sG18 inhibits CNV induced by laser injury. (A) Laser injury to the choroids and Bruch membrane. Left panel, schematic representation of the procedure. Right panel, representative image depicting a typical area of CNV (denoted by the dotted line); H&E staining. (B) Representative images from immunostaining of CNVs 7 days after laser injury and treatment with sA18 or sG18; CD31 (red) and NRP1 (green); nuclei are visualized by DAPI (blue). The dotted line denotes the CNV areas. (C) Analysis of CNV detected by Alexa Fluor 594–labeled isolectin B4 (IB4) staining. Left, representative images from IB4 staining of CNV spots treated with sA18 and sG18. Right, quantitative analysis of CNV. Eyes were removed 7 days after laser injury and treatment with vehicle alone or with the indicated additives. Mean area of CNV was first measured for each mouse (4 injured areas/eye). The results reflect group means (7 mice/group) and are expressed as percent mean neovascular area compared with vehicle alone. (D) VEGF neutralizing antibody and sG18 reduce CNV after laser injury. The results reflect quantitative measurement of neovascular areas stained by Alexa Fluor 568-labeled IB4 and reflect group means ± SEM. Groups of 7 mice were treated with neutralizing antibody to human/mouse VEGF (B20.4.1, 2μg/eye), sG18 (10 μg/eye), and appropriate controls; the eyes were removed 7 days after injury. (E) Representative images depicting monocyte/macrophage infiltration of CNV areas (denoted by the dotted line) detected by immunostaining with antibodies to the monocyte/macrophage marker F4/80 (red); nuclei are stained with DAPI (blue). Eyes were removed 7 days after laser injury and treatment with sA18 or sG18. (F) Quantitative analysis of monocyte/macrophage infiltration in CNV areas detected by immunostaining with F4/80 antibodies and analysis (ImageJ). The results are expressed as group means ± SEM. Each group consisted of 5 mice treated in the eye with sA18 or sG18.