OBJECTIVETo date, studies on the roles of angiopoietin-2 (ang-2) during the retinal vascular development focused on either double transgenic mice with inducible expression of ang-2 or ang-2 gene knockout mice or ang-2 gene deficient mice, and others focused on cell culture. Few studies have been done in animals with normal genetic expression or oxygen-induced animal model. In this study, the hyperoxia-induced model of retinopathy was established to investigate the expression of ang-2 in the retinae of model rats with retinopathy of prematurity (ROP) and explore the expression relationship between ang-2 in the retinae and retinal vascular development.

METHODSEighty newborn Sprague-Dawley rats were randomly divided into hyperoxia and normoxia control groups, then each group was further divided into 4, 7, 10, 14 and 20 days subgroups. Beginning on day 1 of life, 40 rats in hyperoxia group were exposed to hyperoxia [(75+/-2)% O2] for 7 days, and subsequently were moved into room air, thus ROP model was set up. An additional 40 rats were raised in room air served as age matched controls. Rats of postnatal day4 (d 4), d 7, d 10, d 14 and d 20 were sacrificed respectively. Rats left eyes were obtained, the anterior half of the eyeball and the vitreous were removed, then the retinae were gently scraped off the choroid, and used to delineate retinal vascular development by adenosine diphosphatase (ADPase) staining. Right eyes were embedded in paraffin and frozen sections were stained with immunohistochemistry to measure the expression of ang-2 in the retinae. Meanwhile, the number of new vascular cell nuclei extending into the internal limiting membrane in cross-sections was counted after HE staining to assess retinal neovascularization.

RESULTSThe retinal vasculature of the rats began to develop after birth, grew to most of the retinae on the 7th day, and was developed around 20th day. There was a large area of persistent central retinal vaso-obliteration in the retinal vasculature in rats after exposure to hyperoxia for 7 days, and the developed vessels were distorted. Hyperoxia induced neovascularization occurred on the 10th day and reached maximum neovascular response on d 14. The result of HE staining showed that the highest number of neovascular nuclei per cross section extending into the vitreous in hyperoxia model was seen on d 14 as compared with that of the same age of control groups and subretinal hematocele was observed in hyperoxia model of d 10 group. The expression of ang-2 in control group was the strongest in the retina on the 7th day, and was the least on the 20th day. In hyperoxia group, the expression of ang-2 on the 7th day was the weakest, the optical density value (OD value) was 82.106 62+/-6.302 40, significantly less than that of the age-matched normoxia control group, 115.448 33+/-9.373 10, and lower than that of hyperoxia model d 10, 124.425 95+/-10.572 09, and d 14, 117.685 20+/-10.578 48 (P<0.001). After the rats had been returned to room air for 3 days, the expression of ang-2 increased significantly and reached the peak on d10, it's OD value was 110.07 039+/-12.59 962, the result had significant difference as compared with the age-matched control group, 124.42 595+/-10.572 09 (P<0.05) and this strong expression remained till d 14 in model group.

CONCLUSIONHyperoxia induced neovascularization and inhibited the expression of ang-2 in the retinae; and the neovascularization is in accordance with the change of angiopoietin-2 expressed in retinae, suggesting that there is a close relationship between the expression of ang-2 and retinal vascular development.