Polyethylene glycol solutions rapidly restore and maintain axonal continuity, neuromuscular structures, and behaviors lost after sciatic nerve transections in female rats

Abstract

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.

Polyethylene glycol treated allografts not tissue matched nor immunosuppressed rapidly repair sciatic nerve gaps, maintain neuromuscular functions, and restore voluntary behaviors in female rats

“…Female Sprague-Dawley rats weighing 250–300g (n = 79) were anesthetized and maintained with a small inhaled isoflurane anesthetic system (Handlebar Anesthesia, Austin, TX), as previously described for this and other basic surgical procedures in sterile conditions (Mikesh et al, 2018). A 25–35mm incision was made in the skin to expose the sciatic nerve parallel to the biceps femoris muscle.…”

“…Extracellular compound action potentials (CAPs) were evoked prior to and immediately following surgical procedures in all animals to assess electrophysiological function and continuity (Riley et al, 2015; Mikesh et al, 2018). Stimulating and recording electrodes were placed just proximal and just distal to the autograft or allograft and a CAP was recorded to assess whether axonal continuity was re-established across the graft.…”

“…Animals receiving an autograft or allograft were behaviorally tested using the Sciatic Function Index (SFI) and blindly tested and scored, as described in the previous paper (Mikesh et al, 2018). Animals were handled and trained prior to surgery to acclimate rats to the testing procedures.…”

“…Soleus muscles were taken from Unoperated rats (n=2) and the operated sides of PEG-fused (n=7) or Negative Control (n= 4) rats 7, 21, or 42d PO and labelled with antibodies specific to nerves (SV2 and 2H3, DSHB) and conjugated bungarotoxin to label acetylcholine receptors, as previously described (Kang et al, 2007; Mikesh et al, 2018). …”

“…In contrast to the poor outcomes from conventional repair techniques, animals with 5–10mm nerve gaps repaired with viable PEG-fused interposition autografts or (especially) allografts exhibit: ( 1 ) excellent restoration of sensory and motor functions beginning as early as three days postoperatively, ( 2 ) reduction or prevention of Wallerian degeneration of axons, and ( 3 ) reduction or prevention of muscle fiber atrophy or deterioration. However, as reported for single transections in a previous paper (Mikesh et al, 2018), we have not thoroughly examined the structure and/or function of severed donor graft axons or host distal axons, nor neuromuscular junctions (NMJs) and muscle fibers in this model. We have also never quantitatively evaluated the time course of behavioral recovery at various PO times and whether the restored behavior is mediated by: 1 ) immediate reinnervation by axons that are artificially PEG-fused and then continuously maintained, versus 2 ) delayed reinnervation by natural regeneration of axons proximal to the lesion site.…”