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C.P. Lin, P. Zamiri, J.M. Runnels, M. Poureshagh, A. Clermont, S.E. Bursell; Imaging Molecular Expression in the Retinal Vascular Endothelium by in vivo Immunofluorescence Microscopy . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3464.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The endothelial cells (EC) of the retinal vasculature form the inner blood/retina barrier that regulates molecular and cellular exchange between the circulation and the retinal tissues. Leukocyte adhesion and infiltration into the retina under various disease conditions depend on the interaction between cell adhesion molecules on the EC and on the leukocyte surfaces. Imaging molecular expression on the vascular EC will therefore allow us to visualize early disease markers displayed by the EC to the immune cells in the circulation. Methods: Fluorescent antibodies against mouse EC surface molecules, such as anti–VCAM (vascular cell adhesion molecule)–1 conjugated to Cy5.5, were delivered systemically by tail vein injection. After allowing time for unbound antibodies to clear from the circulation, labeled antibodies on the retinal EC were imaged by a confocal scanning laser ophthalmoscope (SLO) that was specifically built for mouse eye imaging. In addition, the SLO can be quickly converted to an in vivo confocal microscope, allowing imaging the skin vasculature of the mouse ear to verify antibody binding and clearance. Results: Immunofluorescence images are obtained for the vasculature of live mice both in the skin and in the eye, after injection of fluorescent antibodies at a dose of 10–20 µg per mouse (0.5–1 µg/g of body weight). The clearance time depends on the specific antibodies and ranges from a few hours to more than 24 hours. Superior images are obtained in the skin compared to the retina due to the limited numerical aperture of the eye. Nonetheless molecular expression on the mouse retinal vascular EC can be visualized in the living eye. Conclusions: In vivo immunofluorescence microscopy can be used to image early disease markers, and to follow the progression of diseases such as diabetic retinopathy, over time, in live animals without sacrificing them at multiple time points. By simultaneous imaging of leukocyte trafficking in retinal vessels using acridine orange and two–color detection, it is possible to correlate the molecular processes involved in leukocyte adhesion and infiltration into the retina.
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