Complete severance of major peripheral mixed sensory-motor nerve proximally in a mammalian limb produces immediate loss of action potential conduction and voluntary behaviors mediated by the severed distal axonal segments. These severed distal segments undergo Wallerian degeneration within days. Denervated muscles atrophy within weeks. Slowly regenerating (∼1 mm/day) outgrowths from surviving proximal stumps that often nonspecifically reinnervate denervated targets produce poor, if any, restoration of lost voluntary behaviors. In contrast, in this study using completely transected female rat sciatic axons as a model system, we provide extensive morphometric, immunohistochemical, electrophysiological, and behavioral data to show that these adverse outcomes are avoided by microsuturing closely apposed axonal cut ends (neurorrhaphy) and applying a sequence of well-specified solutions, one of which contains polyethylene glycol (PEG). This "PEG-fusion" procedure within minutes reestablishes axoplasmic and axolemmal continuity and signaling by nonspecifically fusing (connecting) closely apposed open ends of severed motor and/or sensory axons at the lesion site. These PEG-fused axons continue to conduct action potentials and generate muscle action potentials and muscle twitches for months and do not undergo Wallerian degeneration. Continuously innervated muscle fibers undergo much less atrophy compared with denervated muscle fibers. Dramatic behavioral recovery to near-unoperated levels occurs within days to weeks, almost certainly by activating many central nervous system and peripheral nervous system synaptic and other plasticities, some perhaps to a greater extent than most neuroscientists would expect. Negative control transections in which neurorrhaphy and all solutions except the PEG-containing solution are applied produce none of these remarkably fortuitous outcomes observed for PEG-fusion.
Complete crush- or cut- severance of sciatic nerve axons in rats and other mammals produces immediate loss of axonal continuity. Loss of locomotor functions subserved by those axons are not restored for months, if ever, by outgrowths regenerating at ~1 mm/d from the proximal stumps of severed axonal segments. The distal stump of a severed axon typically begins to degenerate in 1–3 days. We have recently developed a PEG-fusion technology consisting of sequential exposure of severed axonal ends to hypotonic Ca2+-free saline, methylene blue (MB), polyethylene glycol (PEG) in distilled water, and finally to Ca2+-containing isotonic saline. We examined factors that affect the PEG-fusion restoration of axonal continuity within minutes as measured by conduction of action potentials and diffusion of an intracellular fluorescent dye across the lesion site of rat sciatic nerves completely cut- or crush-severed in the mid-thigh. We also examined factors that affect the longer-term PEG-fusion restoration of lost behavioral functions within days to weeks as measured by the Sciatic Functional Index. We report that exposure of cut-severed axonal ends to Ca2+-containing saline prior to PEG-fusion and stretch/tension of proximal or distal axonal segments of cut-severed axons decrease PEG-fusion success. Conversely, trimming cut-severed ends in Ca2+-free saline just prior to PEG-fusion increases PEG-fusion success. PEG-fusion prevents or retards the Wallerian degeneration of cut-severed axons as assessed by measures of axon diameter and G ratio. PEG-fusion may produce a paradigm-shift in the treatment of peripheral nerve injuries.
During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs), however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout – Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors.
Schwann cells (Scs) are integral to the formation and function of the peripheral nervous system (pnS). Exemplifying their importance, the loss or dysfunction of SCs is a feature of a myriad of diseases and conditions that compromise the PNS. Thus, it remains essential to understand the rules that govern the proliferation, differentiation and reconnection of Schwann cells with peripheral axons. Here, we examined the consequences of locally and acutely ablating terminal Schwann cells (tSCs) at the adult mouse neuromuscular junction (nMJ) by using mice expressing diphtheria toxin receptor (DtR) preferentially in tScs compared to myelinating Scs followed by local application of diphtheria toxin (DTX). After DTX application, tSCs died but, importantly and contrary to expectations, their associated motor axons did not fully degenerate. Within 3 weeks, tSCs returned and reestablished coverage of the synapse with increased numbers. Furthermore, the post-synaptic muscle fibers displayed increased distinct clusters of acetylcholine receptors and axon terminals exhibited numerous terminal varicosities. The lack of degeneration of bare motor axon terminals and the morphological remodeling that occurs upon the return of tScs to the nMJ may have wider implications for the mechanisms governing tSC occupancy of the adult NMJ and for conditions that adversely affect tScs. Schwann cells (SCs), the glia of the peripheral nervous system (PNS), serve a variety of important roles 1-3. Terminal Schwann cells (tSCs) are a specialized group of non-myelinating SCs that cap the axon terminal of α-motor neurons at the neuromuscular junction (NMJ) 4,5. There is growing evidence that tSCs are affected in peripheral neuropathies, with some disease states resulting in tSC loss 6. It is therefore important to have a better understanding of the roles of tSCs at the healthy NMJ, the consequences of tSC disorder and death, and how the nervous system recovers from tSC injury. The NMJ, long used as a model synapse because of its large size and ease of accessibility, has three major components: the pre-synaptic motor axon terminal, the post-synaptic muscle fiber, and the tSCs, with evidence suggesting a fourth cell type, the kranocyte 7. Using the advantages of the NMJ as a model synapse, experiments selectively ablating tSCs have offered insight into the normal functions of tSCs and the consequences of glial cell death at the NMJ. The first such study involved the use of a tSC specific antibody in frogs to activate the complement system, which caused the selective ablation of tSCs 8. This study showed that in frogs, tSCs are not required for the acute function of the motor axon, but are necessary for the long-term integrity and maintenance of the NMJ. In addition, this study showed that when SCs were ablated in developing tadpoles, NMJs failed to form properly. In the following decades, a group of studies have utilized anti-ganglioside antibodies and a similar complement-mediated ablation of axons and tSCs in mice to mimic Guillain-Barré Syndrome 9. ...
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