Ketamine enhances human neural stem cell proliferation and induces neuronal apoptosis via reactive oxygen species-mediated mitochondrial pathway.
Ketamine enhances human neural stem cell proliferation and induces neuronal apoptosis via reactive oxygen species-mediated mitochondrial pathway.
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DOI：
10.1213/ane.0b013e3182860fc9
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发表时间：
2013-04
影响因子：
5.7
通讯作者：
Bosnjak ZJ
中科院分区：
文献类型：
--
作者：
Bai X;Yan Y;Canfield S;Muravyeva MY;Kikuchi C;Zaja I;Corbett JA;Bosnjak ZJ
越来越多的证据表明，氯胺酮在各种发育中的动物模型中会造成神经毒性，这导致了人们对儿科麻醉安全性的严重担忧。然而，氯胺酮是否以及如何诱导人类神经细胞毒性尚不清楚。人类胚胎干细胞(HESCs)体外神经发生的概述可以研究氯胺酮对神经干细胞(NSCs)和发育中的神经元的毒性效应，这在人类是不可能实现的。在本研究中，我们评估了氯胺酮对人胚胎干细胞来源的神经干细胞和神经元的影响。人胚胎干细胞通过神经干细胞直接分化为神经元。用不同剂量的氯胺酮处理神经干细胞和两周龄神经元，持续时间不同。用Ki67免疫荧光染色和溴脱氧尿苷比色法检测NSC的增殖能力。用原位末端标记法和半胱氨酸天冬氨酸氨基转移酶3活性测定分析神经细胞凋亡情况。研究了线粒体相关的神经细胞凋亡途径，包括线粒体膜电位、细胞色素c在细胞内的分布、线粒体的分裂和活性氧的产生。氯胺酮(100μM)作用6h可促进神经干细胞增殖。然而，只有在氯胺酮处理24小时后，才能观察到明显的神经元凋亡。此外，氯胺酮降低线粒体膜电位，增加细胞色素c从线粒体释放到胞浆中。与未处理的对照相比，氯胺酮也促进了线粒体的分裂和ROS的产生。重要的是，ROS清除剂Trolox显著减弱了氯胺酮诱导的ROS产生和神经元凋亡的增加。这些数据首次证明：(1)氯胺酮促进神经干细胞增殖并导致神经元凋亡；(2)线粒体参与氯胺酮诱导的神经元毒性，而Trolox可以预防这种毒性；(3)干细胞相关神经发生系统可能为快速筛选麻醉剂的神经毒性、研究其潜在机制以及避免这种毒性的预防策略提供一个简单而有前途的体外模型。
Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models, leading to a serious concern regarding the safety of pediatric anesthesia. However, if and how ketamine induces human neural cell toxicity is unknown. Recapitulation of neurogenesis from human embryonic stem cells (hESCs) in vitro allows investigation of the toxic effects of ketamine on neural stem cells (NSCs) and developing neurons which is impossible to perform in humans. In the present study we assessed the influence of ketamine on the hESC-derived NSCs and neurons. hESCs were directly differentiated into neurons via NSCs. NSCs and two-week-old neurons were treated with varying doses of ketamine for different durations. NSC proliferation capacity was analyzed by Ki67 immunofluorescence staining and bromodeoxyurindine assay. Neuroapoptosis was analyzed by TUNEL staining and caspase 3 activity measurement. The mitochondria-related neuronal apoptosis pathway including mitochondrial membrane potential, cytochrome c distribution within cells, mitochondrial fission, and reactive oxygen species (ROS) production were also investigated. Ketamine (100 μM) increased NSC proliferation after 6 h exposure. However, significant neuronal apoptosis was only observed after 24 h of ketamine treatment. In addition, ketamine decreased mitochondrial membrane potential and increased cytochrome c release from mitochondria into cytosol. Ketamine also enhanced mitochondrial fission as well as ROS production compared with no-treatment control. Importantly, Trolox, a ROS scavenger, significantly attenuated the increase of ketamine-induced ROS production and neuronal apoptosis. These data for the first time demonstrate that (1) ketamine increases NSC proliferation and causes neuronal apoptosis; (2) mitochondria are involved in ketamine-induced neuronal toxicity which can be prevented by Trolox; and (3) the stem cell-associated neurogenesis system may provide a simple and promising in vitro model for rapidly screening anesthetic neurotoxicity and studying the underlying mechanisms as well as prevention strategies to avoid this toxic effect.