ABSTRACT
JOURNAL/nrgr/04.03/01300535-202504000-00033/figure1/v/2024-07-06T104127Z/r/image-tiff Behavioral recovery using (viable) peripheral nerve allografts to repair ablation-type (segmental-loss) peripheral nerve injuries is delayed or poor due to slow and inaccurate axonal regeneration. Furthermore, such peripheral nerve allografts undergo immunological rejection by the host immune system. In contrast, peripheral nerve injuries repaired by polyethylene glycol fusion of peripheral nerve allografts exhibit excellent behavioral recovery within weeks, reduced immune responses, and many axons do not undergo Wallerian degeneration. The relative contribution of neurorrhaphy and polyethylene glycol-fusion of axons versus the effects of polyethylene glycol per se was unknown prior to this study. We hypothesized that polyethylene glycol might have some immune-protective effects, but polyethylene glycol-fusion was necessary to prevent Wallerian degeneration and functional/behavioral recovery. We examined how polyethylene glycol solutions per se affect functional and behavioral recovery and peripheral nerve allograft morphological and immunological responses in the absence of polyethylene glycol-induced axonal fusion. Ablation-type sciatic nerve injuries in outbred Sprague-Dawley rats were repaired according to a modified protocol using the same solutions as polyethylene glycol-fused peripheral nerve allografts, but peripheral nerve allografts were loose-sutured (loose-sutured polyethylene glycol) with an intentional gap of 1-2 mm to prevent fusion by polyethylene glycol of peripheral nerve allograft axons with host axons. Similar to negative control peripheral nerve allografts not treated by polyethylene glycol and in contrast to polyethylene glycol-fused peripheral nerve allografts, animals with loose-sutured polyethylene glycol peripheral nerve allografts exhibited Wallerian degeneration for all axons and myelin degeneration by 7 days postoperatively and did not recover sciatic-mediated behavioral functions by 42 days postoperatively. Other morphological signs of rejection, such as collapsed Schwann cell basal lamina tubes, were absent in polyethylene glycol-fused peripheral nerve allografts but commonly observed in negative control and loose-sutured polyethylene glycol peripheral nerve allografts at 21 days postoperatively. Loose-sutured polyethylene glycol peripheral nerve allografts had more pro-inflammatory and less anti-inflammatory macrophages than negative control peripheral nerve allografts. While T cell counts were similarly high in loose-sutured-polyethylene glycol and negative control peripheral nerve allografts, loose-sutured polyethylene glycol peripheral nerve allografts expressed some cytokines/chemokines important for T cell activation at much lower levels at 14 days postoperatively. MHCI expression was elevated in loose-sutured polyethylene glycol peripheral nerve allografts, but MHCII expression was modestly lower compared to negative control at 21 days postoperatively. We conclude that, while polyethylene glycol per se reduces some immune responses of peripheral nerve allografts, successful polyethylene glycol-fusion repair of some axons is necessary to prevent Wallerian degeneration of those axons and immune rejection of peripheral nerve allografts, and produce recovery of sensory/motor functions and voluntary behaviors. Translation of polyethylene glycol-fusion technologies would produce a paradigm shift from the current clinical practice of waiting days to months to repair ablation peripheral nerve injuries.
ABSTRACT
Pseudomonas aeruginosa is a pathogen that forms robust biofilms which are commonly associated with chronic infections and cannot be successfully cleared by the immune system. Neutrophils, the most common white blood cells, target infections with pathogen-killing mechanisms that are rendered largely ineffective by the protective physicochemical structure of a biofilm. Visualisation of the complex interactions between immune cells and biofilms will advance understanding of how biofilms evade the immune system and could aid in developing treatment methods that promote immune clearance with minimal harm to the host. Scanning electron microscopy (SEM) distinguishes itself as a powerful, high-resolution tool for obtaining strikingly clear and detailed topographical images. However, taking full advantage of SEM's potential for high-resolution imaging requires that the fixation process simultaneously preserve both intricate biofilm architecture and the morphologies and structural signatures characterising neutrophils responses at an infection site. Standard aldehyde-based fixation techniques result in significant loss of biofilm matrix material and morphologies of responding immune cells, thereby obscuring the details of immune interactions with the biofilm matrix. Here we show an improved fixation technique using the cationic dye alcian blue to preserve and visualise neutrophil interactions with the three-dimensional architecture of P. aeruginosa biofilms. We also demonstrate that this technique better preserves structures of biofilms grown from two other bacterial species, Klebsiella pneumoniae and Burkholderia thailandensis.
