THE ROLE OF GLUTAMATE ON THE GENERATION OF TINNITUS

谷氨酸对耳鸣产生的作用

• 批准号: 11671701
• 负责人: OGAWA Kaoru
• 金额: $ 0.83万
• 依托单位: KEIO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671701/
• 关键词: TINNITUS; MECHANISM; GLUTAMATE; ACECHYLCHOLONE; MICRODIALYSIS

In the present research, we investigated a new technique for the quantitative measurement of glutamate in the perilymph using in vivo microdyalysis. NADH produced by a reaction of GDH and NAD was used as a indicator of glutamate concentration. In this method, however, we could not detect a change of the glutamate concentration induced by aminoglycoside ototoxicity. Further study will be planned for improving the sensitivity of this technique.In addition, we measured the concentration of acetylcholine (Ach) in the perilymph in order to figure out the effects of cochlear efferent nervous system on the generation of tinnitus. Ach has been considered as a major neurotransmitter in the cochlear efferent nerve endings. In order to measure Ach release in perilymph, we employed in vitro microdialysis. Ach level of the samples was determined by the HPLC-ECD with the enzyme column on which acetylcholine esterase and choline oxidase were immobilized. The change of Ach level were measured before and after 50 μM AMPA, a non-NMDA glutamate receptor agonist, was added to the perfusate instead of the electrical stimulation on the floor of 4th ventricle. The basal Ach level was 22±4 pico mole/ml (mean±SD), when the Ringer solution contained 10 M physostigmin. The level of Ach increased 2-3 folds immediately after 50 μM AMPA was administered (n=4). Basal Ach level was consistent with Ach-like activity without electrical stimulation on the previous report with bioassay. These results indicate that microdialysis is useful for monitoring Ach release in perilymph. In addition, preliminary data suggest that the afferent stimulation, such as the administration of AMPA, may induce the release of Ach from the efferent nerve endings. Further investigation will be needed to figure out the relationship between Ach release induced by AMPA and the generation of tinnitus.

在本研究中，我们研究了一种利用 GDH 和 NAD 反应产生的体内 NADH 定量测量外淋巴中谷氨酸的新技术，然而，在该方法中，我们可以使用 GDH 和 NAD 反应产生的 NADH 作为谷氨酸浓度的指示剂。未检测到氨基糖苷类耳毒性引起的谷氨酸浓度的变化，将计划进一步研究以提高该技术的灵敏度。此外，我们测量了乙酰胆碱（Ach）的浓度。为了弄清楚耳蜗传出神经系统对耳鸣产生的影响，Ach 被认为是耳蜗传出神经末梢的主要神经递质。为了测量外淋巴中的 Ach 释放，我们采用了体外微透析。采用固定有乙酰胆碱酯酶和胆碱氧化酶的酶柱，通过HPLC-ECD测定样品的Ach水平。在灌注液中添加 50 μM AMPA（一种非 NMDA 谷氨酸受体激动剂）代替第四心室底电刺激前后，测量 Ach 水平的变化。基础 Ach 水平为 22±4 picomol/ml。 (平均值±SD)，当林格溶液含有10 M毒扁豆碱时，在加入50 μM AMPA后Ach水平立即增加2-3倍。基础 Ach 水平与之前生物测定报告中无电刺激的 Ach 活性一致。此外，初步数据表明，微透析可用于监测外淋巴中的 Ach 释放。刺激，例如给予 AMPA，可能会诱导传出神经末梢释放 Ach，需要进一步研究以弄清楚 AMPA 诱导的 Ach 释放与耳鸣产生之间的关系。