【作者】 褚玉霞；

【导师】 赵志奇；

【作者基本信息】 复旦大学 ， 神经生物学， 2010， 博士

【摘要】 强直刺激大鼠坐骨神经可诱发脊髓背角痛敏神经元反应的长时程增强(long-term potentiation, LTP)和痛觉的行为敏化。本研究主要探讨了小胶质细胞在强直刺激坐骨神经诱导的大鼠脊髓LTP的产生和痛觉中枢敏化中的作用。既往电生理研究证实,强直电刺激大鼠坐骨神经、皮肤自然伤害性刺激或神经损伤,均可在脊髓背角引起C反应和A反应场电位的LTP,反映了痛觉信息传递的中枢敏化。引起脊髓LTP的强直电刺激能引起动物的痛觉过敏行为,包括触诱发痛和热痛敏,但机制尚不完全明确。近年来的研究证明,脊髓胶质细胞,尤其是小胶质细胞在病理性疼痛的发生发展中发挥重要作用。炎性痛和神经病理痛情况下均有小胶质细胞的激活。预先应用胶质细胞代谢抑制剂后,强直刺激坐骨神经诱发脊髓长时程抑制(long-term depression,LTD),而不是LTP,同时可缓解痛觉过敏行为,表明小胶质细胞参与脊髓伤害性反应的长时程增强,然而其具体机制尚不清楚。P2X7受体在胶质细胞上大量表达,参与神经元和胶质细胞的对话。P2X7受体激活后,可促进在海马LTP形成中起关键作用的谷氨酸、白介素-1beta (interleukin-1β, IL-1β)、肿瘤坏死因子α(tumor necrosis factorα, TNF-α)的释放。P2X7受体拮抗剂抑制神经损伤诱发的脊髓神经元的自发放电和痛行为。P2X7基因敲除的小鼠慢性炎症痛及神经病理痛均消失。因此,P2X7受体很可能是介导小胶质细胞参与脊髓LTP产生的关键介质。本研究采用脊髓场电位的电生理记录、伤害性机械痛行为学测试、免疫组织化学及Western blot等方法,探讨小胶质细胞的P2X7受体在强直刺激坐骨神经诱发的脊髓LTP及长时程的痛觉过敏行为中的作用。主要结果如下：强直刺激坐骨神经诱发大鼠脊髓场电位C反应的长时程增强和持续的触诱发痛；强直刺激前1小时鞘内注射小胶质细胞代谢抑制剂美满霉素阻断LTP的产生；强直刺激前1小时至强直刺激后7天每天1次鞘内连续注射美满霉素缓解大鼠的触诱发痛行为。以上表明,小胶质细胞参与强直刺激坐骨神经诱发的脊髓LTP及痛觉敏化行为的产生。强直刺激前0.5小时鞘内分别注射P2X7受体拮抗剂oxATP和BBG,均可阻断脊髓LTP的产生；强直刺激前7天鞘内注射的P2X7受体的小干扰RNA片段,也阻断脊髓LTP的产生；离体脊髓切片上预先应用BBG灌流1小时,可阻断强直刺激李骚束所诱发的脊髓兴奋性突触后场电位的LTP,而不影响基础反应。以上表明,P2X7受体在脊髓场电位的LTP产生中具有至关重要的作用。强直刺激前0.5小时鞘内分别注射oxATP和BBG及强直刺激前7天鞘内注射P2X7受体小干扰RNA片段,在强直刺激后3、5和7天均可部分缓解强直刺激所诱发的双侧触诱发痛;强直刺激前0.5小时鞘内注射BBG可明显抑制强直刺激后2小时Fos的表达上调。表明拮抗P2X7受体功能可显著抑制脊髓痛敏神经元的活动过度增强。免疫组化结果表明,P2X7受体与小胶质细胞标志物OX-42共标,而与星形胶质细胞标志物GFAP及神经元标志物NeuN无共标；强直刺激坐骨神经激活脊髓小胶质细胞和明显上调P2X7受体的表达,且均可被强直刺激前0.5小时鞘内注射P2X7受体拮抗剂BBG所抑制。以上表明脊髓LTP的阻断是小胶质细胞P2X7受体的特异性的作用。强直刺激坐骨神经可诱发脊髓p38丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)磷酸化、其下游分子IL-1β的表达及在LTP中发挥关键作用的AMPA受体的GluR1亚基表达的显著增加,且均可被强直刺激前0.5小时鞘内注射P2X7受体拮抗剂BBG所抑制；强直刺激前0.5小时鞘内注射IL-1β特异性抗体IL-1ra可阻断脊髓LTP的产生,并且可抑制GluR1的表达上调。以上表明,P2X7受体参与脊髓伤害性反应长时程增强的产生是通过IL-1β信号通路激活而实现的。免疫组化结果表明,小胶质细胞和星形胶质细胞分别在强直刺激后第3天和第7天被激活；Western blot结果表明,强直刺激可导致P2X7下游分子IL-18及其受体的表达增加。以上过程均可被强直刺激前1小时至强直刺激后7天每天1次连续注射小胶质细胞抑制剂美满霉素抑制。免疫双标结果显示,IL-18绝大部分与小胶质细胞标志物Iba-1共标,少量与星形胶质细胞标志物GFAP共标,而与神经元标志物NeuN无共标,而IL-18受体仅与星形胶质细胞标志物GFAP共标。以上结果表明,小胶质细胞和星形胶质细胞之间的相互作用参与病理性痛的维持,此过程很可能是由IL-18信号通路介导。综上所述,本研究表明,小胶质细胞上的P2X7受体在强直刺激坐骨神经诱发的脊髓场电位的LTP及持续性痛的产生中具有至关重要的作用,而此作用是通过IL-1β信号通路实现的；小胶质细胞可能通过P2X7受体下游信号分子IL-18激活星形胶质细胞,参与病理性痛的维持。更多还原

