Fig. 4

HT combined with ciMSC-EV application reduces HI-induced endothelial activation and leukocyte accumulation. C57BL/6 mice were exposed to HI on postnatal day 9 (P9) followed by 4 h HT or NT. ciMSC-EVs were delivered intranasally on day 1, 3 and 5 after HI. Endothelial activation was assessed via western blot analyses for the adhesion molecules ICAM-1 (A) and VCAM-1 (B) in whole hemisphere protein lysates derived from 160-µm-thick tissue sections at the striatal level at day 7 after HI. Data were normalized to the reference protein GAPDH and to sham animals. Leukocyte accumulation was determined in CD45-stained tissue sections (C, scale bar: 20 µm). CD45 positively stained areas were quantified in the striatum and in the cortex (D). Neutrophil accumulation and localization was analyzed via immunohistochemistry in sections stained for Ly6G (red) and pan-Laminin (green) (E arrows indicate Ly6G neutrophils in the intra/perivascular space, rhombi indicate intraparenchymal neutrophils, scale bar: 20 µm). Neutrophil accumulation was quantified by counting Ly6G positive cells in the striatum and cortex (F) and the percentage of intraparenchymal and intra/perivascular neutrophils was quantified in HI-injured animals (G). Representative images in A and B were cropped and scaled from original full length western blots provided in Additional file 1: Fig. S4. Representative images in C and E are derived from the striatum n = 11 (sham), n = 10 (NT), n = 9 (HT), n = 11 (HT + EV), *p < 0.05, **p < 0.01, ***p < 0.001. HI = hypoxia–ischemia, NT = normothermia/vehicle, HT = hypothermia/vehicle, HT + EV = hypothermia/ciMSC-EV