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Unlike A Conditioning Crush Lesion, Conditioning Electrical Stimulation Promotes Functional Sensory And Motor Nerve Regeneration In A Non-inflammatory Manner
Christine A. Webber, Ph.D., Jenna-Lynn Senger, Ph.D., M.D., Leah Acton, Susanne Lingrell, K. Ming Chan, M.D..
University of Alberta, Edmonton, AB, Canada.

PURPOSE: Prior to a nerve repair, conditioning electrical stimulation (CES) promotes regeneration associated gene (RAG) expression at the neuronal cell bodies and promotes functional nerve regeneration as well as, or beyond that of a conditioning crush lesion (CCL) (Senger et al., 2017; 2019). Forty years of research has established that the inflammatory response evoked by the CCL injury prior to the cut and coaptation is essential to the conditioning response. It is not known how CES promotes nerve regeneration. We propose that CES promotes functional nerve regeneration in a non-injurious, and non-inflammatory manner.
METHODS: Sprague Dawley rats were equally divided into three cohorts (n=10/cohort): i) CES, ii) CCL (positive control) and iv) sham-ES (negative control). CES, CCL and sham-ES conditioning were delivered one week prior to nerve cut/coaptation. The conditioning site and the corresponding dorsal root ganglion neurons were harvest at 1 day, 3 day and 14 days post-conditioning (thus the 14 day cohort was the only one to receive cut/coaptation). Immunocytochemistry for macrophages and neurofilament to investigate inflammation and Wallerian degeneration, respectively was performed and this data was corroborated by qRT-PCR analysis. Immunocytochemistry and qRT-PCR analysis were performed on the neuronal cell bodies to study the RAG expression following conditioning. In a separate experiment, CCR2-/- (macrophage depleted transgenic mice) and wild type C57/Bl mice (n=6/cohort) were conditioned with CES, CCL or no-ES. Previous studies have shown that CCL does not evoke a conditioning effect in the CCR2-/- mice indicating the inflammatory response is necessary for this effect. These experiments were designed to determine if macrophages are necessary for CES to promote nerve regeneration.
RESULTS: Rats treated with CCL alone at all time points evoked an inflammatory response. Unlike CES and sham-ES, there was significant macrophage infiltration at 1 and 3 days in the CCL animals, whereas both CES and CCL demonstrated accelerate nerve regeneration at 14 days post-conditioning. Furthermore, although CCL induced Wallerian degeneration distal to the conditioning site, CES and sham-ES did not cause degeneration of the distal axons. At the neuronal cell bodies, qRT-PCR and immunocytochemistry demonstrated there was an increase in inflammatory (IL-6) and injury markers (ATF3) at 24 hours post-injury in the CCL cohort only whereas the RAG upregulation (pCREB, GAP43, BDNF) of CES and CCL was similar at 3 days. These data suggest that CES and CCL may upregulate RAGs through independent but converging pathways. In the mouse study, the CES treated CCR2-/- (macrophage depleted) mice both showed the same upregulation of pCREB, GAP-43 and BDNF as their corresponding C57/Bl wildtype mice, suggesting CES does not require the immune response to evoke the conditioning effect.
CONCLUSION: CES significantly improves regeneration and reinnervation beyond that attainable with current clinical standards. It is a clinically feasible method of improving outcomes for patients with peripheral nerve injury as it does not evoke an inflammatory response or Wallerian degeneration. Further investigation is required to delineate the molecular and cellular mechanisms associated with the ability of CES to promote regeneration without evoking an immune response.


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