【作者】 徐锋；

【导师】 陈向东；

【作者基本信息】 华中科技大学 ， 麻醉学， 2023， 博士

【摘要】 目的:围术期神经认知功能障碍(Perioperative neurocognitive disorder,PND)是术后常见的并发症,与长时程麻醉引起的神经毒性密切相关。本研究探索长时程七氟烷麻醉诱发PND的神经病理学机制。方法:首先,采用长时程七氟烷麻醉诱发PND动物模型,并采用神经行为学评估PND动物模型的认知功能及负性情绪。其次,通过RNA-seq、神经炎症、细胞凋亡、局部场电位及细胞形态学等检测来评估中枢神经毒性的表现。接着,采用突触标记物检测、突触染色、高尔基染色、形态学分析来明确长时程七氟烷麻醉对海马区突触的影响。接着,通过小胶质细胞功能及形态学评估、补体系统检测、免疫荧光染色技术来明确补体介导小胶质细胞突触修剪在PND中的作用。最后,通过脑室注射C1q的中和抗体、使用PLX3397删除中枢小胶质细胞及腹腔注射美洛昔康抑制中枢神经炎症,并结合RNA-seq、神经行为学及分子生物学去验证小胶质细胞、补体及神经炎症在PND中的作用。结果:(1)长时程七氟烷麻醉可以明显诱发实验动物认知功能损伤及抑郁样行为,并激活海马区NF-κB炎症通路,增加TNF-α、IL-1β及IL-6的表达;长时程七氟烷麻醉尚不引起细胞凋亡及Caspase-3、bcl-2相关凋亡通路的激活;长时程七氟烷麻醉可诱发海马区脑电爆发抑制及C-fos表达降低。(2)长时程七氟烷麻醉后,RNA-seq发现:突触及树突棘相关基因明显下调;PND动物模型海马神经突触数量、突触标记物(PSD95、SYN1及SYP)的转录水平及蛋白表达水平、树突棘数量均明显降低。(3)在PND动物模型中,小胶质细胞激活后,呈现出形态学的改变,并伴随着吞噬及迁移能力的增强;海马组织中补体C1qa及C3表达增加,C1qa标记的突触数量增加,激活的小胶质细胞吞噬突触的数量也明显增加;使用补体C1q的中和抗体可抑制小胶质细胞激活,降低C1qa标记的突触数量,挽救突触丢失,改善PND动物模型的认知功能障碍及抑郁样行为;使用PLX3397删除PND动物模型的小胶质细胞后,RNA-seq发现突触相关基因明显上调;Phospho-NF-κB p65、IL-1β及TNF-α的表达水平降低,补体C1qa的表达及补体标记的突触数量降低,突触丢失减少,PND动物模型的神经行为得到改善。(4)使用美洛昔康抑制PND动物模型的神经炎症后,RNA-seq发现C1q复合体及突触修剪相关基因下调;补体C1qa的表达及补体标记的突触数量降低,突触丢失减少。结论:本研究表明,长时程七氟烷麻醉会激活中枢神经炎症、补体系统及小胶质细胞,通过神经炎症及补体介导小胶质细胞病理性突触修剪,导致突触丢失,引发认知功能障碍及抑郁样行为。这是PND的神经病理学基础。更多还原

【Abstract】 Objective: Perioperative neurocognitive disorder(PND)is a common postoperative complication,which is closely associated with neurotoxicity induced by prolonged anesthesia.In this study,we investigated the neuromorphopathological causes of PND driven by prolonged sevoflurane anesthesia.Methods: First,prolonged sevoflurane anesthesia was employed to create an animal model of PND,and rats with the condition had their cognitive dysfunction and negative emotions assessed using neurobehavioral methods.Second,RNA-seq,neuroinflammation,apoptosis,local field potential,and cell morphology assays were used to evaluate the manifestation of central neurotoxicity.Next,synaptic marker,synaptic staining,Golgi staining,and morphological analysis were utilized to clarify the effects of prolonged sevoflurane anesthesia on synapses in the hippocampus.Then,using immunofluorescence staining,complement detection,and evaluation of the functional and morphological characteristics of the microglia,the involvement of complement-mediated microglial synaptic pruning in PND was defined.Finally,intracerebroventricular injection of a C1 q neutralizing antibody,depletion of microglia via PLX3397,and inhibition of neuroinflammation with intraperitoneal injection of meloxicam,along with RNA-seq,neurobehavioral,and molecular biology,were employed to confirm the roles of microglia,complement,and neuroinflammation in PND.Results:(1)Prolonged sevoflurane anesthesia resulted in cognitive impairment and anxiety-like behavior in rats.It also activated the NF-κB inflammatory pathway in the hippocampus and increased the expression of TNF-α,IL-1β,and IL-6.Prolonged sevoflurane anesthesia did not yet lead to apoptosis and activation of caspase-3 and bcl-2-related apoptotic pathways.Prolonged sevoflurane anesthesia induced the burst suppression pattern and reduction of C-fos expression in the hippocampus.(2)After prolonged sevoflurane anesthesia,RNA-seq revealed that synaptic and dendritic spine-related genes were significantly down-regulated.The number of hippocampal synapses,transcript levels,and protein expression levels of synaptic markers(PSD95,SYN1,and SYP),as well as the number of dendritic spines,were drastically decreased after prolonged sevoflurane anesthesia.(3)In the PND animal model,microglia(iba1)were activated,underwent morphological changes,and had enhanced phagocytosis and migration ability.C1 qa and C3 expression in hippocampal tissue rose,the number of C1qa-labeled synapses elevated,and the number of activated microglia phagocytosed synapses also significantly increased.The C1 q neutralizing antibodies inhibited microglia activation,reduced the number of C1qa-labeled synapses,rescued synapse loss,and improved cognitive dysfunction and anxiety-like behavior in the PND animal model.After the depletion of microglia in the PND animal model via PLX3397,RNA-seq revealed a significant upregulation of synapse-related genes;the expression levels of Phospho-NF-κB p65,IL-1β,and TNF-α were reduced;the expression of C1 qa and the number of complement-labeled synapses was lowered;the synaptic loss was decreased,and neurobehavior in the PND animal model was improved.(4)After using meloxicam to inhibit neuroinflammation in the PND animal model,RNA-seq revealed that C1 q complex and synaptic pruning-related genes were down-regulated;the expression of C1 qa and the number of complement-labeled synapses were reduced,and synaptic loss was decreased.Conclusion: This study demonstrates prolonged sevoflurane anesthesia activates neuroinflammation,the complement system,and microglia and causes synaptic loss through pathological microglial synaptic pruning that is mediated by neuroinflammation and complement,which results in cognitive dysfunction and anxiety-like behaviors and is the neuropathological basis of PND.更多还原