Muscle reinnervation in rats treated with vitamin E]

During muscle reinnervation, a transitory phase of polyinnervation occurs. In reinnervated muscles of vitamin E deficient rats, sprouting and polyinnervation are increased with respect to reinnervated controls. In this work, polyinnervation was observed in reinnervated extensor digitorum longus (edl) muscle of rats treated with pharmacological doses of vitamin E. Sciatic nerve was crushed and edl muscle was examined electrophysiologically at 30, 40 and 60 days after denervation. The percentage of polyinnervated cells in controls peaked at 30 days and thus it decreased. In muscles of vitamin E treated rats, the time course of percentage of polyinnervated muscle cells was qualitatively the same, but it was decreased at all times.     

Changes due to age in the regenerating axons of the adult rat]

Muscle reinnervation after nerve crush was observed in rats at different ages with a combined technique that simultaneously demonstrates nerve endings and endplates. At early times of reinnervation the amount of sprouting was higher in older rats than in younger rats; according to this finding an enhanced number of polyinnervated endplates was found in older rats. A similar enhancement of sprouting and polyinnervation was observed during muscle reinnervation of vitamin E deficient rats, supporting the proposed analogy between vitamin E deficiency and aging.     

Specific innervation of muscle fiber types in a developmentally polyinnervated muscle

In neonatal rabbit soleus muscle, different motor units were found to contract with widely varying time courses. Analysis of these data suggest that individual motor units are largely homogeneous for muscle fiber type despite the presence of extensive polyinnervation at birth. We suggest that (1) neonatal motor neurons are effectively differentiated into specific types insofar as they preferentially innervate muscle fibers which give rise to different contraction times, and (2) muscle fibers begin their physiological differentiation into twitch types while still polyinnervated. Possible mechanisms underlying the development of a specific pattern of neuromuscular innervation are discussed.     

Synapse elimination by fiber type and maturational state in rabbit soleus muscle

We have compared the development of fast and slow motor innervation in the neonatal rabbit soleus, a muscle which contains two distinct motor unit types during the early period of polyneuronal innervation. The innervation state of individual muscle fibers was ascertained using an intracellular electrode; a fluorescent dye was then injected into particular fibers to permit subsequent identification of histochemical type. We found no significant difference in the time course of synapse elimination for fast and slow motor units as judged by the percentage of fibers remaining polyneuronally innervated at two ages: 7-8 days, when most fibers are multiply innervated, and 10-11 days, when the level of polyinnervation is low. In a second experiment, we examined a phenomenon in which compound end-plate potentials were occasionally seen in muscle fibers at an age (17-23 days) well past the major episode of synapse elimination. We present evidence that this apparent polyinnervation in fact derives from an electrode-induced electrical coupling artifact and that genuinely polyinnervated fibers are very rare at this stage, if present at all.     

Slowing of synapse elimination by alpha-bungarotoxin superfusion of the neonatal rabbit soleus muscle

To examine the role of postsynaptic activity in regulating the rate of neuromuscular synapse elimination, contractile activity of neonatal rabbit soleus muscles was decreased by chronic superfusion of alpha-bungarotoxin (alpha-BGT) over their surfaces. Superfusion was begun at 6 days postnatal and continued for a variable duration (2 to 5 days) before muscles were analyzed. The percentage of polyinnervated fibers was assessed both physiologically and anatomically for alpha-BGT-treated muscles and their contralateral muscles, in addition to normal and control muscles of the same age. Within muscles exposed to alpha-BGT, polyinnervation was significantly greater than that for muscles from each of the control groups. The anatomical assay further revealed that the retention of polyinnervation in alpha-BGT-treated muscles was most pronounced near the muscle's surface, although end plates at the center were also affected. This finding, coupled with evidence that only a small percentage of the muscle fibers were completely inactivated, suggests that the activity block was also most pronounced near the surface and relatively low at the muscle's center. The percentage of end plates at which synapse elimination was delayed was greater than the estimated percentage whose activity was completely blocked, suggesting that synapse loss was slowed even in muscle fibers retaining some postsynaptic activity. These observations indicate that the rate of synapse elimination depends on the levels of functional acetylcholine receptors. This process could be mediated in a graded fashion by changes in postsynaptic activity (subthreshold or suprathreshold) or by a nonelectrical effect of blocking postsynaptic receptors.     

