Modulatory action of Botulinum toxin type A on neuropathic pain: behavioral and immunohistochemical studies in an animal model.

S Luvisetto. National Research Council of Italy, Cell Biology and Neurobiology Institute (IBCN), 00143, Italy

Botulinum neurotoxin type A (BoNT/A) is currently used to treat numerous medical conditions. The potential use of BoNT/A to treat pain is mainly derived from the observation that patients treated with BoNT/A for musculoskeletal disorders often experienced reduction of pain. Although the mechanisms of the analgesic effect of BoNT/A are still not completely elucidated, many clinical investigations have shown that the analgesic effects may be attributed not only to the effect of muscle relaxation but also to the neurotransmitters’ release inhibition along nociceptive pathways. In this study we analyzed the effects of BoNT/A in developing, maintaining and recovering from neuropathic pain. As model of neuropathic pain we considered the Chronic Constriction Injury (CCI) of the sciatic nerve, both in mice and rats. We examined the onset of allodynic and hyperalgesic states and, through the footprint walking tracks analysis and the weight bearing, the functional recovery of the injured limb. BoNT/A was able to counteract CCI-induced neuropathic pain and this effect was present after a single intraplantar (IPL) or intrathecal (IT) injection of BoNT/A. Analgesic effects were already evident starting 24 hours after the administration of BoNT/A and were long-lasting, being present 81 or 25 days after IPL injection of the higher dose in mice (15 pg/paw) and rats (75 pg/paw), respectively, and 35 days after IT injection in rats (75 pg/rat). BoNT/A-injected mice showed a quicker recovery of the walking pattern and weight bearing. As revealed by immunofluorescence and western blot analysis of the expression of Cdc2 and GAP-43 regeneration proteins and of S100beta and GFAP Schwann cells (SC) proteins, behavioral improvements were accompanied by structural modifications in the sciatic nerve. Moreover, the effects of IPL injection of BoNT/A on CCI-induced allodynia were correlated with the expression of cleaved-SNAP25 (cl-SNAP25) in tissues along nociceptive pathway, including hindpaw, sciatic nerve, dorsal root ganglia (DRG), and spinal cord. The expression of cl-SNAP25 has been analyzed by immunostaining and confocal microscopy of cl-SNAP25 alone and in colocalization with GFAP, a protein marker expressed in epidermal and hair follicles keratinocytes, in dermal fibroblasts, in nonmyelinating SC, and in spinal cord astrocytes. We analyzed also colocalization of cl-SNAP25 with CD11b, a protein marker of spinal cord microglia, and with NeuN, marker of neuronal cell nuclei. After IPL injection of BoNT/A in the hindpaw of CCI-induced neuropathic mice, we observed an extensive staining for cl-SNAP25 in all tissue examined. The observed staining of cl-SNAP25 in sections of hindpaw, sciatic nerve, DRG and spinal cord is a strong indication for an axonal transport of BoNT/A along the peripheral nerve to spinal cord. In conclusion, this research demonstrated long-lasting anti-allodynic and anti-hyperalgesic effects of BoNT/A in a model of neuropathic pain, accompanied by an acceleration of regenerative processes in the injured nerve; these effects can be accounted for by BoNT/A trafficking along axonal processes, away from the peripheral site of injection, and by entering neurons and glial cells in the central nervous system. These results may have important implications in the use of BoNT/A in pain therapy.