【Abstract】 Tetanic stimulation of the sciatic nerve induced long-term potentiation (LTP) of spinal cord neurons and pain behavioral hypersensitivity. The present work was to investigate the role of microglia in the modulation of central sensitization of spinal pain pathway induced by tetanic stimulation of the sciatic nerve (TSS) in rats.LTP, considered as the substrate of central sensitization in spinal pain pathway, can be induced by TSS, natural noxious stimulation of skin or nerve injury. Furthermore, the tetanic stimulation with the identical parameters used in electrophysiological recording induced hypersensitized pain behavior, including allodynia and hyperalgesia. However, the exact mechanisms of the development of spinal LTP remain obscure.Numerous works have proved that glia in the spinal cord, especially microglia, play a vital role in the development of pathological pain. Microglia can be activated in both inflammatory and neuropathic pain. Pre-application of glial metabolic inhibitor prevents the induction of spinal LTP and alleviates hypersensitized pain behavior, indicating the involvement of microglia in the induction of LTP of spinal nociceptive responses.P2X7 receptor (P2X7R) is excessively expressed in glia and considered as a pivotal mediator in the crosstalk between neuron and glia. The activation of P2X7R by exocytic ATP promoted significant release of glutamate, which is necessary for the induction of hippocampal LTP, and proinflammatory cytokines, such as interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α). It has been reported that spontaneous firing of spinal cord neurons and pain hypersensitivity are significantly suppressed by selective P2X7R antagonists. Moreover, P2X7 knockout mice showed no chronic inflammatory and neuropathic pain. Therefore, P2X7R may be a vital molecule in mediating the involvement of microglia in the induction of spinal LTP.By means of electrophysiological recording, mechanical allodynia test, immunohistochemistry and Western blot, the present study was to investigate the role of P2X7R in microglia in the induction of spinal LTP and long-term pain hypersensitivity induced by TSS. The main findings were as follows:Spinal LTP of C-fiber-evoked field potentials was induced by TSS and was blocked by intrathecal administration of microglial metabolic inhibitor minocycline 1 h before tetanic stimulation; the mechanical allodynia induced by TSS was partially alleviated by consecutively intrathecal administration of minocycline 1 h before and 7 days once daily after tetanic stimulation. These findings indicate that spinal microglia are involved in the induction of spinal LTP and pain hypersensitivity induced by TSS.Pre-injection of P2X7R antagonist oxATP or BBG 0.5 h before tetanic stimulation and P2X7-siRNA 7 days before TSS blocked spinal LTP; preincubation of spinal slices with BBG 1 h before tetanic stimulation blocked the spinal LTP of field excitory postsynaptic potentials (fEPSPs) induced by high frequency stimulation of Lissauer’s tract without affecting the baseline responses, suggesting a pivotal role of P2X7R in the induction of spinal LTP.Pre-injection of P2X7R antagonist oxATP or BBG 0.5 h before tetanic stimulation and P2X7-siRNA 7 days before TSS partially alleviated bilateral allodynia on day 3,5 and 7 after tetanic stimulation; pre-injection of BBG 0.5 h before tetanic stimulation suppressed the upregulation of Fos expression 2 h after TSS, indicating the significant inhibition of excessive excitation of spinal pain sensitization neurons by the antagonism of P2X7R. Immunohistochemistry results showed that P2X7R was predominantly colocalized with microglia marker OX-42 but not with astrocyte marker GFAP and neuron marker NeuN; the activation of microglia and the upregulation of P2X7R induced by TSS was suppressed by preadministration of P2X7 antagonist BBG 0.5 h before tetanic stimulation, indicating a specific role of P2X7R in the induction of spinal LTP.The upregulation of phospharylated p38 mitogen-activated protein kinase (MAPK)、IL-1βand GluR1 induced by tetanic stimulation was reversed by pre-injection of BBG 0.5 h before tetanus; the IL-1βantibody IL-1ra blocked the induction of spinal LTP and inhibited the increased expression of GluR1, indicating that IL-1βsignaling pathway was involved in the induction of spinal LTP after the activation of P2X7R.Imunohistochemistry results demonstrated that microglia and astrocytes were activated on day 3 and day 7 after TSS, respectively; Western blot results showed that the increased expression of interleukin-18 (IL-18), downstream molecule of P2X7R, as well as interleukin-18 receptor (IL-18R) was significantly inhibited by consecutive application of microglial metabolic inhibitor minocycline 1 h before and 7 days after TSS; IL-18 was predominantly colocalized with microglia marker Iba-1 and slightly with astrocyte marker GFAP but no colocalization with neuron marker NeuN; IL-18R was almost completely colocalized with astrocyte marker GFAP. These results suggest that the IL-18 signaling pathway contributes to the exacerbation of pathological pain by mediating the interaction of microglia and astrocytes.In conclusion, the present work demonstrated that P2X7R in microlia played a vital role in the induction of spinal LTP and persistent pain hypersensitization and IL-1βsignaling pathway was involved in the process. The interaction of microglia and astrocytes, mediated by a downstream molecule of P2X7R IL-18, contributed greatly to the maintenance of pathological pain.更多还原