死亡相关蛋白激酶1介导皮层丘脑环路损伤引起卒中后疼痛的机制研究

Central post-stroke pain (CPSP) is the most common central pain caused by cerebral stroke and seriously affects the patients’ post-stroke rehabilitation and quality of life. So far, there is no effective treatment for CPSP. It has been suggested that the pathogenesis of CPSP is associated with central disinhibition, central sensitization and sensory imbalance, but the specific molecular signaling pathways and neural circuits are still not fully elucidated. Our previous work demonstrate that Death Associated Protein Kinase 1 (DAPK1) is a key molecular mediates neuronal death in ischemic stroke, and the development of CPSP relied on the damage of cortex-thalamic projecting neurons by cerebral ischemia induced DAPK1-tau death signaling. DAPK1 gene knockout or exogenous blocking DAPK1 death signal can not only relieve cerebral ischemic injury, but also alleviate CPSP. Based on the previous work, the proposed study will use genetically modified mice to perform focal cortical ischemia induced CPSP mice model, combined with applying neural tracing, optogenetics, electrophysiological recording and pain behavior detection methods, we will study the interaction of DAPK1 with downstream substrate in the cortical-thalamus projecting fibers, DAPK1 signaling dependent cortex-thalamic reticular neuronal (TRN) synapse injury, TRN neuronal excitability, the releasing of GABA neurotransmitter, thalamic ventral basal (VB) neuronal excitability. We will recover that cerebral ischemia induced DAPK1 death signaling promotes the injury of cortex-thalamic TRN-thalamic VB neuronal circuit and finally produce CPSP. Our study will further replenish the central disinhibition theory of CPSP, and provide new idea and strategy for the clinical treatment of CPSP.

卒中后疼痛（CPSP）是脑卒中引起的最常见中枢性疼痛，缺乏有效治疗，严重影响患者卒中后康复与生活质量。CPSP的发病与中枢去抑制、中枢敏化及感觉失衡有关，但具体分子机制不明。我们前期工作表明，死亡相关蛋白激酶1（DAPK1）是介导卒中后神经元死亡的关键分子，敲除DAPK1基因或外源性阻断DAPK1死亡信号，不仅可以减轻脑缺血损伤，也能缓解CPSP。本项目拟在此基础上，运用转基因小鼠局灶性皮层缺血CPSP模型，结合病毒示踪、光遗传学、电生理以及痛行为学检测方法，检测皮层丘脑投射纤维DAPK1与下游底物的相互作用、皮层丘脑投射与丘脑网状结构TRN神经元突触损伤、TRN神经元兴奋性与GABA递质释放、丘脑腹侧基底部VB神经元兴奋性。本研究将揭示缺血性卒中后DAPK1死亡信号通过损伤皮层-丘脑TRN神经元-丘脑VB神经元环路引起CPSP的机制，为临床治疗CPSP提供新策略。

卒中后疼痛（CPSP）是脑卒中引起的最常见中枢性疼痛，严重影响患者卒中后康复过程与生活质量。由于CPSP的发病机制不明，临床治疗非常棘手，主要以缓解症状为主。因此，阐明CPSP的病理生理机制，针对性开发有效的治疗策略非常重要。以往研究表明，CPSP的发病与中枢去抑制、中枢敏化及感觉失衡有关，但具体的神经环路与分子机制仍然未阐明。.我们前期工作表明，死亡相关蛋白激酶1（DAPK1）是介导卒中后神经元死亡的关键分子.，敲除DAPK1基因或外源性阻断DAPK1死亡信号，不仅可以减轻脑缺血损伤，也能缓解CPSP。在此基础上，我们进一步运用DAPK1激酶结构域条件性敲除（DAPK1-KD-/-）小鼠建立局灶性皮层缺血CPSP模型，结合病毒示踪、光遗传学、电生理以及痛行为学检测方法，检测皮层丘脑投射纤维DAPK1与下游底物的相互作用、皮层丘脑投射与丘脑网状结构TRN神经元突触损伤、TRN神经元兴奋性与GABA递质释放、丘脑腹侧基底部VB神经元兴奋性。经过本课题的开展研究，我们初步阐明了：（1）缺血部位与CPSP的发生具有一定的关联，且大脑皮层缺血与CPSP的发生具有联系；（2）CPSP发生后，皮层到丘脑的投射减少，丘脑VB神经元的兴奋性明显增加，而TRN神经元兴奋性减少，异常的皮层丘脑投射减少可能与丘脑TRN神经元兴奋性减少，VB神经元兴奋性增加以及CPSP的发生有关；（3）CPSP小鼠丘脑代谢水平增加，丘脑中GABA能中间神经元被激活，提示GABA能中间神经元的激活可能与丘脑代谢水平增加以及与CPSP发生有关；（4）死亡相关蛋白激酶1（DAPK1）可以磷酸化Caytaxin介导CPSP小鼠皮层神经元死亡；（5）抑制 Caytaxin 的表达，突触前信号传递减小，突触后受体也受到抑制，神经元活性降低；敲除 DAPK1 的激酶域，可以增加Caytaxin蛋白水平，从而参与神经元活性的维持。本研究初步揭示缺血性卒中后DAPK1通过磷酸化Caytaxin引起的死亡信号损伤皮层-丘脑TRN神经元-丘脑VB神经元环路引起CPSP的机制，为临床以Caytaxin为靶点治疗CPSP提供新策略。