Figure 5. Topical tacrolimus increases formation of collateral lymphatic vessels.

(a) Left panel: representative longitudinal immunofluorescent × 40 images of LYVE-1 vessels (green) bridging the surgically created tail wounds of control and early-treated tacrolimus mice harvested 6 weeks after lymphatic injury; inset shows area where longitudinal sections were obtained. Right panel: quantification of bridging lymphatic vessel density (LVD) in the wounded portion of the tail in control versus early or late-treated tacrolimus mice (P<0.001 for both; n=6/group). (b) Left panel: representative ICG (left panels) and × 40 immunofluorescent images of LYVE-1⁺ vessels (right panel showing area in red box) in control and tacrolimus-treated animals 4 weeks following PLND. Right panel: quantification of collateral lymphatic LVD in the anterolateral thigh region of animals treated with control or tacrolimus (P<0.001; n=6/group). (c) qPCR of RNA harvested from control and early-treated tacrolimus mouse tail tissues harvested 6 weeks after lymphatic injury, demonstrating relative expression of VEGF-C (P=0.264), TGF-β1 (P=0.006) and IFN-γ (P=0.022; n=6/group). (d) Protein concentrations from hindlimb tissues for each group using enzyme-linked immunosorbant assay for VEGF-A (P=0.711), VEGF-C (P=0.233), TGF- β1 (P<0.001), IFN-γ (P=0.022), IL-4 (P=0.034) and IL-13 (P=0.009; n=6/group). Experiments were repeated two to three times. All data represent mean±s.d. with P≤0.05 considered as significant. Data analysed by two-tailed student's t-test. Scale bars, 100 μm.