慢性复合应激对大鼠学习记忆损伤及其机制的初步研究
发布时间：2018-12-14 10:54
【摘要】：应激是指机体在受到各种因素刺激时所出现的非特异性的全身反应。应激源可以刺激机体引发应激反应，释放激素和其他细胞因子，促进机体适应刺激；然而，当应激过度或机体不能适应调节时，则会产生有害的病理生理学改变。大脑是应激作用的主要靶器官之一，动物研究已发现应激可以诱导脑部结构和功能的改变，如神经细胞结构的改变，动物认知、行为的改变等。现代自然、社会环境中，个体与各种应激因素相接触，如寒冷、酷热、潮湿等外部环境理化因素；营养缺乏、感觉剥夺等个体内环境因素；还有如竞争失败、丧失亲人等心理社会环境因素。因此研究慢性复合应激对机体的影响尤其是对心理和认知功能的影响则显得极为重要。 以往研究表明：慢性复合应激能够对机体多系统尤其是中枢神经系统（central never system, CNS）产生损伤作用，其可能机制包括影响下丘脑-垂体-肾上腺轴（hypothalamic-pituitary-adrenal axis, HPA）、影响谷氨酸（glutamine, Glu）及其受体的释放、影响突触结构及其可塑性等。 小胶质细胞是中枢神经系统的一个重要的免疫驻留细胞，已有文献报道持续活化的小胶质细胞能够产生大量的炎性因子从而对神经元的代谢、关键蛋白的表达以及神经元的存活产生影响。研究显示内外部环境的微小改变即能刺激并导致小胶质细胞活化从而对中枢神经系统产生影响。基于此我们推测，小胶质细胞活化可能也在慢性复合应激致学习记忆损伤中发挥重要作用。 目的： 通过建立大鼠慢性复合应激动物模型，研究慢性复合应激对大鼠学习记忆能力的影响及其机制，重点阐明小胶质细胞活化在慢性复合应激所致学习记忆损伤中的作用和分子调控机制，为慢性复合应激损伤的防治提供一定的实验依据。 方法： 1．建立慢性复合应激动物模型：40只断乳的SD雄性大鼠随机分为四组：对照组（Con）；慢性复合应激组（MStress）（禁食/禁水，昼夜颠倒，鼠笼倾斜45度，单笼孤独饲养，限制活动，4℃冷应激，20℃强迫游泳）；对照给药组（Con+Mino）；慢性复合应激给药组（MStress+Mino）。 2．水迷宫检测大鼠的空间记忆能力，膜片钳技术检测大鼠海马离体脑片LTP诱导水平。 3．免疫组织化学染色检测小胶质细胞活化状态；ELISA法检测细胞因子表达；TUNEL染色检测海马部位神经元细胞凋亡情况；Western Blot检测Erk1/2、GluR1等蛋白表达情况。 结果： 1．慢性复合应激能够诱导学习记忆能力降低 通过给予动物7种不同的应激刺激建立大鼠慢性复合应激的动物模型，隐蔽平台实验结果显示，慢性复合应激组大鼠到达平台的潜伏期和对照组相比显著延长（P0.05）；空间探索实验结果显示，慢性复合应激组大鼠在目的象限的探索时间和对照组相比显著缩短（P 0.05）；膜片钳技术检测发现慢性复合应激组大鼠海马离体脑片LTP诱导水平显著低于正常对照组（P0.05）。 2．慢性复合应激能够诱导小胶质细胞活化和海马神经元细胞凋亡 免疫组织荧光化学法观察发现，慢性复合应激组大鼠海马部位部分小胶质细胞胞体变粗，突触减少，，呈阿米巴样，即活化状态，海马神经元细胞凋亡增加。统计结果发现慢性复合应激组小胶质细胞活化数量和海马神经元细胞凋亡数量与对照组相比显著增加（P 0.05）。ELISA法检测结果发现慢性复合应激组炎性因子TNF-α、IL-1β表达水平显著高于对照组（P0.01）。 3．慢性复合应激能够诱导海马p-Erk1/2、GluR1表达水平的降低 Western Blot检测结果发现，慢性复合应激可导致大鼠海马p-Erk1/2表达水平降低，GluR1表达降低（P 0.05）。 4.抑制小胶质细胞活化能够降低其相关炎性因子的表达并逆转海马p-Erk1/2、GluR1表达的改变。 给予米诺环素干预后，小胶质细胞活化水平受到显著抑制（P 0.05），其炎性因子TNF-α、IL-1β表达水平也显著降低；经Western Blot检测发现p-Erk1/2、GluR1表达水平均显著恢复（P 0.05）； 5.抑制小胶质细胞活化能够保护海马神经元细胞，并提高海马LTP诱导水平及大鼠的学习记忆能力。 给予米诺环素干预后，慢性复合应激大鼠海马神经元细胞凋亡数量显著减少；海马离体脑片LTP诱导水平较单纯慢性复合应激组显著升高（P0.05）；水迷宫实验检测大鼠空间记忆能力显著高于慢性复合应激组（P0.05）。结论： 1、慢性复合应激能够导致大鼠海马神经元凋亡及突触可塑性改变，并导致学习记忆能力降低。 2、慢性复合应激可能通过激活小胶质细胞从而诱导海马神经元的凋亡，影响学习记忆能力。 3、活化的小胶质细胞可能通过抑制海马神经元Erk1/2磷酸化水平和GluR1的表达，从而抑制LTP的诱导水平；同时通过抑制海马神经元Erk1/2磷酸化水平诱导海马神经元凋亡，最终影响学习记忆能力。
[Abstract]:Stress refers to the non-specific systemic reaction of the body when it is stimulated by various factors. The stress source can stimulate the body to induce stress reaction, release the hormone and other cytokines, and promote the body to adapt to the stimulation; however, when the stress is excessive or the body is unable to adapt to the regulation, harmful pathological and physiological changes can be generated. The brain is one of the main target organs of stress, and animal studies have found that stress can induce changes in the structure and function of the brain, such as changes in the structure of nerve cells, changes in animal cognition, behavior, etc. In the modern natural and social environment, the individual is in contact with various stress factors, such as the physical and chemical factors of the external environment such as the cold, the heat and the humidity, the lack of nutrition, the sensory deprivation, and the like, and also the psychological and social environment factors such as the failure of the competition and the loss of their loved ones. Therefore, it is very important to study the effect of chronic compound stress on the body, especially on the psychological and cognitive function. Previous studies have shown that chronic compound stress can damage the body system, especially the central nervous system (CNS), and its possible mechanism includes the effects of the hypothalamic-pituitary-adrenal axis (HPA), and the release of glutaamine (Glu) and its receptor. Release, Effect of Synaptic Structure and Plasticity The microglia are an important immune-resident cell of the central nervous system, and it has been reported that the sustained activated microglia can produce a large amount of inflammatory factors, which can lead to the metabolism of