Down-regulation of VEGF, Src, PLD1, or PKCγ blocks hypoxia-induced retinal neovascularization. (A) After exposure to 75% oxygen, the mice pups were returned to normoxia, administered 1 μg of Scr or VEGF siRNA at P12 and P13 by intravitreal injections, and retinas were isolated at P15 and analyzed by double immunofluorescence staining for CD31 and Tie2 (top panel). Retinal vascularization was quantified by measuring the ratio of CD31-positive area to retinal area (bottom panel). The left column shows the histochemical staining of the respective retinal sections. The far right column shows the higher magnification (original magnification ×40) of retinal sections shown in the third column. (B-D) After exposure to 75% oxygen, the mice pups were returned to normoxia, administered 1 μg of Scr, Src, PLD1, or PKCγ siRNA at P13, P14, and P15 by intravitreal injections, and at P17 pups were anesthetized, perfused with FITC-dextran, sacrificed, enucleated, retinas were isolated, and flat mounts of whole retina were examined for retinal neovascularization (B). Neovascular tufts were highlighted with red (second and fifth rows). The third and sixth rows show the magnified section of a selected area of the images shown in the first and third rows, respectively. The images were captured using an Inverted Zeiss fluorescence microscope (AxioVision AX10), and fluorescence intensity in the total retinal area (C) and the area of neovascular tufts to total retinal area (D) was analyzed by Nikon NIS-Elements software Version AR3.1. Values are mean ± SD. *P < .01 versus normoxia plus scrambled siRNA control. **P < .01 versus hypoxia plus scrambled siRNA. RGC indicates retinal ganglion cell; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; and PR, photoreceptors.