Abstract: Complete transection of peripheral mixed nerves immediately produces loss of sensory perception, muscle contractions and voluntary behavior mediated by the severed distal axons. In contrast to natural regeneration (~1 mm/d) of proximal axons that may eventually reinnervate denervated targets, re-innervation is restored within minutes by PEG-fusion that consists of  neurorrhaphy and a sequence of well specified hypo- and isotonic calcium-free or calcium-containing solutions, the anti-oxidant methylene blue (MB) and the membrane fusogen polyethylene glycol (PEG). In this study, we examined the relative efficacy of PEG-fusion with no MB (0%), 0.5% MB, or 1% MB on the recovery of voluntary behaviors by female Sprague-Dawley rats with a complete mid-thigh severance of their sciatic nerve bathed in extracellular fluid or calcium-containing isotonic saline. The recovery of voluntary behaviors is the most relevant measure of success of any technique to repair peripheral nerve injuries. We assessed recovery by the sciatic functional index, a commonly used measure of voluntary hindlimb behaviors following complete sciatic transections. We reported that both 1% MB and 0.5% MB in sterile distilled water in our PEG-fusion protocol with neurorrhaphy significantly increased the rate and extent of behavioral recovery compared to PEG plus neurorrhaphy alone. Furthermore, 0.5% MB was as effective as 1% MB in voluntary behavioral recovery as assessed by the sciatic functional index. Since sterile 1% MB is no longer clinically available, we therefore recommend that 0.5% MB be included in upcoming human clinical trials to evaluate the safety and efficacy of PEG-fusion. All animal procedures were approved by the University of Texas Institutional Animal Care and Use Committee (AUP-2019-00225) on September 9, 2020.

Key words: axotomy, methylene blue, nerve repair, neurorrhaphy, peripheral nerve injury, polyethylene glycol fusion, sciatic nerve