Fig. 5

ciMSC-EV treatment mitigates limitations of HT on secondary neuroinflammation and enhances neurotrophic growth factor expression. HI-induced brain injury was induced in postnatal day 9 (P9) mice, followed by 4 h NT or HT. Intranasal ciMSC-EV administration was performed 1, 3 and 5 days after HI followed by western blot, immunohistochemistry and real-time PCR analyses 7 days after HI. Western blot analyses for Iba-1 and GFAP (A) were performed in tissue lysates of the entire hemisphere derived from 160 µm thick tissue sections at the striatal level to quantify microglia (B) and astrocyte (C) activation. Data were normalized to the reference protein GAPDH and sham animals. Immunohistochemistry was performed for Iba1 (green) and GFAP (red) (D, scale bar: 100 µm). Microglia (E) and astrocyte (F) activation were analysed by quantification of positively stained areas in the cortex and striatum. The expression of pro- and anti-inflammatory cytokines (G) and neurotrophic growth factors (H) was measured via real-time PCR in brain tissue lysates of the entire hemisphere derived from 160 µm thick tissue sections at the striatal level. Beta-2-microglobulin served as a housekeeping gene and fold change values were calculated compared to sham animals. Representative images in A were cropped and scaled from original full length western blots provided in Additional file 1: Fig. S4. Representative images in D 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