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Anja Schlecht, Peter Wieghofer, Peipei Zhang, Markus Gruber, Guenther Schlunck, Hansjuergen Agostini, Marco Prinz, Clemens Lange; Gene Expression Profile of Microglia at Sites of Laser-Induced Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1487.
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© ARVO (1962-2015); The Authors (2016-present)
Myeloid cells (MC), such as resident microglia (MG) and infiltrating blood-derived monocytes (MO), are key players in the formation of choroidal neovascularization (CNV). However, the specific discrimination between MG and MO is challenging and the precise function of MG during CNV development remains unclear. In this study, we performed RNA-Seq analysis of MG cells with and without laser-induced CNV to further unveil their function in CNV development.
Adult C57Bl6/J wildtype (WT) mice were used for RNA-Seq analysis. Three laser spots were applied by an Argon laser at equal distance from the optic disc and with a wavelength of 532 nm, a power of 150 mW, a fixed diameter of 100 µm and a duration of 100 ms to induce CNV formation (n=50), while untreated littermates (n=40) served as controls. Retinal MG cells were sorted by FACS (gating strategy: CD45+CD11b+Ly6C-Ly6G-) on the third day after laser injury to perform RNA-Seq followed by Gene Ontology (GO) Cluster Analysis. Protein expression of the most outstanding factor of affected clusters was investigated by ELISA (n ≥ 7 mice per group) and immunohistochemistry.
RNA-Seq analysis demonstrates that the expression profile of MG cells strongly changes following laser-induced CNV formation. GO Cluster Analysis revealed that amongst others the clusters of “angiogenesis” and “cell quantity” were strongly upregulated in MG following CNV induction when compared to unlasered controls. SPP1, also known as Osteopontin, belongs to both GO terms and was highly upregulated following CNV formation (log fold-change = 7.06, adjusted P Value = 2.9512E-18). Immunohistochemistry for SPP1 showed an increased immunofluorescence in retinal MG around laser-induced CNV compared to untreated eyes. SPP1 protein expression was significantly (P = 0.0003) increased in the choroid of lasered mice compared to controls, as revealed by ELISA protein analysis.
Our study demonstrates that induction of CNV via laser leads to significant changes in the expression profile of MG cells. Specific factors affected by these changes could help to further understand the role of MG cells in the development of CNV and may potentially play a role in therapeutic approaches.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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