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Kathryn L Pepple, Leslie Wilson, Russell N Van Gelder, Kevin Rolnick; Inflammatory cell type-specific bioluminescence for quantitative scoring of uveitis in vivo. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1724.
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© ARVO (1962-2015); The Authors (2016-present)
Most methods for studying inflammatory cells in the eye are performed on post-mortem tissues. This prohibits use for longitudinal analysis in individual animals. Our goal is to develop an in vivo bioluminescence assay for the quantitative detection of inflammatory cell type subsets in eyes of mice with experimental uveitis.
Transgenic animals expressing luciferase were generated using the cre-lox system in the albino C57Bl6 background. Cell type-specific luciferase expression in B-cells (CD-19 Cre), T-cells (LCK-cre), monocytes/macrophages (Lyz2-cre), and neutrophils (S100A8-cre) was generated using established lines. Each Cre line was crossed to ROSA26-loxp-STOP-loxp-Luciferase, and experimental uveitis was induced in the double heterozygotes using the primed mycobacterial uveitis (PMU) model. Bioluminescence of the inflamed (right) and uninflamed (left) eyes were captured using an IVIS spectru and compared longitudinally. Cellular infiltrates were also determined by flow cytometry (CD45, CD3, CD19, CD11b, CD11c, Ly6C and Ly6G).
Increased bioluminescence was detected in the inflamed eye on day 1 after uveitis induction (see figure, panel A). Over time, the T-cell and B-cell signal increased while the S100A8 signal decreased. These results were consistent with the flow cytometry results that determined that on PMU day one, the CD45+ cells in the eye are >90% neutrophils, ~10% T-cells, and <1% B-cells. In contrast on day 21, PMU eyes contain <20% neutrophils, ~ 50% T-cells, and ~2% B-cells. The signal from the Lyz2-cre line did not change significantly over the course of inflammation. These results are consistent with the flow results identifying that macrophages were ~ 5% of the CD45+ cells on both days 1 and 21. All left eyes demonstrated a bioluminescent signal above background despite the absence of clinical inflammation. Ex-vivo imaging of enucleated eyes did not detect a signal in fellow eyes (panel B).
In vivo bioluminescence detects inflammatory cells in eyes with uveitis. Longitudinal changes in cell populations determined by flow cytometry are reflected in the quantitative bioluminescent signal. The difference between the bioluminescent signal associated with inflammation and background is limited, but optimization of the signal to noise ratio could expand the utility of this technique for use in models of eye disease with less robust inflammation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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