Bone marrow-derived mesenchymal stem cell transplantation does not improve quality of muscle reinnervation or recovery of motor function after facial nerve transection in rats

Recently, we devised and validated a novel strategy in rats to improve the outcome of facial nerve reconstruction by daily manual stimulation of the target muscles. The treatment resulted in full recovery of facial movements (whisking), which was achieved by reducing the proportion of pathologically polyinnervated motor endplates. Here, we posed whether manual stimulation could also be beneficial after a surgical procedure potentially useful for treatment of large peripheral nerve defects, i.e., entubulation of the transected facial nerve in a conduit filled with suspension of isogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) in collagen. Compared to control treatment with collagen only, entubulation with BM-MSCs failed to decrease the extent of collateral axonal branching at the lesion site and did not improve functional recovery. Post-operative manual stimulation of vibrissal muscles also failed to promote a better recovery following entubulation with BM-MSCs. We suggest that BM-MSCs promote excessive trophic support for regenerating axons which, in turn, results in excessive collateral branching at the lesion site and extensive polyinnervation of the motor endplates. Furthermore, such deleterious effects cannot be overridden by manual stimulation. We conclude that entubulation with BM-MSCs is not beneficial for facial nerve repair.     

Synapse elimination,the size principle,and Hebbian synapses

Synapse elimination at the vertebrate neuromuscular junction reduces a polyinnervated population of muscle fibers to a monoinnervated state. The function of this developmental phenomenon (if any) is unproven. A theoretical analysis of Hebbian (correlation) rules connecting presynaptic and postsynaptic activity and synaptic strength at the neuromuscular junction is presented. The following points are demonstrated: (1) Correlational competition leads to the reduction of polyinnervation to a stable monoinnervated state; (2) the competition gives rise to the size principle over a wide range of the plausible parameter space; (3) over a significant subrange, the competition selectively eliminates topographically incorrect synapses; and (4) in cases in which topographic projection errors overwhelm the system, both error correction and the development of the size principle are disrupted. Correlational competition may explain contradictory experimental results concerning the effects of stimulating or silencing subpopulations of motor neurons. It may also explain an otherwise puzzling instance of a breakdown in the size principle seen in humans undergoing neural regeneration. Taken together, these findings suggest a novel hypothesis for the function of synapse elimination at the neuromuscular junction: the establishment of the size principle. © 1995 John Wiley & Sons, Inc.     

Abnormal enwrapment of intramuscular axons by distal Schwann cells with defective basal lamina in the muscular dysgenic mouse embryo

In the muscular dysgenic (mdg/mdg) mouse embryo, both muscle and nerve are affected early during embryogenesis, from Embryonic Day 13 (E13). We now find that the mutation affects not only the degree of differentiation of the muscle and the pattern of motor innervation but also the relationship between Schwann cell and axon. We studied the sciatic nerve of normal and mdg/mdg embryos between E13 and E18 at the ultrastructural level. We found that in mdg/mdg nerve, (1) Schwann cells do not totally enwrap the growing axons in their most distal part, close to the growth cone, and (2) the terminal Schwann cells do not correctly surround the nerve endings and seal the corresponding synaptic contacts. Moreover, both types of mutant Schwann cell lack a normal electron-dense basal lamina. We found that there is an excess of axons relative to the Schwann cell population in the intramuscular portions of the mdg/mdg sciatic nerve. Our observations point toward a possible defect of the mechanism of migration and maturation of Schwann cells. Such a defect may in turn affect primarily or secondarily the mutual influences between Schwann cell and axon and lead to some or all of the major abnormalities observed in the mdg/mdg neuromuscular system, namely, multifocal polyinnervation, immature axon-myotube contacts, and abnormal T-tubule-sarcoplasmic reticulum junctions.     