Subject(s)
Biofilms , Neutrophils , Microscopy, Electron, ScanningABSTRACT
Klebsiella pneumoniae biofilm formation is associated with chronic and relapsing infections. Scanning electron microscopy (SEM) is a powerful tool for characterizing biofilm structure and studying their formation. Reliable visualization of biofilm structure requires careful sample preservation, otherwise there may be loss of non-covalent interactions that are susceptible to damage during the dehydration and washing preparation steps. However, no standard procedure has been adopted in the literature to fix K. pneumoniae biofilm for scanning electron microscopy studies. This lack of standardization makes it challenging to compare results between studies and determine the degree to which native structures have been preserved. To advance this critical area of study, we investigated different scanning electron microscopy fixation methods for K. pneumoniae biofilm preservation. Our study reveals the impact preparation steps can have on retaining in biofilm architecture observed using scanning electron microscopy. Using fixation methods developed through our studies, we show that although species that overproduce capsular extracellular polysaccharides produced more robust biofilms, K. pneumoniae can form a developed biofilm in the absence of capsular polysaccharides.
ABSTRACT
Ablation/segmental loss peripheral nerve injuries (PNIs) exhibit poor functional recovery due to slow and inaccurate outgrowth of regenerating axons. Viable peripheral nerve allografts (PNAs) as growth-guide conduits are immunologically rejected and all anucleated donor/host axonal segments undergo Wallerian degeneration. In contrast, we report that ablation-type sciatic PNIs repaired by neurorrhaphy of viable sciatic PNAs and a polyethylene glycol (PEG)-fusion protocol using PEG immediately restored axonal continuity for many axons, reinnervated/maintained their neuromuscular junctions, and prevented much Wallerian degeneration. PEG-fused PNAs permanently restored many sciatic-mediated behaviors within 2-6 weeks. PEG-fused PNAs were not rejected even though host/donors were neither immunosuppressed nor tissue-matched in outbred female Sprague Dawley rats. Innate and adaptive immune responses to PEG-fused sciatic PNAs were analyzed using electron microscopy, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction for morphological features, T cell and macrophage infiltration, major histocompatibility complex (MHC) expression, apoptosis, expression of cytokines, chemokines, and cytotoxic effectors. PEG-fused PNAs exhibited attenuated innate and adaptive immune responses by 14-21 days postoperatively, as evidenced by (a) many axons and cells remaining viable, (b) significantly reduced infiltration of cytotoxic and total T cells and macrophages, (c) significantly reduced expression of inflammatory cytokines, chemokines, and MHC proteins, (d) consistently low apoptotic response. Morphologically and/or biochemically, PEG-fused sciatic PNAs often resembled sciatic autografts or intact sciatic nerves. In brief, PEG-fused PNAs are an unstudied, perhaps unique, example of immune tolerance of viable allograft tissue in a nonimmune-privileged environment and could greatly improve the clinical outcomes for PNIs relative to current protocols.
Subject(s)
Adaptive Immunity/physiology , Immunity, Innate/physiology , Polyethylene Glycols/administration & dosage , Sciatic Nerve/immunology , Sciatic Nerve/transplantation , Sciatic Neuropathy/therapy , Adaptive Immunity/drug effects , Allografts/immunology , Allografts/transplantation , Animals , Female , Immunity, Innate/drug effects , Male , Rats , Rats, Sprague-Dawley , Sciatic Neuropathy/immunology , Transplantation, Homologous/methodsABSTRACT
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.
