Fig. 4: Adoptive transfer of splenic hCD68–GFP⁺ monocytes and imaging of CX3CR1⁺ tissue-resident macrophages reveals different levels of clearance to MLNs after MI at P1 versus P7.

Schematic of the adoptive cell transfer approach using adult hCD68–eGFP transgenic mice as splenic GFP⁺ monocyte donors, for intramyocardial delivery into recipient neonatal CD1 mice at the time of MI surgery to assess immune cell trafficking (a). Immunostaining for CD68 and endogenous GFP fluorescence in tissue sections derived from MLNs of P1 and P7 mice that underwent MI, determining the presence of cleared CD68⁺GFP⁺ macrophages (b′ white arrows and c′ white arrowheads). CD68⁺GFP⁺ macrophages were substantially reduced in MLNs after MI at P1 compared to after MI at P7 (compare b and c). Visualization of endogenous GFP⁺ macrophages in MLNs from hCD68–eGFP mice confirmed minimal clearance at P1 after MI, which appeared increased at P7, compared to the respective intact controls that contained resident MLN GFP⁺ macrophages (compare d and e and compare f and g). Similar visualization in CX3CR1–eGFP mice also confirmed minimal clearance at P1 after MI, which increased at P7 (compare h and i and compare k and j). Quantification of macrophage numbers in the MLNs validated these observations and indicated that the difference in clearance at P1 versus P7 was significant (l,m). F4/80⁺ macrophages visualized within afferent lymphatic lumens of MLNs after MI at P7 but not at P1 and evidence of macrophage drainage disruption after P7 MI in the Lyve1^−/− mutant setting (n). b′–k′ indicate magnified view of panel boxes. Data are presented as mean ± s.e.m. In l, n = 4 for P8, n = 8 for P1MI7dpi, n = 5 for P14 and n = 7 for P7MI7dpi. In m, n = 7 for P8, n = 8 for P1MI7dpi, n = 9 for P14 and n = 10 for P7MI7dpi. Magnification boxes are illustrative. Quantification was conducted across the entire MLN area within 10-μm sections. Significant differences were calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. Scale bars, 50 μm for b and c; 0.5 mm for d–k; 20 μm for d′–k′; and 250 μm for n.

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