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Clayton C. Tokunaga, Sr., Yi-Hao Chen, Sr., Mei Cheng, Wendy Dailey, Kimberly A. Drenser; Effect of Intravitreal Injection of Norrin upon Vascular Growth in Mice with Oxygen Induced Retinopathy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3120.
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Retinopathy of prematurity, diabetic retinopathy and other well-known retinal diseases usually manifest with abnormal vessel growth and neovascularization brought about by ischemic changes in the retina tissue. Norrin, a secreted protein, is suggested to be involved in retinal angiogenesis by activation of Wnt-signaling pathway.The purpose of this study is to determine the effects of intravitreal injection of Norrin on vascular development in mice with oxygen induced retinopathy (OIR).
An OIR model using C57BL/6J mice neonates was used in this study. Briefly, at postnatal day (P) 7 mice were exposed to 75% oxygen for 5 consecutive days. At P12 mice were removed from the oxygen chamber and returned to room air. At P14 they received an intravitreal injection of 1 µl (200ng) of Norrin or 1 µl of Phosphate Buffered Saline (PBS) in the right eye. At P17 mice were euthanized and the eyes were enucleated and fixed in 4% paraformaldehyde (PFA) for 1 hour. Retinas were then isolated, the vasculature stained with 500 µl lectin solution overnight, and whole mounted flat in slides.Images of the retinal mounts were taken at 5x magnification to document the superficial vascular plexus and preretinal neovascular tufts, using a fluorescent microscope. To quantify the vascular and avascular areas, as well as neovascularization, we used Adobe Photoshop CS5 to merge pictures and calculate the areas based on the total number of pixels. The avascular area was established as a percentage of total retinal area.
The analysis of retinal whole mounts at P17 revealed that the percentage of avascular area were decreased by 11% in the PBS group compared to the opposite non-treated eye and decreased by 19% in the Norrin treated group compared to the non-treated eye. When comparing both groups, Norrin treated eyes showed avascular areas reduced by 24% (p<0,03, n=8). These data suggest that Norrin intravitreal injection significantly reduced vascular loss and development of neovascularization, although injury alone might have some effect in preventing vasobliteration and neovascularization to a smaller scale.
Our data suggest that exogenous Norrin treatment can diminish the damaging vascular effects of oxygen-induced retinopathy and promote secondary vascularization.
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