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A. Doelemeyer, T. Bensaoula, C. Chatenay-Rivauday, H. Ryckelynck, A. Ottlecz, G.N. Lambrou; Automated Objective Quantification of Vascular Morphology in Rodent Oxygen-induced Retinopathy . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3618.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To design an automated method to quantify vascular abnormalities in the retinas of rat neonates with oxygen-induced retinopathy (OIR) using a digital image analysis of retinal flat mounts. To evaluate different parameter(s) of retinal vascular morphology for their relevance to detect abnormal vessel alterations during the development of retinal neovascularization (NV). Methods: OIR is one of the most frequently used model to study ischemic retinal NV. Briefly, animals are exposed to hyperoxia (75% O2, 25% N2) for 5 days (Postnatal days P7 to P12 ), and then returned to room air. At P17, animals are injected into the left ventricle with fluorescein dextran for visualization of retinal vasculature. Retinas are dissected and flat mounted for observation. Image acquisition is performed with a fluorescent microscope (Olympus AG) equipped with a digital camera (Color View II and Analysis 3.2 software, SIS). In the image of the flat mount the outline of the whole retina is traced manually and the total area is computed. All subsequent computations are performed automatically. Vessel segments are separated from the background by a local thresholding procedure, resulting in a binary image. For the vessel trees a skeleton is computed, reducing the vessels to a one-pixel wide line. In this outline, vessel bifurcations easily are detected. The number of bifurcations and their density are computed and used as parameters to assess vascular changes. In the original binary image, areas of non-perfusion are computed using a region growing procedure. The number of areas, their individual size, the total area of non-perfusion and the relative fraction of the total retinal area are computed as additional parameters. Results: The above parameters were measured in 16 retinas obtained from rat neonates exposed to hyperoxia. The results for the main parameters are given below as means ± SEM: Total area [mm2]: 31.35 ± 0.35 Total non-perfused area[%]: 51 ± 2.3 Number of bifurcations: 4678 ± 106 Conclusions: The new method for automated image analysis of retinal vasculature permits objective quantification of neovascular changes observed in the rodent model of OIR. It provides a rapid and operator-independent assessment of the pathological vascular alterations seen in ischemic retinopathies. Therefore, it will be instrumental in screening new compounds for future therapeutic ways. Further studies in groups of rats with different stages of disease will provide better insight into which parameters or combinations thereof are best suited to detect vascular changes in the OIR model.
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