2043 Impact of NETosis on Neuroma Formation

INTRODUCTION: NETosis, is a form of neutrophil cell death hallmarked by expulsed decondensed chromatin which limits infection by entrapping pathogens. Recent investigation in central nervous system trauma has demonstrated that aberrant NETosis activation is associated with impaired vascular remodeling, amplification of the inflammatory response, increased fibrotic deposition, and impaired neurologic recovery. By contrast, there is limited interrogation of NETosis in peripheral nervous system trauma. METHODS: One-hundred and fifty-two adolescent male mice (n=7/group) underwent rapid-stretch injury of different biomechanically determined grades: sham injured (surgical exposure alone), elastic (<15% persistent length deformation), inelastic (permanent length change), and rupture. NETosis formation was modulated by: 1) perineural DNAse I administration to degrade NETs and 2) PAD4 knockout which prevents NETosis formation. Sciatic nerves were harvested at 6 hours, day 2, 7, 14, and 48 post-injury. Behavioral tests evaluated motor and sensory function. Microstructure and cellular composition were evaluated by immunostaining and fluorescence activated cell sorting. RESULTS: A positive association of increased NETosis formation and nerve injury severity presented at 6 hours post-injury (p<.01). Modulation of NETosis demonstrated a shift in inflammatory cell phenotype and attenuation of inflammatory cell recruitment, in the acute timeframe after injury (day 7, p<.01). Improved sensory recovery was observed by day 14, whereby statistical difference is no longer observed compared to baseline (p=.16). At day 48, NETosis modulation demonstrated increased axon regeneration (p<.05; NF200 and MBP indicative of large myelinated fibers) in rupture injuries. Reduced fibrosis (anti-Col1a, -Col3a) is associated with NETosis modulation. CONCLUSIONS: NETosis appears to be related to injury severity and neuroma formation in rapid-stretch peripheral nerve injuries. Reduction of NETosis improves regeneration and decreases fibrotic deposition. Further evaluation of NETosis in humans is necessary for future clinical translation.