Fig. 6

Intranasal ciMSC-EV delivery overcomes limitations of HT on HI-induced impaired oligodendrocyte maturation and myelination. Postnatal day 9 (P9) mice were exposed to HI followed by 4 h NT or HT. Intranasal ciMSC-EV administration was performed 1, 3 and 5 days after HI. Immunohistochemistry, real-time PCR and western blot analyses were performed 7 days after HI. Oligodendrocyte proliferation was assessed in Olig2/Ki67 double stained tissue sections (A, Olig2 (red), Ki67 (green), scale bar: 50 µm). The total amount of oligodendrocytes (B) and proportion of proliferating oligodendrocytes (C) was quantified. Oligodendrocyte maturation was analyzed via immunohistochemistry through co-staining of Olig2 with either PDGFR alpha for immature oligodendrocytes (D, left) or CC1 for mature oligodendrocytes (D, right), each indicated by arrows (D, scale bar: 20 µm). The proportion of Olig2/PDGFR alpha (E) and CC1/Olig2 (F) double-positive cells was quantified. Real-time PCR (G) and western blot analyses (H) were performed in tissue lysates derived from ipsilateral hemispheres of 160 µm thick tissue sections at the striatal level for markers expressed by mature and myelinating oligodendrocytes. For real-time PCR, beta-2-microglobulin served as a housekeeping gene and fold change values were calculated compared to sham animals (G). For western blot analysis data were normalized to the reference protein GAPDH and sham animals. Representative images in A and D are derived from the external capsule of the white matter. Representative images in H were cropped and scaled from original full length western blots provided in Additional file 1: Fig. S4. 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