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P. Russ; In Vivo Imaging of Neutrophils in the Mouse Retinal Circulation . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3031.
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Purpose: The biochemical mediators of neutrophil migration remain unclear. In this study we sought to develop an in vivo method to visualize the movement of neutrophils in the retinal circulation of a mouse model of endotoxin induced uveitis (EIU). The model system will be used in investigations of the biochemical pathways responsible for neutrophil migration. Methods: Uveitis was induced in BALBc mice by intravenous injection of lipopolysaccharide (LPS). An inverted fluorescence microscope was used to image leukocytes in the retinal circulation of control and EIU mice. The microscope was equipped with a xenon flash light source and filter cubes for detection of fluorescein and Cy3. In addition, the apparatus included a 3CCD camera and image capture card capable of acquiring sequences of 12bit color images at 9 frames per second. This allowed capture of flowing and rolling leukocytes as well as those that were stationary. Mice were anesthetized and their pupils dilated before receiving an intravenous injection of acridine orange to label the circulating cells. Retinal wholemounts were also scanned with a dual–wavelength flat–bed scanner with laser excitation wavelengths of 532 (Cy3) and 635 (Cy5). Results:In vivo images of leukocytes in the retinal circulation in control and EIU mice were obtained. Images of retinal wholemounts from the same animals were produced using a flat–bed scanner. Both the in vivo and in vitro images indicated increased leukocyte adhesion in the vessels of LPS–treated mice, and increased rolling of leukocytes was seen in vivo. Conclusions: As expected, LPS induced uveitis increased leukocyte adhesion in the retinal circulation. Importantly, we have shown that it is possible to image leukocytes in the retinal circulation using an inverted fluorescence microscope and a flat–bed scanner. This provides a method to quantify the amount of adhesion and migration.
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