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W. Culp, Jr., S. Budd, P. Geisen, X. Lu, Y. Saito, M. E. Hartnett; The Role of Vegf164 in Intravitreous Neovascularization Using a Rat Model of Oxygen Induced Retinopathy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2637.
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We have previously shown that neural retinas from rat pups in the 50/10 oxygen-induced retinopathy (50/10 OIR) model upregulate vegf164 mRNA and vegf120 and vegf164 protein at a time when maximal intravitreous neovascularization (IVNV) is occurring. This correlation led us to investigate which cells produce this isoform, and how fluctuating oxygen concentrations contribute to the production of vegf164 and pathological neovascularization in the neural retina.
The 50/10 OIR model exposes newborn rat pups to repeated cycles of 24 h of 50% oxygen alternating with 10% oxygen creating pathology similar to retinopathy of prematurity (ROP) in humans; controls were animals exposed to room air (RA). Animals were euthanized at postnatal day 0 (p0), p6, p12, p14, p18, and p25, neural retinas were dissected, and real-time PCR was used to determine expression of vegf120, vegf164, and vegf188 isoforms normalized to actin. Vegf protein from neural retina tissue lysate was also analyzed on p12, p14, and p18 by ELISA. In situ hybridization probing for vegf164 was performed on p8, p12, and p14 neural retina sections. p8 and p14 sections were stained for glial fibrillary acidic protein (gfap), and p14 neural retina sections were stained with pimonidazol (Hypoxyprobe).
p12 and p18 vegf120 and vegf164 mRNA expression was 2-fold higher compared to RA controls, and p25 vegf188 mRNA expression was 5-fold higher compared to RA controls. p12, p14, and p18 vegf protein expression was more than 6-fold higher compared to RA controls (ANOVA, p<0.001), and p14 vegf protein expression was greater than p12 and p18 (post-hoc-tests p<0.001). p8 in situ hybridization for vegf164 mRNA expression in both RA and 50/10 OIR was primarily in the ganglion cell layer, whereas p14 vegf164 mRNA expression was primarily in the inner nuclear layer in 50/10 OIR but not in RA. Gfap positive labeling in p14 50/10 OIR localized to areas corresponding with vegf164 mRNA expression seen with in situ hybridization, and also corresponded to areas that stained positive for hypoxia.
Repeated fluctuations in oxygen, a risk factor for severe ROP, upregulated both vegf120 and vegf164 mRNA, and vegf protein. Importantly, the upregulation of vegf coincided with the time of maximal IVNV in this model. Cells spanning the inner nuclear layer (Müller cells) show the greatest signal for vegf164 mRNA in the 50/10 OIR on p14. Therefore, knocking down vegf164 in a cell specific manner may represent a therapeutic approach for ROP.
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