Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Characterization of Fatty Acid Binding Protein 7 (FABP7) in the Murine Retina.

PURPOSE: To characterize the mouse retina lacking fatty acid binding protein (FABP7-/-). METHODS: Immunohistochemistry (IHC) was performed in 8-week-old mice to localize FABP7 in the retina. Retinal thickness was measured using image-guided spectral-domain optical coherence topography images. Electroretinography was carried out to assess retinal function. Fundus photography and fundus fluorescein angiography were performed on FABP7-/- and littermate wild-type (WT) mice, and retinal vascular changes were calculated using Singapore I Vessel Assessment (SIVA) analysis. Blood glucose levels were measured in the 8-week-old WT and FABP7-/- mice. In addition, retina was processed for trypsin digestion and retinal flat mounts for isolectin staining. Transcript levels of FABP7, VEGF, GFAP, and Na+K+ATPase were quantified using real-time PCR, and protein expression was analyzed by IHC and Western blot. RESULTS: Fatty acid binding protein 7 is expressed in the inner nuclear layer, outer plexiform layer, and photoreceptor inner segments. No significant difference in retinal thickness and ERG responses was observed between FABP7-deficient and WT retinas. FABP7-/- mice have significantly decreased retinal venular caliber retinal arteriolar fractal dimension compared with WT littermates. FABP7-/- mice showed significant increased areas of fluorescein leakage in the retina. FABP7-/- mice exhibited elevated high blood glucose levels compared with WT mice. Trypsin digested FABP7-/- mice retina showed increased acellular strands and endothelial cell drop outs, and reduced microvasculature branching compared with WT retina. FABP7-/- mice retina also have increased GFAP and VEGF expression. CONCLUSIONS: Fatty acid binding protein 7 is expressed in the retina and might play an important role in maintaining retinal vasculature.