Elucidation of the Mechanisms for Glutamate Release in Ischemic Neuronal Injury

越来越多的证据表明，细胞外谷氨酸被谷氨酸转运体持续摄取并在浓度上进行调节。尤其是GLT-1在前脑区特异而广泛地分布。最近，GLT-1被报道在缺血等条件下向细胞外间隙释放谷氨酸，为了验证这一假说并确定GLT-1在缺血时的功能，我们通过抑制谷氨酸转运体的方法测定了幼稚和缺血条件下沙土鼠的细胞外谷氨酸浓度。另外，烧灼GLT-1基因缺失及其野生型小鼠大脑中动脉造成局灶性脑缺血。结果：(1)采用微透析法，注入谷氨酸转运蛋白抑制剂TPDC，收集沙土鼠脑细胞外液。我们证实，在抑制谷氨酸转运体的过程中，细胞外谷氨酸增加。这一结果表明，谷氨酸转运体在调节胞外谷氨酸浓度方面起着重要作用。下一步，我们对预先注入tPDC的沙土鼠造成短暂性全脑缺血，但不能证实对动物海马区CA1区神经元的显著保护作用。(2)我们使用GLT-1基因敲除小鼠来检测缺血神经元死亡是否加速，梗塞范围是否扩大。造成永久性局灶性脑缺血，脑梗塞体积无差异(纯合子：12.6±10.3，野生型：12.6±6.8，平均±SD mm^3)。由于有可能是最初的缺血损伤的严重程度而不是谷氨酸毒性影响了神经细胞的死亡，我们正在采用比永久性缺血更轻的缺血再灌注模型来测量脑梗塞的体积。

Elucidation of the Mechanisms for Glutamate Release in Ischemic Neuronal InjuryThere have been an accumulating evidence that extracellular glutamate is contineously taken up and regulated in concentration by glutamate transporters. Especially, GLT-1 is specifically and widely distributed in forebrain area. Recently, GLT-1 has reported to release intracellular glutamate toward extracellular space in a condition such as ischemia.To test this hypothesis and make sure the function of GLT-1 during ischemia, we determined extracellular glutamate concentration in gerbils with naive and ischemic condition by inhibition of glutamate transporter. In addition, we produced a focal cerebral ischemia by cauterization of the middle cerebral artery in mice with GLT-1 gene deleted and its wild type.Results :(1) We infused tPDC, an inhibitor of a glutamate transporter and collected extracellular fluid of gerbil brain by a microdialysis method. We confirmed that extracellular glutamate increased during an inhibition of a glutamate transporter. This result indicates that a glutamate transporter plays an important role in regulating extracellular glutamate concentration. As the next step, we induced a transient global ischemia to gerbils which were pre-infused with tPDC.We could not confirm the significant protective effect against neurons in CA1 sector of the hippocampus of the animals.(2) We used GLT-1 gene knockout mice to examine whether ischemic neuronal death is accelerated and an infarct size is augmented. By producing a permanent focal cerebral ischemia, there was no differences in infarct volumes (homozygotes : 12.6±10.3, wild type : 12.6±6.8, Mean±SD mm^3). Since there is a possibility that the severity of an initial ischemic insult but not glutamate toxicity affected the neuronal cell death, we are ongoing to measure the infarct volumes by adopting ischemia-reperfusion models which are less severe than permanent ischemia.