片段睡眠现象对觉醒功能的影响及机制研究

发布时间：2018-02-13 18:28

本文关键词： 片段睡眠睡眠结构觉醒脑电功率片段睡眠觉醒唤醒反应睡眠潜伏期片段睡眠下丘脑觉醒神经元 orexin　出处：《第二军医大学》2012年博士论文　论文类型：学位论文

【摘要】：【目的】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，以及小鼠脑电功率谱在片段睡眠剥夺前后的变化。探讨片段睡眠小鼠动物模型的效果，并分析片段睡眠现象导致的相应睡眠结构变化后小鼠脑电功率以及睡眠稳态调节功能的变化。【方法】成年雄性C57Bl/6J小鼠放置睡眠记录电极、进行连续信号采集。随机分为片段睡眠剥夺组和环境对照组，分别在基线、片段睡眠剥夺1天和片段睡眠剥夺4周等3个时间点分析小鼠睡眠结构。所有片段睡眠剥夺组小鼠采用我们在美国宾夕法尼亚大学设计建立的模型，进行持续片段睡眠剥夺。睡眠记录结果采用SleepSign软件自动分析后人工修正，并用此软件分析不同睡眠期的脑电功率。【结果】1.摇床运动制造片段睡眠现象的效果：和基线状态时相比(1404±139)，小鼠24h觉醒状态转换次数在片段睡眠剥夺第1天明显增加（1989±133，p0.05），睡眠剥夺第28天继续显著增加（2155±188，p0.001）。2.片段睡眠剥夺对小鼠睡眠结构的影响：片段睡眠剥夺组小鼠总觉醒指数较对照组（30.2±1.7）明显增加，其中在剥夺第1天（43.9±2.1，p=0.003）和第28天（43.8±3.8，p=0.004）效果持续存在，并且在恢复睡眠2周后恢复到基线水平（29.2±3.0）。3.片段睡眠剥夺对小鼠脑电功率谱的影响：NREM睡眠期的delta功率在基线（0.37±0.03）较低，片段剥夺第1天（0.42±0.02，p0.05）和第28天（0.55±0.02，p0.01）均较基线明显升高。【结论】我们建立的小鼠模型可以完整展示出片段睡眠现象的特征睡眠结构改变，并且能够对小鼠脑电功率造成相应改变，反应出小鼠睡眠稳态调节的作用，可以作为下一步研究的基础。【目的】观察不同时间片段睡眠剥夺后，小鼠对不同刺激的唤醒阈值，以及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，并观察恢复睡眠对上述损伤的逆转情况。【方法】成年雄性C57Bl/6J小鼠放置睡眠记录电极、进行连续信号采集。随机分为片段睡眠剥夺组和环境对照组，参照第一部分建立片段睡眠剥夺模型，分别在基线、片段睡眠剥夺1周、片段睡眠剥夺2-4周和恢复睡眠2周等4个时间点进行5%高浓度CO2刺激、声音刺激和气流刺激等3种不同刺激后唤醒潜伏期实验，以及小鼠多次睡眠潜伏期测试。唤醒潜伏期测试及多次睡眠潜伏期测试中睡眠/觉醒状态识别均采用人工判断。【结果】1. CO2刺激唤醒潜伏期变化：和基线（74.00±18.69）相比，唤醒潜伏期在片段睡眠剥夺1周（116.40±11.14，p=0.003）、片段睡眠剥夺4周（154.00±7.01，p=0.000）显著升高。在恢复睡眠2周后恢复至基线水平（101.00±10.41，p=0.062）。2.声音、气流刺激阈值：结果显示片段睡眠剥夺后，小鼠被两种刺激的唤醒阈值均升高（p0.01），表明片段睡眠现象的唤醒功能损伤不仅限于高浓度CO2刺激，而是一种全面损伤。3.片段睡眠剥夺后小鼠多次睡眠潜伏期：和基线相比（9.44±0.94min），1周片段睡眠剥夺可以明显降低小鼠的入睡潜伏期(5.89±0.60min，p=0.049)，4周片段睡眠剥夺的同样较基线明显降低(3.80±0.84min，p=0.001)。2周恢复睡眠后，入睡潜伏期同基线睡眠没有显著差异（p=0.10)。【结论】片段睡眠现象可以明显损伤小鼠的唤醒反应能力和觉醒维持能力，其中觉醒维持能力可以在恢复睡眠后得到恢复，唤醒反应能力不能得到完全逆转。【目的】观察下丘脑orexin能神经元在不同时间片段睡眠剥夺和恢复睡眠前后的变化，探讨orexin能神经元损伤在片段睡眠现象造成觉醒功能损伤中的作用。【方法】成年雄性C57Bl/6J小鼠随机分为正常对照组（CT）、SF片段睡眠剥夺组（SF），各组又分CO2持续刺激组（CO2）及空气组（RA）。即共分为四组：CT-CO2组，，SF-CO2组, CT-RA组, SF-RA组，每组10只小鼠。片段睡眠剥夺时间为4周，片段睡眠剥夺方法同前。小鼠经麻醉后处死，采用免疫组织化学法检测小鼠下丘脑觉醒神经元相应神经递质表达以及Fos的表达，蛋白质免疫印迹技术（Western-blot）测定下丘脑orexin的表达。【结果】1.片段睡眠剥夺后各组下丘脑Orexin蛋白表达变化：Orexin阳性细胞分布以下丘脑外侧区(LHA)、穹隆周区及其邻近区域为主。经统计分析，各组orexin阳性细胞数无统计学差异（p0.05）。2.片段睡眠剥夺后各组下丘脑Fos蛋白表达变化：在下丘脑外侧区(LHA)，CT-RA组LHA表达Fos阳性细胞较少，CT-CO2，SF-RA，SF-CO2三各组较对照组相比Fos阳性细胞数均增加。3.片段睡眠剥夺后各组下丘脑Fos阳性的orexin神经元表达变化：阳性率在CT-CO2组最高，双标细胞占总orexin阳性细胞的50.53%，在CT-RA组次之，为39.33％，在SF-CO2组为26.28%，而在SF-RA组最低，仅占21.16%。SF-RA组与CT-RA组及CT-CO2组的阳性细胞率相比差异均具有统计学意义(p0．05)，SF-RA组与CT-RA组及CT-CO2组的阳性细胞率相比差异均具有统计学意义(p0.05)。4. Orexin在小鼠下丘脑的表达变化：Western-blot方法比较CT-RA组及SF-RA组两组之间小鼠下丘脑orexin蛋白含量的变化。两组之间下丘脑的orexin蛋白含量之间不存在统计学差异（p0.05）。【结论】片段睡眠剥夺后orexin蛋白的表达量没有明显的改变，但活性的orexin神经元数量减少，表明片段睡眠现象可以造成小鼠下丘脑orexin能神经元激活能力受到了损伤，可能是片段睡眠导致觉醒功能损害的主要作用递质。
[Abstract]:Objective To establish a mouse model of sleep deprivation in mice , observe the effects of sleep deprivation on the sleep structure of mice and the changes of brain electrical power spectrum of mice before and after the deprivation of sleep deprivation . Sleep deprivation group mice were randomly divided into two groups : sleep deprivation group and environment control group . The sleep structure of mice was analyzed at 3 time points , such as baseline , segment sleep deprivation , 1 day and 4 weeks of sleep deprivation , respectively . All segments of sleep deprivation group used the model we established at the Pennsylvania University of Pennsylvania . The results of sleep recordings were manually corrected after automatic