吸入麻醉药致发育神经元树突生长发育异常的GABAAR-Calpain通路作用机制及干预研究

Inhalational anesthetics are commonly administered in the clinical anesthesia and widespread concern was placed on their influence on the developmental neural system. Calpain plays a vital role in the regulation of dendrite arborization and synapse formation, which are essential for wiring the neural circuitry.Our previous animal researches have shown that isoflurane induced developmental apoptotic neurodegeneration by disruption of the cytoplasmic calcium homeostasis. The increase of intracellular calcium could lead to the calpain activity, which implies that abnormality of calpain activity might be involved in the abnormal developmental neuron dendrite growth and cognitive dysfunction induced by inhalational anesthetics. The brain slices，primary-cultured neurons of hippocampi and neonatal rats are selected for this experimental programs. This project intends to explore the dendrite growth, dendritic spine motility and morphology, morphological and functional plasticity at excitatory synapses and calpain activity. The role of calpain in regulation of dendrite growth, dendritic spine formation and learning and memory explosure to inhalation aneshetic is also investigated. Combined with the anti-virus technology, the probable targets for prevention and treatment of the neurodegeneration are selected. All the designed experiments are conducted to elucidate the role of GABAAR-calpain signaling pathway in the developmental neurotoxicity triggered by inhalational anesthetics and provide a new prospective for prophylaxis and medication of the neurotoxitiy.

吸入麻醉药对患儿发育期神经系统认知功能障碍受到广泛的关注。树突分支以及突触形成是神经回路的必需成分,而Calpain在树突生长发育及突触形成中起重要作用。申请者前期研究发现，吸入麻醉药异氟烷可通过GABAA受体致发育期神经元内钙稳态失衡，引起神经元凋亡等退行性病变。神经元内钙离子浓度增加可导致Calpain异常激活，提示Calpain激活可能参与了吸入麻醉药致发育期神经元树突生长发育异常及认知功能障碍。本项目拟选择胎鼠或者新生大鼠，从单通道、细胞、整体动物水平，探讨吸入麻醉药作用下发育期神经元树突及树突棘生长分化、突触形态及功能改变、Calpain活性变化、Calpian调节树突发育突触形成及学习记忆相关蛋白表达变化并确定参与这种变化的信号通路；结合逆转录病毒技术，筛选出可能的干预靶点，揭示Calpain异常在吸入麻醉药致发育期神经元毒性中的作用，为预防和治疗这种神经毒性提供崭新的思路。

项目背景： 麻醉药对发育期神经元的毒性作用越来越受到人们的广泛关注。由于人类神经系统发育从妊娠第5周开始，持续至婴幼儿，其中妊娠后期及新生儿期是神经元突触形成和突触重塑的关键时期2。大量的临床和临床前研究证实，在大脑发育期暴露于吸入麻醉药（异氟烷、七氟烷、地氟烷）可能会产生永久性认知功能缺陷。而手术室工作人员,尤其是对孕妇,长期接触手术室的麻醉气体同样可能带来风险。因此，吸入麻醉药对发育期大脑毒性机制是我们临床医师和学者叩待解决的问题。.主要研究内容：吸入麻醉致发育期神经元生长分化、突触可塑及神经环路形等方面进行研究。.重要结果：研究发现吸入麻醉可导致发育期神经元突触相关蛋白（PSD-95、Synaptic等）、受体（NR2A、NR2B受体）及磷酸化相关激酶（CDC42、SIRT1、ERK1/2等）表达改变，导致发育期神经元突触可塑性改变及神经元凋亡。筛选出干预的靶点。发表SCI论文5篇，中文核心期刊4篇。还有一篇在麻醉顶级杂志Britain Journal of Anesthesia审稿中。.关键数据：突触蛋白及突触外蛋白的提取，膜蛋白及胞浆蛋白的提取.科学意义：本项目从离子通道、神经元及整体动物水平，探讨吸入麻醉药作用下发育期海马神经元的电生理特征、细胞内钙离子的改变及其对神经突触及环路形成的影响，并深入GABAAR-Calpain-KCC2信号通路在发育期神经中枢系统中所起的作用及对认知功能障碍的影响，为广大孕妇和婴幼儿安全合理使用吸入麻醉药提供重要的理论依据。该项目不仅深入研究了吸入性全身麻醉药致发育神经元毒性作用机制，而且对预防和治疗吸入性全身麻醉药致发育神经元毒性作用有重要意义。