Subject(s)
Neuromuscular Junction/physiology , Neuronal Plasticity/physiology , Schwann Cells/physiology , Animals , Cell Differentiation/physiology , Cell Proliferation/physiology , Diphtheria Toxin/pharmacology , Female , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron , Schwann Cells/drug effects , Synapses/physiology , Tamoxifen/pharmacologyABSTRACT
Synapses undergo transition from polyinnervation by multiple axons to single innervation a few weeks after birth. Synaptic activity of axons and interaxonal competition are thought to drive this developmental synapse elimination and tested as key parameters in quantitative models for further understanding. Recent studies of muscle synapses (endplates) show that there are also terminal Schwann cells (tSCs), glial cells associated with motor neurons and their functions, and vacant sites (or vacancies) devoid of tSCs and axons proposing tSCs as key effectors of synapse elimination. However, there is no quantitative model that considers roles of tSCs including vacancies. Here we develop a stochastic model of tSC and vacancy mediated synapse elimination. It employs their areas on individual endplates quantified by electron microscopy-based analyses assuming that vacancies form randomly and are taken over by adjacent axons or tSCs. The model reliably reproduced synapse elimination whereas equal or random probability models, similar to classical interaxonal competition models, did not. Furthermore, the model showed that synapse elimination is accelerated by enhanced synaptic activity of one axon and also by increased areas of vacancies and tSCs suggesting that the areas are important structural correlates of the rate of synapse elimination.
Subject(s)
Axons/physiology , Neuromuscular Junction/physiopathology , Schwann Cells/physiology , Synapses/physiology , Animals , Computer Simulation , Female , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron, Scanning , Motor Neurons/physiology , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/innervation , Stochastic ProcessesABSTRACT
Polyethylene glycol repair (PEG-fusion) of severed sciatic axons restores their axoplasmic and membrane continuity, prevents Wallerian degeneration, maintains muscle fiber innervation, and greatly improves recovery of voluntary behaviors. We examined alterations in spinal connectivity and motoneuron dendritic morphology as one potential mechanism for improved behavioral function after PEG-fusion. At 2-112 days after a single-cut or allograft PEG-fusion repair of transected or ablated sciatic nerves, the number, size, location, and morphology of motoneurons projecting to the tibialis anterior muscle were assessed by retrograde labeling. For both lesion types, labeled motoneurons were found in the appropriate original spinal segment, but also in inappropriate segments, indicating mis-pairings of proximal-distal segments of PEG-fused motor axons. Although the number and somal size of motoneurons was unaffected, dendritic distributions were altered, indicating that PEG-fusion preserves spinal motoneurons but reorganizes their connectivity. This spinal reorganization may contribute to the remarkable behavioral recovery seen after PEG-fusion repair.
