Investigating Mechanisms Responsible for the Pro-Regenerative Effects of Conditioning Electrical Stimulation

Abstract

Though capable of regeneration, repair surgeries following peripheral nerve injury are often incomplete or entirely unsuccessful. The slow rate of regeneration (1-3mm/day) of a proximal injury requires up to two years to reach the distal target, whereas significant muscle atrophy occurs after 6-12 months; therefore, even if the regenerating axons reach these muscles they may no longer be amenable to reinnervation (Ginsell and Keating, 2014). Despite the advancements in surgical techniques, over half of patients with peripheral nerve injury experience lifelong deficits that can severely impact quality of life (Ruijs et al., 2005). The conditioning crush lesion (CCL), a technique in which a crush injury is performed one week prior to a nerve transection and repair surgery, increases the rate of nerve growth 2-4-fold (Richardson and Issa, 1984). CCL has not been clinically translated as it requires an intentional nerve injury which evokes an invasive inflammatory response. The Webber laboratory has demonstrated that conditioning electrical stimulation (CES) upregulates the same regeneration-associated genes (RAGs) as CCL to accelerate nerve regeneration and promote sensorimotor functional recovery (Senger et al., 2018; Senger et al., 2019). Prior to clinical application of CES we sought to confirm that unlike CCL, CES is non-injurious and non-inflammatory. Infiltrating and resident macrophages did not play a role in the pro-regenerative effects of CES, with decreased IBA-1 and dectin-1 immunofluorescence compared to CCL. Furthermore, macrophage ablation reduced the conditioning effect associated with CCL but not CES. We also investigated the heat-shock protein, alphaB-crystallin, previously found to support remyelination. Proteomics and mass spectrometry identified a five-fold decrease in alphaB-Crystallin protein expression in CES compared to CCL sensory neurons, which was not reproduced in Western Blot Analysis. AlphaB-crystallin null mice displayed increased inherent pCREB expression, all other regeneration-associated genes were not different from wildtype mice, and the conditioning effect was unaffected.