Exogenous Pleiotrophin Applied to Lesioned Nerve Impairs Muscle Reinnervation

Pleiotrophin (PTN) is a heparin-binding growth factor involved in nerve regeneration after peripheral nerve injury. After crush injury, PTN is found in distal nerve segments in several non-neural cell types, including Schwann cells, macrophages, and endothelial cells, but not in axons. To further clarify the role for PTN in nerve regeneration, we investigated the effects of PTN applied to lesioned peripheral nerve in vivo. PTN in a dose of 1 mg/kg impaired muscle reinnervation. Thus, gastrocnemius muscle failed to recover its contractile properties as assessed by in situ maximal isometric tetanic force. PTN also decreased non-neural cell densities and delayed macrophage recruitment in the distal crushed nerve. These results are discussed in the light of recent evidence that PTN is a multifunctional polypeptide.     

The expression patterns of β1,4 galactosyltransferase I and V mRNAs, and Galβ1-4GlcNAc group in rat gastrocnemius muscles post sciatic nerve injury

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of β1,4-galactosylation is performed by a family of β1,4-galactosyltransferases (β1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. β1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides and play roles in sciatic nerve regeneration after sciatic nerve injury. In the present study, the expression of β1,4-galactosyltransferase (β1,4-GalT) I, V mRNAs and Galβ1-4GlcNAc group were examined in rat gastrocnemius muscles after sciatic nerve crush and transection. Real time PCR revealed that β1,4-GalT I and V mRNAs expressed at a high level in normal gastrocnemius muscles and decreased gradually from 6 h, reached the lowest level at 2 weeks, then restored gradually to relatively normal level at 4 weeks after sciatic nerve crush. In contrast, in sciatic nerve transection model, β1,4-GalT I and V mRNAs decreased gradually from 6 h, and remained on a low level at 4 weeks in gastrocnemius muscles after sciatic nerve transection. In situ hybridization indicated that β1,4-GalT I and V mRNAs localized in numerous myocytes and muscle satellite cells under normal conditions and at 4 weeks after sciatic nerve crush, and in a few muscle satellite cells at 4 weeks after sciatic nerve transection. Furthermore, lectin blotting showed that the expression level of the Galβ1–4GlcNAc group decreased from 6 h, reached the lowest level at 2 weeks, and restored to relatively normal level at 4 weeks after sciatic nerve crush. RCA-I lectin histochemistry demonstrated that Galβ1–4GlcNAc group localized in numerous membranes of myocytes and muscle satellite cells in normal and at 4 weeks after sciatic nerve crush, and in a few muscle satellite cells at 2 and 4 weeks after sciatic nerve transection. These results indicated that the expressions of β1,4-GalT I, V mRNAs and Galβ1–4GlcNAc group were involved in the process of denervation and reinnervation, which suggests that β1,4-GalT I, V mRNAs and Galβ1-4GlcNAc group may play an important role in the muscle regeneration.     

Transdifferentiation from striated muscle of medusae in vitro

We have established an in vitro transdifferentiation and regeneration system which is based entirely on mononucleated striated muscle cells. The muscle tissue is isolated from anthomedusae and activated by various means to undergo cell cycles and transdifferentiation to several new cell types. In all cases DNA-replication is initiated and the division products are smooth muscle cells, characterized by their ultrastructure and monoclonal antibodies, and nerve/sensory cells, characterized by their ultrastructure and FMRFamide-staining. Both cell types are found at a 1:1 ratio after the first division. The nerve cells stop to replicate, whereas the smooth muscle cells continue and keep producing in each successive division a smooth muscle cell and a nerve cell. The observed data indicate that smooth muscle cells behave like stem cells. Depending on the destabilization and culturing methods, some isolated muscle tissue will form a bilayered fragment and within only two cell cycles manubria (the feeding and sexual organ) or tentacles will regenerate. In this case six to eight new non-muscle cell types have been formed by transdifferentiation.     

