Relationship of VEGF and IGF-1 to Neovascularization in Acidosis-Induced Retinopathy by Individual Retinal Analysis

Abstract

Abstract: : Purpose: Acidosis-induced retinopathy (AIR) in the neonatal rat provides an alternative model for retinopathy of prematurity (ROP). It is possible that classic oxygen-induced retinopathy (OIR) and AIR may be mediated by different pathways. We studied the relationship of VEGF and IGF-1 mRNA to the emergence of neovascularization in AIR, by analyzing individual retinae. Methods: Newborn Sprague-Dawley rats raised in 6 expanded litters of 25 were assigned to 5, 8 or 10 days of acidosis or non-acidotic controls. Using our established AIR model, acidosis was induced by twice daily gavage with NH₄Cl (10 mM/kg) beginning on the second day of life until day 8. All animals were raised in room air. Total RNA was isolated from individual right retinae and analyzed for VEGF and IGF-1 mRNA expression using Northern blots and quantitative real-time RT-PCR. VEGF and IGF-1 mRNA levels were normalized to 36B4 (housekeeping gene). These ratios were normalized to 3 day room air retinae and compared using non-parametric statistical methods. Results: VEGF and IGF-1 mRNA expression increased over time from day 5 to day 10 in both AIR (VEGF: p= 0.0001 by Northern and real-time RT-PCR, n= 49; IGF-1: p=0.0001 by Northern, n=48) and controls (VEGF: p< 0.002 by Northern and real-time RT-PCR, n=36; IGF-1: p= 0.02 by Northern, n=37). Comparing AIR and controls at each time point, VEGF mRNA levels were increased in AIR at day 8 by both methods (p< 0.0005 by Northern and real-time RT-PCR, n=31), which precedes maximal neovascularization in the AIR model. IGF-1 mRNA levels were similar between AIR and controls at days 5, 8 and 10. Conclusions: Acidosis-induced retinopathy is associated with increased VEGF mRNA, which is temporally related to the emergence of neovascularization. Individual retina Northerns and real-time RT-PCR are both feasible and useful techniques in studying mRNA expression levels in rat models of ROP. Although different models of ROP, OIR and AIR appear to share an increase in VEGF mRNA, prior to the emergence of neovascularization.

Keywords: retinopathy of prematurity • neovascularization • gene/expression