星形胶质细胞形态及表达TSP-1在七氟烷致发育期神经突触损伤中的机制研究

Sevoflurane reduces the activity of brain by enhancing GABA-receptor-mediated inhibition， which is recognized as the most generally used volatile anaesthetics. The mechanism of long term potention (LTP) and memory impairment, which caused by synaptic injury induced by inhaling clinical concentrations of sevoflurane during neurodevelopmental period, is still unclear. Tripartite synapse, in which synapses are defined as comprising the presynaptic and postsynaptic specializations of neurons and the perisynaptic astrocyte, plays a critical role during the formation of excitatory synapse, for the reason that the astrocyte ensheaths the synapse could induce the formation of synapse and maintain its functional surrounding environment. GABAergic signaling mediates the morphology of the immature astrocytes and regulates the activity of MAPK. Thrombospodin-1 (TSP-1) , expressed by immature astrocyte, promotes the formation of synapse. While the expression of TSP-1 is effected by MAPK signaling pathway. Therefore, we postulate that activation of GABAA and GABAB receptor in immature astrocyte by sevoflurane could influence the morphology of astrocyte and the expression of TSP-1, finally lead to synaptic injury and memory deficit.This study tries to investigate the principle of how sevoflurane damage the morphology and gene expression of astrocytes in the developing brain through detecting the related target with the techniques of molecular biology, electrophysiology, and electron microscopy and so on. We expect to find out a specific target on astrocyte to prevent the toxicity of sevoflurane and safeguard its clinical use for early childhood.

七氟烷激活抑制性神经元GABA受体，降低大脑兴奋性，临床婴幼儿麻醉应用最为广泛，尚无更好替代药物。神经发育期七氟烷麻醉可致神经突触形成受损、LTP抑制及记忆损伤，其机制不明。突触旁星形胶质细胞与突触前后神经元形成的三重组分突触在兴奋性神经突触中占有重要地位，诱导神经突触发育成熟并维持其行使功能的微环境。GABA途径激活，促进未成熟星形胶质细胞突起生长、成熟分化，调节MAPK活性；未成熟星形胶质细胞分泌TSP-1，促进神经突触的形成；MAPK在星形胶质细胞中调控TSP-1表达。申请者推测七氟烷可通过激活星形胶质细胞GABA受体途径,影响星形胶质细胞形态及其表达分泌TSP-1水平，从而抑制突触形成，导致记忆损伤。本研究从星形胶质细胞着手，用分生、电生理、超微电镜技术检测相关指标，探讨七氟烷致发育期神经突触损伤机制，为特异性干预星形胶质细胞减轻七氟烷导致发育期神经损伤提供理论依据。

七氟烷作用于大脑抑制性神经元GABA受体，降低大脑兴奋性活动，是目前小儿麻醉副作用最少、应用最多的吸入性麻醉药。流行病学统计显示，在婴幼儿期接受麻醉治疗可导致患儿学龄期学习能力、记忆功能受损。动物实验证实，在神经发育期吸入七氟烷可引起不同程度神经元的损伤及发育异常，抑制海马长时程增强（LTP，long-term potentiation），导致动物学习记忆能力及社会行为能力受损。中枢神经系统抑制性神经元表达GABA受体，主要分为配体门控Cl-通道GABAA受体及代谢性受体GABAB受体两种亚型。七氟烷对这两种亚型的GABA受体均有作用。星形胶质细胞中也存在GABA受体的表达且被七氟烷激活，但星形胶质细胞如何参与七氟烷导致的神经损伤尚不清楚。TSP-1在未成熟星形胶质细胞表达并分泌，促进中枢神经系统形成正常超微形态的突触。七氟烷是否改变星形胶质细胞表达分泌TSP-1尚无人报道。本研究在原代培养星型胶质细胞中，初步探讨了七氟烷对星形胶质细胞功能及表达TSP-1的影响。首先，我们应用划痕实验技术观察到给予2.5%七氟烷6小时可提高星形胶质细胞迁移能力，这种现象在transwell实验中被进一步证实。接下来，我们应用western blot技术检测了星形胶质细胞迁移相关的激酶ERK、P38的表达水平及活性形式，发现给予2.5%七氟烷6小时可导致星形胶质细胞pERK、pP38水平上升，即ERK、P38活性升高。为了验证这一反应是否通过GABA途径实现，我们将原代培养星形胶质细胞分别用GABAA受体抑制剂Picrotoxin（100μM）及GABAB受体抑制剂CGP35348（100μM）预处理30分钟后，给予2.5%七氟烷6小时，应用western blot检测ERK、P38，发现两种抑制剂都能逆转七氟烷导致的ERK、P38激活。同时我们应用划痕实验及transwell检测星形胶质细胞迁移能力，发现两种受体抑制剂都可以逆转七氟烷导致的迁移能力上升。除此之外，我们应用realtime-PCR技术检测了给予2.5%七氟烷可导致TSP-1的mRNA水平上升。本研究证实，七氟烷确实可以影响星形胶质细胞迁移功能及TSP-1表达，导致星形胶质细胞功能紊乱，为研究七氟烷所致神经损伤提供了新的路径。