吸入麻醉药通过影响CRMP蛋白干扰新生大鼠脑突触发育的机制研究

吸入麻醉药可以干扰突触发育而诱导发育神经细胞毒性，但机制未明。我们近期蛋白质组学研究证明，七氟醚麻醉可以降低幼鼠皮质与突触发育相关的坍塌反应调节蛋白(CRMP)1和4的表达。预实验结果发现异氟醚也降低CRMP1/4表达，激活GSK-3β，增加CRMP2的磷酸化。而右美托咪啶可以抑制异氟醚诱导的发育幼鼠神经毒性并恢复CRMP1/4的表达，降低CRMP2的磷酸化。因此我们推论吸入麻醉药可能通过影响CRMP蛋白的功能，干扰突触发育而诱导神经毒性。本课题通过免疫染色观察吸入麻醉药对发育海马神经元树突和轴突的形态学影响及其与CRMP1/2/4表达和分布的关系，通过药理学方法研究吸入麻醉药影响CRMP1/2/4的信号通路，通过膜片钳检测脑片LTP和行为学研究探讨CRMP1/2/4变化是否介导吸入麻醉药诱导的认知功能障碍，以明确吸入麻醉药干扰突触发育的机制，为麻醉药毒性作用的防治提供新靶点。

Inhaled anesthetics can induce neurotoxicity in developing brain by disturbing synaptic development, the mechanism is underdetermined. Our proteomic research suggested sevoflurane decrease the protein expression of collapsing response mediator protein (CRMP) 1 and CRMP4 in neonatal rat cortice which closely related with synaptic development. Recently, our primary experimental results showed that isoflurane also decreased the protein expression of CRMP1 and CRMP4, activated GSK-3β and increased phosphorylation of CRMP2, while dexmedetomidine reduced isoflurane-induce neuroapotosis and increased expression of CRMP1 and CRMP4 and inhibited phosphorylation of CRMP2. Therefore, we presumed inhaled anesthetics may disturb synaptic development and induce neurotoxicity by interfering with the function of CRMP proteins. In this project, we will study 1) the effects of inhaled anesthetics on morphology changes of dendrites and axons development in hippocampal neurons of neonatal rats and their relationship with distribution and expression of CRMP1/2/4 protein by using immunostaining methods; 2) the signal pathway of inhaled anesthetics influencing CRMP1/2/4 proteins by using pharmacological approaches; 3) whether CRMP1/2/4 proteins will be involved in inhaled anesthetics-induced cognitive dysfunction by using patch clamp technique to measure LTP in hippocampus slices and behavior methods to measure learning and memory of rats. These results will better explain how inhaled anesthetics disturbing synaptic development and also provide the new clues about interventions against the anesthetics-induced neurotoxicity in developing brain.