Differential Gene Expression in Nerves Repaired Under Low and High Tension

Abstract

PURPOSE: Tension has been shown to be detrimental to nerve regrowth and reinnervation after primary nerve repair, likely via local ischemia and aberrant wound healing. We hypothesize that an improved understanding of the mechanisms underlying pathologic nerve healing may provide insight into potential targets for optimizing outcomes after nerve repair. The purpose of this study was to use RNA sequencing to identify differentially expressed genes in nerves repaired under minimal and significant tension. METHODS: Sciatic nerve transection was performed in male Lewis rats. The nerve was repaired via either a minimal-tension, interrupted epineural repair with 9-0 Nylon sutures, or a repair under tension for which a 9-mm segment of nerve was excised before interrupted epineural repair. The contralateral nerve served as each subject’s matched control. Nerve specimens were harvested at 14 weeks postoperatively. Following RNA extraction, sequencing was performed using Illumina HiSeq 2500. Reads were mapped back to the genome, and differentially expressed genes were identified using EBSeq. All analyses were conducted using R (R Development Core Team, 2012), a publicly available platform. RESULTS: Nerve repair was performed in 17 subjects (n = 8 minimal tension, n = 9 tension). Accounting for outliers, and using the hypothesis that all 3 groups (control, minimal tension, and tension) were different, 37 differentially expressed genes were identified. Many of the identified genes code for transcription factors and/or are involved in cell-signaling pathways. Col10a1, Crebzf, Prr34, Rsl1, and Setd4 were all found to be differentially upregulated in the tension group. Tfrc, a transferrin receptor upregulated in immunodeficiency syndromes, was downregulated in the tension group. For some genes, such as Bl6 and Naf1, expression in the tension group was more similar to that in the uninjured group. Heat maps allowed for qualitative visual comparison among uninjured control, minimal tension, and tension groups. CONCLUSIONS: To our knowledge, this is the first study to compare molecular signatures of nerves repaired using minimal and significant tension. Further study of these up- and downregulated genes could help identify targets for optimizing nerve healing and regeneration. Similar methodologies using RNA-Seq could be applied to studying neuroma in continuity and other nerve pathology that often requires intervention.