慢性失眠鼠模型的建立及其神经生物学机制的初步研究

Chronic insomnia (chronic insomnia, CI) is an incurable and health threatening disease with high prevalence, however, its neurobiological mechanism remains obscure. The further insight will be given into the mammal models which are not established. Theoretically, the occurrence and development of CI are defined within "3P" model. It has been hypothesized that adult insomnia originated from the early life and stress mediates its entire process. Our project seeks to establish a stable and replicable mouse model, which is characteristic of human CI and reaching the strict evaluation standards, by means of successive multiple-generation screening the animals with natural sleepless and/or administration of stress during prenatal or/and postnatal onwards. The further neurobiological mechanisms of CI will be investigated based on the aforementioned models. Due to the intendancy of excessive arousal (hyperarousal) hypothesis to clarify human CI, our project will focus on the activated status of sleep-wake modulating neurons and the expression of receptors relavant to the crucial cerebral excitatory / inhibitory neurotransmitters, along with the changes of activities in the central pathway of hypothalamic-pituitary-adrenal axis hormones and the changes of astrocytic functions and signaling pathways of adenosine, using molecular methods including immunological double labelling, aiming to disclose the primary mechanism of central hyperarousal under the CI. The project is expected to establish ideal replicable murine model and to preliminarily elucidate the neurobiological mechanisms of CI which will constitute the critical basis for further studies and therapeutic strategies.

慢性失眠（chronic insomnia, CI）具有患病率高、健康影响大、治愈困难的特点，但其神经生物学机制不明，目前缺少可用于深入研究的哺乳类动物模型。理论上，CI的发生发展满足“3P”模型。有假设认为成人失眠有“生命早期起源”，应激介导了全过程。本项目拟对自然睡眠少的动物连续多代筛选和/或对动物出生前后不同时期应激建立符合人类CI特征、满足严格的评估标准、可稳定复制的鼠模型。利用这种模型再进一步探讨CI的神经生物学机制。鉴于用过度觉醒假设解释人类CI的情况越来越多，本项目将利用免疫双标记等多种方法探讨睡眠－觉醒调节神经元激活状态及关键脑区兴奋/抑制性神经递质受体的表达、下丘脑－垂体－肾上腺轴激素的中枢通路活性、星型胶质细胞功能和腺苷信号系统的变化，以期了解CI状态下导致中枢过度觉醒的初步机制。项目的完成将提供理想的可复制的鼠模型，初步阐明CI的神经生物学机制。

慢性失眠（CI）可导致患者躯体不适（疲劳、不安、僵硬、触痛或头痛）、情绪、认知、社交功能损害，降低患者的生活质量。然而，由于缺乏合适的动物模型可供研究，对 CI 的病因、发生和维持及相关神经生物学机制的了解不足，导致 CI 的临床治疗大多基于对症层面。本研究采用适当的应激方式、适宜的动物品系，从睡眠、行为、神经内分泌等多个方面评估了CI 的动物模型，同时利用该模型开展了一系列相关神经生物学机制研究。本研究开发了一套无创睡眠记录系统，视频监测并评估小鼠的睡眠状况，再进行系统的行为学评估，构建可操作性CI 小鼠模型，采用ELISA、免疫组化、Western Blot、高效液相色谱法、RNA-scope、光/化学遗传学等技术探究CI的神经生物学机制。由于软件开发耗时较长，且受新冠疫情影响，本课题组同时继续了衰老机制和临床慢性失眠的探索。研究结果显示：（1）胚胎期炎症应激不仅可以导致母鼠中老年期出现认知下降，而且还导致其子代也表现出学习记忆损害、海马突触可塑性损害以及海马表观遗传学改变，比如Arc、Syt-1水平升高、中年以后 H3S10p下降和H3K9me3水平的升高、线粒体功能损害等等，一直持续到老年，且这些神经生物学指标的变化与学习记忆能力损害有显著相关性。青春期应激或丰富环境可以相应地加重或延缓胚胎期炎症应激所致的不良效应。（2）临床慢性失眠患者的认知水平下降，血液中睡眠相关生物学标志物也不同程度的改变且与睡眠质量相关。（3）胚胎期炎症导致子鼠认知和睡眠质量下降，HPA轴中枢通路过度活跃和大脑睡眠觉醒相关脑区神经递质表达异常。