SDF1-CXCR4通过媒介胶-胶、胶-神对话参与中枢卒中后疼痛的分子细胞机制研究

Central post-stroke pain (CPSP) is one of the most intractable central neuropathic pain that is insensitive to all available therapeutic approaches, deeply affecting the quality of life in survival patients. To date, the underlying mechanisms of CPSP remain unknown due to lack of animal studies. More recently, we successfully established experimentally hemorrhage-induced CPSP by intra-thalamic collagenase IV (ITC) injection. It was found that both microglial and astrocytes were significantly activated around the hemorrhagic core. Intra-thalamic injection of specific inhibitor of the two glial cells, respectively, can effectively reverse the ITC-induced CPSP. Moreover, intra-thalamic injection of SDF1, a chemokine, resulted in a similar behavioral response to ITC that could be reversed by co-injection of a selective antagonist against its receptor CXCR4, suggesting critical role of SDF1-CXCR4 signaling in induction and maintenance of CPSP. The hypothesis of the present project is that SDF1-CXCR4 mediates glial-glial and glial-neuronal cross-talk through trans-membrane signaling, causing synaptic allostasis of thalamocerebral connections by neuroinflammatory niche, leading to CPSP. Herein, we will use behavioral pharmacology, electrophysiological recordings and cellular/molecular biology to study the roles of SDF1-CXCR4 signaling in induction and maintenance of CPSP. It is expected to provide experimental and theoretic basis for for prevention and treatment of CPSP.

中枢卒中后疼痛(Central post-stroke pain, CPSP)是临床最顽固的一类慢性中枢神经病理性痛，对现有药物几乎都不敏感，严重影响了病人的生命质量。由于研究稀少，CPSP的发病机制不清。本课题组前期利用丘脑内注射胶原酶的方法成功建立并评价了大鼠CPSP模型，发现丘脑损伤周边区星型胶质细胞和小胶质细胞激活，注射胶质细胞抑制剂显著翻转CPSP；此外丘脑注射趋化因子SDF1可诱导出同样的痛行为，而阻断其受体CXCR4可以翻转SDF1和胶原酶诱导的痛行为，提示SDF1-CXCR4信号参与CPSP发病。本项目的假说是SDF1-CXCR4信号介导胶质细胞-胶质细胞和胶质细胞-神经元的对话，造成炎症微环境导致丘脑-皮层通路突触稳态失调，参与CPSP的发生和维持。为论证该假说，本项目从细胞、组织和整体动物水平研究，旨在阐明CPSP的发病机制，为临床防治CPSP提供实验和理论依据。

本项目提出的假说是丘脑出血性卒中引起出血部位及邻近脑组织损伤和神经炎症累及损伤了脊髓背角脊丘束（STT）投射神经元轴突终末而发生逆行性溃变，最终导致其脊髓背角STT投射神经元胞体变性死亡，进而引起脊髓背角继发性神经炎症和中枢敏化，这个脊髓背角继发性胶-胶和胶-神互作过程也可能由趋化因子SDF1及其受体CXCR4介导并引起脊髓中枢敏化和屈肌反射易化，出现机械性痛敏。本项目应用脑立体定位注射IV型胶原酶溶液（ITC）法建立大鼠中枢卒中后疼痛（central post-stroke pain, CPSP）模型，注射生理盐水（ITS）做对照，然后应用细胞免疫荧光技术、免疫印迹技术、多电极阵列胞外记录技术和鞘内置管给药技术等研究了丘脑出血损伤后脊髓背角是否发生因STT投射轴突终末损伤而出现逆行性神经元胞体变性和继发性神经炎症进而使CPSP加重和慢性化问题。本项目在ITC注射第7天大鼠双侧后肢出现稳定的机械性痛敏后开始采样，获得以下主要发现：（1）丘脑出血性卒中可引起脊髓背角继发性逆行性神经元胞体凋亡变性、变性神经元胞体毗邻突触结构变性；与ITS组比较，电生理记录显示脊髓背角三类神经元大量丢失（以有自发基础放电型的WDR和NS神经元为主）。（2）丘脑出血性卒中可引起脊髓背角继发性神经炎症，伴随小胶质细胞和星形胶质细胞增生，趋化因子SDF1及其受体CXCR4表达上调。（3）与ITS组比较，丘脑出血性卒中可引起脊髓背角健在的神经元敏化，这些神经元大多数是没有自发基础放电的WDR和NS神经元。（4）注射ITC第7天实施鞘内置管术，恢复3天后（即ITC注射后第10天）开始鞘内药理学试验，发现鞘内分别给予小胶质细胞抑制剂米诺环素、氟代柠檬酸和CXCR4拮抗剂AMD3100均可以抑制小胶质细胞和星形胶质细胞的活化增生，趋化因子SDF1及其受体CXCR4过表达，并且可以缓解双侧机械性痛。