Early CALP2 expression and microglial activation are potential inducers of spinal IL‐6 up‐regulation and bilateral pain following motor nerve injury

Previous work from our laboratory showed that motor nerve injury by lumbar 5 ventral root transection (L5‐VRT) led to interleukin‐6 (IL‐6) over‐expression in bilateral spinal cord, and that intrathecal administration of IL‐6 neutralizing antibody delayed the induction of mechanical allodynia in bilateral hind paws. However, early events and upstream mechanisms underlying spinal IL‐6 expression following L5‐VRT require elucidation. The model of L5‐VRT was used to induce neuropathic pain, which was assessed with von Frey hairs and the plantar tester in adult male Sprague–Dawley rats. Calpain‐2 (CALP2, a calcium‐dependent protease) knockdown or over‐expression and microglia depletion were conducted intrathecally. Western blots and immunohistochemistry were performed to explore the possible mechanisms. Here, we provide the first evidence that both IL‐6 and CALP2 levels are increased in lumbar spinal cord within 30 min following L5‐VRT. IL‐6 and CALP2 co‐localized in both spinal dorsal horn (SDH) and spinal ventral horn. Post‐operative (PO) increase in CALP2 in ipsilateral SDH was evident at 10 min PO, preceding increased IL‐6 at 20 min PO. Knockdown of spinal CALP2 by intrathecal CALP2‐shRNA administration prevented VRT‐induced IL‐6 overproduction in ipsilateral spinal cord and alleviated bilateral mechanical allodynia. Spinal microglia activation also played a role in early IL‐6 up‐regulation. Macrophage/microglia markers ED1/Iba1 were increased at 30 min PO, while glial fibrillary acidic protein (astrocyte) and CNPase (oligodendrocyte) markers were not. Increased Iba1 was detected as early as 20 min PO and peaked at 3 days. Morphology changed from a small soma with fine processes in resting cells to an activated ameboid shape. Depletion of microglia using Mac‐1‐saporin partially prevented IL‐6 up‐regulation and attenuated VRT‐induced bilateral mechanical allodynia. Taken together, our findings provide evidence that increased spinal cord CALP2 and microglia cell activation may have early causative roles in IL‐6 over‐expression following motor nerve injury. Agents that inhibit CALP2 and/or microglia activation may therefore prove valuable for treating neuropathic pain. Interleukin‐6 (IL‐6) up‐regulation in dorsal root ganglion (DRG) neurons may contribute to the neuropathic pain induced by motor nerve injury. In this study, we provide evidence that early up‐regulation of IL‐6 in spinal cord is mediated by increased calpain‐2 and microglial cell activation, sug