AIM:To evaluate the effect of adeno-associated virus (rAAV)mediated encoding endostatin(ES) gene transfection for inhibiting experimental corneal neovascularization (CNV).METHODS:Sixty healthy SD rats were sutured on the superior cornea.Animals were randomly divided into 4 groups(A,B,C,D).50μL of rAAV-ES solution was injected into subconjunctiva immediately after sutured in the group A.After scraping off epithelium,the cornea was soaked in the rAAV-ES solution for 10minutes in the group B.After scraping off epithelium,the cornea was soaked in the rAAV-ES solution for 10minutes and 50μL of rAAV-ES solution was injected into subconjunctiva in the group C.50μL of saline was injected into subconjunctiva in the group D.Eyes were examined by a slit-lamp biomicroscope and a surgical microscope was used to monitor angiogenesis in response to rAAV-ES transduction.The size of CNV area was measured and calculated.The ES gene expression in inflammatory cornea of rats was detected by immunohistochemistry after transduction with rAAV-ES.The sections were stained with hematoxylin and eosin to measure the CNV density.RESULTS:The occurrence and development of CNV were observed by slit-lamp microscope after transduction with rAAV-ES.After suturing,CNV increased gradually,peaked on day 14,and decreased gradually,degenerated on day 21 after suture induction.Analysis of variance of repeated data showed that there was crossover effect of time and treatment factors(F=175.810,P<0.01).At each transducted group,there was a significant difference between any two groups(F=2243.816,P<0.01).The size of CNV areas of group C was significantly smaller than that of the group A,B and D at each time point.Accordingly,the size of CNV areas of group D was the largest,but the size of CNV areas of group C was the smallest after transduction with rAAV-ES.At each time point,there was a significant difference between any two groups(F=1060.854,P<0.01).Inhibition ratio of CNV was increased gradually after transduction,and the highest inhibition ratio of CNV was detected in group C on day 28 after suture induction.CNV density was lower in transducted group than in control group.The ES gene expression in inflammatory cornea of rats was detected by immunohistochemistry after transduction with rAAV-ES.Only corneal epithelium was stained brown in group A,some of corneal epithelium and NV endothelial cells in superficial stroma of cornea were stained brown in group B,and most of corneal epithelium and NV endothelial cells in superficial stroma of cornea were stained brown in group C,but corneal cells were not stained at all in group D.The sections were stained with hematoxylin and eosin to measure the CNV density.CNV density was lower in transducted group than in control group.CONCLUSION:There was a significant inhibitory effect on neovascularization in rat inflammatory CNV model after rAAV-ES transduction.The inhibitory effect on CNV was higher by use of combined delivery approaches than by use of either one,i.e.the cornea was soaked in the rAAV-ES solution after scraping off epithelia or the rAAV-ES viral particles were injected into subconjunctiva.