睡眠剥夺对大鼠学习能力的影响及机制实验研究

发布时间：2018-05-13 11:58

本文选题：睡眠剥夺 + 学习　；参考：《天津医科大学》2007年硕士论文

【摘要】： 目的睡眠是脑的高级功能之一，是生命赖以维持的基本条件。睡眠剥夺是由于各种原因导致的睡眠不足的状态，睡眠剥夺能够对机体产生广泛的影响。本研究利用小平台水环境法制备大鼠睡眠剥夺动物模型，探讨不同持续时间睡眠剥夺对大鼠学习能力的影响并初步分析其可能机制。方法 1实验动物及分组选用体重240～260g的健康成年SD大鼠，雌雄各半。随机分为正常对照组、大平台对照组、睡眠剥夺2d、4d、6d组。 2睡眠剥夺模型制备制作30cm×30cm×30cm睡眠剥夺箱，内置一直径6.3cm的小平台，高8cm，箱中注水，水面低于平台面约1.0cm。利用此法制备大鼠睡眠剥夺2d、4d、6d组模型。大平台对照组平台直径为18cm，大鼠置于大平台环境6天，其余条件同睡眠剥夺组。正常对照组笼养。 3一般状态观察观察睡眠剥夺后大鼠精神状态、皮毛、饮水饮食、反应能力等改变。 4学习能力测试大鼠学习能力测试在三等分“Y”型迷宫中进行，环境为安静暗室。Y迷宫每臂顶端设1个信号灯，以此提示“安全区”，通电后安全区灯亮，该区内无电流刺激，其余区域被通以36V交流电。实验时，将大鼠放入迷宫，先适应5分钟，然后开始实验。规定大鼠受电击后从起步区直接逃至亮臂安全区为“正确反应”。实验中每次通电刺激间隔时间为30秒，大鼠连续10次反应正确时，即为达标，记录大鼠达标时所需的总训练次数，以此代表学习能力。 5海马组织超微结构透射电镜检查取SD-6d大鼠海马组织制作电镜标本，观察超微结构改变。 6海马组织神经递质测定将大鼠迅速断头，取双侧海马。高效液相色谱法检测五羟色胺、去甲肾上腺素、谷氨酸、γ-氨基丁酸含量；碱性羟胺比色法检测乙酰胆碱含量。 7统计学分析方法各组数据以(?)±s表示。利用单因素方差分析进行假设检验。组间差异利用Dunnett-t检验，检验水准α=0.05。结果 1大鼠睡眠剥夺后一般状态的改变 SD组动物在实验早期表现出一定的兴奋性行为，，如活动增多，对外界声、光等刺激敏感。而在实验中后期(SD-4d以后)，SD动物行为由兴奋逐渐转为抑制，并表现出情绪上的“激惹”现象。 2睡眠剥夺对大鼠学习能力的影响与CC组比较，SD-4d组和SD-6d组大鼠学习能力下降，达标所需训练次数增多(p＜0.01)；SD2d组学习能力提高，达标所需训练次数减少(p＜0.05)。 3睡眠剥夺大鼠海马超微结构改变海马神经元细胞水肿，暗细胞变，向凋亡或类凋亡方向过渡；部分神经元出现核染色质溶解，胞质内粗面内质网和核糖体明显减少或缺失，线粒体减少，结构模糊；部分突触水肿，突触小泡减少；血脑屏障结构明显异常，内皮细胞暗调型或细胞变性甚至出现胞质断裂等严重损伤的表现，星形胶质细胞足突严重水肿；在神经元内及血脑屏障结构等处均可见到髓磷体明显增多。 4睡眠剥夺对海马神经递质含量的影响乙酰胆碱：SD-4d组、SD-6d组大鼠海马Ach含量低于CC组，具有显著性差异(p＜0.05，p＜0.01)。去甲肾上腺素：SD-4d组、SD-6d组大鼠海马NE含量低于CC组(p＜0.05)；TC组及SD-2d组NE含量高于CC组(p＜0.05)。五羟色胺：高效液相法测得SD-2d组、SD-4d组大鼠海马5-HT含量明显高于CC组和TC组(p＜0.01，p＜0.05)；SD-6d组大鼠海马5-HT含量明显低于CC组和TC组(p＜0.01)；TC组5-HT含量与CC组相比无显著差异。谷氨酸、γ-氨基丁酸及二者比值：SD-2d组大鼠海马GLU含量高于CC组(p＜0.01)，SD-6d组大鼠海马GLU含量低于CC组(p＜0.05)，与TC组比较，SD-2d组和SD-4d组GLU含量增加(p＜0.01，p＜0.01)，SD-6d组GLU含量减少(p＜0.05)。SD各组大鼠海马GABA含量均高于CC组(p＜0.05，p＜0.01)；与CC组比较，SD-4d组和SD-6d组GLU／GABA值显著降低(p＜0.01)；SD-2d和TC组GLU／GABA值无显著变化。结论 1．较长时间(4天以上)睡眠剥夺会造成大鼠学习能力降低，表现为Y型迷宫训练达标所需训练次数增多。 2．睡眠剥夺引起大鼠海马结构损伤，表现为神经元细胞水肿，向凋亡或类凋亡方向过渡；突触水肿，突触小泡减少；血脑屏障结构明显异常；髓磷体明显增多。 3．睡眠剥夺可以造成大鼠海马多种神经递质(乙酰胆碱、去甲肾上腺素、五羟色胺、谷氨酸、γ-氨基丁酸及GLU／GABA比值)分布异常。 4．睡眠剥夺引起的海马结构损伤及神经递质含量异常是SD损害动物学习能力的可能机制。
[Abstract]:objective
