Fig. 4

ciMSC-EVs prevent HI-induced vessel loss and impaired endothelial proliferation while reducing endothelial activation and leukocyte infiltration. Postnatal day 9 (P9) C57BL/6 mice were exposed to HI followed by i.n. administration of vehicle (0.9% NaCl, Veh) or ciMSC-EVs 1, 3, and 5 days after HI. At P16, vessel density and endothelial proliferation were quantified via immunohistochemistry for the pan-vessel/endothelial cell marker CD31 and the proliferation marker Ki67 (a) in the cortex (b) and striatum (c). Vessel densities were quantified by unbiased automated software-based object detection (b). Endothelial cell proliferation was determined by counting CD31/Ki67 double-positive cells (c). Endothelial activation was determined by western blot analysis for the adhesion molecule VCAM-1 (d). Data were normalized to the reference protein GAPDH and to sham-operated mice (d). Leukocyte infiltration was analyzed via immunohistochemistry for the pan-leukocyte marker CD45 (e). Representative images in a and e are derived from the striatum (scale bar in a: 100 µm, scale bar in e: 50 µm). Representative western blot images in d show examples of protein abundance in tissue lysates obtained from (1) sham-operated, (2) HI-injured vehicle-treated, and (3) HI-injured ciMSC-EV-treated animals. Images were cropped and scaled for illustration purposes from original western blot images provided in Suppl. Figure 6. *p < 0.05, **p < 0.01, ***p < 0.001, n = 8–10/group