PROJECT SUMMARY/ABSTRACT Reconstruction of peripheral nerve gap injuries continues to pose a challenge in the clinic due to their significant long-term functional morbidity. Multiple surgical approaches have been developed to promote nerve regeneration across a damaged nerve with a gap. In the clinic, nerve autografts are considered the standard of care in treating nerve gaps due to their superior capacity in promoting nerve regeneration. However, the use of nerve autografts is limited by their availability, increased surgery time, loss of nerve function from the lost nerve and donor site morbidity. Acellular nerve allografts (ANAs) have been proposed as a clinical alternative to autografts in repairing nerve gaps due to their capability in promoting regeneration across short gaps (< 3 cm). ANAs are allogenic nerves that are chemically processed to remove the cells, reducing immunogenicity, while maintaining the extracellular matrix (ECM) that will allow cell migration into the ANA and subsequent nerve regeneration. However, unlike autografts, ANAs do not support regeneration across long defects (> 3 cm). The reason why long ANAs do not promote regeneration or regenerate poorly is not completely understood. Previous studies have demonstrated the critical role of inflammatory cells, such as macrophages and T cells, in regulating and promoting the early stages of nerve regeneration in ANAs (before 4 weeks post-surgery). Specifically, macrophages will promote angiogenesis and T cells will promote axonal myelination. However, when compared to short ANAs capable of robust regeneration, preliminary data shows an increase in the expression of pro-inflammatory cytokines and changes in the morphology of blood vessels in long ANAs at the later stages of nerve regeneration (between 4- and 8- weeks post-surgery). The increase in the expression of pro-inflammatory cytokines and the changes in the morphology of blood vessels happen after angiogenesis has occurred and axons have started migrating into the ANAs. These changes suggest that a pro-inflammatory environment develops inside long ANAs in the later stages of nerve regeneration, and this correlates with changes in the morphology of blood vessels and a decrease in the number of myelinated axons. We hypothesize the state of chronic inflammation in long ANAs is disrupting the regenerative microenvironment in long ANAs. In brief, the proposed research aims to characterize and compare the inflammatory microenvironment that develops in the later stages of nerve regeneration in long ANAs with the microenvironment in the regenerated short ANAs and autografts. This project also explores the effect of reducing inflammation in long ANAs, using a pharmacological intervention, on axonal regeneration. An integral part of this study is to identify the cell subpopulations promoting the pro- inflammatory microenvironment in long ANAs. An in-depth analysis of these cell subpopulations may provide specific molecular target...