Fig. 5

ciMSC-EV treatment decreases HI-induced microglia activation, associated with alterations of typical M1/M2 marker expression. 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. Analyses were performed at P16. Microglia density and activation were analyzed by quantification of Iba-1 immunoreactivity via immunohistochemistry (a–c) and western blot (d). Iba-1 immunoreactivity, as a measure of microglia activation, was quantified by measurement of positively stained areas (b) and mean fluorescence intensities (MFI) on Iba-1-positive areas (c) in the cortex and striatum. For western blot analyses, data of Iba-1 were normalized to the reference protein GAPDH and to sham-operated animals (d). A broad set of pro-inflammatory M1-phenotype-associated (e) and anti-inflammatory M2-phenotype-associated (f) molecules was analyzed via real-time PCR in brain tissues obtained from 160-µm-thick tissue sections at the striatal level. Beta-2-microglobulin served as housekeeping gene, and fold change values compared to sham animals were calculated (e, f). Representative images in a are derived from the striatum (scale bar: 50 µm). Higher magnification images of single cells derived from depicted rectangles in low magnification images are provided in insets to demonstrate morphological changes exemplarily (a). Representative western blot images in d show examples of protein abundance in tissue lysates obtained from sham-operated, HI-injured vehicle-treated, and 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