Neuron-derived chemokine CCL3 in spinal dorsal horn mediates brachial plexus avulsion-induced neuropathic pain and related central sensitization

Neuropathic pain (NP) induced by brachial plexus avulsion (BPA) brings up devastating impact to patients’ life, however, the mechanism underlying BPA-induced NP is still poorly understood. BPA can cause direct injury to the corresponding innervated dorsal horn of spinal cord. Herein, we intend to uncover the role of chemokine C–C motif chemokine ligand 3 (CCL3) in the ipsilateral spinal dorsal horn (SDH) during the development of BPA-induced NP and the related mechanism on mediating central sensitization. A novel BPA mouse model was established and pain behavioral tests, including von Frey filaments test, cold plate test and CatWalk Gait Analysis, were conducted. RNA sequencing was used to detect the differentially expressed genes in ipsilateral cervical SDH after BPA. Qualitative and quantitative analysis was used to evaluate the expression of CCL3 and its receptor CCR1 in ipsilateral SDH through Western blot and immunohistochemistry (IHC) technology. The analgesic function of CCL3 was evaluated through using pharmacological inhibition of CCR1, neuron-specific knockdown of CCL3 and chemogenetic test. CCL3-related signaling was also explored using Western blot and pharmacological methods. CCL3 was identified highly expressed in the ipsilateral SDH of BPA mice, which was further confirmed by Western blot. Interestingly, CCL3 was found predominantly expressed in both activated c-fos Td−Tom and CaMKII positive neurons in SDH, and chemogenetic inhibition of these neurons could relieve the pain of BPA mice. The pain of BPA mice could also be relieved through using antagonist of CCR1 and genetic knockdown of CCL3. TLR2-IRF5-CCL3 was identified as the possible signaling pathway according to transcriptomics data, and our results support the potential involvement of the analgesic role of TLR2-IRF5-CCL3 signaling, which is consistent with a regulatory contribution of this axis by Western blot and pharmacological methods. Gliosis was also found happening due to direct injury on the SDH through using IHC, which could be mitigated by antagonist of CCR1. This study supports the involvement of CCL3 in modulating BPA-induced NP via a potential TLR2-IRF5-CCL3 signaling pathway that is consistent with our experimental observations during the process of spinal central sensitization. The findings also provide evidence for the role of neuroinflammation and glial cell activation in driving central sensitization under chronic pain, particularly following BPA exposure.