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E. Aguilar, M.I. Dorrell, M.R. Ritter, C.M. Aderman, A.C. H. Smith, J. Friedlander, E. Banin, M. Friedlander; T2–TrpRS Inhibits Neovascular Tuft Formation and Enhances Revascularization in Oxygen Induced Retinopathy as Assessed by a New, Rapid Method of Quantification . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4167.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The mouse model of oxygen–induced retinopathy (OIR) has been used extensively to study mechanisms of neovascularization (NV) and effects of angiostatics. We present a novel method to quantify vascular changes, including vascular obliteration and pre–retinal neovascular tufts, using retinal whole mounts, confocal fluorescent microscopy, and computerized image analysis. We then used this method to assess the effects of the angiostatic compound T2–TrpRS on OIR. Methods: C57BL/6J mice were transiently exposed to hyperoxic conditions (75% O2) between post–natal day 7 (P7) and P12. Retinas were isolated, blood vessels were visualized by staining with isolectin Griffonia Simplicifolia, and confocal images of retinal whole–mounts were acquired. Readily available image processing software was used to measure and follow two main parameters in each retina: area of vascular obliteration and extent of pre–retinal NV. This method was compared to the standard method of OIR quantification in which the number of pre–inner limiting membrane (pre–ILM) nuclei is counted in serial sections of whole eyes. To test the angiostatic effects of T2–TrpRS on pathological NV, mice were injected intravitreally after return to normoxia, and the effects on obliterated areas and on neovascular tuft formation were assessed. Results: The extent of vascular obliteration and of abnormal NV can be accurately quantified using this method. Fellow eye and inter–observer variabilities were low, and quantification of pre–retinal neovascular tufts correlated well with pre–ILM nuclei counts using standard methods. Using this quantification method, T2–TrpRS was found to inhibit the area of neovascular tufts by >90% at P17. At the same time, it enhanced the revascularization process, reducing the obliterated areas by 50% compared to control–injected retinas. Conclusions: Confocal imaging and computerized image analysis of retinal whole–mounts provides a highly reproducible means for quantifying vascular changes associated with OIR. The observations using T2–TrpRS demonstrate the importance of a quantification system that allows analysis of effects on vascular obliteration as well as on pre–ILM NV. T2–TrpRS was found to be a potent inhibitor of pathological angiogenesis while also promoting physiological revascularization, demonstrating its potential utility as a drug for the treatment of ischemic and neovascular eye diseases.
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