusion of the aneural regenerated rat muscle was followed by a second round of muscle damage and regeneration, demonstrating that the regenerative process is accompained by survival and/or proliferation of myogenic cells with structural and functional characteristics of satellite cells (or, in other words, “stem cells”).9 Conclusive evidence of aneural satellite cell activation, proliferation, fusion and progression to small muscle fibers was obtained by gel electrophoresis which revealed the transient presence of the embryonic isoforms of the myosin light and heavy chains (MLCemb, MHCemb, respectively) in regenerating rat muscles,10 and by immunolabeling aneurally regenerating muscle cryosections (Fig. 3) with an antibody that recognizes epitopes of the MHCemb (Figs. 4 and 5).11,12 Note that in Fig. 4 the size of the regenerated muscle fibers increases up to 10 days (Fig. 4, A-C), at least, but that the immunostain starts to fade in several muscle fibers at this time of regeneration (Fig. 4, C). At 30 days of regeneration in aneural muscles (Fig. 3, A), indeed, the majority of the muscle fibers are negative for MHCemb protein and only some small myofibers are positive (Fig. 5, A). It is interesting to note that in rat muscle denervated for 30 days (Fig. 3, B) only one small round muscle fiber with a central nucleus is stained (Fig. 5, B) with the anti-MHCemb antibody that we are using for our studies in rodents and human muscles,13 while all other larger denervated muscle cells, with subsarcolemmal nuclei, are negative (Fig. 5, B). In our opinion, the anti-body we are using recognizes epitopes of the embryonic myosin heavy chains that are not re-expressed in adult muscle fibers during the first month of denervation, at least in the rat muscles.13 The same result was observed when the marcaine damage and the myogenic response occurred in rat muscles permanently denervated for 4 to 6 months.13 Further evidence of muscle fiber regeneration is the detection of myogenin and MRF4 gene expression in response to long-term muscle denervation,14,15 in spite of the down-regulation of activity-dependent genes in long-term denervated rat muscle.15 In conclusion, we are confident that expression of the MHCemb is the result of a cellular process of muscle regeneration.

Bottom Line: Whether in mammals, humans included, this is a result of persistent de novo formation of muscle fibers is an open issue we would like to explore in this review.Although embryonic isoforms of acetylcholine receptors are known to be re-expressed and to spread from the end-plate to the sarcolemma of muscle fibers in early phases of muscle denervation, we suggest that the MHCemb positive muscle fibers we observe result from the activation, proliferation and fusion of satellite cells, the myogenic precursors present under the basal lamina of the muscle fibers.Some of the mandatory procedures, are ready to be translated from animal experiments to clinical studies to meet the needs of persons with long-term irreversible muscle denervation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurorehabilitation, Foundation San Camillo Hospital , I.R.C.C.S., Venice, Italy.

ABSTRACT
Despite the ravages of long term denervation there is structural and ultrastructural evidence for survival of muscle fibers in mammals, with some fibers surviving at least ten months in rodents and 3-6 years in humans. Further, in rodents there is evidence that muscle fibers may regenerate even after repeated damage in the absence of the nerve, and that this potential is maintained for several months after denervation. While in animal models permanently denervated muscle sooner or later loses the ability to contract, the muscles may maintain their size and ability to function if electrically stimulated soon after denervation. Whether in mammals, humans included, this is a result of persistent de novo formation of muscle fibers is an open issue we would like to explore in this review. During the past decade, we have studied muscle biopsies from the quadriceps muscle of Spinal Cord Injury (SCI) patients suffering with Conus and Cauda Equina syndrome, a condition that fully and irreversibly disconnects skeletal muscle fibers from their damaged innervating motor neurons. We have demonstrated that human denervated muscle fibers survive years of denervation and can be rescued from severe atrophy by home-based Functional Electrical Stimulation (h-bFES). Using immunohistochemistry with both non-stimulated and the h-bFES stimulated human muscle biopsies, we have observed the persistent presence of muscle fibers which are positive to labeling by an antibody which specifically recognizes the embryonic myosin heavy chain (MHCemb). Relative to the total number of fibers present, only a small percentage of these MHCemb positive fibers are detected, suggesting that they are regenerating muscle fibers and not pre-existing myofibers re-expressing embryonic isoforms. Although embryonic isoforms of acetylcholine receptors are known to be re-expressed and to spread from the end-plate to the sarcolemma of muscle fibers in early phases of muscle denervation, we suggest that the MHCemb positive muscle fibers we observe result from the activation, proliferation and fusion of satellite cells, the myogenic precursors present under the basal lamina of the muscle fibers. Using morphological features and molecular biomarkers, we show that severely atrophic muscle fibers, with a peculiar cluster reorganization of myonuclei, are present in rodent muscle seven-months after neurectomy and in human muscles 30-months after complete Conus-Cauda Equina Syndrome and that these are structurally distinct from early myotubes. Beyond reviewing evidence from rodent and human studies, we add some ultrastructural evidence of muscle fiber regeneration in long-term denervated human muscles and discuss the options to substantially increase the regenerative potential of severely denervated human muscles not having been treated with h-bFES. Some of the mandatory procedures, are ready to be translated from animal experiments to clinical studies to meet the needs of persons with long-term irreversible muscle denervation. An European Project, the trial Rise4EU (Rise for You, a personalized treatment for recovery of function of denervated muscle in long-term stable SCI) will hopefully follow.

No MeSH data available.


Related in: MedlinePlus