Pain Research Center and Department of Physiology, Zhongshan Medical School of Sun Yat-Sen University, Guangzhou, China.
Department of Anesthesiology, Cancer Center, Sun Yat-Sen University, State Key Laboratory of Oncology in South China, Collaborative, Innovation Center for Cancer Medicine, Guangzhou, China.
J Neurochem. 2018 Apr;145(2):154-169. doi: 10.1111/jnc.14317.
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.
先前我们实验室的工作表明，腰 5 脊神经根切断术（L5-VRT）导致双侧脊髓中白细胞介素 6（IL-6）过度表达，鞘内给予 IL-6 中和抗体可延迟双侧后爪机械性痛觉过敏的诱导。然而，L5-VRT 后脊髓中 IL-6 表达的早期事件和上游机制仍需阐明。我们使用 L5-VRT 模型诱导神经病理性疼痛，并用 von Frey 毛发和足底测试仪在成年雄性 Sprague-Dawley 大鼠中进行评估。鞘内进行钙蛋白酶 2（CALP2，一种钙依赖性蛋白酶）敲低或过表达和小胶质细胞耗竭。通过 Western blot 和免疫组织化学来探索可能的机制。在这里，我们首次提供证据表明，L5-VRT 后 30 分钟内，IL-6 和 CALP2 水平均在腰椎脊髓中升高。IL-6 和 CALP2 在脊髓背角（SDH）和脊髓腹角中均有共定位。术后（PO）同侧 SDH 中 CALP2 的增加在 PO 后 10 分钟即可观察到，而 IL-6 的增加则在 PO 后 20 分钟可见。鞘内给予 CALP2-shRNA 可阻止 VRT 诱导的同侧脊髓中 IL-6 的过度产生，并减轻双侧机械性痛觉过敏。脊髓小胶质细胞的激活也在早期 IL-6 的上调中起作用。PO 后 30 分钟，巨噬细胞/小胶质细胞标志物 ED1/Iba1 增加，而神经胶质纤维酸性蛋白（星形胶质细胞）和 CNPase（少突胶质细胞）标志物则没有。Iba1 的增加早在 PO 后 20 分钟即可检测到，并在 3 天达到峰值。形态从静止细胞中小的胞体和细的突起改变为激活的阿米巴样形状。使用 Mac-1-箭毒肽耗竭小胶质细胞可部分阻止 IL-6 的上调，并减轻 VRT 诱导的双侧机械性痛觉过敏。总之，我们的研究结果提供了证据表明，脊髓中 CALP2 和小胶质细胞激活的增加可能在运动神经损伤后 IL-6 过度表达中具有早期因果作用。因此，抑制 CALP2 和/或小胶质细胞激活的药物可能对治疗神经病理性疼痛有价值。
