Figure 4.

The Depletion of Macrophages by Intravenous Injection of Clodronate Liposomes (CLs) Attenuates the Functional Recovery of the Heart Post-AMI after Nes⁺cMSC Treatment

(A) The percentages of CD11b⁺CD14⁺ monocytes in blood and CD11b⁺F4/80⁺ macrophages in spleen and heart were analyzed by flow cytometry at 1, 3, and 7 days after CL injection. (B) Schematic of the strategies used for the systemic depletion of macrophages, AMI model establishment, and cardiac function analysis. One day before establishment of the AMI model (−1 day), mice were systematically depleted of macrophages using anionic clodronate liposomes. At 0 day, the AMI model was generated by permanent ligation of the LAD coronary artery. One minute later, the ischemic area was identified and saline (vehicle-treated control) or Nes⁺cMSCs were intramyocardially injected into the infarct border zone. Cardiac function and the degree of infarct were analyzed by echocardiography and TTC staining, respectively, at 3 weeks post-AMI. (C) The survival rate of mice was analyzed after macrophage depletion and Nes⁺cMSC treatment; n = 15–30. (D) Heart function was evaluated by echocardiography at 3 h (baseline) and 3 weeks post-AMI, and LVEF, LVFS, LVEDV, and LVESV were measured; n = 10–15. (E and F) Five heart sections (1 mm thick) from the various groups were stained with 1% TTC for visualization of the infarct area (pale) and viable myocardial area (brick red). Scale bar, 10 mm (E). Comparison of the relative scar areas among the study groups. The ratio of the length of the infarct band to the total length of the LV was calculated; n = 5 (F). Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. CL Control, clodronate-free liposomes (negative control); CL Anionic, clodronate-containing liposomes.