Role of interstitial cell migration in generating position-dependent patterns of nerve cell differentiation in Hydra

The role of interstitial cell migration in the formation of newly differentiated nerve cells was examined during head regeneration in Hydra magnipapillata. When distal tissue was removed from the body of a wild-type strain (105), nerve cell differentiation occurred at a rapid rate during the first 48 hr of regeneration, slowing after this point. Rapid nerve cell differentiation was due primarily to migration of interstitial cells, some of which appeared to be nerve cell precursors, into the regenerating head. The migration decreased considerably after the first 48 hr of regeneration. In reg-16, a mutant strain deficient in head regeneration, no migration of interstitial cells and hence no new nerve cell differentiation were observed in the regenerating tip. However, the interstitial cells of reg-16 were observed to migrate into regenerating tissue of strain 105. These observations suggest that the migration of nerve cell precursors plays an important role when the new nerve net is being established during head regeneration.     

Tissue engineering of peripheral nerves: A comparison of venous and acellular muscle grafts with cultured Schwann cells

Bioengineering is considered to be the laboratory-based alternative to human autografts and allografts. It ought to provide "custom-made organs" cultured from patient's material. Venous grafts and acellular muscle grafts support axonal regeneration only to a certain extent because of the lack of viable Schwann cells in the graft. We created a biologic nerve graft in the rat sciatic nerve model by implanting cultured Schwann cells into veins and acellular gracilis muscles, respectively. Autologous nerve grafts and veins and acellular muscle grafts without Schwann cells served as controls. After 6 and 12 weeks, regeneration was assessed clinically, histologically, and morphometrically. The polymerase chain reaction analvsis showed that the implanted Schwann cells remained within all the grafts. The best regeneration was seen in the control; after 12 weeks the number of axons was increased significantly compared with the other grafts. A good regeneration was noted in the muscle-Schwann cell group, whereas regeneration in both of the venous grafts and the muscle grafts without Schwann cells was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. The venous grafts with Schwann cells showed less fibrous tissue and disorganization than the veins without Schwann cells, but failed to show an excellent regeneration. This might be attributed to the lack of endoneural-tube-like components serving as scaffold for the sprouting axon. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biologic graft with basal lamina tubes as pathways for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

The Role of Genetically Modified Mesenchymal Stem Cells in Urinary Bladder Regeneration

Recent studies have demonstrated that mesenchymal stem cells (MSCs) combined with CD34⁺ hematopoietic/stem progenitor cells (HSPCs) can function as surrogate urinary bladder cells to synergistically promote multi-faceted bladder tissue regeneration. However, the molecular pathways governing these events are unknown. The pleiotropic effects of Wnt5a and Cyr61 are known to affect aspects of hematopoiesis, angiogenesis, and muscle and nerve regeneration. Within this study, the effects of Cyr61 and Wnt5a on bladder tissue regeneration were evaluated by grafting scaffolds containing modified human bone marrow derived MSCs. These cell lines were engineered to independently over-express Wnt5a or Cyr61, or to exhibit reduced expression of Cyr61 within the context of a nude rat bladder augmentation model. At 4 weeks post-surgery, data demonstrated increased vessel number (~250 vs ~109 vessels/mm²) and bladder smooth muscle content (~42% vs ~36%) in Cyr61OX (over-expressing) vs Cyr61KD (knock-down) groups. Muscle content decreased to ~25% at 10 weeks in Cyr61KD groups. Wnt5aOX resulted in high numbers of vessels and muscle content (~206 vessels/mm² and ~51%, respectively) at 4 weeks. Over-expressing cell constructs resulted in peripheral nerve regeneration while Cyr61KD animals were devoid of peripheral nerve regeneration at 4 weeks. At 10 weeks post-grafting, peripheral nerve regeneration was at a minimal level for both Cyr61OX and Wnt5aOX cell lines. Blood vessel and bladder functionality were evident at both time-points in all animals. Results from this study indicate that MSC-based Cyr61OX and Wnt5aOX cell lines play pivotal roles with regards to increasing the levels of functional vasculature, influencing muscle regeneration, and the regeneration of peripheral nerves in a model of bladder augmentation. Wnt5aOX constructs closely approximated the outcomes previously observed with the co-transplantation of MSCs with CD34⁺ HSPCs and may be specifically targeted as an alternate means to achieve functional bladder regeneration.     

