Effects of Green Tea Extracts on Oxygen–Induced Retinal Neovascularization in the Neonatal Rat

Abstract

Abstract: : Purpose: To investigate the preventive effects of green tea extracts (GTE) on oxygen–induced neovascularization in the neonatal rat as retinopathy of prematurity model. Methods: Neovascularization was induced by maintaining Sprague–Dawley (SD) neonatal rats in 80% oxygen from birth till post–natal day 12 (P12), interrupted daily by 30 minutes in ambient air, and followed by a progressive return to 80% oxygen. Rats were then placed in ambient air until P18. Green tea leaves (500 g) were extracted with 5 L of methanol for 2 hr under reflux. After evaporation, the methanolic extract was dissolved in water and extracted with 2L of n–hexane 3 times. The lower layer was extracted with 2 L of n–butanol 3 times. Three layers were evaporated to give n–hexane soluble fraction (50 g), n–butanol soluble fraction (M–B, 85 g) and water soluble fraction (M–W, 30 g), respectively. M–B (20 g) was applied to a Sephadex LH–20 column and eluted with water (DM–W, 5.6 g) and then 50% acetone (DM–A50, 12.3 g). The rats (n=49) were treated once daily from P6 to P17 via oral administration of GTE or distilled water (DW) at 50 µL/10 g body weight. At P18, the animals were sacrificed, the retinal samples were prepared for flat mount and stained with ADP–ace. Retinal neovascularization was scored (NV) and avascular areas were measured as a % of total retinal area (%AVA) by using NIH image. Statistical analysis was performed using Welch's t–test for NV and %AVA. Results: The NV scores were 5.7±0.8 in DW, 4.0±1.0 in M–B, 3.9±1.6 in M–W, 4.5±1.0 in DM–W, and 4.3±1.3 in DM–A50 treatment group, respectively. The %AVAs were 23±2% of DW, 19±5% in M–B, 14±3% in M–W, 37±10% in DM–W, and 9±4% in DM–A50, respectively. Conclusions: These data suggest that oxygen–induced retinal NV in the neonatal rat is suppressed by a green tea M–W fraction, probably containing several types of water soluble antioxidants.

Keywords: retinal neovascularization • retinopathy of prematurity • antioxidants