吸入麻醉药致发育期神经元毒性的线粒体动力学机制及干预研究

Inhalational anesthetics are commonly administered in the clinical anesthesia and widespread concern was placed on their influence on the developmental neural system. The previous animal researches have shown that isoflurane induced developmental apoptotic neurodegeneration by disruption of the cytoplasmic calcium homeostasis. The dysregulation of calcium could lead to the imbalance of mitochondrial dynamics, which implies that abnormality of mitochondrial dynamics might be involved in the developmental neurotoxicity induced by inhalational anesthetics. The isolated mitochondria，primary-cultured neurons of hippocampi and neonatal rats are selected for this experimental programs. For mitochondrial and cytoplasmic calcium measurements, 5-day-cultured rat hippocampal neurons are preloaded with Rhod-2 and fluo-4AM,respective indicator of mitochondrial and cytoplasmic calcium, and then are imaged under laser scanning confocal microscope. To assay the dynamic change of mitochondrial morphology, the neurons are stained with MitoTracker after exposed to inhalational anesthetics.The mRNA and protein are assessed by real-time PCR and Western blot respectively after the neurons and animals are treated with inhalational anesthetics. Also, pathways of cell signaling transduction relevant to the imbalance of mitochondrial dynamics induced by inhalational anesthetics are 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 mitochondrial dynamics disruption in the developmental neurotoxicity triggered by inhalational anesthetics and provide a new prospective for prophylaxis and medication of the neurotoxitiy.

q吸入麻醉药在临床应用广泛，它对患儿发育期神经系统的影响受到广泛的关注。申请者前期基础研究发现，吸入麻醉药异氟烷可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。神经元内钙稳态失衡可导致线粒体动力学异常，提示线粒体动力学异常可能参与了吸入麻醉药致发育期神经元毒性。本项目拟选择胎鼠或者新生大鼠，从线粒体、细胞、整体动物水平，探讨吸入麻醉药作用下发育期神经元线粒体和细胞内Ca2+的变化、线粒体动力学的改变状况、线粒体动力学调节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，揭示线粒体动力学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。

随着吸入麻醉药在小儿手术中广泛应用，它对小儿发育期神经系统的影响受到越来越多的关注。申请者前期研究发现吸入麻醉药可导致发育期钙稳态失衡，继而可能会导致线粒体动力学异常。本课题主要从线粒体、细胞、整体动物水平，探讨吸入麻醉药作用下发育期神经元线粒体动力学改变情况，以及参与调节蛋白表达变化，筛选出参与该过程相关信号通路及干预靶点，使用相应工具药物或病毒对其进行干预。结果表明，异氟烷可导致发育期神经元线粒体形态改变，粒体动力学相关蛋白drp-1上调及其上游calpain活性增高，激活NF-κB/IκB通路引起神经炎症反应，上调PTEN致Tau蛋白磷酸化增加，抑制p-CamkIIα/cdc42/PAK3信号通路，导致神经元凋亡，突触发生障碍和认知功能障碍。使用相关工具药物或病毒干预后可有效缓解和逆转异氟烷对发育期神经元毒性，改善认知功能障碍。以上研究为预防和治疗吸入麻醉药致发育期神经元毒性提供新的思路。