Abstract: Chronic denervation is one of the key factors that affect nerve regeneration. Chronic axotomy deteriorates the distal nerve stump, causes protein changes, and renders the microenvironment less permissive for regeneration. Some of these factors/proteins have been individually studied. To better delineate the comprehensive protein expression profiles and identify proteins that contribute to or are associated with this detrimental effect, we carried out a proteomic analysis of the distal nerve using an established delayed rat sciatic nerve repair model. Four rats that received immediate repair after sciatic nerve transection served as control, whereas four rats in the experimental group (chronic denervation) had their sciatic nerve repaired after a 12-week delay. All the rats were sacrificed after 16 weeks to harvest the distal nerves for extracting proteins. Twenty-five micrograms of protein from each sample were fractionated in SDS-PAGE gels. NanoLC-MS/MS analysis was applied to the gels. Protein expression levels of nerves on the surgery side were compared to those on the contralateral side. Any protein with a P value of less than 0.05 and a fold change of 4 or higher was deemed differentially expressed. All the differentially expressed proteins in both groups were further stratified according to the biological processes. A PubMed search was also conducted to identify the differentially expressed proteins that have been reported to be either beneficial or detrimental to nerve regeneration. Ingenuity Pathway Analysis (IPA) software was used for pathway analysis. The results showed that 709 differentially expressed proteins were identified in the delayed repair group, with a bigger proportion of immune and inflammatory process-related proteins and a smaller proportion of proteins related to axon regeneration and lipid metabolism in comparison to the control group where 478 differentially expressed proteins were identified. The experimental group also had more beneficial proteins that were downregulated and more detrimental proteins that were upregulated. IPA revealed that protective pathways such as LXR/RXR, acute phase response, RAC, ERK/MAPK, CNTF, IL-6, and FGF signaling were inhibited in the delayed repair group, whereas three detrimental pathways, including the complement system, PTEN, and apoptosis signaling, were activated. An available database of the adult rodent sciatic nerve was used to assign protein changes to specific cell types.  The poor regeneration seen in the delayed repair group could be associated with the down-regulation of beneficial proteins and up-regulation of detrimental proteins. The proteins and pathways identified in this study may offer clues for future studies to identify therapeutic targets.

Key words: chronic axotomy, chronic denervation, delayed repair, distal nerve, functional recovery, nerve regeneration, peripheral nerve, prolonged denervation, proteome, sciatic nerve, sciatic nerve transection