七氟醚暴露后胶质细胞依赖的TSP1/NLs信号异常致突触发育障碍的机制研究

Exposure to general anesthetics at the developmental stage induces neurological damages, and the underlying molecular mechanisms remain unknown. In 2016, the US Food and Drug Administration (FDA) issued a safety warning for the use of general anesthetics. Our study found that the number of astrocytes and proportion of inhibitory synapses were significantly increased in the prefrontal cortex after exposure to a commonly used GABAA mimic agent sevoflurane at the embryonic stage. It has been reported that activities of GABAA receptor in the embryonic stage regulate the differentiation of radial glial cells to astrocytes. However, how the glial cells regulate neuronal synaptic differentiation and balance is not known. Recent studies have shown that glial cells secrete TSP1 which interacts with adhesion molecules NLs to form synaptogenic complexes and further induce synaptogenesis. Therefore, it is likely that NL1/NL2 participates in the control of synaptic differentiation and indeed, our preliminary data showed that the expression of NL1 was significantly down-regulated after sevoflurane exposure at the embryonic stage. Therefore, we hypothesized that sevoflurane exposure at embryonic stage may activate GABAA receptor and promote TSP1 secretion from astrocytes. The up-regulated TSP1, in turn, interacts with synaptic complexs mediated by NLs and then causes the brain excitatory and inhibitory imbalance that eventually leads to neurologic function impairment. We aim to investigate the neurotoxic mechanism of sevoflurane exposure at embryonic stage by using knockout mice and specific blocking agents through sophisticated morphological, molecular and behavioral assessments. We hope that our research outcome will provide theoretical guidance for clinical application of general anesthetics including sevoflurane.

全麻药发育期暴露可致神经损害，其机制缺乏深入研究，2016年FDA对全麻药使用发出安全警告。我们研究发现，常用全麻药GABAA受体激动剂七氟醚胚胎期暴露致前额叶皮质星形胶质细胞数目和抑制性突触比例显著增加。有报道，胚胎期GABAA受体活动调控放射状胶质细胞向星形胶质细胞分化。但胶质细胞通过哪个信号调控突触分化和平衡？最新研究表明，胶质细胞分泌TSP1与粘附分子NLs形成突触发生复合体，诱导突触发生，NL1/NL2参与控制突触分化。我们预实验发现七氟醚诱导前额叶皮质NL1表达显著下调。据此我们推测：胚胎期七氟醚暴露激活GABAA受体，促进星形胶质细胞发育，上调TSP1分泌，通过NLs介导的突触复合体致兴奋性和抑制性突触失衡，导致神经功能异常。本项目拟应用基因敲除小鼠和特异性阻断剂，通过形态学、行为学、分子生物学等方法研究胚胎期七氟醚暴露的神经毒性机制，预期成果为七氟醚临床应用提供理论指导。

项目的背景：七氟醚是妊娠期非产科手术首选的吸入全麻药，但从1999年开始，大量研究表明临床常用全麻药对发育的大脑具有神经毒性，损害远期神经功能，导致神经行为学异常。我们前期研究发现，妊娠期七氟醚麻醉可致子代认知障碍和情绪异常，前额叶皮质星形胶质细胞和抑制性突触显著增加，突触粘附分子NL1表达显著上调。因此，我们提出：胚胎期七氟醚暴露激活GABAA受体，致星形胶质细胞提前发育，分泌TSP1，通过NL1/NL2致兴奋性和抑制性突触的分化失衡，是发育期神经毒性的分子机制。.主要研究内容：证明放射状胶质细胞上的GABAA受体是否介导了胚胎期七氟醚暴露对星形胶质细胞发育及TSP1分泌的影响；证明TSP1是否介导了胚胎期七氟醚暴露所致的前额叶皮质抑制性突触和兴奋性突触失衡；证明TSP1/NLs信号异常是否介导了胚胎期七氟醚暴露所致的前额叶皮质突触分化失调。.重要结果和关键数据：出生前七氟醚暴露促进PFC抑制性神经传递系统，兴奋/抑制（E/I）比例显著下调；星形胶质细胞提前发育，星形胶质细胞中TSP1分泌过多，激活TSP1/NLs信号，致抑制性突触显著增多，抑制性/兴奋性突触分化失衡，证实了七氟醚暴露后胶质细胞依赖的TSP/NLs信号致突触发育障碍的重要分子机制。而且，我们利用七氟醚可促进GABA能突触发育和传递，治疗GABA能缺陷相关的疾病。我们选择了精神分裂症，双相障碍和重性抑郁，临床前和临床研究均证实了很好的疗效。.科学意义：本研究为全麻药胚胎期神经毒性提供理论依据及干预靶点,并进行了成果的临床转化。在精神分裂症、双相障碍和重性抑郁的治疗中，均取得了很好的治疗效果。