Orexinergic神经元在异氟烷麻醉觉醒效应中的机制研究

Approximately 230 million people undergo general anesthesia every year in the world, but the mechanism of general anesthesia is still unclear. Our previous studies found that isoflurane could inhibit activation of orexinergic neurons, while exogenous administration of orexin could promote arousal from isoflurane anesthesia. Another study demonstrated that isoflurane inhibit the 5-HT neurons in dorsal raphe nucleus (DRN), suggesting 5-HT neurons in DRN might involved in anesthesia-arousal. Our pilot experiments revealed that orexin neurons promote anesthesia arousal by activation of 5-HT neurons in the DRN. Together with these pilot studies and other lines of evidence, we therefore put forward the hypothesis of this project: Orexinergic neurons in perifornical area (Pef) activates 5-HT neurons in the dorsal raphe nucleus (DRN), then increase the projection from 5-HT neurons to the cortex during isoflurane anesthesia, thereby promoting anesthesia arousal. To address this unresolved question, we will critically test this hypothesis with a combination of optogenetics, in vivo microdialysis, neuropharmacology, electrophysiology and high performance liquid chromatography on mice expressed the Cre recombinase under the promoter of the gene encoding different neuron markers. These studies will provide an important theoretical basis for further investigation of general anesthesia, thus establishing novel targets for the new narcotic and wake-promoting drugs development.

全球每年有约2.3亿人接受全身麻醉，但全麻药物作用机制还不清楚。项目组前期研究发现异氟烷能够抑制Orexinergic神经元活性，外源性给予摄食素 (Orexin)能够促进异氟烷麻醉觉醒。另有研究证实，异氟烷可抑制中缝背核(DRN)的5-HT神经元，提示DRN区5-HT神经元可能参与麻醉-觉醒。预实验结果显示Orexinergic神经元可以通过激活DRN区5-HT神经元促进麻醉觉醒。综上我们提出假说：异氟烷麻醉中，下丘脑穹窿周围核(Pef)区Orexinergic神经元激活DRN区5-HT神经元，导致5-HT神经元到皮层的投射增加，从而促进麻醉觉醒。本项目拟采用光遗传、在体微透析、电生理、高效液相色谱等技术深入阐明Orexinergic神经元在异氟烷麻醉觉醒中作用机制。研究结果将为进一步揭示全麻机制提供重要理论依据，同时也为新型麻醉和促觉醒药物的研发提供新的作用靶标。

全球每年约有超过2.3亿人接受全身麻醉，但全麻药物的机制尚不清楚。已有研究表明，orexin能神经元的激活有助于动物全身麻醉后的苏醒。此外，有研究表明，接收orexin神经元投射的5-HT能神经元也参与了睡眠-觉醒调节。本项目采用神经药理学、脑电分析、频谱分析、免疫组化、动物行为学等技术深入阐明Orexinergic神经元在异氟烷麻醉觉醒中作用机制。将Orexin A(30或100 pmol)、Orexin B(30或100 pmol)及其拮抗剂SB-334867和TCS- OX2-29(5或20μg)微注射入大鼠中缝背核(DRN)，分析其对诱导和觉醒时间的影响。同时记录并分析脑电图的变化，阐明异氟烷麻醉下DRN微注射orexin对皮层兴奋性的影响。通过对orexin给药后c-Fos表达的免疫组化分析，检测5-HT能神经元的激活。结果表明，与生理盐水组相比，注射orexin及其拮抗剂对大鼠诱导时间均无影响，而注射orexin-A(100 pmol)可增强觉醒。而给予orexin1型受体拮抗剂SB- 334867(20μg)延长了异氟烷麻醉的苏醒时间。异氟烷麻醉下，微注射orexin-A可诱导脑电图觉醒模式，并降低爆发抑制比。异氟烷麻醉抑制5-HT能神经元的活动，表现为与假手术组相比c-fos免疫反应阳性的5 -HT能神经元数量减少，这种抑制作用被orexin-A部分逆转。电生理学研究结果表明灌注异氟烷会抑制DRN区5-HT神经元活性，给予orexin-A会激活异氟烷灌注期间DRN的5-HT神经元活性。综上所述，我们的研究结果表明，orexinergic神经元的促进异氟烷麻醉觉醒作用通过DRN的5-HT能神经元发挥作用。这些研究将为进一步研究全麻提供重要的理论依据，其①是对以往由核团为主构成的“神经环路理论”进行完善;②证实上行激活系统在麻醉-觉醒中作用机制的新途径;③为诠释全麻药物麻醉-觉醒调控机制提供新的理论依据④为新型麻醉和促觉醒药物的研发提供新的作用靶标。