(A) Confocal images show phosphorylated VEGF-R2 (Y1175) immunolabeling in an obstructed capillary (top row, see DiI-coated microsphere) and one that recanalized (bottom row, note DiI labeling of GFP-labeled endothelium without presence of microsphere). (B) Histogram shows % pVEGF-R2 colocalization with endothelium at the site of an obstructed or recanalized capillary 3 hr after microsphere injection. Sham capillaries were measured from mice injected with vehicle solution but without microspheres. Inset shows the distribution of pVEGF-R2 colocalization values for all recanalized capillaries. Based on this distribution, we used a 20% cut off (dotted line) to separate the two distinct populations. Average eight capillaries per mouse, range 5–11. Note that recanalized capillaries exhibit significantly higher or lower pVEGF-R2 colocalization. n = 83 vessels total with 3 sham and seven injected mice, one way ANOVA F(3,77)=22.93, p<0.0001, unpaired t-test to compare groups. *p<0.05, ****p<0.0001. (C) Box and whisker plots (+denotes mean) show normalized density of microspheres in the cortex of saline and VEGF injected (i.c.v.) mice 24 hr after microsphere injection. n = 5 mice per group, unpaired t-test. *p<0.05. Microsphere density: Vehicle 7.97 ± 1.3 per mm3 versus VEGF injected 22.27 ± 2.1 per mm3. (D) Summary and timeline of VEGF-R2 knockdown or inhibition experiments. E) Left: Immunolabeling for pVEGF-R2 in Tek-GFP mice shows reduced vascular expression 3 hr after injection of 50 mg/kg SU5416. Right: Quantification of vascular pVEGF-R2 in Tamoxifen-treated TekCreERT2 X Kdr+/fl mice [four mice per group; unpaired t-test, t(6)=3.32, p=0.01] or Tek-GFP mice injected with SU5416 [four mice per group; unpaired t-test t(6)=3.17, p=0.02]. (F) Capillary fates 21 days after obstruction in vehicle or tamoxifen treated Tek CreERT2 X Kdr+/fl mice based on in vivo time lapse imaging (n = 6 mice per group, vehicle = 75 capillaries, Tamoxifen = 81 capillaries, Average 10 capillaries per mouse, range 5–16). Unpaired t-test to compare % recanalized, t(10)=3.88, p=0.003.**p<0.01. G) Left: Representative images of cortical microspheres in coronal brain sections from vehicle or Tamoxifen-injected Tek CreERT2 X Kdr+/fl mice 21 days after microsphere injection. Scale bar 500 µm. Right: Normalized density of microspheres in the cortex of vehicle and Tamoxifen-injected mice at 4 and 21 days after microsphere injection (n = 4–9 mice; unpaired t-test at 4 days t(6)=4.43, p=0.004; and 21 days t(12)=2.74, p=0.017.+is mean. *p<0.05, **p<0.01). Microsphere densities (/mm3) at +4 days: Vehicle 16.2 ± 1.5, Tamoxifen injected 10.3 ± 5, +21 days: Vehicle 6.38 ± 3, Tamoxifen injected 1.5 ± 0.4. (H) In vivo determination of capillary fates after 21 days in vehicle or SU5416 injected Tek-GFP mice [n = 4 mice per group at 4 days, n = 5 for vehicle and n = 9 for tamoxifen injected at 21 days, vehicle = 84 capillaries; SU5416 = 90 capillaries; Average 20 capillaries per mouse, range 16–30, unpaired t(7)=2.73, p=0.03]. I) Left: Images show cortical microspheres in brain sections from vehicle and SU5416 treated Tek-GFP mice 21 days after microsphere injection. Scale bar 500 µm. Right: Normalized density of microspheres in the cortex of vehicle or SU5416 injected mice at 4 and 21 days [n = 4 mice per group; unpaired t-test at 4 days t(6)=6.97, p=0.004, and 21 days t(6)=2.92, p=0.02]. Microsphere densities (/mm3) at +4 days: Vehicle 69.2 ± 1.4, SU5416 injected 31.94 ± 4.2, +21 days: Vehicle 21.59 ± 5.3, SU5416 injected 12.21 ± 1.33. *p<0.05, **p<0.01, and ***p<0.001 compared to vehicle injected. Error bars are S.E.M.