the neurons, the expression of the key proteins, and the survival of the neurons. The study shows that minor changes in the internal and external environment, i.e., can stimulate and cause the activation of microglia to produce the central nervous system On the basis of this, the activation of microglia may also play a role in learning and memory damage induced by chronic complex stress to act Objective: To study the effect of chronic compound stress on learning and memory of rats and its mechanism by establishing the animal model of chronic compound stress in rats. and can be used for preventing and treating the chronic compound stress injury. certain The experimental results were as follows: 1. Establishment of a chronic compound stress animal model: 40 SD male rats with broken milk were randomly divided into four groups: control group (Con), chronic compound stress group (Mstress) (fasting/ forbidden water, day and night, rat cage). 45 degree oblique, single cage alone, limited activity, cold stress at 4 & deg; C, forced swimming at 20 & deg; C); control administration group (Con + Mino); chronic compound stress administration group (MS stress + Mino. 2. Water maze test of rat's spatial memory ability, patch-clamp technique to detect rat LTP-induced level of the hippocampus from the body. 3. Immunohistochemistry staining to detect the activated state of the microglia; the expression of the cytokines was detected by the ELISA; the apoptosis of the neurons in the hippocampus was detected by the TUNEL staining; and the Western Blot was used to detect Erk1./ 2, GluR1 and other protein expression. Results: 1. Chronic compound stress can induce the ability of learning and memory to reduce the development of rats by giving 7 different stress stimuli to the animal The experimental results of the hidden platform showed that the latency of the rats in the chronic compound stress group was significantly prolonged compared with the control group (P0.05). The experimental results of the space exploration showed that the rats in the chronic compound stress group were in the target quadrant. The time of exploration and the control group were significantly shortened (P 0.05), and the detection of patch clamp technique found that the hippocampus of the rats with chronic compound stress group was isolated from the body of the brain. The P-induced level was significantly lower than that in the control group (P0.05). Combined stress can induce the activation of the microglia and the fluorescent chemical method of the apoptosis in the hippocampal neurons, and the part of the hippocampus in the chronic compound stress group is small. The cell of the glial cell becomes thicker and the synapse is reduced. The results of the statistics show that the number of microglia in the chronic complex stress group and the hippocampal God Compared with the control group, the number of apoptotic cells increased significantly (P 0.05). The level of IL-1 expression was significantly higher in the control group than in the control group (P0.01). The results of the Western Blot test that could induce the level of the expression of p-Erk1/ 2 and GluR1 in the hippocampus found that chronic complex stress could lead to the formation of p-Erk1/ 2 in the rat's hippocampus. Erk1/ 2 expression level decreased, GluR1 expression decreased (P 0.05). 4. Inhibition of microglia activation can reduce its correlation The expression of inflammatory factors and the reversal of the expression of p-Erk1/ 2 and GluR1 in the hippocampus were significantly inhibited (P 0.05), and the level of expression of IL-1 in the inflammatory factors decreased significantly. The expression level of p-Erk1/ 2 and GluR1 was found to be significantly higher than that of P-Erk1/ 2 (P 0.05). It can protect the neurons of the hippocampus and improve the induced level of the LTP in the hippocampus and the learning and memory ability of the rats. The results showed that the level of LTP in the hippocampus from the hippocampus was significantly higher than that of the chronic composite stress group (P0. 