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

本研究旨在建立2小时局灶性缺血再灌注大鼠脑N0_2-Tyr升高过程中硝基酪氨酸(NO_2-Tyr) >形成和衰减的动态，并探讨INOS和nNOS在升高过程中的作用。我们首先用n0 -单甲基- l-精氨酸阻断NO_2-Tyr的形成，用水解/高效液相色谱法观察N0_2-Tyr的衰变，测定再灌注后24小时大鼠脑内NO_2-Tyr的半衰期。得到的值分别为2.16和。梗死笔区和梗死核心区分别有2.25个角-s。使用相同的水解/高效液相色谱方法，我们测量了在存在和不存在氨基胍（每天两次，腹腔注射100 mg/kg）的情况下，从闭塞2小时到再灌注开始后72小时的N0_2-Tyr水平。在氨基胍不存在的情况下，再灌注开始后1小时，梗死周围区和梗死核心区N0_2-Tyr水平分别为0.95*0.34%和0.52* 0.34%。升高的水平持续了48小时，然后下降。梗死笔区N0_2-Tyr水平最高，其次为梗死核心区，其次为尾丘门区。氨基胍在24-48小时内显著降低N0_2-Tyr的形成（抑制率高达90%）。此外，使用相同的水解高效液相色谱法，我们测量了19只雄性CS7BIack/6J小鼠在再灌注0.5小时后闭塞2小时，在7-硝基茚唑存在和不存在的情况下，N0_2-Tyr水平（100 mg/kg，闭塞前15分钟腹腔注射）。7-吲哚唑显著降低了N02-Tyr的生成（0.10A 0.07%）。组（0.18A 0.05%）。因此，我们得出结论，nNOS和iNOS分别在再灌注早期和后期主要负责N0_2-Tyr的形成。这些结果对脑梗死患者的治疗时间窗和NOS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.