Subject(s)
Motor Neurons/metabolism , Neural Conduction , Sciatic Neuropathy/etiology , Sciatic Neuropathy/metabolism , Action Potentials , Allografts , Animals , Cell Count , Dendrites/metabolism , Disease Models, Animal , Electrophysiological Phenomena , Female , Immunohistochemistry , Motor Neurons/cytology , Nerve Regeneration , Polyethylene Glycols , Rats , Recovery of Function , Sciatic Neuropathy/rehabilitationSubject(s)
Facial Nerve Injuries , Facial Paralysis , Animals , Antioxidants , Facial Nerve , Nerve Regeneration , Polyethylene Glycols , RatsSubject(s)
Facial Nerve Injuries , Animals , Axons , Facial Nerve , Nerve Regeneration , Polyethylene Glycols , RatsABSTRACT
BACKGROUND: Nervous system injuries in mammals often involve transection or segmental loss of peripheral nerves. Such injuries result in functional (behavioral) deficits poorly restored by naturally occurring 1-2 mm/d axonal outgrowths aided by primary repair or reconstruction. "Neurorrhaphy" or nerve repair joins severed connective tissues, but not severed cytoplasmic/plasmalemmal extensions (axons) within the tissue. NEW METHOD: PEG-fusion consists of neurorrhaphy combined with a well-defined sequence of four pharmaceutical agents in solution, one containing polyethylene glycol (PEG), applied directly to closely apposed viable ends of severed axons. RESULTS: PEG-fusion of rat sciatic nerves: (1) restores axonal continuity across coaptation site(s) within minutes, (2) prevents Wallerian degeneration of many distal severed axons, (3) preserves neuromuscular junctions, (4) prevents target muscle atrophy, (5) produces rapid and improved recovery of voluntary behaviors compared with neurorrhaphy alone, and (6) PEG-fused allografts are not rejected, despite no tissue-matching nor immunosuppression. COMPARISON WITH EXISTING METHODS: If PEG-fusion protocols are not correctly executed, the results are similar to that of neurorrhaphy alone: (1) axonal continuity across coaptation site(s) is not re-established, (2) Wallerian degeneration of all distal severed axons rapidly occurs, (3) neuromuscular junctions are non-functional, (4) target muscle atrophy begins within weeks, (5) recovery of voluntary behavior occurs, if ever, after months to levels well-below that observed in unoperated animals, and (6) allografts are either rejected or not well-accepted. CONCLUSION: PEG-fusion produces rapid and dramatic recovery of function following rat peripheral nerve injuries.
Subject(s)
Neuroprotective Agents/pharmacology , Neurosurgical Procedures , Polyethylene Glycols/pharmacology , Sciatic Nerve/drug effects , Sciatic Nerve/injuries , Allografts , Animals , Axons/drug effects , Axons/pathology , Disease Models, Animal , Female , Male , Neuromuscular Junction/pathology , Neurosurgical Procedures/methods , Random Allocation , Rats, Sprague-Dawley , Sciatic Nerve/pathology , Suture Techniques , Wallerian Degeneration/prevention & controlABSTRACT
Many publications report that ablations of segments of peripheral nerves produce the following unfortunate results: (1) Immediate loss of sensory signaling and motor control; (2) rapid Wallerian degeneration of severed distal axons within days; (3) muscle atrophy within weeks; (4) poor behavioral (functional) recovery after many months, if ever, by slowly-regenerating (â¼1mm/d) axon outgrowths from surviving proximal nerve stumps; and (5) Nerve allografts to repair gap injuries are rejected, often even if tissue matched and immunosuppressed. In contrast, using a female rat sciatic nerve model system, we report that neurorrhaphy of allografts plus a well-specified-sequence of solutions (one containing polyethylene glycol: PEG) successfully addresses each of these problems by: (a) Reestablishing axonal continuity/signaling within minutes by nonspecific ally PEG-fusing (connecting) severed motor and sensory axons across each anastomosis; (b) preventing Wallerian degeneration by maintaining many distal segments of inappropriately-reconnected, PEG-fused axons that continuously activate nerve-muscle junctions; (c) maintaining innervation of muscle fibers that undergo much less atrophy than otherwise-denervated muscle fibers; (d) inducing remarkable behavioral recovery to near-unoperated levels within days to weeks, almost certainly by CNS and PNS plasticities well-beyond what most neuroscientists currently imagine; and (e) preventing rejection of PEG-fused donor nerve allografts with no tissue matching or immunosuppression. Similar behavioral results are produced by PEG-fused autografts. All results for Negative Control allografts agree with current neuroscience data 1-5 given above. Hence, PEG-fusion of allografts for repair of ablated peripheral nerve segments expand on previous observations in single-cut injuries, provoke reconsideration of some current neuroscience dogma, and further extend the potential of PEG-fusion in clinical practice.