05) The experimental results showed that the spatial memory of the rats was significantly higher than that of the chronic compound stress group (P0.05). Chronic compound stress can lead to the changes of neuronal apoptosis and synaptic plasticity in the hippocampus of the rat, and lead to a decrease in learning and memory capacity. 2. The chronic compound stress can induce the apoptosis of the hippocampal neurons by activating the microglia, and the ability of learning and memory. 3. The activated microglia may inhibit the level of Erk1/ 2 phosphorylation and the expression of GluR1 in the hippocampal neurons, thus inhibiting the induction level of LTP.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2378492
[Abstract]:Stress refers to the non-specific systemic reaction of the body when it is stimulated by various factors. The stress source can stimulate the body to induce stress reaction, release the hormone and other cytokines, and promote the body to adapt to the stimulation; however, when the stress is excessive or the body is unable to adapt to the regulation, harmful pathological and physiological changes can be generated. The brain is one of the main target organs of stress, and animal studies have found that stress can induce changes in the structure and function of the brain, such as changes in the structure of nerve cells, changes in animal cognition, behavior, etc. In the modern natural and social environment, the individual is in contact with various stress factors, such as the physical and chemical factors of the external environment such as the cold, the heat and the humidity, the lack of nutrition, the sensory deprivation, and the like, and also the psychological and social environment factors such as the failure of the competition and the loss of their loved ones. Therefore, it is very important to study the effect of chronic compound stress on the body, especially on the psychological and cognitive function. Previous studies have shown that chronic compound stress can damage the body system, especially the central nervous system (CNS), and its possible mechanism includes the effects of the hypothalamic-pituitary-adrenal axis (HPA), and the release of glutaamine (Glu) and its receptor. Release, Effect of Synaptic Structure and Plasticity The microglia are an important immune-resident cell of the central nervous system, and it has been reported that the sustained activated microglia can produce a large amount of inflammatory factors, which can lead to the metabolism of the neurons, the expression of the key proteins, and the survival of the neurons. The study shows that minor changes in the internal and external environment, i.e., can stimulate and cause the activation of microglia to produce the central nervous system On the basis of this, the activation of microglia may also play a role in learning and memory damage induced by chronic complex stress to act Objective: To study the effect of chronic compound stress on learning and memory of rats and its mechanism by establishing the animal model of chronic compound stress in rats. and can be used for preventing and treating the chronic compound stress injury. certain The experimental results were as follows: 1. Establishment of a chronic compound stress animal model: 40 SD male rats with broken milk were randomly divided into four groups: control group (Con), chronic compound stress group (Mstress) (fasting/ forbidden water, day and night, rat cage). 