Fig. 5

RIPK1 alleviates myocardial reperfusion injury in mice. (A) Representative TTC-Evan’s Blue stained sections of heart from each group. Brick red-stained area represents the area at risk (AAR), whereas the white area indicates the infarcted size (IS). (B) Ratio of AAR/LV, IS/ARR. Data are expressed as mean ± SD (n = 10 for each), and were analyzed using Mann-Whitney U test. ^*P < 0.05 vs. WT + I/R group. (C) Representative cross-sectional images of the left ventricular (LV) showing no-reflow regions indicated by the absence of thioflavin-S fluorescence within the ischemic area that consists of both no-reflow (yellow arrows) and reflow zones (green arrows) following IR. (D) Ratio of no reflow area/LV. Data are expressed as mean ± SD (n = 10 for each), and were analyzed using Mann-Whitney U test. ^*P < 0.05 vs. WT + I/R group. (E) Survival analysis for mice subjected to 45 min ischemia and following 6 h perfusion. (^*P < 0.05 vs. WT + I/R group, Kruskal Wallis test with Dunn post-hoc test for survival analysis). (F) Heart sections were obtained from WT and RIPK1^flox/flox mice under sham or 45 min ischemia and following 6 h reperfusion. The sections were immunohistologically analyzed by staining with antibody against F4/80, CD68 and CD11b. (G) Quantitative analysis of inflammatory cells was performed. Data are expressed as mean ± SD (n = 5 for each), were analyzed using Mann-Whitney U test or two-tailed Student t test. ^*P < 0.05 vs. WT + I/R group, Scale bar = 20 μm and 25 μm. (H-I) Immunoblot analysis and quantification by densitometry of Caspase-8, FADD and c-GSDMD in the heart from RIPK1^flox/flox mice post-myocardial reperfusion. Data are expressed as mean ± SD (n = 3), were analyzed using Mann-Whitney U test or two-tailed Student t test. ^*P < 0.05 vs. WT + I/R group. (J) LDH activity in serum from WT, RIPK1^flox/flox, WT + I/R, RIPK1^flox/flox+I/R groups, ^*P < 0.05 vs. WT + I/R group. (K-L) Percent CD31-positive area per field was obtained by image processing using ImageJ. Data are expressed as mean ± SD (n = 3), were analyzed using Mann-Whitney U test or two-tailed Student t test. ^**P < 0.05 vs. WT + I/R group. Scale bar = 50 μm