Effects of extracellular calcium and surgical techniques on restoration of axonal continuity by polyethylene glycol fusion following complete cut or crush severance of rat sciatic nerves

Abstract

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.

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

“…Ephaptic activation of CAPs has not been observed in this or previous studies (Britt et al, 2010; Bittner et al, 2012, 2016a,b;2017a,b; Riley et al, 2015; Ghergherehchi et al, 2016). …”

“…Data was recorded in spreadsheets and graphs were prepared in Graphpad Prism 7. PEG-fused animals were deemed to have a successful recovery if they achieved an SFI score of -59 or better by 42d PO, a value 3SD greater than obtained by Negative Control animals at 42d PO (Ghergherehchi et al, 2016); significant recovery at p < 0.05 would be an SFI score of − 75 or better in this and previous studies.…”

“…1 d–f). [Other publications have also shown diffusion of intra-axonal dyes across the lesion site (Bittner et al, 2012; Ghergherehchi et al, 2016)]. 2 Distal segments of singly-transected axons that are continuously maintained from 0-42d PO, i.e., reducing or preventing axonal Wallerian degeneration as assessed by gross anatomical inspection (Fig. 3) and TEM of axonal cross sections (Figs.…”

“…Runs without stops or hesitation producing three consecutive footprints of the left and right foot were scored and recorded. Two successful runs were collected for each rat per testing day (Britt et al, 2010; Ghergherehchi et al, 2016). For both the normal and the experimental sides, assessors blinded to their treatment measured six variables for the normal and experimental footprints: footprint length, total toe spread, and intermediate toe spread as previously reported (Carlton and Goldberg, 1986).…”

“…A two-tailed Student’s t-test, a test for homoscedastic data, was used to compare mean SFI scores for each treatment group on a given post-operative day. Two-way ANOVAs were used to analyze means and standard errors of SFI scores (Bittner et al, 2012; Riley et al, 2015; Ghergherehchi et al, 2016; Mikesh et al, 2018). …”