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G.S. Robinson; The VEGF R2 Inhibitor SU5416 Reduces Ischemia-Induced Retinopathy: A Novel Method to Quantitate Retinal Neovascularization . Invest. Ophthalmol. Vis. Sci. 2003;44(13):562.
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Purpose: To develop a rapid and sensitive method to measure and statistically evaluate the effects of drug candidates in animal models of retinal disease. Methods: Oxygen-induced retinopathy was induced in C57BL/6J mice as described by Smith et al (Smith et al. 1994, IOVS 35, 101-111). From P12-P17 mixed litters of mice were treated subcutaneously (s.c.) with 25 mg/kg/day (N= 8), 50 mg/kg/day (N=8) SU5416, or vehicle (N=8). Mice were anesthetized and euthanized with cardiac perfusion of 4% paraformaldehyde, eyes enucleated, and fixed overnight in 4% paraformaldehyde before flat mounting retinas. Retinal flatmounts were stained with Hoeschst 33342 dye before coplanar optical analysis. An optical slice with a defined position with respect the ganglion cell layer was selected and Nearest Neighbor Deconvolution (NNDC) was performed to remove out-of-focus light from the in-focus image of vitreal endothelial nuclei. Results: We observed a 9.3 fold increase in vitreal endothelial nuclei in vehicle treated mice in hyperoxic conditions (401 +/- 64) as compared with normoxic controls (43 +/- 6). Treatment with SU5416 at 25 mg/kg/day and 50 mg/kg/day reduced retinal neovascularization by 8% (369 +/- 25) and 55% (181 +/- 15, p<0.05), respectively. These results matched published results as determined by endothelial cell counting. These results were obtained in a 2-3 day time period vs. a 3-6 week period necessary for endothelial cell counting. In addition, retinal hemorrhaging could be assessed and was reduced to normoxic control levels with both treatments of SU5416. Conclusions: We have developed a simple, fast and robust imaging procedure to quantitate retinal neovascularization and have used this model to analyze the effect of VEGF R2 inhibition on retinal neovascularization in a murine model of ROP. Our results confirm and expand published results obtained using the standard counting method. This new optical procedure provides a simple and accurate method to evaluate the effects of drug candidates in animal models of retinal disease with a more efficient use of resources.
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