85 The Role of NETosis in Axon Regeneration Following Rapid-Stretch Nerve Injury

Faculty Mentor: Mark Mahan and Sama Noroozi (Neurosurgery, University of Utah)

Research Abstract

Traumatic peripheral nerve injuries, specifically the rapid-stretch injuries from events like car accidents or severe limb traumas, often result in poor nerve regeneration despite surgical intervention and can result in lifelong disability, including permanent motor and sensory impairments [1-3].^[1][2][3]

Despite the genetic capacity of the peripheral nerves to support axonal regeneration, clinical outcomes remain poor in severe rapid-stretch injuries due to the formation of neuromas—scar tissue within nerves that disrupts axon regrowth.^[4][5] Following an these rapid-stretch injury, neutrophils rapidly accumulate at the wound site, releasing inflammatory factors, and neutrophil extracellular traps (NETs) to clear debris and pathogens. NETs are composed of decondensed DNA and antimicrobial proteins, forming a web-like structure that traps pathogens and delays tissue healing when the NET formation is excessive or inappropriate.^[6][7] This research project was aimed towards elucidating the role of NETs and the process of NETosis in axon regeneration and neuroma formation. Dr. Mark Mahan’s lab developed an animal model that replicates the rapid-stretch injuries, allowing the study of associated functional and histopathological outcomes.^[8] Specifically for understanding the role of NETs in axon regeneration following severe stretch injuries, this study focused on two experimental approaches targeting NET formation:

1. PAD4 Knockout (KO): PAD4 is a key enzyme in NET formation.^[9] Using PAD4 KO animals, which lack the ability to produce NETs, we will investigate if blocking NETosis can improve nerve regeneration.
2. DNase I Treatment: DNase I, an enzyme that degrades the DNA backbone of NETs, will be used to explore whether the breakdown of NETs supports axon regrowth in these injury models.^[10]

To evaluate axon regeneration, we used immunohistochemistry (IHC) with NF200, a marker that shows neurofilaments in axons. In the severe injuries, where axons degenerate, there was reduced NF200 expression. Successful axon regeneration was indicated by the reappearance of NF200-positive filaments in nerve sections.^[11] Axonal regeneration was quantified by comparing NF200 counts across the proximal, middle, and distal nerve segments in different experimental groups. I used ImageJ/Fiji software to quantify axonal regeneration by analyzing the NF200-positive axons in sections of the injured nerve. Specifically, the Cell Counter plugin within ImageJ, which allowed for the manual counting of NF200-stained axons in the segments of the nerve (proximal, middle, and distal). This approach provided a systematic way to compare the axonal regeneration across different experimental groups. To ensure accuracy, images of each nerve section were captured at consistent magnification and orientation. The Cell Counter plugin enables tagging each counted axon individually, minimizing overlap and ensuring precise quantification of the NF200 expression in each nerve segment. This quantification approach facilitates statistical analysis of axonal regeneration, providing a detailed understanding of how NET inhibition affects different segments of the nerve following the rapid-stretch injury. Successful axon regeneration was observed by the reappearance of the NF200-positive filaments after the injury site, which means axons have crossed the injured site and reached their target muscles. Our results suggest that degrading NETs early after injury is associated with better axon regeneration in rapid-stretch injuries.