Neutrophil Extracellular Traps Proliferate in Response to Peripheral Sciatic Nerve Crush Injury

Introduction: Wallerian degeneration (WD) is the process by which the peripheral nervous system (PNS) repairs injury to itself. This effort is coordinated by Schwann Cells (SCs), and their activation of the neuroinflammatory response initiating repair. Neutrophils are necessary for complete repair. However the exact molecular mechanisms their action is not fully understood. Neutrophil extracellular traps (NETs) represent a novel pathway by which neutrophils interact in response to trauma. The role of NETs in peripheral nerve injury has not been explored. This study aimed to explore the presence and role of NETs in response to crushed and transected sciatic nerve, to determine how NETs interact with the PNS traumatic landscape with the hope of augmenting their behavior towards a clinical target for improved regeneration.

Methods: 10 biologic replicates of Wild-type, C57BL/6 mice were split into both sciatic nerve crush injury and sciatic nerve transection injury. Sciatic nerves were harvested at 6, 12, 24, and 72 hours post injury after perfusion with 1x PBS and 4% PFA. Nerves were cryosectioned and stained for immunohistochemistry (IHC) analysis. Images were manually counted within 500 microns of the injury site for the presence of neutrophil extracellular traps, denoted by co-localization of DAPI, myeloperoxidase, and citrulinated histone H3. Results were pooled and matched analysis was undertaken for descriptive statistics regarding presence of immune cells.

Results: IHC analysis demonstrates a 2 fold increase around the crush nerve injury site, within 500 microns of injury. Analysis demonstrated a a statistically significant increase in NET population towards the peak of neutrophil action in PNI. NETs accumulated near the perineurium, rather than within the endoneurium of the injury site. Macrophage accumulation at the endoneureum followed NET accumulation and dispersal at peak of 3 days post injury.

Conclusion : For the first time, NETs have been shown to associate with crush site in peripheral nerve injury. While the immune response is essential to Wallerian degeneration, NETs may yet present a useful target for exogenous biologic augmentation to improve regeneration after peripheral nerve injury.