TGF-β/SMAD 参与小胶质细胞活化调控术后认知功能障碍发病机制的作用研究

Postoperative cognitive dysfunction (POCD) is one of the most common diseases after surgery. It is remains a difficult condition to manage, due to lack of knowledge on pathophysiological mechanisms. It is related to many complications which impact people’s life seriously. During cerebral injury, microglia secrete many soluble factors, such as cytokines, contribute to neuroinflammation cascades and various aspects of immune responses in the CNS. According to previous study, neuroprotective drugs and interventions may inhibit the acute phase of inflammatory response and then reduce memory deficits. However, they were not effective enough to regulate neuroinflammation and cognitive decline precisely. Based on our recent research, after induction with isoflurane, over-activated CX3CR1+ microglia could produce inflammatory factors considerably in aged mice, which involved in the memory encoding deficits. However, CX3CR1CreER/+: R26iDTR/+ mice showed lower IPSCs after isoflurane. Together with emerging genetic and functional evidence implicates that aged microglia showed decreased activity of TGF-β/SMAD signaling pathway, we proposed a hypothesis that aging, anesthesia and surgery together promote excessive onset of microglia activation. After that, inhibition of central TGF-β/SMAD signaling pathways will lead to neuroinflammation cascades and neuronal synaptic plasticity deficits, which could induce POCD. Combined with pharmacological tools, multi-types of transgenic mice will be used in the current study. Electrophysiology, RT-PCR, FACS, HPLC-MS analysis and other molecular biology techniques will also be used to prove that over-activated microglia in hippocampus could induce POCD via the modulation of TGF-β/SMAD signaling pathways. We look forward this project will not only provide a new theoretical basis for the microglia function in the pathogenesis of POCD, but also a new candidate target for clinical treatment of POCD.

术后认知功能障碍(POCD)是患者术后严重的长期并发症之一，然而发病机制不清造成缺乏有效治疗手段。大脑脆性增加基础上神经炎症级联反应的发生是老龄患者高发POCD的主要原因，但如何实现阻断炎症级联反应尚待阐明。我们近期研究发现，异氟醚麻醉引起老年小鼠海马依赖的学习记忆功能下降，与诱发小胶质细胞过度活化及促炎性因子释放显著增加有关。而选择性敲除小胶质细胞可减少抑制性突触递质释放调节突触可塑性，结合单细胞转录组学及功能学证据提示小胶质细胞中老化基因与TGF-β通路活性下调相关，提示: 麻醉药物及手术创伤刺激过度活化小胶质细胞致神经炎症级联反应，调控TGF-β/SMAD信号转导通路介导突触可塑性受损，诱发POCD。本课题以多品系转基因小鼠为平台，采用双光子、膜片钳、HPLC-MS等技术，为小胶质细胞参与POCD的作用机制提供新的理论依据，也为临床以小胶质细胞为靶点防治POCD提供新的研究方向。

术后认知功能障碍（POCD），是以学习记忆损伤为主要表现的术后神经系统并发症，发病率19.2%-46%，发生后死亡风险增加40%，是国际医学研究的热点及难点。麻醉手术引起的神经炎症级联反应的发生是老龄患者高发POCD的主要原因，但如何实现阻断炎症级联反应尚待阐明。我们基于前期研究发现，提出了如下关键科学问题：1.围术期炎症介导的小胶质细胞活化过程的时空特征如何？2.能否直接有效地精细调控小胶质细胞实现防治POCD的发生？3.小胶质细胞如何介导突触受损及可塑性改变进而调控POCD 的？本课题采用转基因、病毒学、单细胞测序、电生理等技术对上述科学问题进行探索，主要发现：①利用“异氟烷剖腹探查手术”及“缺血再灌注”模型均可稳定诱导术后认知功能损伤，同时术后早期认知相关脑区海马、皮层内存在小胶质细胞过度激活并伴有突触吞噬功能增强；②基于药理学手段进行小胶质特异性消融能够改善术后小胶质细胞的过度活化并逆转术后学习记忆损伤；③基于单细胞测序手段探究发现术后小胶质细胞可进一步划分为“静息态“、“中间态”、”激活态”三种不同亚型，其中激活态小胶细胞介导的突触吞噬功能增强是参与认知功能改变的关键环节；④发现小胶质细胞内Clec7a蛋白通过增强小胶质细胞吞噬兴奋性神经突触引发术后认知损伤。 选择性降低小胶质细胞Clec7a够改善术后小胶质细胞对于兴奋性神经元的吞噬作用并逆转术后学习记忆损伤，可望成为POCD防治的潜在靶点。以上结果表明：老龄动物海马及皮层脑区内小胶质细胞敏感性增高，在麻醉药物及手术创伤刺激后转化为“静息态“、“中间态”、”激活态”三种不同状态；麻醉手术通过引起小胶质细胞内Clec7a蛋白表达升高，从而介导吞噬兴奋性神经突触作用增强是导致术后认知损伤的关键环节。以小胶质细胞为靶点利用药理学手段或基因调控均可改善术后认知损伤的发生，以上结果为POCD的发病机制和临床防治提供新思路和新方法。