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Updated: Jul 12, 2026
Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease
Published on: August 26, 2017
In vivo immunostaining demonstrates macrophages associate with growing and regressing vessels
Jikui Shen1, Bing Xie, Aling Dong
1Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-9277, USA.
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.
Insights
This study introduces improved methods for assessing retinal neovascularization (NV) in mice. Techniques using anti-PECAM1 antibodies enhance visualization and quantification of new blood vessels and associated cells, aiding research into eye diseases.
Area of Science:
- Ophthalmology
- Vascular Biology
- Immunology
Background:
- Retinal neovascularization (NV) is a hallmark of several blinding eye diseases.
- Accurate assessment of NV is crucial for understanding disease progression and evaluating treatments.
- Current methods for quantifying NV can be limited in resolution and specificity.
Purpose of the Study:
- To develop and validate improved techniques for the qualitative and quantitative assessment of retinal neovascularization (NV).
- To enhance the structural analysis and quantification of NV in a mouse model of ischemic retinopathy.
Main Methods:
- Oxygen-induced retinopathy model in mice.
- Intravitreous injection of FITC-labeled or unlabeled anti-platelet endothelial cell adhesion molecule 1 (PECAM1) antibodies.
- Retinal flat-mounting and immunohistochemical staining with secondary antibodies for visualization.
Main Results:
- Labeled anti-PECAM1 antibodies provided high-resolution imaging of retinal NV, revealing sprout structures.
- New vessels were observed originating from superficial and deep retinal capillaries, as well as large vessels.
- Unlabeled anti-PECAM1 with labeled secondary antibodies enabled selective NV staining, facilitating image analysis.
- Double labeling identified bone marrow-derived cells, including macrophages, associated with NV and regressing vessels.
- Placental growth factor (PlGF) was localized to growing and regressing vessels.
Conclusions:
- The described techniques improve the measurement and structural analysis of retinal NV.
- These methods allow for the identification and quantification of bone marrow-derived cells, including macrophages, in retinal NV.
- Findings support the role of macrophages in the growth and regression of ocular blood vessels.