Subject(s)
Nerve Regeneration/drug effects , Peroneal Nerve/drug effects , Peroneal Nerve/transplantation , Polyethylene Glycols/pharmacology , Sciatic Nerve/drug effects , Sciatic Neuropathy/therapy , Allografts/drug effects , Animals , Axons/drug effects , Axons/physiology , Axotomy , Disease Models, Animal , Female , Muscle, Skeletal , Nerve Fibers/drug effects , Neural Conduction/drug effects , Neuromuscular Junction/drug effects , Peripheral Nerve Injuries/pathology , Peripheral Nerve Injuries/therapy , Random Allocation , Rats , Rats, Sprague-Dawley , Recovery of Function/drug effects , Sciatic Nerve/pathology , Sciatic Nerve/physiology , Sciatic Nerve/surgery , Sciatic Neuropathy/chemically induced , Transplantation, Homologous , Wallerian Degeneration/prevention & controlABSTRACT
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 (â¼1 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.
Subject(s)
Axons/drug effects , Axons/physiology , Nerve Regeneration/drug effects , Neuromuscular Junction/drug effects , Neuromuscular Junction/physiology , Polyethylene Glycols/pharmacology , Sciatic Nerve/drug effects , Animals , Axotomy , Disease Models, Animal , Female , Nerve Regeneration/physiology , Neural Conduction/drug effects , Rats , Recovery of Function , Sciatic Nerve/physiology , Sciatic Nerve/surgery , Sciatic Neuropathy/chemically induced , Sciatic Neuropathy/drug therapy , Sciatic Neuropathy/pathology , Wallerian Degeneration/drug therapy , Wallerian Degeneration/pathologyABSTRACT
Transection of nerve axons (axotomy) leads to rapid (Wallerian) degeneration of the distal portion of the severed axon whereas the proximal portion and the soma often survive. Clinicians and neuroscientists have known for decades that somal survival is less likely for cells transected nearer to the soma, compared to further from the soma. Calcium ion (Ca(2+)) influx at the cut axonal end increases somal Ca(2+) concentration, which subsequently activates apoptosis and other pathways that lead to cell death. The same Ca(2+) influx activates parallel pathways that seal the plasmalemma, reduce Ca(2+) influx, and thereby enable the soma to survive. In this study, we have examined the ability of transected B104 axons to seal, as measured by uptake or exclusion of fluorescent dye, and quantified the relationship between sealing frequency and transection distance from the axon hillock. We report that sealing frequency is maximal at about 150µm (µm) from the axon hillock and decreases exponentially with decreasing transection distance with a space constant of about 40µm. We also report that after Ca(2+) influx is initiated, the curve of sealing frequency versus time is well-fit by a one-phase, rising exponential model having a time constant of several milliseconds that is longer nearer to, versus further from, the axon hillock. These results could account for the increased frequency of cell death for axotomies nearer to, versus farther from, the soma of many types of neurons.
Subject(s)
Axons/pathology , Neurons/pathology , Animals , Apoptosis , Axons/ultrastructure , Axotomy , Calcium/metabolism , Calcium Signaling , Cell Line, Tumor , Cell Membrane/metabolism , Fluorescent Dyes , Models, Neurological , Neurites , Neurons/ultrastructure , RatsABSTRACT
Synaptic connections in the nervous system are rearranged during development and in adulthood as a feature of growth, plasticity, aging, and disease. Glia are implicated as active participants in these changes. Here we investigated a signal that controls the participation of peripheral glia, the terminal Schwann cells (SCs), at the neuromuscular junction (NMJ) in mice. Transgenic manipulation of the levels of membrane-tethered neuregulin1 (NRG1-III), a potent activator of SCs normally presented on motor axons, alters the rate of loss of motor inputs at NMJs during developmental synapse elimination. In addition, NMJs of adult transgenic mice that expressed excess axonal NRG1-III exhibited continued remodeling, in contrast to the more stable morphologies of controls. In fact, synaptic SCs of these adult mice with NRG1-III overexpression exhibited behaviors evident in wild type neonates during synapse elimination, including an affinity for the postsynaptic myofiber surface and phagocytosis of nerve terminals. Given that levels of NRG1-III expression normally peak during the period of synapse elimination, our findings identify axon-tethered NRG1 as a molecular determinant for SC-driven neuromuscular synaptic plasticity.
