Experimental Study On Pathogenesis In Oxygen-induced Retinopathy

Retinopathy of prematurity (ROP) is a proliferative retinopathy and a major cause of blindness in children. It is reported that the incidence of ROP is 20.3% in prematurity in China. Hyperoxic damage to immature retinal blood vessels, result in retinal ischemia,hypoxia and a compensatory induction of leaky blood vessels(angiogenesis).Vasoproliferation lead to the growth of retinal blood vessels through the inner limiting membrane into the vitreous cavity,often resulting in vitreous hemorrhage , tractional retinal detachment and loss of vision. The culprit of the pathogenesis in the ROP is the formation of abnormal new blood vessels in retina, of which the mechanism is not identified so far. In our investigation, we develop the mouse model of oxygen-induced retinopathy that mimics human ROP, moreover we can understand the mechanism of ROP.Part oneFluorescein angiography and quantification of retinal neovascularizationmodel in the mouse[Objective]To establish the retinal neovascularization model in mice by variable oxygen environment and measure the retinal neovascularization by high molecular weight fluorescein angiography and histological section.[Methods]1.Animal model: Newborn C57BL/6J mice were randomly divided into experimental and control groups. At postnatal day 7, mice in the experimental group were exposed to hyperoxia(75%±2%O2) for 5 days and then returned to normoxia(room air) to induce retinal neovascularization. Control mice were raised in room air.2.Retinal angiography with high-molecular-weight fluorescein: At postnatal days 12, 14, 17 (P12,14,17),High molecular weight fluorescein isothiocyanate dextran were perfused through the left ventricle directly,then the eyes were enucleated and fixed with paraformaldehyde. The retina was separated from the eyecup and flat mounted on a gelatin coat slide.The vasculature was examined under fluorescent microscope and photographed.3.Quantification of retinal neovascularization: At postnatal days 12> 14, 17 (P12,14,17),the mice were killed ,the eyes were enucleated and fixed, and then stained with hematoxylin and eosin.The nuclei of vascular endothelial cells which broke through internal limiting membrane(ILM) were counted.[Results]1. Consecutive retinal neovascularization can be induced in C57BL/6J mice after exposed hyperoxia.2. The entire retinal vascularization was visualized with fluorescent microscope.By focusing through the diffent layer of retina, the superficial, deep and connecting vessels were seen.The retinal vascularization occurred at the junction between the perfused and nonperfused retina and the exudation,hemorrhage and microaneurism were seen.3. The histological section showed that the nuclei of vascular endothelial cells which broke through internal limiting membrane (ILM) gradually increased at diffent time point.[Conclusion]1. When C57BL/6J mice that exposed to hyperoxia returned to relative hypoxia ,all the retinas occur neovascularization which resemble human ROP.2. The position and morphologic change of retinal neovascularization has been observed in the mice by retinal angiography with high-molecular-weight fluorescein.lt is a good method for foundation research of retinal neovascularization.3. Retinal angiography and histologic section results show that the retinal neovascularization gradually increase from pl2 to pi7 in the OIR model.Part twoThe expression and signification of vascular endothelial growth factor and pigment epithelium derived factor in the oxygen-induced retinpathy[Objective]To investigate the expression of Vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in the OIR mice model,which facilicate to understand possible pathogenesis mechanism of retinopathy of prematurity.[Methods]1.Animal model: Newborn C57BL/6J mice were randomly divided into experimental and control groups. At postnatal day 7,mice in the experimental group were exposed to hyperoxia (75%±2%O2) for 5 days and then returned to normoxia(room air) to induce retinal neovascularization.Control mice were raised in room air.2.Expression of VEGF^ PEDFmRNA: At postnatal days \2, U> 17(P(K 2> 5 after hyperoxia ),the mice were killed and the expression of VEGF >PEDFmRNA were semi-quantitative measured by RT-PCR.3. Expression of VEGF , PEDF protein: At postnatal days 12. 14, 17(P0> 2> 5 after hyperoxia ),the mice were killed and the expression of VEGF, PEDFmRNA were measured by Western blot analysis.[Results]l.With relative anoxia in retina, the level of VEGF elevated and retinal PEDF reduced,leading to VEGF-to-PEDF ratio progressively increased. At pl7,it reached the maximal change.2.The level of VEGF mRNA elevated and retinal PEDF mRNA reduced. At pl4,it reached the maximal change. The changes in the PEDF -. VEGF mRNA level were detected prior to that of the protein level.3.Consistent with severity of retinal neovascularization, VEGF-to-PEDF ratio gradually increased.The time course of the VEGF/PEDF ratio change was correlated with the development and progression of retinal neovascularization.[Conclusion]l.At the protein level:The time course of VEGF expression change is positivety correlated with the progression of retinal neovascularization.Reversely ,retinal PEDF levels are negatively correlated with the progression of retinal neovascularization. The time course of the PEDF down-regulation is consistent with the increased VEGF expression. The time course of the VEGF/PEDF ratio change is correlated with the development and progression of retinal neovascularization.2.At the RNA level: The time course of the PEDF mRNA down-regulation is consistent with the increased VEGF mRNA expression,similar with the change of protein. Moreover the change of VEGF , PEDF mRNA is prior to that of protein.The experiment results suggest that the regulation of VEGF and PEDF occues at RNA level.