Sleep is one of the advanced functions of the brain. It is the basic condition for the maintenance of life. Sleep deprivation is a state of sleep deprivation caused by various reasons. Sleep deprivation can exert a wide influence on the body. This study uses a small platform water environment to prepare rat sleep deprivation animal models and discuss the effect of sleep deprivation on different duration of sleep. The influence of learning ability of rats was analyzed and its possible mechanism was preliminarily analyzed.
Method
1 experimental animals and groups
Healthy adult SD rats weighing 240 to 260g were randomly divided into normal control group, large platform control group, sleep deprived 2D group, 4D group and 6D group.
The preparation of 2 sleep deprivation model
The 30cm x 30cm x 30cm sleep deprivation box was built with a small platform with a constant diameter of 6.3cm, high 8cm, water injection in the box, and the water surface was lower than the platform of about 1.0cm. to make rats sleep deprived of 2D, 4D, 6D group model. The platform diameter of the large platform control group was 18cm, the rats were placed in the large platform environment for 6 days, and the rest were kept in the same condition as the sleep deprivation group. Normal control group cage.
3 general state observation
The changes of mental state, fur, drinking water and reactivity after sleep deprivation were observed.
4 learning ability test
The rats' learning ability test was carried out in the three equal "Y" maze labyrinth, the environment for the quiet dark room.Y maze at the top of each arm set up 1 signal lights, which prompted the "safety zone", the safety zone light after the electricity, the area without current stimulation, the rest of the area was switched to 36V alternating current. Experiment, the rats in the maze, first adapt to 5 minutes, and then start the experiment It is required that the rats receive a correct response from the start area to the bright arm safety area after the electric shock. The interval of the stimulation is 30 seconds each time in the experiment. When the rats are correct for 10 consecutive times, the rats are up to the standard to record the total training times required when the rats reach the standard, which represents the learning ability.
Ultrastructural transmission electron microscopic examination of 5 hippocampal tissue
The hippocampal tissues of SD-6d rats were made to observe the ultrastructural changes by electron microscopy.
