CCR2-RFP cells infiltrating the spinal cord ventral horn. A1–A3, Round and elongated CCR2-RFP cells lacking CX3CR1-GFP. Some of these cells are isolated (top arrow), but more frequently interact with microglia (bottom arrow). B1–B3, Many CCR2-RFP cells are positive for CD3ε, a T-cell marker. C1, D1, E1, F1, Confocal images (2D projections) of different types of dual-labeled CCR2-RFP/CX3CR1-GFP cells. C2, D2, E2, F2, CX3CR1-GFP expression. C3, D3, E3, F3, CCR2-RFP and CX3CR1-GFP merge images. C4, C5, D4, D5, E4, E5, F4, F5, CCR2-RFP content inside these cells, confirmed by surface rendering CX3CR1-GFP cells in Imaris and rotating the 3D volume. F4, F5, The surface of a blood vessel (stained by background VGLUT1 labeling) is also surface rendered. C, Rounded cell with low CX3CR1 fluorescence. D, Cell with few processes. E, Cell with multiple process and high CX3CR1 fluorescence and microglia-like morphology. F, Perivascular cell. G, Time course of CCR2-RFP/CX3CR1-GFP dual-expressing cells after tibial (red line and dots) or sciatic (blue line and dots) injuries. Data points represent individual animals (average of n = 23.2 sections per animal/data point). Lines indicate averages at each time point. Scale bars indicate SE (n = 4 animals per time point). Black and gray asterisks, respectively, represent significant differences with controls or between injury types at individual time points: **p < 0.01; ***p < 0.001. A significant infiltration of dual-labeled cells occurs after both injuries starting at 3 d and lasting for 8 weeks, but a peak increase in entry at 14 d occurs only after sciatic injuries. H, Genetic lineage labeling demonstrating cells derived from resident microglia (Iba1 and tdTomato colabeled) or sources other than resident microglia (labeled only with Iba1) 21 d after sciatic injuries. These include cells in close apposition to MN cell bodies (arrow). I–L, Change in microglia and CCR2-RFP cell numbers in CCR2 heterozygotes compared with CCR2 KOs 21 d after sciatic nerve injury and repair (STR). CCR2 heterozygotes are CX3CR1-EGFP:CCR2-RFP dual-heterozygotes. In all cases, n = 6 animals (each represented by a different dot). Errors bars indicate SE. *p < 0.05 (t tests). ***p < 0.001 (t tests). Deltas indicate effect sizes. Scale bars: A–F, 10 μm; H, 20 μm.
