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M.R. Kuebbeler, P.S. Muether, I. Semkova, M. Beyer, N. Kociok, A.M. Joussen; Stationary and Migrated Immunocompetent Cells in LPS–Induced Uveitis of GFP+–Chimeric Mice . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2587.
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The generation of GFP+–chimeric mice allows evaluating ratios of residential and attracted cells in the retina. This study is to determine to which extent local retinal – and bloodstream derived immunocompetent cells participate in the inflammatory response associated with LPS–induced uveitis.
C57/B6 mice were stably transplanted with whole bone marrow from b–actin promoted GFP+–heterozygous donors after lethal irradiation. Transformation rates were evaluated using FACS–analysis. Autoimmune–uveitis was induced via foodpad–injection of LPS. 24h after LPS injection, animals were enucleated and retinal flatmount preparation was conducted. Transcardial perfusion with concanavalin A was performed for differentiation of extravasal retinal cells. Extravasal cells were counted via an image analysis software. For colocalization and differentiation studies, macrophages and dendritic cells were labelled with F4/80– and CD11c antibodies, respectively, and evaluated using fluorescence– and confocal microscopy.
FACS analysis of peripheral blood draws showed an average of 80% GFP+–transformation rate after bone marrow substitution. Perfused retinal flatmounts of non–uveitic control animals without uveitis were free of extravasal GFP–positive cells as well as dendritic cells confirmed by CD11c staining. Diffuse GFP–negative cells positive for F4/80 were detected. LPS–induced uveitis led to influx and extravasation of GFP–positive cells as well as an increase in GFP–negative cells positive for F4/80–macrophage and CD11c dendritic cell staining.
The high amounts of GFP–negative cells positive for macrophage and dendritic cell staining in uveitic retinas was not explained the low number of GFP–negative cells transplanted. These findings suggest an activation of residential immunocompetent retinal cells together with an influx of bloodstream–derived cells.
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