Effect Of Hydroxyl Fasudil On Neuronal Apoptosis And Bcl-2, Caspase-3Protein Expression In Retinal Ischemia-Reperfusion Injury In Rabbit

Objective To establish a kind of reliable animal model which can simulate humantraits of RIRI, and after injecting fasudil hydrochloride into rabbits’ ear veins, itrespectively observes retinal morphological changes by HE staining method, detects thechange of nerve cell apoptosis by using the method of TUNEL, detects the changes ofprotein expression of Bcl-2and Caspase-3apoptosis genes and the influences of fasudilhydrochloride by using the method of IHC, thus to further explore and discuss theneuroprotection and mechanism of action to RIRI by fasudil hydrochloride, and toprovide theoretical basis for the clinical treatment of RIRI.Materials and Methods1Sectionalization of experimental animals: This study takes54healthy male purebred big-eared Japanese rabbits as the research objects, and dividesthem into three groups randomly. There are: normal group (6rabbits), injured group (24rabbits), and treatment group (24rabbits), and among them, injured group and treatmentgroup are divided into6groups according to the different time points（6h、12h、24h、48h、72h、120h）of reperfusion.2Set up RIRI animal models: Rabbits in injuredgroup and treatment group are all caused retinal ischemia by the method of anteriorchamber perfusion, after ischemia for1h, drop the intra-ocular pressure to normal levelquickly, thus to finish the making of RIRI animal model.3The way and method of dose:three groups are injected through the ear venous, treatment group is injected fasudilhydrochloride with the dose of10mg/kg, normal group and injure group are injectedwith same quantity of BSS.4Specimen making and method: each group shall killanimals and extract eyeballs6h,12h,24h, and48h.72h and120h after reperfusion,observe the morphological change of retina by HE staining method of retinal tissue indifferent time periods, detect the change of nerve cell apoptosis by TUNEL method andthe change of the expression of apoptosis gene Bcl-2, Caspase-3protein by IHC.Result1The morphologic change of retina: The degree of retinal damage in treatment group is obviously lighter than injure group.2The change of retinal neuron apoptosis: at point-in-time of6h,12h,24h,48h,72h, the pair wise comparisons of apoptotic cell counting results in three group at same point-in-time are statistically significant (P<0.01), but there is no statistically significant comparison of thr ee groups at120h (P>0.05); injure group: the comparisons of apoptotic cell counting results at any point-in-time are statistically significant (F=13.06, P<0.01); treatment group: the comparisons of apoptotic cell counting results at any point-in-timeare statistically significant (F=22.54, P<0.01).3The expression of apoptosis geneBcl-2protein: at the point-in-time of6h,12h,24h,48h,72h, the pair wise comparisons of the expression of Bcl-2protein in three group at same point-in-time arestatistically significant (P<0.01), but there is no statistically significant comparisonof three groups at120h (P>0.05); injure group: the comparisons of he expressionof Bcl-2protein at any point-in-time are statistically significant (F=15.14,P<0.01);treatment group: the comparisons of he expression of Bcl-2protein at any point-in-time are statistically significant (F=17.06,P<0.01).4The expression of apoptosisgene Caspase-3protein: at the point-in-time of6h,12h,24h,48h,72h, the pair wise comparisons of the expression of Caspase-3protein in three group at same point-in-time are statistically significant (P<0.01); injure group: the differences of positive cell counting results at any point-in-time are statistically significant (F=65.860,P<0.01); treatment group: the differences of positive cell counting results at anypoint-in-time are statistically significant (F=276.550,P<0.01).Conclusion1fasudil hydrochloride can reduce the damage of retinal ischemia reperfusion, and play an important role in maintaining the morphology and functionof retina;2fasudil hydrochloride can inhibit the apoptosis of retinal neuron, its mechanism of action may associate with the improved expression of Bcl-2and the lowered expression of Caspase-3, and protect retinal neuron from retinal ischemia reperfusion damage obviously.