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In Vivo Immunostaining Demonstrates Macrophages Associate with Growing and Regressing Vessels

¹From the Departments of Ophthalmology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

PURPOSE. The purpose of this study was to identify ways to improve qualitative and quantitative assessments of retinal vessels and neovascularization (NV).

METHODS. At postnatal day (P) 17, mice with oxygen-induced ischemic retinopathy were injected intravitreously with one of a variety of FITC-labeled or unlabeled antibodies and humanely killed 12 hours later. Retinas were flat mounted (retinas from eyes injected with labeled antibodies) or incubated with secondary antibody and then flat mounted (retinas from eyes injected with unlabeled antibodies).

RESULTS. Retinas from eyes injected with labeled anti-platelet endothelial cell adhesion molecule 1 (PECAM1) showed good resolution of the fine structure of retinal NV, including filopodia at the tips of sprouts. New vessels originated from superficial retinal vessels, something that is widely recognized, but they also arose from deep retinal capillaries and from large retinal vessels, which is not generally known. Retinas from eyes injected with unlabeled anti-PECAM1 antibody and then incubated with labeled secondary antibody showed selective staining of retinal NV with little or no background, greatly facilitating identification and quantification of the NV by image analysis software. Double labeling with anti-PECAM1 antibody and one of three other antibodies—anti-CD45, F4/80, or anti-CXCR4—showed exquisite localization of various populations of bone marrow–derived cells with respect to the vasculature and demonstrated close association of macrophages with NV and regressing vessels. Double labeling with anti-PECAM1 antibody and anti-placental growth factor (PlGF) showed high levels of PlGF in growing and regressing vessels but no detectable signal elsewhere in the retina.

CONCLUSIONS. This study describes techniques that facilitate measurements and detailed structural analysis of retinal NV and that allow identification and quantification of populations of bone marrow–derived cells and support the view that macrophages contribute to the growth and regression of vessels in the eye.

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