Variation of ginkgolides and bilobalide contents in leaves and cell cultures of<Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

Ginkgolides (GK) and bilobalide are valuable compounds that belong to the lactone terpene. The contents of these metabolites were determined by HPLC from female and male tree ofGinkgo biloba L. The productivity ofG. biloba cells was also compared with the corresponding individual trees. High variations in the ginkgolides and bilobalide were observed from different individuals, plant parts, and cultured cells. The ginkgolides and bilobalide contents were different depending on the plant parts. Callus was obtained from various plant tissues, and NAA was better at callogenesis than 2,4-D in both the female and male trees. The plants and their corresponding cells showed considerable variation in their ginkgolides and bilobalide concentrations. The ginkgolides and bilobalide contents were not correlated with the production between dominant trees and their corresponding cells. Light irradiation enhanced the production of GK-A and GK-B, however, the concentration of bilobalide decreased under dark conditions.     

Suppression of pacemaker activity by Ginkgo biloba extract and its main constituent, bilobalide in rat sino-atrial nodal cells

Effects of Ginkgo biloba extract (GBE) and bilobalide (a main constituent) on the pacemaker activity and the underlying ionic currents in rat sino-atrial (SA) nodal cells were investigated using patch-clamp techniques. Both GBE and bilobalide depressed the pacemaker activity in a concentration-dependent manner. At both 0.03 mg/ml GBE and 0.3 microM bilobalide, a negative chronotropic effect was produced. Dysrhythmias often occurred. The L-type Ca(2+) current (I(Ca)) and the hyperpolarization-activated inward current (I(f)) decreased by 69.7+/-3.2% (n=6, P<0.001) and by 12.6+/-2.1% (n=7, P<0.05) at 0.03 mg/ml GBE, and by 51.2+/-3.3% (n=6, P<0.01) and by 19.8+/-2.2 % (n=6, P<0.05) at 0.3 microM bilobalide, respectively. The delayed rectifier K(+) current (I(K)) also decreased. The inhibition was 12.3+/-2.0% (n=6, P<0.05) at 0.03 mg/ml GBE, and was 28.0+/-2.9% (n=6, P<0.05) at 0.3 microM bilobalide. These results indicate that cardiac ionic channels contributing to the pacemaking are highly sensitive to GBE and bilobalide, which can sufficiently modify the spontaneous activity in rat SA nodal cells.     

Improved Neurological Outcome by Intramuscular Injection of Human Amniotic Fluid Derived Stem Cells in a Muscle Denervation Model

PurposeThe skeletal muscle develops various degrees of atrophy and metabolic dysfunction following nerve injury. Neurotrophic factors are essential for muscle regeneration. Human amniotic fluid derived stem cells (AFS) have the potential to secrete various neurotrophic factors necessary for nerve regeneration. In the present study, we assess the outcome of neurological function by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model.ResultsNT-3 (Neurotrophin 3), BDNF (Brain derived neurotrophic factor), CNTF (Ciliary neurotrophic factor), and GDNF (Glia cell line derived neurotrophic factor) were highly expressed in AFS cells and supernatant of culture medium. Intra-muscular injection of AFS exerted significant expression of several neurotrophic factors over the distal end of nerve and denervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with a reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology with high expression of desmin and acetylcholine receptors. Up to two months, AFS produced significant improvement in electrophysiological study and neurological functions such as SFI (sciatic nerve function index) and Catwalk gait analysis. There was also significant preservation of the number of anterior horn cells and increased nerve myelination as well as muscle morphology.ConclusionIntramuscular injection of AFS can protect muscle apoptosis and likely does so through the secretion of various neurotrophic factors. This protection furthermore improves the nerve regeneration in a long term nerve anastomosis model.     

