电刺激Meynert基底核对AD转基因小鼠保护作用的研究

发布时间：2018-06-19 03:22

本文选题：阿尔茨海默病 + 脑深部电刺激　；参考：《清华大学》2016年博士论文

【摘要】：目前阿尔茨海默病(Alzheimer’s disease,AD)主要依靠药物控制疾病进展,但现有药物的疗效相当有限。脑深部电刺激(deep brain stimulation,DBS)是藉由轻度连续性电脉冲对脑内特定核团进行慢性神经调控电刺激,现主要应用于运动障碍疾病的治疗。近年来,有关于DBS治疗AD患者的个案报道,取得良好的疗效。本课题主要探讨DBS对AD动物模型治疗的最佳刺激模式,并进一步研究DBS治疗AD的相关机制。我们选用APP/PS1双转基因小鼠,以Meynert基底核(NBM)为刺激靶点。首先采用不同刺激强度(10 Hz,50 Hz,100 Hz,130 Hz)、刺激开始时间(4月龄,6月龄,9月龄,12月龄)及刺激持续时间(7天,14天,21天,28天),应用水迷宫实验,包括定位航行及空间探索实验来确定DBS治疗AD的最优化参数。同时用尼氏及NeuN染色标记神经元,TUNEL及Hoechest染色标记凋亡神经细胞,Western blot法检测凋亡相关蛋白,相关试剂盒检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性、丙二醛(MDA)含量及乙酰胆碱酯酶(AChE)、乙酰胆碱转移酶(ChAT)活性,以探讨DBS治疗AD的相关机制。进一步检测磷酸化各信号通路因子并应用相关信号通路抑制剂来验证DBS治疗AD可能的信号转导通路。Morris水迷宫实验显示,100 Hz刺激频率、4月龄开始刺激及刺激持续21天较各部分其他组小鼠的逃避潜伏期明显缩短,穿越象限/平台时间及次数明显增多。我们进一步发现,上述参数组合可显著下调小鼠海马可溶性Aβ40及Aβ42含量。因此,我们得出最优刺激参数,即以NBM为刺激靶点,早期(4月龄)开始持续21天的高频(100 Hz)刺激,并用于后续实验。本课题结果显示,DBS可增加APP/PS1小鼠海马组织神经元存活,减轻细胞凋亡,下调凋亡相关蛋白caspase-3、caspase-8及Bid表达,但不影响caspase-9,Bax及Bcl-2表达。同时,DBS显著增加APP/PS1小鼠SOD、GSH-Px活性,下调MDA含量,并使ChAT活性增加,AChE活性下降。DBS显著上调磷酸化Akt,并下调磷酸化ERK水平。且ERK抑制剂U0126及PI3K抑制剂LY294002均可削弱DBS对APP/PS1小鼠缩短逃避潜伏期,增加穿越象限/平台时间及次数,以及下调Aβ的效应。我们的研究表明,早期(4月龄)开始持续21天的高频(100 Hz)DBS可通过激活PI3K/Akt通路并抑制ERK通路,发挥对AD小鼠改善认知功能、增加神经元存活、减少细胞凋亡、减轻氧化应激及调节ACh系统的效应。本研究提示NBM-DBS对AD有潜在的疗效,可望成为一种新的AD治疗手段。
[Abstract]:At present, Alzheimer disease (AD) mainly depends on drugs to control the progress of the disease, but the efficacy of existing drugs is very limited. Deep brain stimulation (DBS) is a kind of chronic neuroregulatory electrical stimulation of specific nuclei in the brain, which is mainly used in the treatment of motor disorders. In recent years, there have been cases of DBS for AD patients, and good results have been obtained. The aim of this study was to explore the best stimulation mode of DBS for AD animal model, and to further study the mechanism of DBS in the treatment of AD. We selected APP / PS1 double transgenic mice and used Meynert basal nucleus NBMs as the stimulus target. At first, different stimulation intensities of 10 Hz ~ 50 Hz ~ 50 Hz ~ 100 Hz ~ 100 Hz ~ (1) ~ (130 Hz) were used. The time of stimulation was 4 months old, 6 months old, 9 months old and 12 months old) and the duration of stimulation was 7 days, 14 days, 21 days and 28 days. The water maze test was used. It includes navigation and space exploration experiments to determine the optimal parameters for DBS treatment of AD. The apoptosis-related proteins were detected by Tunel and Hoechest staining, and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected by the relevant kit. The content of malondialdehyde (MDA) and the activity of acetylcholinesterase (ache), acetylcholinesterase (acetylcholinesterase) (acetylcholinesterase) were determined to explore the mechanism of DBS in the treatment of AD. Further detection of phosphorylated signal pathway factors and application of related signal pathway inhibitors to verify the possible signal transduction pathway in AD treated by DBS .Morris water maze experiment showed that 100 Hz stimulation frequency and stimulation began at 4 months old and continued for 21 months. The escape latency of mice in each part of the day was significantly shorter than that in other groups. The time and frequency of traversing quadrant / platform increased obviously. We further found that the above parameters could significantly down-regulate the contents of soluble A 尾 40 and A 尾 42 in hippocampus of mice. Therefore, we obtained the optimal stimulation parameters, that is, using NBM as the stimulus target, the early age of 4 months) began to last 21 days of high frequency 100 Hz) stimulation, and used in the follow-up experiment. The results showed that DBS could increase the survival of hippocampal neurons in APP / PS1 mice, reduce apoptosis and down-regulate the expression of caspase-3, caspase-8 and bid, but did not affect the expression of caspase-9, Bax and Bcl-2. At the same time, DBS significantly increased the activity of SODH-Px, down-regulated the content of MDA, increased the activity of chat and decreased the activity of ache. DBS upregulated the phosphorylation of Akt and down-regulated the level of phosphorylated ERK. Both ERK inhibitor U0126 and PI3K inhibitor LY294002 could reduce the escape latency, increase the time and times of crossing quadrant / platform, and down-regulate A 尾 in APP / PS1 mice. Our study has shown that high frequency 100HzndsDBS, which lasts for 21 days, can improve cognitive function, increase neuronal survival and reduce apoptosis in AD mice by activating the PI3K / Akt pathway and inhibiting ERK pathway. Alleviate oxidative stress and regulate ach system. This study suggests that NBM-DBS has a potential therapeutic effect on AD and is expected to be a new therapy for AD.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R749.16

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