Fig 2: Interference with SPP1 on protein level changes the outcome of experimental choroidal neovascularization. (A) Experimental scheme. Six weeks old Cx3cr1CreER:Rosa26-tdTomato mice were exposed to TAM to induce microglia-specific Tomato labeling. Eight weeks later three focal argon laser burns were applied to each retina to induce microglia activation and subsequent choroidal neovascularization (mCNV). Mice were sacrificed at CNV d3 for immunofluorescence stainings. (B) Representative immunofluorescence images for collagen IV (white), Tomato (red), and SPP1 (green) in Cx3cr1CreER:Rosa26-tdTomato mice on d3 after CNV induction. Arrows point to a Tomato+SPP1+ rMG while asterisks indicate a Tomato+SPP1- rMG. Representative pictures of seven mice out of two independent experiments are displayed. (C) SPP1 levels in lesioned RPE tissue (mCNV) of wildtype mice compared to untreated controls d3 after CNV induction as measured by ELISA (control: n = 7, CNV: n = 8 from two independent experiments). Bars represent means ± s.e.m. ***p < 0.0001, Unpaired t-test. (D) Experimental scheme. Choroidal neovascularization was laser induced at day 0 (d0) in Cx3cr1GFP/+ mice. At d1, 50 ng anti-SPP1 (dissolved in 1 µL PBS injection volume) was injected intravitreally into one eye and the same amount of IgG control antibody (again dissolved in PBS) into the contralateral eye. Mice were sacrificed at d7 after evaluation of fundus and fluorescence angiography. (E) Exemplary funduscopic image of lesions directly after laser burn-induced mCNV at day 0 (left) and d7 (right) under treatment with IgG control (top row) or anti-SPP1 antibody (bottom row). Fluorescence angiography indicates leakage of intraperitoneally applied Fluorescein. Representative Imaris reconstruction of collagen IV immunoreactivity and corresponding quantification of CNV size in mice that underwent laser treatment and subsequent injection of either anti-SPP1 into the left eye (squares) or IgG isotype controls into the right eye (circles). Each symbol represents the mean lesion size within one eye of one mouse, based on Collagen IV area of individual lesions imaged by confocal microscopy and normalized to the IgG control. Corresponding values from left and right eye are displayed as pairs. Bar graphs represent the overall mean value of seven mice in total. All values were normalized to the IgG control mean value. Bars represent mean ± s.e.m. ***p < 0.001, ratio paired t-test.