Morphological changes in rat skeletal muscle following reinnervation

Morphological changes appearing in the course of muscle regeneration after reinnervation of denervated M. soleus (slow) and M. tibialis anterior (fast) rat skeletal muscle were investigated. It was found that pathological changes typical for denervation atrophy (seen on the 10th day after crushing the sciatic nerve) and symptoms of regeneration (beginning about the 15th day) were much more pronounced in the soleus than in the tibialis muscle. Some stages of regeneration in the soleus muscle could be distinguished. The contractile material destructions were the first pathological changes that disappeared after the beginning of regeneration. In the second stage other denervation changes disappeared and intensive regeneration of muscle fibres was observed. In the next stage regeneration slowed down, and the reduction of the excess of muscle nuclei was visible. Four months after crushing the nerve, regeneration proceeded to completion with only some traces of the passed processes: in the soleus muscle, chains of sarcolemmal nuclei, satellite cells and newly formed muscle fibres were more often seen than in contralateral muscle; in the tibialis, collagen depots were present around the vessels and between muscle fascicles.     

Cytotactin is involved in synaptogenesis during regeneration of the frog neuromuscular system.

The expression of cytotactin, an extracellular matrix glycoprotein involved in morphogenesis and regeneration, was determined in the normal and regenerating neuromuscular system of the frog Rana temporaria. Cytotactin was expressed in adult brain and gut as two major components of Mr 190,000 and 200,000 and a minor form of higher molecular weight, but was almost undetectable in skeletal muscle extract. However, cytotactin was concentrated at the neuromuscular junctions as well as at the nodes of Ranvier. After nerve transection, cytotactin staining increased in the distal stump along the endoneurial tubes. In preparations of basal lamina sheaths of frog cutaneous pectoris muscle obtained by inducing the degeneration of both nerve and muscle fibers, cytotactin was found in dense accumulations at original synaptic sites. In order to define the role of cytotactin in axonal regeneration and muscle reinnervation, the effect of anti-cytotactin antibodies on the reinnervation of the basal lamina sheaths preparations was examined in vivo. In control preparations, regenerating nerve terminals preferentially reinnervate the original synaptic sites. In the presence of anti-cytotactin antibodies, axon regeneration occurred with normal fasciculation and branching but with altered preterminal nerve fibers pathways. Ultrastructural observations showed that synaptic basal laminae reinnervation was greatly delayed or inhibited. These results suggest that cytotactin plays a primordial role in synaptogenesis, at least during nerve regeneration and reinnervation in the adult neuromuscular system.     

Extracellular vesicles from human umbilical cord mesenchymal stem cells improve nerve regeneration after sciatic nerve transection in rats

Peripheral nerve injury results in limited nerve regeneration and severe functional impairment. Mesenchymal stem cells (MSCs) are a remarkable tool for peripheral nerve regeneration. The involvement of human umbilical cord MSC‐derived extracellular vesicles (hUCMSC‐EVs) in peripheral nerve regeneration, however, remains unknown. In this study, we evaluated functional recovery and nerve regeneration in rats that received hUCMSC‐EV treatment after nerve transection. We observed that hUCMSC‐EV treatment promoted the recovery of motor function and the regeneration of axons; increased the sciatic functional index; resulted in the generation of numerous axons and of several Schwann cells that surrounded individual axons; and attenuated the atrophy of the gastrocnemius muscle. hUCMSC‐EVs aggregated to rat nerve defects, down‐regulated interleukin (IL)‐6 and IL‐1β, up‐regulated IL‐10 and modulated inflammation in the injured nerve. These effects likely contributed to the promotion of nerve regeneration. Our findings indicate that hUCMSC‐EVs can improve functional recovery and nerve regeneration by providing a favourable microenvironment for nerve regeneration. Thus, hUCMSC‐EVs have considerable potential for application in the treatment of peripheral nerve injury.