Characterization of early myocardial inflammation in ischemia-reperfusion injury

Figures

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  Figure 1. Establishment of mouse IR model. (A), schema of in vivo protocol. The present study population comprised 60 mice which were used for the characterization of myocardial inflammation during the first week after IR. (B), graphic image of myocardial IR injury induction. (C), typical LGE image on day 7 in IR group. (D), representative echocardiography on day 1 in sham and experimental group. (E), immunofluorescence staining detect the infiltration of leukocytes (Bar=150 μm).

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  Figure 2. The immune response induced by IR injury at the first week. Representative images of histological changes in the ischemic myocardium. (A), for each time point, images are shown of staining with H&E (Bar=1mm, 50μm). (B), macrophages in mice post-IR were evaluated by CD68 staining (Bar=50 μm). (C-F), plasma levels of IL-1β (C), TNF-α (D), Arg-1 (E) and TGF-β (F) were also determined by ELISA (n = 3-5). *p < 0.05 VS. Sham.

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  Figure 3. T2-weighted imaging sequence detect inflammation signal in the mouse heart during the first week post-IR. (A), one mouse equipped with ECG (☆) and respiratory (◇) are being prepared to scan under 7.0 T MRI. (B), real-time ECG and respiratory rate in a mouse. (C), the typical T2W images from the same mouse before and after IR. (D), quantitative the mean T2W signal intensity of the LV myocardium in each group, (n = 5). *p < 0.05 VS. Sham, ** p < 0.01, *** p < 0.001.

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  Figure 4. Characterization of temporal dynamic of the macrophages and neutrophil in the heart after IR. (A), gating strategy for cardiac macrophages, M1 (CD11b+F4/80+Gr1-CD206-) and M2 (CD11b+F4/80+Gr1-CD206+) macrophages at indicated time point after myocardial IR injury. Represent flow cytometric images of M1 and M2 macrophages in the post-IR heart. (B-E), the percentages of macrophages (B), M1 (C), M2 (D) and neutrophils (E) were determined in the hearts of sham group and reperfusion groups (n = 5-6, each). *p < 0.05 VS. Sham, ** p < 0.01, *** p < 0.001.

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  Figure 5. Liner correlation between myocardial T2W signal and inflammation mediator. (A-D), scatter plot of myocardial T2W signal and IL-1β (A), TNF-α (B), TGF-β (C) and Arg-1 (D). (E), heat map of neutrophils, MI macrophages, M2 macrophages and macrophages among myocardial T2W signal, ** p < 0.01, *** p < 0.001.

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  Figure 6. Myocardial IR induced edema at 3 h post-IR. (A), WGA staining cardiomyocyte hypertrophy, (n = 3) (Bar=400 μm, 50μm). (B) (1-6), ultrastructural changes in mouse myocardium. (1), sham group ventricular myocardium showing normal myofilament architecture and mitochondria (Bar=2μm). (2), anomalous myofilaments and mitochondria are displayed after IR 3 h (Bar=2μm). (3-4), Vessel with no apparent gaps, but several large vacuoles apparent (Bar=2μm, 500nm). (5-6), section transverse to myocardium showing an RBC in the extracellular space (Bar=2μm, 500nm). (C), western blotting detect VEGF and p-Src protein expression, (n = 3). Quantitative of VEGF protein (D), p-Src protein (E) and cardiomyocytes area (F). RBC, red blood cell; VEGF, vascular endothelial growth factor; p-Src, phosphor-Src, *p < 0.05 VS Sham, ***p < 0.001.

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  Figure 7. No cell apoptosis happens after 3 h IR. (A), Masson staining of heart sections was performed for each time point (Bar 100 μm). (B), cell apoptosis was measured by TUNEL assay (Bar =200 μm, 50μm) and Western blotting (C) in sham group and 3 h, 1 day after IR induction. Quantitative of cleaved-caspase 3 protein expression level (D), (n = 3). **p < 0.01 VS. Sham.