自发性节律性神经网络电活动协同有序转换调控Zn2+-MMP2/9-BDNF轴介导全身麻醉药神经发育毒性易感窗口期研究

During the early development of central nervous system, the interaction of neuronal electrical activities and extracellular environment plays a significant role in facilitating the maturation of neurons and the formation of neural networks. Our previous studies indicated: the sequential switch of GABA, NMDA, etc. receptors or subtype receptors regulated the spontaneous and rhythmic neural network activities and physiological apoptosis; general anesthetic ketamine inhibited neuronal activities and aggravated physiological apoptosis through blockade of ACh and NMDA receptors in the early developing rat retina; Inhibition of extracellular zinc dependent matrix metalloproteinases 2/9 (MMP2/9), which degrade extracellular matrix, brain derived neurotrophic factor (BDNF), etc. substrates, could alleviate physiological and ketamine-induced neuronal apoptosis. We speculated that general anesthetics acting on GABA or/and NMDA receptors might disturb the balance of Zn2+-MMP2/9-BDNF axe through influencing the sequential switch of the spontaneous and rhythmic neural network activities and resulted in the degradation of BDNF, cytokines, fractalkine, etc. substrates and the abnormal expressions of anti-and pro-apoptosis genes, and caused neuronal apoptosis in the end. To confirm the above mentioned working hypothesis, We will use whole mount retina of neonatal rat as a simplified whole brain experimental model to explore the effects of general anesthetics and the sequential switch of the spontaneous and rhythmic neural network activities on Zn2+-MMP2/9-BDNF axe and microglial related fractalkine-complement system, and their interactions with neural activities, and to provide a new thought and possible targets for the treatment of general anesthesia related window of susceptibility and early development related diseases in the developing central nervous system.

在中枢神经发育早期，神经元电活动与外环境相互作用对神经元成熟与环路形成起重要作用。我们研究发现：GABA、NMDA等受体或受体亚型有序转换调控自发性节律性神经电活动和生理性凋亡；氯胺酮阻断ACh和NMDA受体，抑制视网膜神经电活动，加剧生理性凋亡；抑制锌依赖金属蛋白酶(MMP)2/9裂解细胞外基质、BDNF等底物能减轻生理性和氯胺酮诱发的神经元凋亡。由此推测，作用于GABA或/和NMDA受体的全麻药可能通过影响自发性节律性神经电活动有序转换，改变Zn2+-MMP2/9-BDNF轴平衡，导致BDNF、趋化因子等裂解及凋亡基因表达，引起神经元凋亡。为证实该假设，本项目采用新生大鼠视网膜这一简化整体脑模型，研究自发性节律性神经电活动协同转换及全麻药对Zn2+-MMP2/9-BDNF轴及趋化因子-补体系统的调控及与神经元的相互作用，为全麻药发育易感窗口期及发育相关疾病的干预提供新的思路和治疗靶点。

在大脑发育早期，尤其是突触形成高峰期（出生后7~9天，窗口期），全身麻醉可以引起动物中枢神经神经元大量凋亡。这一现象引起人们对婴幼儿麻醉安全的极大关注。既往的研究表明，细胞外基质-小胶质细胞微环境对突触的形成起着置关重要的调控。然而，微环境是否介导了全麻药物发育早期的神经毒性尚不清楚。本研究应用免疫组化等技术探讨大鼠视网膜细胞外基质重塑和小胶质细胞微环境在全身麻醉诱发神经元凋亡中的作用机制，并探讨了白蛋白的保护作用。全身麻醉药氯胺酮引起时间依赖性（暴露5小时）和空间依赖性（神经节细胞层）的Zn2+集聚，进而上调了Zn2+依赖的金属基质蛋白酶9（MMP9），导致神经元周围细胞外基质成分（层粘连蛋白laminin）的降解，即细胞外基质重塑。细胞外基质-神经元（laminin-laminin受体）“对话”中断导致神经元的“失巢性”凋亡。同时，细胞外基质重塑通过小胶质细胞表面integrin β1受体触发反应性小胶质细胞增生，表现为细胞数量增加、形态改变、促炎性因子（IL-1β、TNF-α）和趋化因子（CCL2、CXCL10）释放。提示细胞外基质重塑介导了氯胺酮引起小胶质细胞微环境改变，导致神经元炎症性病变。尽管小胶质细胞integrin β1受体在发育早期逐渐下调，但氯胺酮明显上调了凋亡高峰期（P7）的小胶质细胞integrin β1受体，提示小胶质细胞在发育早期具有反应“窗口期”。另外，白蛋白通过结合Zn2+下调了MMP9的表达和活性，减少了神经元的“失巢性”凋亡；同时白蛋白促使促炎性小胶质细胞向多核吞噬细胞转化，减少炎症介质的释放。提示白蛋白可能在细胞外基质-小胶质微环境层面上保护了神经元。.总之，在突触形成高峰期，细胞外基质-小胶质细胞微环境参与了全身麻醉药诱发的发育神经毒性。本研究为全身麻醉引起的中枢神经系统毒性及发育早期相关疾病的干预提供了新的思路和治疗途径。