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D.A. Leske, J. Wu, M.P. Fautsch, J.M. Holmes; Individual Retinal Analysis of VEGF and IGF-1 in Hypercarbic Oxygen-Induced Retinopathy . Invest. Ophthalmol. Vis. Sci. 2003;44(13):572.
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Purpose: Hypercarbic oxygen-induced retinopathy (HcOIR) in the neonatal rat provides a more severe model for retinopathy of prematurity (ROP) than classic OIR. We studied both the timecourse of neovascularization and the relationship of VEGF and IGF-1 mRNA to neovascularization in HcOIR, by analyzing individual retinae. Methods: Newborn Sprague-Dawley rats were raised in 10 expanded litters of 25; 4 HcOIR and 6 room air controls. HcOIR litters were exposed to 7 daily cycles of hyperoxia (80% O2 for 20.5 hours) followed by a hypoxic period (10% O2 for 0.5 hours) and a return to 80% O2 over 3 hours. Hypercarbia was maintained throughout the cycles (10% CO2). Rats were recovered in room air and sacrificed at days 8, 10, 13 or 20. Left eyes were ADPase-stained and evaluated in a masked manner for neovascularization. Total RNA was isolated from the corresponding 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: Neovascularization first appeared at day 8, prior to room air recovery (12% incidence), was maximal at days 10 and 13 (75% and 69%), and almost completely resolved by day 20 (8%). Only 2% of 114 room air retinas were graded positive for neovascularization. VEGF and IGF-1 mRNA expression increased over time from day 8 to day 20 in both HcOIR and controls (p=0.0001 for all analyses). Comparing HcOIR and controls at each time point, VEGF mRNA levels were decreased in HcOIR at day 8 (p= 0.0004 by Northern and real-time RT-PCR, n=23). At day 10, VEGF mRNA levels were increased in HcOIR (p= 0.0005 by Northern and real-time RT-PCR, n=21), which corresponds to maximal neovascularization. IGF-1 mRNA levels by Northern were decreased in HcOIR vs. controls at days 8 (p=0.002), 10 (p=0.007) and 13 (p= 0.05) but returned to normal by day 20. Conclusions: We have provided new data on the timecourse of hypercarbic oxygen-induced retinopathy, which are important for planning future therapeutic studies. Regarding molecular techniques, individual retina Northerns and real-time RT-PCR are both feasible and useful in studying mRNA expression levels in rat models of ROP. Increased VEGF mRNA and decreased retinal IGF-1 mRNA are temporally related to the emergence of neovascularization in hypercarbic oxygen-induced retinopathy.
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