Fig. 4.

FG4592 maintains a neuroprotective effect in a HIF-α independent manner in vivo after I/R injury. A and B Representative western blots and statistical analyses of band intensity of HIF-1ɑ protein in peri-infarct brain tissue of mice after 24 h of tMCAO. The mice of YC-1 pretreated groups were injected with YC-1 (10 mg/kg, ip) for 1 hour before tMCAO. All experimental groups were subjected to tMCAO and treated with FG4592 or vehicle after 5 min of reperfusion. Data are presented as mean ± SEM. Sham vs vehicle or FG4592: ***p < 0.001, **p < 0.01. vehicle vs FG4592 or FG4592+YC-1 or YC-1: ##p < 0.01. FG4592 vs FG4592+YC-1 or YC-1: †††p < 0.001 (one-way ANOVA followed by Dunnett’s post-hoc test, n=6). C Representative images of brain TTC staining from mice in each group. Layer thickness = 1 mm, scar bar = 1 cm. D and E The infarct volumes (one-way ANOVA followed by Dunnett’s post-hoc test) and mNSS scores (non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc) of the vehicle (n = 10), FG4592 (n = 11), FG4592+YC-1 (n = 9) and YC-1 groups (n = 10) after 24 h of tMCAO. Data are presented as mean ± SEM. vehicle vs FG4592 or FG4592+YC-1: ***p < 0.001, **p < 0.01. YC-1 vs FG4592 or FG4592+YC-1: ###p < 0.001, ##p < 0.01. F Fluorescent staining of mouse brains injected with AAV-PHD2-shRNA in situ for 28 d (Left: 4×, Scale bar, 1 mm). G and H Representative western blot and protein level of PHD2 in mouse brains. All mice were cortical in situ injected with AAV-PHD2-GFP-shRNA or AAV-Con-GFP-shRNA virus for 28 days (n = 6 in each group). Data are presented as mean ± SEM. AAV-Con-shRNA vs AAV-PHD2-shRNA: ***p < 0.001 (student’s t-test. n = 6). I Representative images of TTC-stained brain slices of mice after 24 hours of tMCAO. All mice were cortical in situ injected with AAV-Con-shRNA or AAV-PHD2-shRNA for 28 days and underwent tMCAO. Layer thickness = 1 mm, scar bar = 1 cm. J and K The quantitative infarct volume and mNSS of AAV-Con-shRNA (n = 9), AAV-Con-shRNA+FG4592 (n = 10), AAV-PHD2-shRNA (n = 10) and AAV-PHD2-shRNA + FG4592 (n = 9) groups after 24 hours of tMCAO. Data are presented as mean ± SEM, AAV-Con-shRNA vs AAV-Con-shRNA+FG4592 or AAV-PHD2-shRNA+FG4592: **p < 0.01, *p < 0.05. AAV-PHD2-shRNA vs AAV-Con-shRNA + FG4592 or AAV-PHD2-shRNA+ FG4592: ###p < 0.001, ##p < 0.01, #p < 0.05 (one-way ANOVA followed by Dunnett’s post-hoc test for infarct volume, non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc for mNSS)