CCR2 Blocker Inhibits Choroidal Neovascularization perfused with fluorescein isothiocyanate-dextran in flat-mount choroid-RPE complex. Scale bar = 100 mm. (C) Quantitative analysis of CNV size. Values are mean 6 SE, vehicle, n = 78 spots, INCB3344, n = 81 spots. *P,0.001. doi:10.1371/journal.pone.0028933.g001 injections of 1 ml INCB3344 (1800 nM), and 7 Sod12/2 mice with CNV received intravitreal injections of 1 ml vehicle. Fundus photography and FA were performed pre- and 1-week-post- treatment. Angiograms were obtained 5 min after the injection of fluorescein sodium solution, and the area of the CNV lesion was measured three times and averaged using Image J for Windows (NIH, Bethesda, Maryland). Statistical analysis Results are expressed as the mean 6 SE with n as indicated. Student’s t test and one-way ANOVA was used for statistical comparison between two or three groups. Differences between the means were considered statistically significant at values of P,0.05. Results Histological evaluation and quantitative assessment of laser-induced CNV Histopathologic analysis showed that the CNV lesions in INCB3344-treated mice were smaller in diameter compared with those in vehicle-treated mice. Both groups had areas of fibro- Figure 2. Macrophages detected by immunohistochemistry of choroid-RPE flat mounts and flow cytometry. (A) Immunohistochem- istry of macrophages in choroid-RPE flat mounts on Day 3. After photocoagulation, a large number of macrophages accumulated at the laser injury sites. INCB3344 suppressed this increase. Scale bar = 100 mm. (B) Left: Overlay histogram of flow cytometric results. Right: Flow cytometric analysis data with F4/80 staining of the macrophages in choroid-RPE on Day 3 after laser photocoagulation (Macrophage numbers per choroid-RPE complex). After photocoagulation, the number of macrophages significantly increased compared with no laser photocoagulation controls (relative to normal control, *P,0.001 n = 5, **P,0.001 n = 5). INCB3344 treatment significantly reduced the number of macrophages compared to the vehicle-treated group (***P,0.001, n = 5). doi:10.1371/journal.pone.0028933.g002 PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28933