Reciprocal actions between generation of reactive oxygen species and nitric oxide, and extracellular release of glutamate and ascorbate during cerebral ischemia-reperfusion

脑缺血再灌注过程中活性氧和一氧化氮的产生与细胞外谷氨酸和抗坏血酸的释放之间的相互作用

• 批准号: 11671513
• 负责人: YUSA Toshiko
• 金额: $ 2.18万
• 依托单位: University of the Ryukyus
• 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-11671513/
• 关键词: cerebral ischemia-reperfusion; glutamate; ascorbbic acid (ascorbate); NOS inhibitor; postischemic brain hypothermia; rat forebrain ischemia; rat hippocampus; microdialysis biosensor; 脳虚血-再灌流; ascorbic acid (ascorbate); 再灌流後脳温低下; 再灌流後脳血流; ascor

Excessive release of excitatory amino acids (glutamate), increased generation of reactive oxygen species and free radical (NO) are regarded as important factors in the pathogenesis of cerebral ischemia-reperfusion injury. Reciprocal actions between these factors also involved. Ascorbate, a major antioxidant in the brain, is highly concentrated in neuropils. Its extracellular release is closely related to that of glutamate, and ascorbate-mediated protection from excitotoxins has been demonstrated in vitro. Thus, 1) after establishment of method to measure the dynamics of extracellular ascorbate release in vivo, 2) the relationship between extracellular release of ascorbate and glutamate, 3) the effect of NOS inhibitor (L-NAME), 4) the effect of extended time of ischemia, 5) the effect of postischemic hypothermia (from 36 to 32 ℃) on extracellular ascorbate and glutamate release were investigated in vivo using a microdialysis biosensor system during the early stage of forebrain ischemia- … More reperfusion in rat.METHODS : Two probes of a microdialysis biosensor electrode were inserted stereotaxically in the bilateral striatum or hippocampus of male Wistar rats. Mean arterial pressure (MAP), cortical CBF measured by laser-Doppler flowmeter, and rectal and cortical temperatures were continuously recorded. Forebrain ischemia-reperfusion was performed by bilateral carotid artery occlusion with hemorrhagic hypotension (MAP=30mmHg) for 10 min followed by reperfusion for 60 min.RESULTS : 1) Using the microdialysis biosensor, continuous real-time measurement of extracellular ascorbate can be obtained in rat striatum in vivo and a novel dialysis electrode can follow the changes in extracellular ascorbate during reperfusion, 2) the marked increase of ascorbate during reperfusion was associated with the rapid decrease in glutamate, 3) in L-NAME treated rats, glutamate was higher than saline treated rats throughout the experiment, 4) the extended time of ischemia (5 min) caused significant inhibition of glutamate re-uptake and ascorbate release during reperfusion, 5) the post-ischemic hypothermia caused significant increase in ascorbate release and glutamate re-uptake during reperfusion along with significantly higher MAP and cortical CBF.CONCLUSION : These results suggest that 1) the heteroexchange of ascorbate with glutamate, 2) L-NAME did not prevent marked intraischemic glutamate accumulation, moreover, increased its level during reperfusion, 3) the changes in extracellular ascorbate could be considered a marker of glutamate re-uptake during the early stage of reperfusion after forebrain cerebral ischemia, 4) the marked extracellular release of ascorbate by post-ischemic hypothermia consistent with the reported ascorbate-mediated protection from glutamate citotoxicity. Less

