Fig. 7

ciMSC-EVs protect from HI-induced astrocyte activation, associated with an increased expression of neural growth factors. 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. Astrocyte density and activation were analyzed by immunohistochemistry (a, b) and western blot (c) for GFAP. GFAP immunoreactivity, as a measure of reactive astrocytes, was quantified by measurement of positively stained areas in the cortex and striatum (b). For western blot analyses, lysates of entire ipsilateral hemispheres derived from 160-µm-thick tissue sections at the striatal level were analyzed (c). Data of GFAP were normalized to the reference protein GAPDH and to sham-operated animals (c). Proliferating astrocytes were quantified in tissue sections stained for Ki67 and GFAP (d). Double-positive cells were quantified in the cortex and striatum (d). Expression of the typical pro-inflammatory/neurotoxic A1-phenotype-associated molecule complement 3 (C3) was evaluated by measurement of mean fluorescence intensities (MFI) of C3 staining on GFAP positively labelled areas (e). Additional markers associated with pro-inflammatory/neurotoxic A1 (f) or anti-inflammatory/regenerative A2 astrocytes (g) and essential neural growth factors (h) were analyzed via real-time PCR in brain tissues, obtained from 160-µm-thick tissue sections from the striatal level. Beta-2-microglobulin served as housekeeping gene, and fold change values compared to sham animals were calculated (f–h). Representative immunofluorescence images are derived from the cortex in a and e and from the striatum in d. Scale bar in a: 100 µm, scale bar in d and e: 20 µm. Arrows in representative images of d indicate GFAP/Ki67 double-positive cells. Representative western blot images in c 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