Glu与GABA递质/受体系统介导椎管内麻醉的镇静作用及其机制研究

The previous studies and our pilot studies have been reported that neuraxial blockade (epidural block or spinal block) has sedative effect on animals and humans, so sedatives during combined epidural/spinal anesthesia should be reduced. However, the underlying mechanism of its sedative effect is not clear. The basis theory of sedation is the reinforcement of inhibitory synaptic transmission (γ-aminobutyric acid, GABA) and the inhibition of excitatory synaptic transmission (glutamate, Glu), both of which exist widely in spinal dorsal horn of mammals. Our pilot study has been reported that subarachnoid bupivacaine block could change the concentration of Glu and GABA in the spinal cord in rats. Therefore, we hypothesized that the sedative effect of neuraxial anesthesia was local anesthetics act on the neurotransmitter/receptor system in spinal dorsal horn, where the inhibitory amino acid and the excitability amino acid existed. The rat model of subarachnoid block will be established and to observe the sedative effect after intrathecal injection of NMDA and GABA receptor agonist/antagonist; To detect spinal cord amino acid concentrations after spinal block. The whole-cell patch-clamp technique is used to record GABA-induced and NMDA-induced currents in isolated dorsal root ganglion neurons and the molecular biology techniques is used to detect the expression of CaM/CAMKⅡα/CREB, GAT-1 and GLT-1. The sedative effect of spinal block or epidural block and its mechanism will be revealed and will provide the basic theory for reasonable use of sedatives during combined anesthesia.

研究表明椎管内麻醉（硬膜外麻醉/蛛网膜下腔阻滞）具有镇静作用，因此联合椎管内麻醉时镇静药量应减少，但其镇静作用机制尚不清楚。镇静的理论基础是主要通过抑制谷氨酸（Glu）和增强γ-氨基丁酸（GABA）的活性而产生镇静作用，课题组在椎管内麻醉镇静作用的预实验中发现蛛网膜下腔注射布比卡因可影响脊髓中Glu和GABA的浓度。因此我们假设蛛网膜下腔注射布比卡因可作用于脊髓Glu和GABA递质/受体系统而产生镇静作用。课题拟建立大鼠蛛网膜下腔阻滞模型，以Glu和GABA递质/受体系统为主线，通过观察NMDA和GABA受体工具药对镇静药物剂量的影响，检测脊髓Glu和GABA浓度、脊髓神经细胞内Ca2+浓度、CaM/CAMKⅡα/CREB及GAT-1、GLT-1蛋白表达，记录神经细胞NMDA及GABA受体诱导电流变化，从整体、细胞和分子水平揭示椎管内阻滞的镇静作用机制，为镇静药的合理使用提供理论基础。

椎管内麻醉联合全身麻醉具有诸多优点而被临床广泛使用。实施联合麻醉时，因对椎管内麻醉的镇静作用认识不足，易导致麻醉过深或过浅，因此研究椎管内麻醉的镇静作用及其机制具有重要临床意义。椎管内麻醉镇静作用的确切机制尚不清楚，其机制可能与改变脊髓多种中枢神经递质/受体有关。本课题组通过建立大鼠蛛网膜下腔阻滞模型，以Glu和GABA递质/受体系统为主线，采用工具药、细胞生物、免疫组织化学及分子生物学等多种技术从整体、细胞和分子不同水平探讨布比卡因蛛网膜下腔阻滞的镇静作用及其机制。结果显示布比卡因蛛网膜下腔阻滞可显著降低丙泊酚和依托咪酯的镇静剂量；布比卡因蛛网膜下腔阻滞可使大鼠脊髓组织中抑制性氨基酸GABA、Gly的含量显著增加，可引起大鼠脊髓组织中GAT1、GlyT1蛋白及GAT1 mRNA、GlyT1 mRNA的表达显著下调，可使大鼠脊髓组织中NMDA受体亚型NR2B、NR2B mRNA及其下游p-CaMKIIα、CaMKIIα mRNA、p-CREB、CREB mRNA表达显著降低。实验揭示了布比卡因蛛网膜下腔阻滞主要是通过影响脊髓组织中抑制性氨基酸（GABA、Gly）及其转运体(GAT1、GlyT1)和兴奋性氨基酸受体（NMDA受体）亚基NR2B及其下游信号通路CAMKⅡα /CREB而发挥的镇静作用。本课题首次提出蛛网膜下腔阻滞在脊髓水平的镇静作用机制，为麻醉镇静药物的合理使用和新型镇静药研发提供理论基础。