Subject(s)
Motor Neurons/physiology , Neuregulin-1/physiology , Neuromuscular Junction/ultrastructure , Neuronal Plasticity/physiology , Schwann Cells/physiology , Synapses/physiology , Alternative Splicing , Amyloid Precursor Protein Secretases/genetics , Animals , Animals, Newborn , Aspartic Acid Endopeptidases/genetics , Axons/chemistry , Axons/physiology , Gene Dosage , Heterozygote , Membrane Proteins/physiology , Mice , Mice, Knockout , Mice, Transgenic , Motor Neurons/chemistry , Muscle, Skeletal/growth & development , Muscle, Skeletal/innervation , Neck Muscles/growth & development , Neck Muscles/innervation , Neuregulin-1/genetics , Neuromuscular Junction/growth & development , Neuromuscular Junction/metabolism , Phagocytosis/genetics , Protein Isoforms/genetics , Protein Isoforms/physiology , Receptors, Cholinergic/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction , Spinal Cord/cytology , Spinal Cord/growth & development , Synapses/ultrastructureABSTRACT
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 is restored only after months, if ever, by outgrowths regenerating at â¼1 mm/day from the proximal stumps of severed axonal segments. The distal stump of a severed axon typically begins to degenerate in 1-3 days. We recently developed a polyethylene glycol (PEG) fusion technology, consisting of sequential exposure of severed axonal ends to hypotonic Ca(2+) -free saline, methylene blue, PEG in distilled water, and finally Ca(2+) -containing isotonic saline. This study examines 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 midthigh. Also examined are 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 Ca(2+) -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 Ca(2+) -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. © 2016 Wiley Periodicals, Inc.
Subject(s)
Calcium/metabolism , Neurosurgery/methods , Polyethylene Glycols/therapeutic use , Recovery of Function/drug effects , Sciatic Neuropathy/drug therapy , Sciatic Neuropathy/surgery , Action Potentials/drug effects , Action Potentials/physiology , Animals , Axons/drug effects , Axons/physiology , Calcium/therapeutic use , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Female , Fluorescent Dyes/pharmacokinetics , Male , Mental Disorders/etiology , Mental Disorders/therapy , Nerve Regeneration/drug effects , Neural Conduction/drug effects , Neuromuscular Junction/drug effects , Neuromuscular Junction/pathology , Rats , Rats, Sprague-Dawley , Sciatic Neuropathy/complications , Time FactorsABSTRACT
Schwann cells (SCs) at neuromuscular junctions (NMJs) play active roles in synaptic homeostasis and repair. We have studied how SCs contribute to reinnervation of NMJs using vital imaging of mice whose motor axons and SCs are transgenically labeled with different colors of fluorescent proteins. Motor axons most commonly regenerate to the original synaptic site by following SC-filled endoneurial tubes. During the period of denervation, SCs at the NMJ extend elaborate processes from the junction, as shown previously, but they also retract some processes from territory they previously occupied within the endplate. The degree of this retraction depends on the length of the period of denervation. We show that the topology of the remaining SC processes influences the branching pattern of regenerating axon terminals and the redistribution of acetylcholine receptors (AChRs). Upon arriving at the junction, regenerating axons follow existing SC processes within the old synaptic site. Some of the AChR loss that follows denervation is correlated with failure of portions of the old synaptic site that lack SC coverage to be reinnervated. New AChR clustering is also induced by axon terminals that follow SC processes extended during denervation. These observations show that SCs participate actively in the remodeling of neuromuscular synapses following nerve injury by their guidance of axonal reinnervation.