Accepted

Spinal cord injury (SCI) is one of the main causes leading to neuropathic pain. Here, we aim to explore the molecular mechanism and function of lncRNA PVT1 in neuropathic pain induced by SCI. The expression of lncRNA PVT1, microRNA (miR) - 186-5p was measured via quantitative reverse transcription PCR (qRT-PCR), and the activation of astrocytes (labeled by GFAP) was detected by immunohistochemistry. Western blot was conducted to detect the expression of chemokine ligand 13 (CXCL13), chemokine receptor 5 (CXCR5), cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP) in spinal cord injury lesions. The levels of inflammatory cytokines (including IL-1β and IL-6) and MDA in tissues were examined via Enzyme-linked immunosorbent assay (ELISA). In vitro experiments were also conducted in primary cultured astrocyte to explore the response of astrocyte to lipopolysaccharide (LPS). What's more, the PVT1-miR-186-5p interaction was verified via the dual luciferase activity assay and RNA immunoprecipitation (RIP) assay. The results demonstrated that the levels of PVT1, CXCL13 and CXCR5 were upregulated, while miR-186-5p were decreased in SCI rats' spinal cord and LPS-mediated astrocytes. In the SCI model, PVT1 depletion significantly alleviated neuropathic pain, astrocytic activation and reduced the expression of neuroinflammatory factors and proteins. The relevant mechanism studies confirmed that PVT1 is a competitive endogenous RNA (ceRNA) of miR-186-5p, targets and inhibits its expression and promotes the expression of CXCL13/CXCR5, while miR-186-5p targets CXCL13. In conclusion, inhibition of lncRNA PVT1 alleviates neuropathic pain in SCI rats by upregulating miR-186-5p and down-regulating CXCL13/CXCR5. The PVT1/miR-186-5p/CXCL13/CXCR5 axis can be used as a new therapeutic target for neuropathic pain.

Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in pain. However, there has not been any direct evidence showing that selective microglial activation in vivo is sufficient to induce chronic pain. Here, we used optogenetic approaches in microglia to address this question employing CX3CR1creER/+: R26LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity in both male and female mice. In addition, activation of microglial ReaChR up-regulated neuronal c-Fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased interleukin (IL)-1β production, which is likely mediated by inflammasome activation and calcium elevation. IL-1 receptor antagonist (IL-1ra) was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation. Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.