兴奋性氨基酸（谷氨酸）的过度释放、活性氧和自由基（NO）的产生增加被认为是脑缺血再灌注损伤发病机制中的重要因素。这些因素之间也存在相互作用。抗坏血酸是大脑中的一种主要抗氧化剂，高度集中在神经髓鞘中。它的细胞外释放与谷氨酸密切相关，抗坏血酸介导的保护作用已在体外被证明。因此，1）在建立了体内测定细胞外抗坏血酸释放动力学的方法之后，2）细胞外抗坏血酸释放与谷氨酸之间的关系，3）NOS抑制剂（L-NAME）的作用，4）延长缺血时间的作用，5）缺血后低温的影响采用微透析生物传感器系统，在体研究了36 ~ 32 ℃条件下脑缺血早期细胞外抗坏血酸和谷氨酸释放的变化。 ...更多信息 方法：将微透析生物传感器电极的两个探针立体定位地插入雄性Wistar大鼠的双侧纹状体或海马。连续记录平均动脉压（MAP）、皮质CBF（激光多普勒血流计测量）以及直肠和皮质温度。采用双侧颈动脉阻断伴出血性低血压的方法进行前脑缺血再灌注（MAP= 30 mmHg）10 min后再灌注60 min。1）利用微透析生物传感器，可以在体内获得大鼠纹状体细胞外抗坏血酸的连续实时测量，并且新型透析电极可以跟踪再灌注过程中细胞外抗坏血酸的变化，（2）再灌注期间抗坏血酸的显著增加与谷氨酸的迅速减少有关;（3）在L-NAME处理的大鼠中，谷氨酸在整个实验期间高于盐水处理的大鼠;（4）缺血时间延长（5 min）导致再灌注期间谷氨酸再摄取和抗坏血酸释放的显著抑制，5）缺血后低温可引起再灌注时抗坏血酸释放和谷氨酸重摄取显著增加沿着MAP和皮质CBF显著升高。这些结果表明：1）抗坏血酸与谷氨酸的异源交换，2）L-NAME不能阻止明显的缺血内谷氨酸积累，此外，在再灌注期间增加其水平，3）细胞外抗坏血酸的变化可被认为是前脑缺血后再灌注早期谷氨酸再摄取的标志物，4）缺血后低温引起的抗坏血酸的显著细胞外释放与报道的抗坏血酸介导的对谷氨酸细胞毒性的保护一致。少

项目成果

Toshiko Yusa: "Effects of nitric oxide synthase inhibition on extracellular glutamate and cerebral blood flow during forebrain ischemia-reperfusion in rat in vivo"Journal of Anesthesia. 14(1). 24-29 (2000)

Toshiko Yusa：“一氧化氮合酶抑制对大鼠体内前脑缺血再灌注过程中细胞外谷氨酸和脑血流的影响”麻醉杂志。

Yusa T: "Continuous real-time measurement of extracellular ascorbate release in the rat striatum in vivo during forebrain ischemis-reperfusion."Neuroscience Letters. 293. 123-126 (2000)

Yusa T：“前脑缺血再灌注期间体内大鼠纹状体细胞外抗坏血酸释放的连续实时测量。”神经科学快报。

Toshiko Yusa: "Effects of nitric oxide synthase inhibition on extracellular glutamate and cerebral blood flow durin forebrain ischemia-reperfusion in rat in vivo"Journal of Anesthesia. 14(1). 24-29 (2000)

Toshiko Yusa：“一氧化氮合酶抑制对大鼠体内前脑缺血再灌注期间细胞外谷氨酸和脑血流的影响”麻醉杂志。

湯佐祚子: "前脳虚血-再灌流によるラット線状体in vivoでのascorbic acid細胞外離"Journal of Anesthesia. 13(suppl). 161(PD-0114) (1999)

Koko Yusa：“由于前脑缺血再灌注导致体内大鼠纹状体的抗坏血酸细胞外脱离”《麻醉杂志》13（增刊）（1999）。

Yusa T, Fujimura T: "Effect of forebrain ischemia-reperfusion on extracellular ascorbic acid release in rat striatum in vivo."Journal of Anesthesia. 13(suppl). 161(PD-0114) (1999)

Yusa T、Fujimura T：“前脑缺血再灌注对体内大鼠纹状体细胞外抗坏血酸释放的影响。”麻醉杂志。