伴认知相关脑区Aβ沉积的脑梗死后认知障碍食蟹猴模型的建立与验证

Post-stroke cognitive impairment animal models are mainly established with rodents whose brain structure and neurological functions significantly differ from human beings, and nonhuman primates have outstanding advantages in cognitive experimental researches. Our previous studies and published articles have found that thalamic Aβ deposit and secondary degeneration following cerebral cortical infarction were associated with post-stroke cognitive impairment, indicating common pathogenesis between vascular cognitive impairment and Alzheimer disease. Recently, our preliminary experiments have observed that obvious cognitive deficits accompanied by Aβ accumulation in the ipsilateral thalamus appeared in cynomolgus monkeys at 6 months after focal cerebral infarction, suggesting the cynomolgus monkeys with Aβ deposit in the cognitive related brain regions after stroke can serves as a candidate to establish animal model of post-stroke cognitive impairment. Therefore, the present project is designed to firstly occlude the distal M1 branch of the middle cerebral artery by bipolar electrocoagulation to establish focal cerebral infarction in cynomolgus monkeys, and evaluate cognitive functions pre-stroke and at 3, 6, and 12 months after stroke. Secondary, we apply multi-modal magnetic resonance imaging to study the secondary change pattern of cognitive related brain regions, ELISA to detect Aβ levels in blood and cerebrospinal fluid, and molecular pathology to analyze Aβ deposit and secondary degeneration in the above regions at the same time points. Finally, we further confirm that Aβ deposit within the cognitive related brain regions are involved in post-stroke cognitive deficits through reducing Aβ generation by BACE1 inhibitor. This project would establish and validate a nonhuman primate animal model of post-stroke cognitive impairment in cynomolgus monkeys with Aβ deposit in the cognitive related brain regions, which would provide suitable experimental tools for clinical translational researches of vascular cognitive impairment.

现有脑梗死后认知障碍模型主要以脑结构和功能与人类相差甚远的啮齿类来复制，而非人灵长类在认知障碍研究中具有突出优势。本课题组前期研究并发表论文观察到，丘脑Aβ沉积和继发性损害与脑皮层梗死后认知损害有关，提示血管性认知障碍和Alzheimer病有共同发病基础。新近在预实验中发现，食蟹猴脑梗死后6月出现认知功能下降和同侧丘脑等认知相关脑区Aβ沉积，提示有望用食蟹猴建立伴认知相关脑区Aβ沉积的脑梗死后认知障碍模型。因此，本项目拟电凝闭塞食蟹猴一侧大脑中动脉，术前和术后3、6和12月评估认知功能和认知相关脑区多模态MRI继发性改变模式，同步采集血液和脑脊液检测Aβ含量，联合分子病理学研究上述脑区Aβ沉积和继发性损害；再用BACE1抑制剂减少Aβ产生，证实认知相关脑区Aβ沉积参与了脑梗死后认知损害，以建立和验证伴认知相关脑区Aβ沉积的脑梗死后认知障碍食蟹猴模型，为临床转化研究提供合适的实验工具。

现有脑梗死后认知障碍模型主要以脑结构和功能与人类相差甚远的啮齿类来复制，而非人灵长类在认知障碍研究中具有突出优势。本项目通过观察年轻食蟹猴局灶性脑梗死后1年认知功能、认知相关远隔脑区（同侧丘脑和海马）继发性改变和Aβ沉积情况，并评估脑梗死后认知障碍与同侧丘脑、海马继发性损害和Aβ沉积的相关性，旨在建立一种可用于脑梗死后认知障碍研究的非人灵长类动物模型，并初步揭示其组织病理学改变。本项目研究结果显示与假手术对照组相比，食蟹猴MCAO术后1年出现迂回取物任务的触壁次数增多和保守反应次数增多等认知损害表现。与假手术对照组相比，MCAO术后1年同侧丘脑腹后外侧核和海马CA3区NeuN+细胞减少，GFAP+和Iba-1+细胞增多。食蟹猴MCAO术后1年同侧丘脑和海马各分区均未发现Aβ斑块沉积，同侧丘脑和海马Aβ40、Aβ42、APP、BACE1蛋白表达水平与假手术对照组相比无统计学差异。MCAO术后1年脑脊液和血浆Aβ40水平、Aβ42水平和Aβ40/Aβ42比值与假手术对照组相比无统计学差异。Spearman秩相关分析提示，认知功能指标（触壁次数）与同侧丘脑和海马CA3区NeuN+细胞数量呈负相关，而与另一个认知功能指标（保守反应次数）相关性无统计学意义；而触壁次数和保守反应次数与同侧丘脑和海马脑组织、脑脊液和血浆Aβ40、Aβ42水平与认知均无显著相关性。延长观察时间至脑梗死后5年，淀粉样蛋白显像18F-flutemetamol PET和免疫组织化学染色未发现MCAO术后5年食蟹猴脑内丘脑、海马、皮层等Aβ沉积。食蟹猴局灶性脑梗死后远隔无缺血损伤的同侧丘脑和海马也发生神经元丢失和胶质细胞增生，但尚未观察到存在细胞外Aβ斑块沉积。脑梗死后认知功能障碍与脑梗死后同侧丘脑和海马神经元丢失相关，暂未发现其与同侧丘脑、海马脑组织、脑脊液和血浆Aβ水平相关，提示卒中后认知障碍可能与阿尔茨海默病有不同的发病机制，但需要观察卒中后更长时间以进一步证实。采用一侧大脑中动脉皮层支闭塞方法制备的食蟹猴局灶性脑梗死模型，可出现认知功能损害，可用于脑梗死后认知障碍的发病机制和药物临床前研究。