Downregulation of Kdr or Flt1 levels blocks hypoxia-induced retinal EC proliferation and neovascularization. (A) After exposure to 75% oxygen, the mice pups were returned to normoxia, administered 1 μg/0.5 μL control, Kdr, or Flt1 siRNA at P12 and P13 by intravitreal injections, and the retinas were isolated at P15 and fixed; cross-sections were made and analyzed by immunofluorescence staining for CD31 and Ki67. The right column shows the higher magnification (×40) of the selected areas (rectangular boxes) of the images shown in the left column. (B) Retinal EC proliferation was measured by counting CD31- and Ki67-positive cells that extended anterior to the inner limiting membrane per section (n = 6 eyes, 3 sections/eye). (C) All the conditions were the same as in panel A except that intravitreal injections of control, Kdr, or Flt1 siRNAs were given at P12, P13, and P15, and at P17 the mice pups were anesthetized, sacrificed, and enucleated, and retinas were isolated and stained with isolectin B4; flat mounts were made and examined for retinal neovascularization. Retinal vascularization is shown in the first row. Neovascularization is highlighted with red in the second row. The third row shows selected rectangular areas of the images shown in the first row under ×10 original magnification. (D-E) Retinal vascularization (D) and the neovascularization (E) were determined as described in “Materials and methods.” (F-J) All the conditions were the same as in panels A-E, respectively, except that mice pups were administered with Kdr and Flt1 siRNA simultaneously at 1 μg each in a total volume of 0.5 μL/eye. The bar graphs represent the quantitative analysis of 6 retinas. The values are presented as mean ± SD. *P < .01 vs normoxia + control siRNA; ^§P < .01 vs hypoxia + control siRNA.