氨基胍对兔缺血-再灌注损伤后视网膜形态学及一氧化氮和一氧化氮合酶表达的影响

背景临床研究表明，多种眼科疾病如青光眼、视网膜中央动脉阻塞、缺血性视神经病变等均可导致视网膜缺血-再灌注损伤（RIRI），严重影响视功能，因而对治疗RIRI药物的研究是非常必要的。目的观察并探讨氨基胍对兔RIRI后形态学的变化，并研究其对一氧化氮（NO）及一氧化氮合酶（iNOS）在视网膜中表达的影响及其机制。方法清洁级日本大耳白兔66只，以随机数字表法分为正常组、RIRI模型组和氨基胍治疗组。用前房灌注生理盐水法升高眼压60min后恢复灌注建立兔RIRI模型，氨基胍治疗组每日在模型兔腹腔内注射氨基胍注射液80mg／kg，而RIRI模型组以同样的方法注射等量生理盐水。RIRI模型组和氨基胍治疗组分别于缺血即时、再灌注后6、24、72h各取2只兔活体行眼底彩色照相及荧光素眼底血管造影（FFA）。各组兔分别于再灌注后1、6、24、72h用空气栓塞法处死并摘除眼球以制备视网膜切片，用TUNEL法检测视网膜组织神经细胞的凋亡变化，通过硝酸还原酶法检测NO浓度，比色法检测iNOS活力。结果各时间点眼底彩色照相及FFA检查结果表明，与RIRI模型组比较，氨基胍治疗组视网膜水肿程度减轻，血管闭塞程度及比例降低，荧光素渗漏量及面积减轻并减少。TUNEL染色凋亡细胞计数检测表明，正常组兔视网膜未见TUNEL阳性细胞，而缺血-再灌注1、6、24、72h后RIRI模型组兔视网膜凋亡细胞计数均明显高于氨基胍治疗组（F分组=2762．37，P=0．00；F时间=894．24，P=0．00）。RIRI模型组和氨基胍治疗组各组内相邻时间点之间TUNEL阳性细胞的差异均有统计学意义（RIRI模型组：q=24．47、36．59、-20．37，P〈0．05；氨基胍治疗组：q=20．94、16．79、-6．92，P〈0．05），再灌注后24h各组TUNEL阳性细胞数量达到高峰。各时间点RIRI模型组兔视网膜NO浓度明显高于氨基胍治疗组（q=3．84、4．01、8．91、3．75，P〈0．05），各组内相邻时间点之间NO浓度的差异均有统计学意义（RIRI模型组：q=4．77、13．40、-10．29，P〈0．05；氨基胍治疗组：q=4．55、9．05、-5．08，P〈0．05），各组24h视网膜中NO浓度达峰值。各时间点RIRI组视网膜iNOS活力均明显高于氨基胍治疗组（q=-3．74、-4．94、-6．53、-3．98，P〈0．05）；各组内相邻时间点间iNOS活力的差异均有统计学意义（RIRI模型组：q=8．43、6．71、-6．39，P〈0．05；氨基胍治疗组：q=4．16、5．08、-3．93，P〈0．05），各组24h iNOS活力达峰值。结论氨基胍对维持RIRI后的视网膜形态和功能起保护作用，其作用机制可能为抑制iNOS的活性，减少NO的生成。

Influence of aminoguanidine on retina morphology and expression of nitric oxide and inducible nitric oxide synthase after retina ischemia-reperfusion injury

Background Many eye diseases such as central retinal artery occlusion,glaucoma and ischemic optic neuropathy,etc, lead to retinal isehemia-reperfusion injury （RIRi） and furthmore visual functional damage. It is necessary to study the treatment of RIRI. Objective This study was to observe and discuss the influence of aminoguanidine on the retina morphological changes and its mechanism after RIRI. Methods Eighty clean healthy male Japanese white rabbits were randomly divided into normal injury group, RIRI group and aminoguanidine （AG） treated group. The model of RIRI was established by infusing saline solution into the anterior chamber to elevate intraocular pressure （lOP） in both RIRI group and AG group. AG was intraperitoneally injected in the models of the AG group,and normal saline solution was used at the same method in the normal group and the RIRI group. The fundus photography and fundus fluorescein angiography（FFA） were performed on the rabbits at the moment of retina ischemia and 6,24 and 72 hours after reperfusion. The parts of rabbits were sacrificed 1,6,24 and 72 hours after reperfusion,followed by the enueleation of the eyeballs. Retinal section was prepared for TUNEL examination to evaluate the apoptosis of retinal cells. Nitric oxide （NO） concentration in retina was detected with nitrate reductase, and the activity of inducible nitric oxide synthase （iNOS） was measured by colorimetric detection. The use of the animals followed the Regulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission. Results The fundus photography and FFA showed that the retinal edema was more mild,and the percentage of vascular occlusion was lower in the AG treatment group than that in RIRI group and the amount and area of fluorescein leakage were also smaller than the treatment group. The numbers of TUNEL positive cells in the AG treatment group were less than those in the RIRI model group at 1,6,24 and 72 hours after experiment （ F分组= 2762.37, P = 0.00 ; F时间= 894.24, P = 0. 00 ）. Numbers of TUNEL positive cells between adjacent time points were significantly different in both RIRI model group and AG treatment group （RIRI group： q = 24. 475,36. 591, -20.37,P〈0. 05 ;AG group：q=20. 94,16.79, -6. 92,P〈0.05） ,with the peak value at 24 hours after experiment. NO contents were significantly higher in the RIRI model group compared to AG group at various time points （q = 3.84, 4.01,8.91,3.75,P〈0.05） ,and those between adjacent time points showed significant differences （RIRI group： q = 4.77,13. 403, - 10. 29, P 〈 0.05 ; AG group ： q = 4.55,9.05, - 5.08, P 〈 0. 05 ）. iNOS activity was significantly elevated in the RIRI model group compared with AG group（q=-3.74,-4.94,-6.53,-3.98 ,P〈0.05） ,and obvious differences also were seen between the adjacent time point in both two groups （ RIRI group ： q = 8.43,6.71, - 6.39,P〈0.05;AG group：q=4.16,5.08,-3.93,P〈0.05）. Conclusions Aminoguanidine can protect the retinal function and morphology from oxidative stress damage afte RIRI by reducing the NO level and inhibiting the iNOS activity in retina.