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Neurobiology of Neuropathic Pain in Neuroma-in-continuity Traumatic Nerve Injury
Wesley S. Warner, BA1; Stewart Yeoh, PhD1; Matthew C. Findlay, BS1; Mark A. Mahan, MD2
1University of Utah, Salt Lake City, UT; 2Department of Neurosurgery, University of Utah, Salt Lake City, UT

Background: Neuropathic pain (NP) is canonically elaborated as a consequence of pathologic neuroinflammation, neural circuitry or threshold remodeling. Putative mechanisms are derived from experimental nerve injury models that lack clinical correlate, such as chronic constriction. There has been no systematic examination of chronic NP in pathophysiologic neuroma-in-continuity injuries, particularly the correlation of NP to cellular composition within the neuroma, dorsal root ganglion (DRG), and spinal cord.
Methods: C57BL/6J mice (n=11) underwent rapid-stretch nerve rupture injury which heals in-continuity. Behavioral assays of von Frey, Dixon up-down method, and guarding were used to assess pain at prolonged timepoints after injury. At week 16 post-injury, sciatic nerve, DRG, and spinal cord were harvested from injured and contralateral sides. Neuronal (NF200, BIII-tubulin, MBP), architectural (COL1a, COL3a, Laminin IV, GLUT1), inflammatory (CD68, Iba1, GFAP, CD4), and canonical pain neuropeptides (CGRP, Substance P) immunofluorescent staining was performed to assess for correlated changes. Na´ve, age-matched C57BL/6J mice (n=7) were used for behavioral and histologic controls.
Results: At 16 weeks, von Frey revealed statistically increased paw withdrawal frequency for both injured and contralateral sides compared to control (p<.0001). No statistical difference between sides was observed (p=.92), consistent with bilateral hypersensitivity. The Dixon up-down method revealed a significantly decreased sensitivity threshold for both injured and non-injured sides (p<.0001), without difference between sides (p=.98). Animals demonstrated significantly increased guarding behavior (p<.001); an assay indicative of spontaneous pain. A novel motor response, involving foot flapping, was unique and consistent to nociception. Neuroma immunofluorescence demonstrated robust disorganized small unmyelinated fibers co-localizing with CGRP. Entropic architectural remodeling also presented - consistent with clinical specimens. Notably, the neuroma microenvironment was hallmarked by a persistent inflammatory residence of macrophages (CD68+, Iba1+), adaptive immune (CD4+) cells, and significant hypercellularity (p<.01). DRG ipsilateral to the injury demonstrated reduction of neuronal staining with pronounced bilateral inflammatory cell presence (Iba1+). Spinal cord histology revealed bilateral loss of large and small diameter axons (NF200, BIII-tubulin), in both the dorsal and ventral columns (p<.05), with time-dependent infiltration of GFAP+ cells in white and grey matter.
Conclusion: Neuromas, and the consequent aberrant neural regeneration, are associated with bilateral hypersensitivity at prolonged timepoints after injury. This pain phenotype correlates with persistent inflammatory cell presence on both injured and non-injured sides at all anatomical levels. The mechanisms underpinning NP development in neuroma-in-continuity lesions remain unexplored; this study supports a role for inflammation in orchestrating and maintaining a microenvironment conducive to the development of NP


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