Effects of Systemic Inhibition of Reactive Oxygen Species on Oxygen-Induced Retinopathy in Neonatal Rats

Abstract

Purpose:: To investigate the effects of intraperitoneal (IP) injections of inhibitors to reactive oxygen species (ROS) on oxygen-induced retinopathy (OIR) in neonatal rats.

Methods:: We used the 50/10 OIR model that exposes Sprague-Dawley newborn rat pups to repeated cycles of 24 h of 50% oxygen alternating with 24 h of 10% oxygen until postnatal 14 day (p14), then to room air until p18. Pups were randomly assigned to receive daily IP injections of either 150 mg/kg N-acetylcysteine (NAC: an ROS scavenger), 10 mg/kg apocynin (an NAD(P)H oxidase inhibitor) or PBS as a control from p12 to p17 daily. At p18, retinas were dissected, and fixed for flat mounts, or collected for western blot analysis. Flat mounted retinas were stained with Griffonia simplicifolia isolectin B4 and scored for retinal intravitreous neovascularization (IVNV) by counting clock hours and for peripheral avascular areas as a percent of total retinal area (%AVA) using Image J (NIH). Activated Caspase-3 and VEGF were measured by western blot. The data were analyzed by Mann-Whitney test using SPSS software.

Results:: There were at least two separate experimental litters. There was a significantly smaller %AVA in apocynin-injected (n=11) compared to control groups (n=11) (20±2% vs 28±3% SE: p=0.01) but no difference in IVNV scores (4.2±0.7 vs 5.3±0.6 SE: p=0.26). There was no difference in NAC-injected (n=6) vs. control (n=5) as to % AVA (19±4% vs 26±3% SE: p=0.20) or IVNV (3.3±0.8 vs 4.0±0.7 SE: p=0.51). Activated Caspase-3 was reduced by apocynin (n=4) significantly (p=0.02) but not by NAC (n=4) compared to controls (n=4). There was no significant difference in VEGF protein from apocynin groups (p=0.08) or NAC (p=1.00) compared to control.

Conclusions:: NAD(P)H oxidase is a source of ROS. Apocynin decreased retinal avascularity, but not IVNV in a rat model of OIR that has relevance to human ROP. However, NAC did not reduce % AVA or IVNV. NAD(P)H oxidase inhibitors as given in this study reduced apoptosis and offer promise in reducing the avascular areas in diseases such as ROP. Further study is warranted.

Keywords: retinopathy of prematurity • retinal neovascularization • oxidation/oxidative or free radical damage