外侧下丘脑谷氨酸能神经元在调控间歇低氧引起微觉醒和上气道开放中的作用及机制研究

Cortical arousals from sleep are a characteristic feature of adult obstructive sleep apnea-hypopnea syndrome (OSAHS). Arousals are vital to reopen the collapsed upper airway and restore airflow. However, frequent arousals contribute to OSAHS pathogenesis by destabilizing ventilatory control and sleep continuity. It is important to identify the mechanisms leading to arousal and upper airway reopen, which are currently only partially understood. Hypoglossal motoneurons (HMNs) innervate the intrinsic and most extrinsic tongue muscles such as genioglossus and exert a critical role in maintaining airway patency. Previously, we found glutamatergic neurons in the lateral hypothalamus (LH) may be involved in arousals and genioglossus muscle electromyograms enhancing caused by intermittent hypoxia, suggesting that glutamatergic neurons may play an important role in arousal of OSAHS. Therefore, we will use Vglut2-IRES-cre mice, fiber photometry, DREADDs, neurons lesion, patch clamp and immunohistochemistry, combine with recording of sleep-wake electroencephalograms and genioglossus electromyograms to clarify the roles and the underlying neurobiology and molecular mechanisms of glutamatergic neurons in LH on arousals and upper airway reopen caused by intermittent hypoxia. The results would find out the precise neural circuitry that mediates arousal of OSAHS, and would provide new insights into therapeutic approaches in OSAHS.

微觉醒是OSAHS重要病理生理特征，虽可有效开放上气道终止呼吸事件，但导致睡眠结构紊乱。目前微觉醒发生机制不详，其与上气道开放的直接关系及内在机制仍待探明。上气道的开放主要由以颏舌肌为主的肌群控制，受舌下神经运动神经元（HMNs）调控。我们发现间歇低氧引起小鼠发生较多微觉醒，伴随颏舌肌肌电增强的同时引起大脑外侧下丘脑（LH）谷氨酸能神经元激活。因此，我们推测间歇低氧异常激活LH谷氨酸能神经元诱发皮层微觉醒，同时兴奋HMNs促进上气道的开放。本课题将使用小鼠睡眠脑电/颏舌肌肌电记录系统、Vglut2-IRES-cre转基因小鼠、光纤钙信号、DREADDs、神经元毁损、全细胞膜片钳和免疫组化等方法，研究LH谷氨酸能神经元在调控间歇低氧引起微觉醒和上气道开放中的作用及其神经生物学和分子生物学机制。研究结果将揭示OSAHS微觉醒发生的神经调控环路，有助于从中枢调控层面寻找药物治疗靶点。

微觉醒是OSAHS重要病理生理特征，虽可有效开放上气道终止呼吸事件，但导致睡眠结构紊乱。目前微觉醒发生机制不详，其与上气道开放的直接关系及内在机制仍待探明。上气道的开放主要由以颏舌肌为主的肌群控制，受舌下神经运动神经元（HMNs）调控。下丘脑中的谷氨酸和组胺是大脑调节觉醒的主要神经物质，其是否参与OSAHS微觉醒及上气道开放的调控目前仍待探明，同时CIH引起的神经功能损伤亦可能破坏睡眠觉醒调节功能。我们推测LH组胺能、谷氨酸能神经元以及神经炎症诱发的神经损伤均参与了微觉醒的调控。我们运用小鼠睡眠脑电/颏舌肌肌电记录系统、CIH模型、Vglut2-IRES-cre转基因小鼠、NLRP3基因敲除小鼠、光纤钙信号、DREADDs、神经元毁损、全细胞膜片钳和免疫组化等方法，发现CIH后LH及舌下神经运动核中的组胺表达水平下降，说明CIH显著影响下丘脑的觉醒系统；在NREM睡眠期，LH谷氨酸能神经元的钙信号显著低于觉醒期和REM睡眠期，当小鼠由NREM睡眠期向觉醒转换转换时，LH谷氨酸能神经元的钙信号均明显增强，而当小鼠由觉醒期转换为睡眠期时，LH的谷氨酸能神经元的钙信号明显减弱，说明LH谷氨酸能神经元参与大脑觉醒的调控；LH谷氨酸神经元对蓝斑核、基底前脑的投射介导了觉醒的促进作用；CIH环境中，小鼠经过恐惧调节测试后环境联系性记忆和声音联系性记忆均较正常状态下小鼠下降，而NLRP3敲除小鼠在CIH环境中的学习记忆功能较正常小鼠增强；NLRP3炎症小体在CIH诱导神经功能损伤中的分子机制与Parkin信号通路依赖的线粒体自噬密切相关，进一步探讨神经炎症与OSAHS睡眠觉醒调节紊乱关系的研究目前在进展中。本课题完善了OSAHS睡眠觉醒从神经递质-神经元投射到神经炎症的综合调控网络，为未来OSAHS睡眠觉醒紊乱的治疗方案特别是药物治疗提供了方向。