Figure 4. Macrophage depletion with clodronate liposomes inhibits regeneration.
(A) Ears were injected with clodronate liposomes (Clo-Lipo) or PBS liposome controls (PBS-Lipo) at D0 immediately before injury, D2 after injury and at D5. Ears were allowed to regenerate and tissue collected at later time points. (B) Wound size was measured over time. PBS-lipo ears close completely by D34 (black line, graph, bottom panel images). Clo-Lipo ears remain open until D70 (grey dotted line, graph, top panel images, n = 6 animals, 12 ears per treatment). (C) Representative ear from Clo-Lipo (top panel) and PBS-Lipo (bottom panel) followed over time. (D–E) H & E stained Clo-Lipo ear at D20 shows variable reduction in cell accumulation past the injury site (black dotted line) with relatively little new growth compared to PBS-Lipo ears. Scale bar = 100 μm. (F–I) H & E stained Clo-Lipo ear at D5 (F) shows a delay in epidermal closure (blue arrows) and loss cartilage plate histolysis (green arrow) compared to PBS-Lipo ears (H). Scale bar = 200 μm. (G) Boxed region in (F) showing accumulation of polymorphonuclear cells (yellow arrowheads). (I) Boxed region in (H) with monocytic cells evident (green arrowheads) and few polymorphonuclear cells present (yellow arrowhead). Scale bar = 10 μm. (J–N) IBA-1 immuno-positive area following macrophage depletion compared to control. Total IBA-1+ area normalized to total DAPI+ area at D5 after the final treatment (J, n = 3 animals per treatment, *unpaired Student's t-test p<0.05). IBA-1+ cells in PBS-lipo tissue at D5 (K) compared to Clo-lipo tissue at D5 (L). IBA1+ cells in PBS-Lipo tissue at D20 (M) and Clo-lipo treated tissue at D20 (N) show a return of positive cells.. Scale bar = 100 μm. Box delineates area of high-magnification images, Scale bar = 50 μm. IBA-1 = red, DAPI = grey, autofluorescent red blood cells (RBC) = orange. Distal = left, Dorsal = top.