Early CALP2 expression and microglial activation are potential inducers of spinal IL-6 up-regulation and bilateral pain following motor nerve injury
Early CALP2 expression and microglial activation are potential inducers of spinal IL-6 up-regulation and bilateral pain following motor nerve injury
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DOI：
10.1111/jnc.14317
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发表时间：
2018-04-01
影响因子：
4.7
通讯作者：
Zang, Ying
中科院分区：
文献类型：
--
作者：
Chen, Shao-Xia;Wang, Shao-Kun;Zang, Ying
本实验室先前的工作表明，腰5腹根切断（L5-VRT）造成的运动神经损伤导致双侧脊髓中白细胞介素-6（IL-6）的过度表达，鞘内注射IL-6中和抗体可延迟双侧后爪机械性异常性疼痛的诱导。然而，L5-VRT后脊髓IL-6表达的早期事件和上游机制需要阐明。采用L5-VRT模型诱导成年雄性Sprague-Dawley大鼠的神经病理性疼痛，用von Frey毛发和足底测试仪进行评估。鞘内进行钙蛋白酶-2（CALP 2，一种钙依赖性蛋白酶）敲低或过表达以及小胶质细胞耗竭。采用免疫印迹和免疫组织化学方法探讨其可能的机制。在这里，我们提供了第一个证据表明，IL-6和CALP 2水平在腰5-VRT后30分钟内增加。IL-6和CALP 2共定位于脊髓背角（SDH）和脊髓腹角。术后（PO）10 min时，同侧SDH中的CALP 2明显增加，20 min时IL-6增加。通过鞘内给予CALP 2-shRNA敲低脊髓CALP 2可防止VRT诱导的同侧脊髓中IL-6的过度产生，并减轻双侧机械性异常性疼痛。脊髓小胶质细胞活化也在早期IL-6上调中发挥作用。巨噬细胞/小胶质细胞标志物ED 1/Iba 1在PO 30分钟时增加，而胶质细胞酸性蛋白（星形胶质细胞）和CN-12（少突胶质细胞）标志物则没有增加。Iba_1在PO后20 min即开始升高，3d达高峰。形态从静止细胞中具有细突起的小索马变为活化的阿米巴样形状。使用Mac-1-皂草素消耗小胶质细胞部分阻止了IL-6的上调，并减弱了VRT诱导的双侧机械性异常性疼痛。总之，我们的研究结果提供了证据，增加脊髓CALP 2和小胶质细胞活化可能有早期的致病作用，在运动神经损伤后的IL-6过表达。因此，抑制CALP 2和/或小胶质细胞活化的药剂可证明对治疗神经性疼痛有价值。
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 30min 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 10min PO, preceding increased IL-6 at 20min 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 30min PO, while glial fibrillary acidic protein (astrocyte) and CNPase (oligodendrocyte) markers were not. Increased Iba1 was detected as early as 20min PO and peaked at 3days. 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.