井穴刺络法改善颅脑创伤性昏迷的腹侧中脑导水管周围灰质-下丘脑环路机制研究

颅脑创伤(TBI)性昏迷的促醒是神经学界的难点。课题组前期已证实手十二井穴刺络法可提高多种中枢神经损伤昏迷患者的意识水平，兴奋TBI大鼠腹侧中脑导水管周围灰质（vPAG）的多巴胺和P2RX7（嘌呤能受体）神经元，脑内P2RX7通路介导该法促醒。结合网状上行激活系统经典通路，本项目提出井穴刺络→vPAG多巴胺神经元（表达P2RX7）→下丘脑结节乳头核（TMN）组胺神经元（表达多巴胺受体）→促醒的科学假说。利用行为学、脑肌电评价促醒效应；采用TET-OFF系统(针灸时段特异表达c-fos)、vPAG多巴胺神经元顺行示踪联合fMOST技术描绘刺络兴奋脑区、vPAG到TMN的投射；利用基因定量、液质联用、免疫染色联合类流式定量明确刺络可兴奋TMN表达多巴胺受体的组胺神经元；vPAG和TMN分别注射P2RX7和多巴胺受体拮抗剂以探讨假说的上下游关系；揭示井穴刺络促醒的科学原理，促进中医急诊医学发展。

Developing the therapies targeting traumatic brain injury (TBI)-induced coma is one of the difficulties in neuroscience society. Our research group has confirmed that bloodletting puncture at hand twelve Jing-Well points (HTWP) can improve the consciousness level of the comatose patients with several central nervous system injuries. This therapy could excite the dopamine and P2RX7 expressed neurons in the ventral periaqueductal gray (vPAG) of the brain in TBI rats, and P2RX7 pathway mediated the revival of TBI comatose rats. Combined with the classic conduction pathway of the ascending reticular activing system, the project proposes a scientific hypothesis that bloodletting puncture at HTWP stimulates dopamine neurons (expressing P2RX7) in vPAG, then excite histamine neurons (expressing dopamine receptor) in hypothalamic tuberomamillary nucleu (TMN) to promote wakefulness. In the project, neural behavioral scoring, EEG and EMG will be detected multidimensionally to quantify the wake-up effect of this therapy. The TET-OFF system (c-Fos expressed specifically in acupuncture period), vPAG dopamine neuron anterograde tracing and fMOST technology will be used to describe the distribution of the activated brain area during bloodletting puncture at HTWP and the anterograde conduction path from vPAG to TMN. The methods of gene quantification, HPLC-MS and multiple immunofluorescence staining combined with TissueFACS quantification will be applied to explicit bloodletting puncture at HTWP activating the histamine neurons expressing dopamine receptors in TMN. Respective injection of P2RX7 and dopamine receptor antagonists in vPAG and TMN nuclei will be used to test the upstream and downstream relationship of the hypothesis，so as to provide scientific evidence for bloodletting puncture at HTWP application in TBI-induced comatose， and promote the development the emergency medicine of traditional Chinese medicine.