Mouse model of oxygen-induced retinopathy (OIR). a The mouse model of oxygen-induced retinopathy (OIR). Neonatal mice and their nursing mother are placed into 75% oxygen from P7 to P12, which induces loss of immature retinal vessels, leading to a central zone of vaso-obliteration (VO). After returning to room air at P12, the central avascular retina becomes hypoxic, inducing vascular regrowth with pathologic neovascularization (NV). At P17, the maximum severity of NV is reached. NV starts to regress shortly after P17 and almost no VO or NV remains visible by P25. b Retinal whole mount stained with isolectin-B4-Alexa (red) displaying a normal vascular development at P17 under normoxic condition. b′ OIR P12 retinal whole mount showing an extensive VO area without NV. b″ OIR P17 retinal whole mount showing a decreased VO with NV at its maximum. c Quantification of vaso-obliteration (VO) by manually outlining the avascular area with image-processing software (Photoshop, Adobe Systems) c′ For computer-aided NV quantification, both the original image and the VO image generated with Adobe Photo-shop were imported into NIH’s free-access ImageJ software. The SWIFT_NV macroset isolates the red color channel, subtracts background fluorescence, and divides the VO image into four quadrants. c″ SWIFT_NV then allows the user to outline NV tufts but not normal vessels by setting a fluorescence threshold for each quadrant. The macroset then quantifies all NV pixels from all four quadrants, reports the result as neovascular total area, and creates an overlay of NV and original image
|
