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Transcriptomic Signatures of Neuroma-in-continuity: Translational Insights into Pathophysiology
Wesley S. Warner, BA1; Chris Stubben, PhD1; Stewart Yeoh, PhD1; Alan R Light, PhD1; Mark A. Mahan, MD2
1University of Utah, Salt Lake City, UT; 2Department of Neurosurgery, University of Utah, Salt Lake City, UT

Background: The cellular and molecular underpinnings of Wallerian degeneration have been robustly explored in models of successful nerve regeneration. In contrast, there is limited interrogation of how the natural regeneration program is corrupted to produce the pathophysiologic neuroma-in-continuity.
Methods: Sixty-one adolescent male C57BL/6J mice (n=5/group) were randomly assigned to 2 injury severities: 1) surgical exposure without experimental injury (sham) 2) rapid-stretch rupture. Sciatic nerves were harvested at 6 hours post-injury, day 2, 7, 14, and 48. Additionally, control nerves were harvested where no surgical intervention was performed (n=6). Total RNA was extracted and underwent next-generation RNA sequencing (RNA-seq). Resulting reads were optimized, trimmed, and aligned to a reference database. A 5% false discovery rate and log2fold change were the cutoff thresholds used to identify differentially expressed genes (DEGs). To isolate the genes unique to the rupture injury (compared to surgically manipulated sham), an interaction model was employed to evaluate injury effect. Global transcript expression profiles were examined through principal component (PCA) and cluster model analysis. DEGs were explored with Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) to identify significant pathways associated with pathological remodeling. To corroborate genetic analysis, nerves were subject to immunofluorescent staining for proteins representative of the prominent biological pathways identified.
Results: Global genetic activity dramatically increased immediately following both injuries; however, by D48, sham injury expression resolved to control levels, but RNA expression remained elevated in rupture injury. PCA demonstrated distinct expression patterns for neuroma injuries at D7 (Figure 1A). DEGs applied to independent analyses of 1) GSEA and 2) IPA, cross-validate one another, identifying significant longitudinal upregulation of inflammatory pathways (p<.01). Overall, significant genetic activity annotated to inflammation, cellular death, fibrotic, and neurodegeneration pathways were identified as critically involved in neuroma development (Figure 1B). Immunofluorescent staining corroborated RNAseq analysis, identifying persistent immune activation within the neuroma.
Conclusion: Pathophysiologic nerve regeneration produces substantially altered genetic profiles both temporally and chronically, demonstrating persistent genetic expression that correlates with the persistent inflammation within neuromas. To our knowledge, this study presents as the first transcriptional study of neuroma-in-continuity pathophysiology, and has identified inflammation, cellular death, fibrotic, and neurodegeneration signatures which diverge from pathways elucidated by traditional models of successful nerve regeneration.


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