The study concerning the formation of peroxynitrite in focal ischemia-reperfusion

本研究旨在建立大鼠局灶性脑缺血再灌流后NO2-Tyr升高过程中硝基酪氨酸(NO2-Tyr&gt；)生成和衰变的动态变化，并探讨iNOS和nNOS在NO2-Tyr升高中的作用。我们首先用N0-单甲基-L-精氨酸阻断NO2-Tyr的形成，并用水解/高效液相色谱法跟踪NO2-Tyr的衰变，首次测定了NO2-Tyr在再灌流开始后24小时在大鼠脑内的半衰期。测定值分别为2.16和2.16。2.25侧梗死区和梗死区核心区的角蛋白-S。采用同样的水解/高效液相色谱方法，在有或无氨基胍(100 mg/kg，每天2次)的情况下，测定缺血2小时至再灌流72小时的NO_2-Tyr水平。在不加氨基胍的情况下，再灌流开始后1h，梗死灶周围和梗死灶核心区的NO2-Tyr水平分别为0.95±0.34%和0.52±0.34%。升高的水平一直持续到48小时，然后下降。脑周梗死区NO2-Tyr水平最高，其次是梗死灶中心、尾壳核。在24-48小时内，氨基胍显著减少NO2-Tyr的形成(抑制率高达90%)。此外，我们使用相同的高效液相色谱水解法，测定了19只雄性CS7BIack/6J小鼠在再灌流0.5h后，在有和没有7-硝基吲唑(100 mg/kg，阻断前15分钟内注射)的情况下，阻断2小时后的NO_2-Tyr水平。与赋形剂处理组相比，7-引入吲唑显著减少了N02-Tyr的生成(0.10A 0.07%)。组(0.18A0.05%)。因此，nNOS和iNOS分别是再灌流早期和晚期NO2-Tyr形成的主要原因。这些结果对脑梗塞患者的治疗时间窗和一氧化氮合酶抑制剂的选择具有重要意义。

The purpose of this study was to establish the dynamics of nitrotyrosine (NO_2-Tyr> formation and decay during the rise of N0_2-Tyr in rat brain subjected to 2-hour focal ischemia-reperfusion, and to evaluate the role of INOS and nNOS in the rise. We first determined the half life of N0_2-Tyr in rat brain at 24 hours after the start of reperfusion by blocking NO_2-Tyr formation with N0-monomethyl-L-arginine and following the decay of N0_2-Tyr by means of a hydrolysis/HPLC procedure. The values obtained were 2.16 and. 2.25 horn-s in pen-infarct and core-of-infarct regions, respectively. Using the same hydrolysis/HPLC procedure, we measured N0_2-Tyr levels from 2-hour occlusion up to 72 hours after the start of reperfusion, in the presence and absence of aminoguanidine (intraperitoneally 100 mg/kg twice a day). In the absence of aminoguamdine, N0_2-Tyr levels in the peri-infarct and core-of-infarct regions reached 0.95*0.34% and 0.52*0 34% respectively, at 1 hour after the start of reperfusion. The elevated levels persisted until 48 hours, then declined. The pen-infarct region showed the highest N0_2-Tyr level, followed by the core of infarct, then the caudoputamen. Aminoguanidine significantly reduced N0_2-Tyr formation (up to 90% inhibition) during 24-48 hours. Further, using the same hydrolysisfHPLC procedure, we measured N0_2-Tyr levels in 19 male CS7BIack/6J mice with 2-hour occlusion following 0.5-hour reperfusion, in the presence and absence of 7-nitro indazole (100 mg/kg, given intrapenitoneally 15 minutes before occlusion). 7-intro indazole significantly reduced N02-Tyr formation (0.10A 0.07%), compared to that in vehicle-treated. group (0.18A 0.05%). We therefore conclude that nNOS and iNOS are predominantly responsible for N0_2-Tyr formation in the early phase and the late phase of reperfusion, respectively. These results have important implications for the therapeutic time window and choice of NOS inhibitors in patients with cerebral infarction.