Determination of neurotransmitter in 6 hippocampal tissue
The rats were quickly broken and the hippocampus was taken. The content of five hydroxytryptamine, norepinephrine, glutamic acid and gamma aminobutyric acid were detected by high performance liquid chromatography, and the content of acetylcholine was detected by alkaline hydroxylamine colorimetric assay.
7 statistical analysis method
The data of each group were expressed as (+) s. Hypothesis test was conducted by one-way ANOVA. Dunnett-t test was used to test the level of alpha =0.05..
Result
Changes in the general state of 1 rats after sleep deprivation
In the early experiment, the animals of group SD showed certain excitatory behavior, such as increased activity, external sound, light and other irritation. In the later period of the experiment (after SD-4d), the behavior of SD animal was gradually turned from excitement to inhibition and showed emotional "irritability".
The effect of 2 sleep deprivation on the learning ability of rats
Compared with the CC group, the learning ability of rats in group SD-4d and group SD-6d was decreased, and the number of training times needed to meet the standard was increased (P < 0.01); the learning ability of the SD2d group was improved and the number of training required to meet the standard was reduced (P < 0.05).
The ultrastructural changes of hippocampus in 3 sleep deprived rats
Hippocampal neurons were edema, dark cell change, transition to apoptosis or apoptosis, nucleus chromatin dissolved in some neurons, apparent decrease or loss of rough endoplasmic reticulum and ribosome in the cytoplasm, mitochondria decreased, structure blurred, partial synaptic edema, synaptic vesicles decreased; blood brain barrier structure was obviously abnormal, endothelial cell tone type The appearance of severe injury, such as cell degeneration or even cytoplasmic breakage, is a serious edema of the astrocyte podocyte, and a significant increase in myelin body can be seen in the neurons and the structure of the blood brain barrier.
The effect of 4 sleep deprivation on the content of hippocampal neurotransmitters
Acetylcholine: in group SD-4d, the content of Ach in hippocampus of SD-6d group was lower than that of CC group, with significant difference (P < 0.05, P < 0.01).
In norepinephrine group SD-4d, the content of NE in hippocampus of SD-6d group was lower than that of CC group (P < 0.05); NE content in TC group and SD-2d group was higher than that in CC group (P < 0.05).
Five serotonin: SD-2d group was measured by high performance liquid chromatography. The content of 5-HT in hippocampus of rats in group SD-4d was significantly higher than that in group CC and TC group (P < 0.01, P < 0.05), and the content of 5-HT in hippocampus of SD-6d group was significantly lower than that of CC group and TC group (P < 0.01).
The ratio of glutamic acid, gamma aminobutyric acid and two groups was higher than that of group CC (P < 0.01), and the content of GLU in hippocampus of group SD-6d was lower than that of group CC (P < 0.05). Compared with group TC (P < 0.05), the content of GLU in SD-2d and SD-4d group was increased (0.01, 0.01), and the content of hippocampus in hippocampus was higher than that of 0. group (0.05). 05, P < 0.01); compared with group CC, the GLU / GABA values of group SD-4d and group SD-6d were significantly lower (P < 0.01); SD-2d / TC group had no significant change in GLU / GABA value.
conclusion
1. sleep deprivation in a longer period of time (more than 4 days) will reduce the learning ability of rats. The result shows that the number of training required for Y maze training is increased.
2. sleep deprivation caused hippocampal damage in rats, characterized by edema of neuron cells, transition to apoptosis or apoptotic direction, synaptic edema, decrease of synaptic vesicles, obvious abnormal structure of blood brain barrier, and significantly increased myelin body.
3. sleep deprivation can cause a variety of neurotransmitters in the hippocampus of rats (acetylcholine, norepinephrine, five serotonin, glutamic acid, gamma aminobutyric acid and GLU / GABA ratio) distribution.
4. sleep deprivation induced hippocampal structural damage and abnormal neurotransmitter content are the possible mechanisms of SD damaging the learning ability of animals.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

【引证文献】