Introduction/Aim: Chronic pain affects 1 in 5 Canadians and costs over $43B annually, yet effective and safe treatment options remain elusive. Recent discoveries have brought to the forefront sex differences in mechanisms of pain as a potential explanation why novel pre-clinical therapeutics have not translated into successfully in clinical trials. Methods: To begin understanding how males and females differ in pain processing, we analyzed gene expression, using RNA sequencing, and DNA methylation, using reduced representation bisulfite sequencing (RRBS), in rodent models of neuropathic pain. Results: Across both sexes, our data reveals peripheral nerve injury (PNI) caused upregulation of 61 genes involved in innate immune responses in spinal cord. In females specifically, we observed PNI-induced downregulation of 5 genes involved in neuronal function and upregulation of two classes of Cathepsins. (C and E). On the other hand, in males, we observed upregulation of 14 genes including those involved in metabolism of purines and glutathione. Additionally we found that PNI leads to methylome remodeling in a sexually dimorphic manner: 125 promoters in rat spinal cord that were differentially methylated in injured males versus females. Discussion/Conclusions: Our data shows robust sex specific DNA methylation and transcriptome signature after PNI. Additionally, our findings leads to the hypothesis that remapping of DNA methylation, with subsequent alterations in the transcriptome, are critically involved in the development of neuropathic pain. We anticipate that future research directed at understanding these differences may lead to effective drug development to combat chronic pain.

Microglia-neuron signalling in the spinal cord is a key mediator of mechanical allodynia caused by peripheral nerve injury. We recently reported sex differences in microglia in pain signalling in mice: spinal mechanisms underlying nerve injury-induced allodynia are microglial dependent in male but not female mice. Whether this sex difference in pain hypersensitivity mechanisms is conserved in other species is unknown. Here, we show that in rats, the spinal mechanisms of nerve injury-induced hypersensitivity in males differ from those in females, with microglial P2X(4) receptors (P2X(4)Rs) being a key point of divergence. In rats, nerve injury produced comparable allodynia and reactive microgliosis in both sexes. However, inhibiting microglia in the spinal cord reversed allodynia in male rats but not female rats. In addition, pharmacological blockade of P2X(4)Rs, by an intrathecally administered antagonist, attenuated pain hypersensitivity in male rats only. Consistent with the behavioural findings, nerve injury increased cell surface expression and function of P2X(4)Rs in acutely isolated spinal microglia from male rats but not from female rats. Moreover, in microglia cultured from male rats, but not in those from female rats, stimulating P2X(4)Rs drove intracellular signalling through p38 mitogen-activated protein kinase. Furthermore, chromatin immunoprecipitation-qPCR revealed that the transcription factor IRF5 differentially binds to the P2rx4 promoter region in female rats vs male rats. Finally, mechanical allodynia was produced in otherwise naive rats by intrathecally administering P2X(4)R-stimulated microglia from male rats but not those from female rats. Together, our findings demonstrate the existence of sexually dimorphic pain signalling in rats, suggesting that this sex difference is evolutionarily conserved, at least across rodent species.