睡眠片段现象对唤醒反应能力的影响及orexin的调节作用

Sleep apnea syndrome is a sleep state-dependent disorder that associated with significant neurobehavioral impairments and neural injury. The impaired arousal responses to respiratory stimuli is a feature of the sleep apnea. Our previous study found that sleep fragmentation in both human and animals could increase arousal thresholds to stimuli and decrease plasma orexin-A levels. However, the mechanism by which sleep fragmentation impairs arousal ability and orexin system is not clear. Accordingly, the purpose of this study is to test the hypothesis that sleep fragmentation could, like intermittent hypoxia, result in impaired arousal responses to respiratory stimuli, and the orexin system may have a neuromodulatory effect on arousal states. The method of sleep fragmentation utilized in this study is designed to stimulate multiple arousals which occur in patients with sleep apnea. In this series of studies, we expect to validate the effects of sleep fragmentation on arousal ability which are obtained from observation of the daytime behavior, and to confirm more objectively on a number of occasions by measurement of the arousal latency of stimuli. We then will apply neurobiological methods to investigate the relationship between arousals index and lateral hypothalamic orexin neuron activity and orexin release, and to evaluate whether suppression of orexin signaling by receptor antagonist could change the arousal response to stimuli. Furthermore, we will explore potential mechanism of orexin neurons injury with a focus on endoplasmic reticulum stress pathways. The proposed studies are designed to identify the hypothesis that sleep fragmentation associate with arousal ability impairment and orexin system play an important role in this process. The result and broad conclusions will increase our understanding of the neurobiological and molecular mechanisms underlying sleep apnea, and bringing us closer to finding therapies to prevent or reduce this disorder.

唤醒反应能力下降是睡眠呼吸暂停综合征（SAS）的主要病理损害之一，已被证明可以在SAS发病中起重要作用。我们前期工作模拟SAS患者睡眠结构特征建立了睡眠片段(SF)模型，通过唤醒潜伏期测试证实SF可以造成唤醒反应能力损伤，并进一步发现SF后大鼠下丘脑orexin（Ox）能神经元功能发生明显改变，但具体机制尚不清楚。本研究采用现代睡眠医学和神经生物学手段，通过对SF后人体和实验动物唤醒反应能力的行为学观察和测试，进一步明确SF对唤醒反应能力的影响。进而应用形态学和分子生物学方法，观察SF前后大鼠下丘脑Ox能神经元数量和活性的改变，以及给予药物干预Ox信号途径后唤醒反应能力的变化，明确Ox在唤醒反应能力损伤中的调节作用，然后观察SF后内质网应激途径关键信号蛋白的表达，探索造成Ox能神经元损伤的分子机制。以期揭示SF导致唤醒反应能力损伤的神经生物学机制，为SAS患者的临床治疗提供新的线索。

研究背景：唤醒反应能力损伤是睡眠呼吸暂停患者的常见现象，探索睡眠呼吸暂停患者患者唤醒反应能力损伤的机制研究对于分析SAS的病因以及改善SAS患者的临床症状都具有十分重要的价值。慢性睡眠片段可能是造成这一损伤过程的重要因素，觉醒相关神经元可能是主要的损伤靶点。.主要研究内容：通过建立片段睡眠剥夺的小鼠动物模型，观察不同时间片段睡眠剥夺对小鼠睡眠结构的影响，以及小鼠对不同刺激的唤醒阈值，持续维持觉醒状态能力的变化，探讨片段睡眠现象对小鼠觉醒功能的损伤情况。.主要结果：对健康雄性小鼠进行4周片段睡眠剥夺（30次/小时）和对照观察，而后进行行为学观察、免疫组化和蛋白分析等。小鼠多次睡眠潜伏期测试证实小鼠平均睡眠潜伏期缩短（P<0.05）。CO2刺激后的唤醒潜伏期明显延长（64±5 s vs. SF4wk 154±6 s, P<0.001）。C-fos阳性的orexin能神经元数量显著减少(P<0.05-0.001)。.结论及研究意义：应用我们设计的摇床作为觉醒刺激源的方法，可以明显增加小鼠的总觉醒指数及睡眠-觉醒转换次数。C02刺激唤醒潜伏期随片段睡眠剥夺时间延长而逐渐延长，睡眠片段现象可以损伤小鼠的唤醒反应能力。小鼠下丘脑Fos阳性的orexin神经元明显较对照组减少，片段睡眠现象可以损伤orexin神经元的激活功能，可能是其造成唤醒反应能力损伤的主要途径。