Microscopic Visualization of the Retina by Angiography with High-Molecular-Weight Fluorescein-Labeled Dextrans in the Mouse

Methods currently available for the examination of the retinal vasculature of laboratory animals have significant drawbacks. Fluorescein angiography of rodent eyes is hampered by a poor view of the peripheral retina and difficulty in performing fundus photography. Methods of staining or filling retinal vessels are unreliable, labor-intensive, or have high backgrounds. We have developed a novel technique that is quick, simple, and accurate. Fluorescein-labeled 2 million molecular weight dextrans are used to fill the retinal vasculature of mice in vivo, followed by removal of the retina, fixation in paraformaldehyde, and examination of the vascular pattern in whole mount preparations by fluorescence microscopy. We found that fluorescein and fluorescein-labeled low-molecular-weight dextrans (40,000-500,000) are not suitable as they leak out of the vasculature to stain the entire retina in whole mount preparations. By contrast, fluorescein-labeled 2 million molecular weight dextrans remain in the vasculature for many months without diffusion or decay. Under low magnification, the entire retinal vasculature can be visualized at one time. By focusing from one plane to another, the superficial, connecting, or deep vascular layers are delineated. The background fluorescence is very low. We have successfully used this technique in over 20 mice per day to document retinal angiogenesis in a model of oxygen-induced proliferative retinopathy.