Conundrums and confusions regarding how polyethylene glycol-fusion produces excellent behavioral recovery after peripheral nerve injuries

Abstract

Current Neuroscience dogma holds that transections or ablations of a segment of peripheral nerves produce: (1) Immediate loss of axonal continuity, sensory signaling, and motor control; (2) Wallerian rapid (1-3 days) degeneration of severed distal axons, muscle atrophy, and poor behavioral recovery after many months (if ever, after ablations) by slowly-regenerating (1 mm/d), proximal-stump outgrowths that must specifically reinnervate denervated targets; (3) Poor acceptance of microsutured nerve allografts, even if tissue-matched and immune-suppressed. Repair of transections/ablations by neurorrhaphy and well-specified-sequences of PEG-fusion solutions (one containing polyethylene glycol, PEG) successfully address these problems. However, conundrums and confusions regarding unorthodox and dramatic results of PEG-fusion repair in animal model systems often lead to misunderstandings. For example, (1) Axonal continuity and signaling is re-established within minutes by non-specifically PEG-fusing (connecting) severed motor and sensory axons across each lesion site, but remarkable behavioral recovery to near-unoperated levels takes several weeks; (2) Many distal stumps of inappropriately-reconnected, PEG-fused axons do not ever (Wallerian) degenerate and continuously innervate muscle fibers that undergo much less atrophy than otherwise-denervated muscle fibers; (3) Host rats do not reject PEG-fused donor nerve allografts in a non-immuno-privileged environment with no tissue matching or immunosuppression; (4) PEG fuses apposed open axonal ends or seals each shut (thereby preventing PEG-fusion), depending on the experimental protocol; (5) PEG-fusion protocols produce similar results in animal model systems and early human case studies. Hence, iconoclastic PEG-fusion data appropriately understood might provoke a re-thinking of some Neuroscience dogma and a paradigm shift in clinical treatment of peripheral nerve injuries.

Keywords: Wallerian degeneration; allograft; autograft; axonal repair; axotomy; nerve regeneration; polyethylene glycol.

Figure 1

Electrophysiological, morphologicical and behavioral results of PEG-fusion. (A, B) Electrophysiological evidence of sciatic nerve continuity within 5 minutes after successful allograft PEG-fusion. (A) CAP (mV) recordings after ablating a 1 cm segment, insertion of a > 1 cm donor segment without (NC: black dashed line) or with PEG-fusion (PEG: blue solid line) of both severed ends microsutured to the proximal or distal ends of the host sciatic nerve. CAP arrow: peak amplitude. As one essential positive control for immediate viability and success of PEG-fusion, we always extracellularly generate action potentials in sciatic nerves in the upper thigh proximal to all lesion sites and extracellularly record those CAPs conducted to the lower leg in all animals prior to, and after, any PEG-fusion procedure (Unop and PEG traces). (B) Through-conduction of CAPs across sites of PEG-fusion is often associated with a twitch and a CMAP of muscles in the calf and foot. Through-conduction of CAPs is lost after single cuts or 0.5–1 cm ablations in the mid-thigh and is not restored unless a lesion is successfully PEG-fused (NC in Figure 1A). Within minutes, PEG-fusion restores through-conducting CAPs from upper thigh to lower limb, as well as twitching and CMAPs of muscles in the calf and foot. CAP amplitudes after PEG-fusion are typically not as large when compared to CAPs initially recorded from the intact nerve, i.e., CAPs are a binary measure of PEG-fusion success because CAP amplitude depends on many variables including electrode placement. CAPs or CMAPs recorded at the time of initial PEG-fusion are not a measure of long-term PEG-fusion success because the PEG-fused ends can separate if not properly microsutured once the animal starts to use the orated limb. CAPs are a much better measure of initial success than CMAPs because CMAPs can be produced in the absence of direct innervation by ephaptic current spread. (C) Darkfield digital micrographs and matching computer-generated composites of transverse hemisections through the lumbar spinal cords of an intact control rat (unop), and a rat with a PEG-fused sciatic nerve allograft at postoperative (PO) day 42 (42 d PEG) following injection of horseradish peroxidase conjugated to the cholera toxin B subunit (BHRP) into the anterior tibialis muscle. Computer-generated (Neurolucida, MBF Bioscience) composites of BHRP-labeled somata and processes were drawn at 480 μm intervals through the entire rostrocaudal extent of the tibialis motor pool. In the unoperated animal, motoneuron labeling is restricted to the lumbar (L) 3 spinal segment. In the PEG-fused animal, labeled motoneurons are present in the L3 segment, but are now also found in the L4 and L5 segments; L4/5 motoneurons typically innervate other muscles of the lower leg and intrinsic foot muscles, but now project to the anterior tibialis. That is, reinnervation by continuously surviving PEG-fused motoneurons with inappropriate spinal to peripheral connections are somehow producing dramatically better behavioral recovery than is ever produced by motoneurons that re-innervate by slowly regenerating outgrowths that presumably must make appropriate connections to restore any lost behavior. Scale bar: 500 μm. (D) Behavioral recovery results as measured by the SFI test for 42 days PO after various surgical procedures in the mid-thigh to rat sciatic nerves as abbreviated in key for mean ± SE scores for (1) Sham operations in which the sciatic nerve is not cut (dotted gray line). (2) Allografts microsutured and PEG-fused after lesioning (solid green line). (3) Single cuts (transections) microsutured and PEG-fused (solid blue line); (4) Single Crush 1–2 mm long made with microforceps and PEG-fused (solid red line). (5) Single Crush 1–2 mm long made with microforceps but no PEG is applied (dotted black line). (6) Single cuts microsutured but no PEG is applied (solid black line). (7) Allografts microsutured but no PEG is applied (dashed black line). SFI scores are usually 0 ± 10 for unoperated animals and –90 to –110 for animals with a complete sciatic transection or ablation of a segment. SFIs for PEG-fusion protocols differ significantly from negative controls, *P < 0.05, **P < 0.01, ***P < 0.001 (detected by one way analysis of variance). Note that in the absence of application of a PEG-containing aqueous solution that behavioral recovery is very poor except for 1–2 mm crush lesions made by microforceps that leave endoneurial sheaths intact/continuous from proximal to distal across the lesion site. This short-length experimental crush lesion made by neuroscientists in mice or rats is essentially impossible to produce naturally in a large mammal and is never seen by clinicians (Green and Wolfe, 2011). PEG: Polyethylene glycol; SA: stimulus artifact; Unop: Unoperated; NC: negative controls; CAP: compound action potentials; CMAP: compound muscle action potentials.

Figure 2

PEG produces fusion of proximal and distal axons if their open, vesicle-free, cut ends are brought into close apposition by microsutures (“PEG-fusion”). If axonal ends are not brought into close apposition, PEG causes the membranes at the cut ends to collapse and seal (“PEG-sealing”). If axons are completely cut, a Ca²⁺-induced accumulation of vesicles (v) occurs naturally to form a plug that seals the severed cut end--or any small hole in an axolemma (Spaeth et al., 2012; Bittner et al., 2016).