The Experimental Study Of Mechanism And Prevention In Oxygen-induced Retinal Neovascularization

Purpose:To study oxygen-induced retinal neovascularization mechanism and prevention methods.Methods:Twenty mice were randomly divided into 2 groups:control group(10 mice)and experimental group(10 mice).Experimental group mice were exposed to an atmosphere of hyperoxia(75%±2%O2) from P7 to P12. On P12d,and returned to room air (21%02) until P17 to induce retinal neovascularization.Those in control were exposed in constant normoxia from P7 to P17.At P17,retinal neovascularization was examined by fluorescence angiography and hematoxylin/eosin staining;Randomly selected control group and experimental group of mice 6,using real-time polymerase chain reaction observed Annexin A2 and TPA mRNA level at P12,P14,P17 points;At P17,ten normal pups and twenty OIR model pups(include ten hydrogen saline treatment pups and control ten normal saline treatment pups) were decapitated and retinal neovascularization was assessed by fluorescence imaging and histopathological examination,VEGF expression was evaluated by realtime PCR and immunofluorescence histochemistry,oxidative stress was measured by MDA assessment.Results:1.Fluorescence angiography showed a pattern of pathological retinal neovascularization such as central non-perfused areas,tortuous and dilated blood vessels and leaky neovascular tufts in experimental mice,but showed normal retinal blood vessels-no fluorescent leakage and non-perfusion area in control group.And the serial HE staining sections confirmed the results. It showed 79.70±7.57 retinal vascular cell nuclei anterior to the internal limiting membrane in experimental mice compared with 0.28±0.12 in control group and there was statistical significance between two group(P<0.01).2. At P12 and P17, the mRNA expression of Annexin A2 and TPA in control and experimental groups had no significant difference(P>0.05); But at P14, the mRNA expression of Annexin A2 and TPA in experimental group was higher than the control group and had significant difference (P<0.01).3.Fluorescence angiography showed Blood vessels between the optic nerve and the peripheral retina were evenly distributed in a radial fashion and had no fluorescence leakage in normal mice. After hyperoxia,large retinal blood vessels expanded irregularly and distorted in shape and showed large non-perfusion areas intervened with large number of new vascular plexus, accompanied by significant leakage of fluorescent. After hydrogen saline treatment, neither significant non-perfusion areas nor neovascularization plexus and nor fluorescence leakage mitigation were observed,The average number of vascular endothelial cell nuclei which break through the internal limiting membrane on each section was 0.90±1.28,79.70±7.57 and 41.00±8.01 respectively in normal group,HSS control group and HSS treat group.There was no statistical difference between normal group and HSS treat group(P>0.05),while there existed statistically significant difierence between HSS control group and HSS treat group(P<0.01).Real-time PCR analysis was performed to assess the mRNA levels of VEGF in the retinas of experimental animals. Low expression of VEGF mRNA was found in normal group(1.00±0.03). HSS control group enhanced VEGF mRNA levels (7.40±0.04). Hydrogen saline treatment decreased VEGF mRNA levels (1.94±0.12). The expression of VEGF mRNA in HSS control group is significantly higher than normal group and HSS treat group(P<0.01). VEGF protein was mainly distributed in the vicinity of retinal internal limiting membrane,ganglion cell layer,inner nuclear layer cell cytoplasm and plasma membrane and pigment epithelial cell layer, as shown by brown or brownish yellow staining. Count the number of positive staining of VEGF per 400 high-power view and VEGF expression in retinal tissue of each group.The average number of positive staining of VEGF was 1.30±0.06,2.92±0.70 and 1.46±0.01 respectively in normal group,HSS control group and HSS treat group.There was no statistical difference between normal group and HSS treat group(P>0.05),while there existed statistically significant difierence between HSS control group and HSS treat group(P<0.01).The content of MDA of retina in each group was detected at P17. Hyperoxia enhanced significantly the content of MDA (22.42±2.24) when compared normal animals (5.17±4.23). Hydrogen saline suppressed the production of MDA (16.07±1.05) partially. There was no statistical difference between normal group and HSS treat group(P>0.05),while there existed statistically significant difierence between HSS control group and HSS treat group (P<0.01).Conclusion:The oxygen-induced ischemic retinopathy mouse model is a successful animal model of retinal neovascularization,which could be a reproducible model of angiogenesis in there search for retinal neovascularization.Annexin A2 and TPA participate in the oxygen induced retinal neovascularization and closely related. Hydrogen saline reduced oxidative stress,decreased VEGF expression and suppressed retinal neovascularization in a model of hyperoxia induced Retinopathy.