Fig. 1

Combination of HT with intranasal ciMSC-EV treatment, but not HT monotherapy improves long-term functional deficits. Postnatal day 9 (P9) C57BL/6 mice were exposed to HI followed by 4 h NT or HT. Repetitive intranasal ciMSC-EV administration was performed at day 1, 3 and 5 after HI. Behavior was evaluated starting 35 days after HI with the Elevated Plus Maze (EPM) test. Representative heat maps of the time spent in different regions of the EPM are shown for each experimental group (A, red rectangles depict open arms). Mean velocities and total distances (B), the time and percentage of distance from the total distance moved (C) in the open arms of the EPM was quantified. Risk assessment behavior was evaluated by analyses of the frequency of head dippings in the open arms of the EPM (D). From day 40 after HI onwards cognitive function was assessed in the Barnes Maze (BM) test, measuring the time needed to enter the escape hole (E, d0 = day of habituation, d1 and d2 training days). On the 4th day spatial reference memory was analyzed in the probe trial, with all holes (including escape hole) closed (F, left: running pattern (escape hole in red), right: time needed to reach the escape hole, mean ± SEM). n = 12 (sham), n = 13 (NT), n = 13 (HT), n = 14 (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