Data_Sheet_1_Neuron-Glia Crosstalk Plays a Major Role in the Neurotoxic Effects of Ketamine via Extracellular Vesicles.docx

Background: There is a compelling evidence from animal models that early exposure to clinically relevant general anesthetics (GAs) interferes with brain development, resulting in long-lasting cognitive impairments. Human studies have been inconclusive and are challenging due to numerous confounding factors. Here, we employed primary human neural cells to analyze ketamine neurotoxic effects focusing on the role of glial cells and their activation state. We also explored the roles of astrocyte-derived extracellular vesicles (EVs) and different components of the brain-derived neurotrophic factor (BDNF) pathway.
Methods: Ketamine effects on cell death were analyzed using live/dead assay, caspase 3 activity and PARP-1 cleavage. Astrocytic and microglial cell differentiation was determined using RT-PCR, ELISA and phagocytosis assay. The impact of the neuron-glial cell interactions in the neurotoxic effects of ketamine was analyzed using transwell cultures. In addition, the role of isolated and secreted EVs in this cross-talk were studied. The expression and function of different components of the BDNF pathway were analyzed using ELISA, RT-PCR and gene silencing.
Results: Ketamine induced neuronal and oligodendrocytic cell apoptosis and promoted pro-inflammatory astrocyte (A1) and microglia (M1) phenotypes. Astrocytes and microglia enhanced the neurotoxic effects of ketamine on neuronal cells, whereas neurons increased oligodendrocyte cell death. Ketamine modulated different components in the BDNF pathway: decreasing BDNF secretion in neurons and astrocytes while increasing the expression of p75 in neurons and that of BDNF-AS and pro-BDNF secretion in both neurons and astrocytes. We demonstrated an important role of EVs secreted by ketamine-treated astrocytes in neuronal cell death and a role for EV-associated BDNF-AS in this effect.
Conclusions: Ketamine exerted a neurotoxic effect on neural cells by impacting both neuronal and non-neuronal cells. The BDNF pathway and astrocyte-derived EVs represent important mediators of ketamine effects. These results contribute to a better understanding of ketamine neurotoxic effects in humans and to the development of potential approaches to decrease its neurodevelopmental impact.

研究背景：已有充分证据表明，在动物模型中，早期暴露于临床相关的全身麻醉药（general anesthetics, GAs）会干扰大脑发育，进而导致持久的认知功能损伤。然而相关人体研究结论尚不明确，且由于存在诸多混杂因素，相关研究开展难度较大。本研究利用原代人神经细胞分析氯胺酮的神经毒性作用，重点关注神经胶质细胞及其激活状态的作用。此外，本研究还探讨了星形胶质细胞衍生的细胞外囊泡（astrocyte-derived extracellular vesicles, EVs）以及脑源性神经营养因子（brain-derived neurotrophic factor, BDNF）通路不同组分的相关功能。

研究方法：采用活/死细胞染色实验、半胱天冬氨酸蛋白酶3（caspase 3）活性检测以及多聚ADP核糖聚合酶1（PARP-1）剪切实验，分析氯胺酮对细胞死亡的影响。通过逆转录聚合酶链反应（RT-PCR）、酶联免疫吸附实验（ELISA）以及吞噬功能实验，检测星形胶质细胞与小胶质细胞的分化情况。利用Transwell共培养体系，分析神经元-神经胶质细胞相互作用对氯胺酮神经毒性的调控作用。此外，本研究还探究了分离纯化的分泌型细胞外囊泡在该细胞串扰中的功能。采用ELISA、RT-PCR以及基因沉默技术，分析BDNF通路不同组分的表达与功能。

研究结果：氯胺酮可诱导神经元与少突胶质细胞发生凋亡，并促进促炎型星形胶质细胞（A1型）与小胶质细胞（M1型）的表型转化。星形胶质细胞与小胶质细胞会增强氯胺酮对神经元的神经毒性作用，而神经元则会加剧少突胶质细胞的死亡。氯胺酮可调控BDNF通路的多个组分：降低神经元与星形胶质细胞的BDNF分泌水平，同时上调神经元p75受体的表达，并在神经元与星形胶质细胞中均增加BDNF反义RNA（BDNF-AS）的表达以及前体脑源性神经营养因子（pro-BDNF）的分泌。本研究证实，经氯胺酮处理的星形胶质细胞分泌的细胞外囊泡在神经元死亡过程中发挥重要作用，且囊泡结合的BDNF-AS参与了该毒性效应。

研究结论：氯胺酮可通过影响神经元与非神经元细胞，对神经细胞产生神经毒性作用。BDNF通路与星形胶质细胞衍生的细胞外囊泡是介导氯胺酮神经毒性效应的重要调控因子。本研究结果有助于进一步阐明氯胺酮在人体中的神经毒性作用机制，并为开发降低其神经发育损伤风险的潜在干预策略提供理论依据。