Delay alters the immune response to nerve repair
Masoud Golshadi, PhD1; Jennifer K. Grenier, PhD1; Michael G. Edwards, PhD2; Jonathan Cheetham, VetMB, PhD, DACVS1
1Cornell University, Ithaca, NY, 2Bioinfo Solutions LLC, Parker, CO
The prolonged delay before surgical reinnervation results in chronic denervation of the distal nerve stump, loss of the association between Schwann cells (SC) and their axonal processes, and a progressive worsening in recovery. In the early stages after injury SCs remove fragmented axonal segments and then recruit macrophages. The macrophages then encourage proliferation of SCs. However, the mechanistic interactions of SCs with key elements of repair, including macrophages, has not been previously explored. This study presents the detail of the interaction between macrophages and SCs after delayed repair.
Mice of both sex were used for in vivo collection of macrophages and SCs from the regenerative nerve bridge. The tibial (distal) and common peroneal (proximal) nerve branches were transected and sutured in silicone nerve conduit, either immediately after injury or after 8 weeks of delay. The in vivo isolation of cells was done using a novel multi-color fluorescence assisted cell sorting. Gene expression analysis was performed using RNA sequencing at day 5 and 14 after repair. Protein expression analysis (Luminex) and in vivo phagocytosis of the recruited macrophages were studied 5 days after repair. Moreover, the number of motor neurons (MN) crossing the injury site was evaluated using retrograde tracer 8 weeks after repair. Based on the gene expression analysis, targeted manipulation of the immune response to delayed repair was used to improve repair.
The immune response was downregulated by delayed repair in both macrophages (dominated by IL10, IL6, Cxcl10) and the distal stump (dominated by IL34); but, upregulated in SCs (dominated by Vegfa, TNF, Adam17). The inflammatory response also showed the same trend of downregulation by delayed repair in macrophages (dominated by Chi3l3, IL10, Saa3) and distal stump (dominated by IL34, Ccr7), but upregulation in SCs (dominated by Saa3, Cd14, TNF). Protein expression analysis showed increase in TNF-α in the delayed repair group (p=0.04). As expected, delayed repair reduced the number of MN crossing the injury site (p=0.0001). No differences in phagocytosis activity of macrophages were observed between the two groups. Targeted immunotherapy, based on the pathway analysis of the gene expression data, promoted recovery after a delay repair (p=0.009).
This study reveals the details of macrophage and SC interaction at the site of nerve injury, and the mechanisms and pathways through which the regenerative nerve bridge will be formed. It also identifies the exogenous delivery of immunomodulatory cytokines to improve recovery after delayed nerve repair.
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