Enhancement of ectopic discharge in regenerating A- and C-fibers by inflammatory mediators Running title: Enhancement of activity in regenerating afferent fibers

Acknowledgements: The authors are grateful to Dr. Peter Reeh for comments and critical review of this manuscript. ABSTRACT Afferent A-and C-fibers regenerating into a nerve following peripheral nerve injury are exposed to inflammatory mediators released by Schwann cells, resident and invading macrophages and other inflammatory cells. Here we tested the hypothesis that ongoing and evoked activity in these afferent fibers are enhanced by a mixture of inflammatory mediators (inflammatory soup, IS) applied to the injured nerve. Using in vivo electrophysiology, regenerating afferent nerve fibers were investigated 7-14 days after sural nerve crush lesion. The ectopic activity was studied before and up to 1.5 hours after topical application of IS to the nerve in 73 C-fibers and 22 A-fibers that were either ectopically active before application of IS (61 C-fibers, 17 A-fibers) or recruited by IS (12 C-fibers, 5 A-fibers). More than half of the C-fibers were activated by IS for up to 90 minutes after its removal. The majority of mechano-(23/38) and heat-sensitive (29/35) C-fibers as well as mechano-sensitive A-fibers (12/17) decreased their activation thresholds and/or increased the response magnitude to mechanical and/or heat stimulation of the nerve. Noxious cold sensitivity, but not non-noxious cold sensitivity, was weakly influenced by IS. Some initially non-responsive C-and A-fibers developed new ectopic properties, i.e. were recruited, and exhibited ongoing activity and/or could be activated by physiological stimuli after application of IS. The results suggest that inflammatory mediators may be critical to enhance ectopic excitability of regenerating afferent nerve fibers. These peripheral mechanisms may be important triggering and maintaining neuropathic pain.