【作者】 孙慧敏；

【导师】 章军建；

【作者基本信息】 武汉大学 ， 内科学， 2010， 博士

【摘要】 背景：阿尔茨海默病(AD)和血管性痴呆(VaD)为最常见的痴呆临床类型,慢性脑低灌注是其共有的重要病理基础,探讨改善慢性脑低灌注导致的血管性认知功能损害的发生机制及其干预方法具有重要的理论与临床意义。丰富环境(Enriched environment, EE)作为一种行为学干预方式,近年来在认知神经科学实验研究中越来越受到关注。大量研究已证实了丰富环境对脑在结构和功能上的有益影响,包括对行为的修正,特别是涉及到复杂的认知功能。但是,迄今为止,丰富环境对血管性认知功能损害是否有效及其可能的分子机制未见研究报道。本实验通过制备大鼠2-VO模型,模拟慢性脑低灌注状态下的认知功能损害,探讨丰富环境干预对血管性认知功能损害的影响,同时检测大鼠海马在慢性脑低灌注状态下与认知功能相关的脑源性神经营养因子(BDNF)、N-甲基-D-天冬氨酸(NMDA)受体亚单位1(NR1)和亚单位2B (NR2B)、胰岛素样生长因子-1(IGF-1)和血管内皮生长因子(VEGF)的蛋白表达水平变化以及丰富环境干预对这种变化的影响。目的：探讨丰富环境是否能改善大鼠慢性脑低灌注导致的认知功能损害及其可能的分子发生机制。方法：健康雄性Wistar成年大鼠,200-220g,随机分为四组：假手术+标准环境组(sham+SE),2-VO手术+标准环境组(2-VO+SE),假手术+丰富环境组(sham+EE)以及2-VO手术+丰富环境组(2-VO+EE)；给予大鼠双侧颈总动脉永久性结扎(2-VO)制备慢性脑低灌注模型后(2-VO或假手术后4周),丰富环境组大鼠给予丰富环境干预4周,丰富环境装置参照Nithianantharaj ah和Hannan描述的方法设置；标准环境组大鼠仍给予标准环境饲养；然后通过目标识别测验(Object recognition test)和Morris水迷宫试验(Morris water maze test),评测大鼠非空间和空间学习记忆能力；以及免疫组织化学、‘放免试验和Western blotting检测海马BDNF、NR1和NR2B、IGF-1及VEGF的蛋白分布和表达水平变化。结果：(1)目标识别测验中,标准环境中的假手术组大鼠对新奇物体的探索时间显著长于对熟悉物体的探索时间,而标准环境中的、2-VO组大鼠对新奇物体和熟悉物体的探索时间几乎相等；给予丰富环境干预的2-VO组和假手术组、以及标准环境中的假手术组大鼠,对新奇物体的辨别比均显著高于机会值。(2).Morris水迷宫测验训练期的第4天到第5天,标准环境下的2-VO组大鼠找到平台的时间显著多于假手术组,给予丰富环境干预的2-VO组大鼠找到平台的时间显著少于标准环境中的2-VO组：训练期的第2天到第4天,给于丰富环境干预的假手术组大鼠找到平台的时间显著少于标准环境中的假手术组；空问探索试验中,给予丰富环境干预的假手术组大鼠在目标象限花费的时间显著多于标准环境中的假手术组。(3)标准环境中的2-VO组大鼠海马BDNF和NR1蛋白水平与假手术对照组比较均显著下降,而丰富环境干预的2-VO组大鼠海马BDNF和NR1蛋白水平与标准环境对照组比较显著增加,NR2B、IGF-1及VEGF蛋白水平在各组大鼠之间则无显著差异。结论：丰富环境干预可以减轻慢性脑低灌注所导致的空间学习记忆和非空间记忆能力损害改善认知功能,并上调慢性脑低灌注所导致的BDNF和NR1蛋白表达水平下降。丰富环境对BDNF和NR1水平的调控可能是其改善慢性脑低灌注所致的血管性认知功能损害的重要分子机制。更多还原

【Abstract】 Background:Alzheimer’s disease (AD) and vascular dementia (VaD) are the two most common types of dementia, chronic cerebral hypoperfusion is the important pathologic basis for both of them. It’s extremely necessary to explore the intervention paradiam of improving the cognitive impairment induced by chronic cerebral hypoperfusion (CCH) and its possible underlying mechanism. As a behavioral intervention paradiam, Enriched environment (EE) has drawn more and more attention in the experimental study of cognitive neuroscience in recent years. A large number of studies have demonstrated the beneficial effects of EE on brain structure and function, especially in modifying the behaviors, particularly in tasks involving complex cognitive functions. However, the impacts of EE on cognitive impairment induced by chronic cerebral hypoperfusion (CCH) and the underlying molecular mechanisms have not been studied. In present study, we investigated the effects of EE on cognitive impairment caused by CCH and examined whether CCH altered the protein levels of brain-derived neurotrophic factor (BDNF),N-methyl-D-aspartate (NMDA) receptor subunit 1 (NR1) and subunit 2B (NR2B), insulin like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) in the hippocampus of rats and whether EE exposure attenuated the effects.Objects:To explore whether EE can improve the cognitive impairment induced by CCH and its possible underlying molecular mechanisms.Methods:Wistar Rats were divided into four groups(sham+SE; 2-VO+SE; sham+EE; 2-VO+EE) that received either permanent bilateral ligation of the common carotid arteries (2-vessel occlusion,2-VO) surgery or sham surgery. Four weeks later, the groups were supplied either EE housing or standard environment (SE) housing for 4 weeks. EE paradiam was designed as described by Nithianantharajah and Hannan. Then all the rats were examined non-spatial recognition memory in the novel object recognition task, spatial learning, and memory ability in the Morris water maze as well as the protein levels of BDNF, NR1, NR2B,IGF-1 and VEGF in the hippocampus.Results:(1) in the object recognition task, regarding the two groups housed in SE, the rats of sham group spending the time in the exploration of novel objects was significantly more than the time of exploration of familiar objects, while the rats of 2-VO group spent almost equal time in the exploration of both objects; The discrimination rate of novel objects were significantly higher than chance in the rats of either 2-VO group and sham group treated by EE, as well as the rats of sham group housed in SE. (2) On the training period of 4 to 5 days in Morris water maze test, the rats of 2-VO group housed in SE spending the time to find the platform was significantly longer than the the rats of sham group, and the rats of 2-VO group treated by EE spending the time to find the platform was significantly less than the rats of 2-VO group housed in SE; Regarding the rats of sham groups, on the training period of 2 to 4 days, the group treated by EE spending the time to find the platform was significantly less than the group housed in SE, and in the space exploration test, the group treated by EE also spending the time in the target quadrant was significantly more than the group housed in SE. (3) The BDNF and NR1 protein levels of 2-VO rats housed in SE were significantly decreased compared with the sham control group, while the BDNF and NR1 protein levels of 2-VO rats treated by EE were significantly increased compare with their counterparts housed in SE; the protein levels of NR2B, IGF-1 and VEGF in each group showed no significant difference.Conclusion:EE exposure can restore cognitive impairment induced by CCH and up-regulates the decreased protein levels of BDNF and NR1. Inversely, BDNF and NR1 may contribute to the beneficial effects of EE on CCH in rats.更多还原