Analysis of perilymphatic glutamate by in vivo microdialysis study

我们应用微透析技术研究淋巴管周围谷氨酸。(1)研究了豚鼠耳蜗高K^+诱发去极化时淋巴周围谷氨酸释放的时间变化及其对Ca^<2+>的依赖性。灌注10 min后，谷氨酸升高出现两个高峰。在没有Ca^<2+>的情况下，第一个峰减弱，而第二个峰的抑制作用最小。(2)研究了豚鼠小脑前下动脉（AICA）封堵过程中耳蜗淋巴周围谷氨酸和耳蜗血流的动态变化。本研究结果提示耳蜗谷氨酸调节系统依赖于耳蜗血流量。(3)观察了卡那霉素和乙酸对内耳毛细胞的损伤作用过程中淋巴周围谷氨酸盐变化的时间过程。在豚鼠皮下注射800 mg/kg卡那霉素负荷剂量，3小时后静脉注射40 mg/kg乙酸，在注射乙酸约2小时后明显发现谷氨酸释放明显。目前的研究结果提供了谷氨酸在氨基糖苷诱导的毒性中起加重作用的额外证据。采用Northern印迹法分析了卡那霉素（KM）诱导大鼠耳蜗谷氨酸-天冬氨酸转运体（GLAST）基因表达的变化。每日1次KM (600 mg/kg/d)，连续20 d， GLAST mrna表达逐渐增加，在第20天达到峰值。虽然GLAST mRNA的表达在KM治疗结束后12天仍处于较高水平，但在2个月内降至正常水平。目前的研究结果表明，在给药期间，毛细胞塌陷释放的高浓度细胞外谷氨酸诱导了GLAST mRNA的表达。

We applied microdialysis technique to investigate the perilymphatic glutamate.(1) The time course of changes in perilymphatic glutamate release and their Ca^<2+>-dependency were studied in the guinea pig cochlea during high K^+-evoked depolarization. Two peaks of glutamate increase were found in response to perfusion for 10 min. In the absence of Ca^<2+> the first peak was diminished, whereas the inhibition of the second peak was minimal.(2) The dynamic changes in perilymphatic glutamate and cochlear blood flow were studied in the guinea pig cochlea during occulusion of anterior inferior cerbellum artery (AICA). The result of this study indicate that the glutamate regulating system in the cochlea is dependnt on cochlear blood flow.(3) The time course of changes in perilymphatic glutamate were observed during the application of kanamycin and ethacrynic acid, which are known to damage the hair cells in the inner ear. In guinea pigs receiving a loading dose of 800 mg/kg of kanamycin subcutaneously, followed three hours later by an intravenous injection of 40 mg/kg of ethacrynic acid, a marked glutamate release was clearly found about 2 hours after the injection of ethacrynic acid. The present findings provideadditional evidence that glutamate acts as an aggravating factor in aminoglycoside-inducedototoxicity.Kanamycin (KM) -induced changes in the gene expression for glutamate-aspartatetransporter (GLAST) in the rat cochlea were analyzed by Northern blotting. With theadministration of KM (600 mg/kg/day) once daily for 20 days, the expression of GLAST mRNAgradually increased and reached a peak on day 20. Although the expression of GLAST mRNA remained at a high level at 12 days after the completion of the KM treatment, it then fellto the normal level within 2 months. The present findings suggest that during the KMadministration, high concentrations of extracellular glutamate released by collapsing haircells induced GLAST mRNA expression.