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Depletion of resident macrophages led to long-term incompetence of heart regeneration. Long-term effect of resident macrophage depletion by injecting CL at 1 mo (–1 m_CL) prior cardiac injury. (A) Revascularization was evaluated at 7 days post cryoinjury (dpci). Endogenous fli1:EGFP fluorescence depicts the vascular endothelial cells. White dotted lines delineate injury areas; scale bars, 100 μm. Quantification of vessel density by ImageJ is shown in the right panel (n ≥ 5; p=0.0082). (B) Neutrophils in injured areas at 7 dpci were examined by Myeloperoxidase (Mpx) immunostaining. White dotted lines delineate injury areas; scale bars, 100 μm. Quantification of neutrophil number in injured areas is listed in the right panel (n ≥ 6; p=0.0605). (C) CM proliferation was evaluated by EdU cell proliferation assay at 7 dpci. White arrows point out the proliferating CMs; scale bars, 100 μm (left panels) and 20 μm (middle panels). Quantification of CM density (n = 6; p=0.0562) and CM proliferation index (n = 6; p=0.8609) in 200 μm adjacent to the injured area is shown in the right panels. (D) Scar resolution was evaluated by AFOG staining at 30 dpci. Healthy myocardium, fibrin, and collagen are visualized in orange, red, and blue, respectively. Quantification of scar area is shown in the right panel (n ≥ 5; p=0.0232). CL, clodronate liposomes; IP, intraperitoneal; CM, cardiomyocyte; AFOG, Acid Fuchsin Orange G. The heart samples under regenerative (–1 m_PBS) or resident macrophage-deficient (–1 m_CL) conditions are indicated by purple or green, respectively. Student’s t-test was used to assess all comparisons by Prism 9.
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