Fig. 5. EE promotes cardiac healing after MI by enhancing the survival of Ly6C^low macrophages and their CCR2⁻MHCII^low subsets.

(A) Immunostaining for CD68 (red) and TUNEL (green) in infarct zones of SE and EE mice at day 14 after MI. Scale bars, 20 μm. The percentage of TUNEL⁺ cells is expressed as TUNEL⁺CD68⁺ cells/CD68⁺ cells. *P < 0.05 versus MI-SE. (B) Immunostaining for CD68 (green) and active caspase-3 (red) in Ly6C^low macrophages sorted from hearts of SE and EE mice at day 7 after MI. Scale bars, 50 μm. (C) Gating strategy for CD11b⁺CD64⁺ macrophages and its subsets in hearts of SE and EE mice after MI. (D) Quantification of CCR2⁻MHCII^low, CCR2⁻MHCII^high, and CCR2⁺MHCII^high macrophages as shown in (C) (n = 6). *P < 0.05 versus MI-SE. (E) Gating strategy for CD11b⁺CD64⁺Ly6C⁻Lyve1-GFP⁺ macrophages in infarct tissues from Lyve1^EGFP-Cre mice at day 14 after MI. Quantification of Lyve1-GFP⁺ macrophages (n = 6). *P < 0.05 versus MI-SE. (F) Immunostaining for CD68 (red), CCR2 (white), and TUNEL (green) in infarct zones of hearts from SE and EE mice at day 14 after MI. Scale bars, 20 μm. The percentage of CD68⁺CCR2⁻TUNEL⁺ cells is expressed as CD68⁺CCR2⁻TUNEL⁺cells/CD68⁺ cells (n = 6). *P < 0.05 versus MI-SE. (G) Representative immunostaining for CD68 (red), CCR2 (white), and TUNEL (green) in CCR2⁻MHCII^low macrophages sorted from infarcted hearts at day 7 after MI from SE- and EE-treated mice. The percentage of TUNEL⁺ cells is expressed as TUNEL⁺CD68⁺ cells/CD68⁺ cells (n = 6). *P < 0.05 versus MI-SE. Data are expressed as means ± SEM. Data were analyzed using Student’s t test. Scale bars, 50 μm.