45 degree oblique, single cage alone, limited activity, cold stress at 4 & deg; C, forced swimming at 20 & deg; C); control administration group (Con + Mino); chronic compound stress administration group (MS stress + Mino. 2. Water maze test of rat's spatial memory ability, patch-clamp technique to detect rat LTP-induced level of the hippocampus from the body. 3. Immunohistochemistry staining to detect the activated state of the microglia; the expression of the cytokines was detected by the ELISA; the apoptosis of the neurons in the hippocampus was detected by the TUNEL staining; and the Western Blot was used to detect Erk1./ 2, GluR1 and other protein expression. Results: 1. Chronic compound stress can induce the ability of learning and memory to reduce the development of rats by giving 7 different stress stimuli to the animal The experimental results of the hidden platform showed that the latency of the rats in the chronic compound stress group was significantly prolonged compared with the control group (P0.05). The experimental results of the space exploration showed that the rats in the chronic compound stress group were in the target quadrant. The time of exploration and the control group were significantly shortened (P 0.05), and the detection of patch clamp technique found that the hippocampus of the rats with chronic compound stress group was isolated from the body of the brain. The P-induced level was significantly lower than that in the control group (P0.05). Combined stress can induce the activation of the microglia and the fluorescent chemical method of the apoptosis in the hippocampal neurons, and the part of the hippocampus in the chronic compound stress group is small. The cell of the glial cell becomes thicker and the synapse is reduced. The results of the statistics show that the number of microglia in the chronic complex stress group and the hippocampal God Compared with the control group, the number of apoptotic cells increased significantly (P 0.05). The level of IL-1 expression was significantly higher in the control group than in the control group (P0.01). The results of the Western Blot test that could induce the level of the expression of p-Erk1/ 2 and GluR1 in the hippocampus found that chronic complex stress could lead to the formation of p-Erk1/ 2 in the rat's hippocampus. Erk1/ 2 expression level decreased, GluR1 expression decreased (P 0.05). 4. Inhibition of microglia activation can reduce its correlation The expression of inflammatory factors and the reversal of the expression of p-Erk1/ 2 and GluR1 in the hippocampus were significantly inhibited (P 0.05), and the level of expression of IL-1 in the inflammatory factors decreased significantly. The expression level of p-Erk1/ 2 and GluR1 was found to be significantly higher than that of P-Erk1/ 2 (P 0.05). It can protect the neurons of the hippocampus and improve the induced level of the LTP in the hippocampus and the learning and memory ability of the rats. The results showed that the level of LTP in the hippocampus from the hippocampus was significantly higher than that of the chronic composite stress group (P0. 05) The experimental results showed that the spatial memory of the rats was significantly higher than that of the chronic compound stress group (P0.05). Chronic compound stress can lead to the changes of neuronal apoptosis and synaptic plasticity in the hippocampus of the rat, and lead to a decrease in learning and memory capacity. 2. The chronic compound stress can induce the apoptosis of the hippocampal neurons by activating the microglia, and the ability of learning and memory. 3. The activated microglia may inhibit the level of Erk1/ 2 phosphorylation and the expression of GluR1 in the hippocampal neurons, thus inhibiting the induction level of LTP.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
【参考文献】
相关期刊论文 前2条
1 吴振宇;刘晓梅;张云;吴广均;王荣梅;;慢性复合式应激对雄性小鼠生育能力的影响及中药的干预作用[J];中国医药导刊;2009年05期
2 孙静;李伟;张俊权;李秀平;王生;;慢性复合应激对大鼠胰岛β细胞功能的影响[J];环境与职业医学;2010年10期
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