Characterization of Transverse Aortic Constriction in Mice Based on the Specific Recruitment of Leukocytes to the Hypertrophic Myocardium and the Aorta Ascendens

Figures

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  Figure 1. Methodological procedure and definition of the immune cell subsets. (a) We sacrificed the mice 3, 6, and 21 days after TAC or sham surgery. (b) Heart-weight/body-weight-index in mg/g was calculated 3, 6, and 21 days after TAC or sham surgery. (c) In addition to the LV tissue, we analyzed the tissue of the aorta ascendens, aorta descendens, spleen, blood, and heart draining lymph nodes. (d) The gating strategy for flow cytometry analyses was performed as illustrated for the tissue of the aorta ascendens. All cell-like events (a) were isolated by using the forward scatter area versus sideward scatter area. Doublets were excluded using forward scatter area versus sideward scatter width (b). We defined the living immune cells (c), using CD45 staining versus Hoechst staining. The CD45+ immune cells were further classified by staining for the surface markers Ly6G and F4/80 to determine neutrophils (d) and macrophages (e). Next, we checked for the Ly6C surface expression of the macrophage population as a function of CD11b to differentiate between the Ly6Chigh (f) and the Ly6Clow macrophages (g). We analyzed the DC compartment based on the population of the CD45+ cells (c) and stained…

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  Figure 2. Innate immune response in the myocardium and the ascending and descending parts of the aorta. (a)–(l) The CD45+ immune cells, neutrophils, Ly6Clow, and Ly6Chigh macrophages in the tissue of the LV, the aorta ascendens and descendens of the mice were quantified via flow cytometry. The particular cell subsets were defined as follows: neutrophils as CD45+ F4/80− Ly6G+ and macrophages as CD45+ F4/80+ Ly6G−. Moreover, the Ly6Chigh and Ly6Clow macrophages were further discriminated according to the respective Ly6C surface expression. ∗The above individual columns indicate the significant differences between the TAC and respective sham group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

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  Figure 3. Innate immune response in the spleen. (a)–(e) The CD45+ immune cells, neutrophils, and Ly6Clow and Ly6Chigh macrophages, and DCs in the spleen of the mice were quantified via flow cytometry. The particular cell subsets were defined as follows: neutrophils as CD45+ F4/80− Ly6G+ and macrophages as CD45+ F4/80+ Ly6G−. Moreover, the Ly6Chigh and Ly6Clow macrophages were further discriminated according to the respective Ly6C surface expression, and the DCs were defined as CD45+ F4/80− Ly6G−and CD11c+. ∗The above individual columns indicate the significant differences between the TAC and respective sham group; ∗P < 0.05, and ∗∗P < 0.01.

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  Figure 4. Innate immune cell quantification and cytokine levels in response to TAC in the blood. (a)–(d) The CD45+ immune cells, neutrophils, and Ly6Clow and Ly6Chigh monocytes in the blood of the mice were quantified using flow cytometry. The particular cell subsets were defined as follows: neutrophils as CD45+ CD115− Ly6G+ and monocytes as CD45+ CD115+ Ly6G−. In addition, the Ly6Chigh and Ly6Clow monocytes were further discriminated according to the respective Ly6C surface expression. (e)–(g) The cytokine levels of IL-6, IL-1β, and TNF-α in the serum of the mice were measured at days 3 and 6 after TAC or after day 3 after sham surgery. ∗The above individual columns indicate the significant differences between the TAC and respective sham group; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Figure 5. TAC provoked the response of the DC compartment in the myocardium, aorta ascendens, aorta descendens, and draining lymph nodes. (a)–(p) The total of the MHC-II+ cells was determined from the fraction of CD45+ Ly6G− cells. The population of cDCs was defined using the CD11c surface staining. CD86 MFI was calculated from the population of all cDCs. The population of CD103+ cDCs was determined based on the results of the CD103 surface staining. ∗The above individual columns indicate the significant differences between the TAC and respective sham group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.