The right retina of each animal was used for image analysis. Imaging was performed within 12 hours of lectin labeling with a fluorescence microscope (BX50F; Olympus Optical Co., Ltd., Tokyo, Japan) equipped with a charge-coupled device (CCD)-digital camera (Photometrics Coolsnap fx; Roper Scientific, Trenton, NJ) and image acquisition software (RS Image, ver 1.01; Roper Scientific). Sequential, overlapping high-resolution images of the entire retina were captured with a 4× objective. Images were manually arranged, merged to construct a montage image of the retina (Photoshop, ver. 7.0; Adobe Systems Inc., San Jose, CA) and analyzed using image-analysis software (ImageJ, ver. 1.30; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Each montage was analyzed in a window measuring 20 × 20 cm on a high-definition LCD computer monitor (17-in. Apple Studio Display; Apple Computer, Cupertino, CA). Avascular areas were manually outlined and measured as a percentage of the total retinal area by a masked observer. Repeat analyses by the same observer were highly concordant (mean difference 2.1% ± 1.9%). The central avascular zone was defined as the capillary-free region surrounding the optic nerve head. Peripheral avascular zones were capillary-free regions not in continuity with the optic nerve head.
A semiquantitative system was used to grade vascular morphology. The retinal montages of P18 cyclic oxygen-exposed rats were compared with reference images of room air-exposed controls. Montages were scored for vascular morphologic abnormalities and tortuosity. Morphologic abnormalities were defined as features not seen in the course of normal retinal vascularization, including regions of exceptionally dense vascular budding, vascular ridges, marked vascular dilatation, and neovascular tufts. Neovascular tufts were defined as vascular projections anterior to the superficial vascular plexus. Each retinal quadrant was divided into thirds to give a total of 12 clock hours of retina, and each clock hour was scored for the presence (1) or absence (0) of any of these morphologic abnormalities.
20 38 42 The tortuosity of retinal vessels was assessed by eye in masked fashion, using reference images for comparison. First-order vessels were considered along their entire length, and an overall grading was determined for each retina as normal (1), minimally increased (2), moderately increased (3), or markedly increased (4).