Pathophysiologic Nerve Regeneration - ABSTRACT / PROJECT SUMMARY Closed-mechanism peripheral nerve injuries are among the most devastating neurologic injuries, often with complete loss of functional use of a limb. Nerve regeneration, i.e., the cascade of regenerative changes after injury, commonly fails in rapid-stretch injuries. Instead, a neuroma forms – where abundant scar tissue replaces the normal pathway for nerve regeneration. Unfortunately, there is little pathophysiologic understanding of how the regenerative cascade that normally promotes nerve regrowth is corrupted into to the abundant fibrosis, chronic inflammation, limited axonal regeneration, and painful consequences of neuromas. The goals of this project are to evaluate the cellular composition, activation state and spatial niches in neuroma formation, validate our preliminary data that NETosis appears to be one of the inciting events in neuroma formation, and treat nerves with DNase I – which appears to improve nerve regeneration after injury. First, the initial response to nerve injuries appears to determine whether the nerve will recover or not. Our preliminary data highlights five expression pathways in neuroma formation: 1) Vascular 2) Neuronal 3) Fibrosis 4) Cell Death 5) Inflammation. Furthermore, we have substantial evidence that inflammation is exuberant and persistent, suggestive of a potentially coordinating role. We will analyze cell types and states, as determined by protein and genetic expression, to help identify the mechanistic details of neuroma formation. NETosis has been established, in other disorders, to lead to impaired vascular remodeling, worsening fibrosis, and amplification of inflammation - aligning with the critical features of neuromas. Our preliminary data indicates that NETs form selectively in severe, neuroma-forming injuries, and not in milder stretch injuries that do not form neuromas. Furthermore, preliminary evidence indicates that genetic reduction in NETosis (PAD4 KO) improved the gross pathology, fibrosis and axonal regeneration after severe nerve injury. We will assess the impact of reducing NETosis on nerves recovering after severe injury, using PAD4 KO animals and Cl-amidine treatment, which reduces NETosis, in comparison to untreated WT animals. Second, we will assess the impact of exacerbating NET formation in lower-severity injuries, using purified NET products or pharmacologically activated neutrophils. Third, we will assess impact of re-introducing NETs in PAD4 KO animals to assess for return of neuroma formation. Lastly, we will investigate DNase I for identification of optimized treatment strategies for severe nerve injuries. Our preliminary data suggests treatment with DNase I over 14 days dramatically improves axonal regeneration. We will investigate dosage, duration and initiation of DNase I treatment, to develop optimal dose strategies. The outcomes of the proposed experiments will (i) address a knowledge gap on mechanisms underlying neuroma formation, (ii) determine how NETs impact nerve regeneration, and (iii) provide essential data for designing future human trials with DNase I for nerve injuries.