analysis using SleepSign software and analyzed with this software to analyze the brain electrical power of different periods of sleep . The effects of sleep deprivation on sleep structure in mice were significantly increased ( 2155 卤 188 , p = 0.004 ) and 28 ( 43.8 卤 3.8 , p = 0.004 ) and 28 ( 43.8 卤 3.8 , p = 0.004 ) . Conclusion : The mouse model established by us can completely display the characteristic sleep structure change of the fragment ' s sleep phenomenon , and can change the brain electric power of mice accordingly , and reflect the effect of sleep homeostasis in mice , which can be used as the basis for the next study . Objective To observe the effect of fragment sleep on the wake - up function of mice after sleep deprivation in different time segments , and to investigate the effect of fragment sleep on the wake - up function of mice , and to observe the reversal of the recovery of sleep on the above - mentioned injury . The sleep deprivation model was established in the first part by reference to the first part . The sleep deprivation model was established by reference to the first part . After 3 different stimuli , such as baseline and segment sleep deprivation , 1 week after sleep deprivation , 2 - 4 weeks of sleep deprivation and 2 weeks of recovery , the sleep latency test was performed in mice . The sleep / wake state recognition in the sleep latency test and multiple sleep latency tests were determined manually . The result is incremented by 1 . Compared with baseline ( 74.00 卤 18.69 ) , the wake - up latency was significantly higher than baseline ( 101.00 卤 10.41 , p = 0.062 ) . Conclusion The sleep deprivation can significantly impair the ability of awaking reaction and the ability to maintain awaking ability of the mice , and the ability of arousal to maintain can be recovered after the recovery of sleep , and the ability of awakening reaction cannot be completely reversed . Objective : To observe the effects of orexin on sleep deprivation and restoration of sleep before and after sleep deprivation and restoration of sleep deprivation in hypothalamic orexin neurons . Methods Twenty - six adult male C _ 2 groups were divided into four groups : CT - CO2 , SF - CO2 , CT - RA and SF - RA . The results were as follows : CT - CO2 , SF - CO2 , CT - RA and SF - RA . All the 10 mice were divided into four groups : CT - CO2 , SF - CO2 , CT - RA and SF - RA . The expression of orexin protein in the hypothalamus of each group was significantly higher than that in the control group ( p < 0.05 ) . The positive rate of the positive cells in the hypothalamus of the hypothalamus ( LHA ) and the CT - RA group was 26.28 % , and the SF - RA group was 26.28 % . The expression of orexin in hypothalamus of mouse was changed : Western - blot was used to compare the changes of orexin protein content between two groups of CT - RA and SF - RA group . There was no statistical difference between the two groups ( p < 0.05 ) . Conclusion There was no significant change in orexin protein expression after sleep deprivation , but the number of active orexin neurons decreased , suggesting that fragment sleep could cause damage to the activation of orexin neurons in the hypothalamus of mice .

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R338

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