Fig 1.
LNT decreases swelling after diphtheria toxin-induced lymphatic ablation.
A) Schematic diagram of the experimental protocol. Transgenic mice underwent activation of the Cre-Lox recombination through a course of intraperitoneal injections of tamoxifen followed by a series of subdermal diphtheria toxin (DT) injections into the dorsum of the hindpaw one week later in order to ablate the local lymphatic system. Two weeks later, the mice underwent ipsilateral popliteal lymph node dissection (PLND). After an additional two week recovery, experimental mice underwent ipsilateral lymph node transplantation (+LNT), while control mice underwent popliteal incision without lymph node transplantation (-LNT). All analysis was done using hindlimbs of mice that were sacrificed 10 weeks after the final surgery. B) Representative whole-mount immunofluorescent image of the mouse hindlimb harvested one week after DT injection. Note ablation of both capillary (podoplanin+α-SMA-) and collecting lymphatics (podoplanin+α-SMA+). Collecting lymphatics are also indicated by the dotted white line. C) Left panel: Representative photograph of hindlimbs after surgery with or without LNT. Note the gross swelling in the hindlimb in the mouse without LNT (-LNT). Right panel: Quantification of change in hindpaw thickness from baseline in the weeks following surgery with or without LNT. Note significant decrease in mice that underwent LNT (+LNT) beginning 2 weeks after surgery.
Fig 2.
LNT decreases the pathological changes of lymphedema.
A) Left panel: Representative H&E stain of the hindlimbs of mice with or without LNT. Cross-sections were obtained 2 mm proximal to the tarsal joint. The dotted black line indicates the area of fibroadipose deposition. The area highlighted by the blue dotted box is shown in high-power view in part B. Right panel: Quantification of the percentage of fibroadipose deposition area of hindlimbs of mice with and without LNT. B) Left panel: Representative high-power view of the areas indicated in the blue dotted boxes in part A. Note the decreased hyperkeratosis and dermal thickness in mice treated with LNT (+LNT). Right panel: Quantification of epidermal and dermal thickness in hindlimbs of mice with and without LNT. C) Left panel: Representative immunofluorescent images of hindlimbs stained for type I collagen (red) and nuclear DAPI (blue). Note decreased type I collagen deposition in mice treated with LNT (+LNT). Right panel: Quantification of type I collagen deposition (measured as a percentage of the total slide stained area) after surgery with and without LNT.
Fig 3.
LNT decreases perilymphatic accumulation of inflammatory cells.
A) Left panel: Representative images of CD45+ cells (red) surrounding subdermal lymphatic vessels (green) in hindlimbs of mice with or without LNT. Right panel: Quantification of CD45+ cells located within a 50 μm radius of lymphatic vessels in the hindlimbs of mice with and without LNT. B) Left panel: Representative images of CD3+ cells (red) surrounding the subdermal lymphatic vessels (green) in hindlimbs of mice in the hindlimbs of mice with and without LNT. Right panel: Quantification of perilymphatic CD3+ cells located within a 50 μm radius of lymphatic vessels in the hindlimbs of mice with and without LNT.
Fig 4.
LNT promotes regeneration of collecting lymphatic vessels.
A) Upper panel: Representative images of ICG lymphangiograms of normal mice (i.e., Cre-Lox mice without DT ablation), mice that had undergone local DT ablation followed by PLND but no LNT (-LNT), and mice that had undergone local DT ablation followed by PLND then LNT (+LNT). Main lymphatic collecting vessels can be seen in the normal hindlimb as white parallel linear structures, whereas no visible collecting vessels are noted in mice with ablated lymphatic circulation and no LNT (-LNT). Also note the dermal reflux as represented by accumulation of white dye diffusely in the mice without LNT (-LNT). In contrast, mice treated with LNT after lymphatic ablation (+LNT) have abnormal hyperplastic lymphatic vessels that converging toward the transplanted node, as indicated by the bright white spot in the blue dotted box. Lower panel: Immunofluorescent image of the transplanted lymph node indicated by the blue dotted box in the upper panel. Lymphatic collecting vessels indicated by podoplanin (green) and α-SMA (red). The inset in the lower left corner represents the magnified view of the lymphatic vessel adjacent to the transplant lymph node as indicated by the white dotted line. B) Representative immunofluorescent images of collecting lymphatics in mice with and without LNT. Note the collapsed lumen and proliferation of α-SMA+ cells in mice without LNT (-LNT). C) Quantification of collecting vessels in a 0.25 mm2 area, collecting vessel diameter, and α-SMA thickness in mice with and without LNT. Note the significant increase in number of collecting vessels and luminal diameter with a corresponding decrease in α-SMA thickness in mice treated with LNT (+LNT).
Fig 5.
LNT increases migration of DCs to inguinal lymph node.
A) Schematic diagram of the experimental protocol. Ten weeks after surgery with or without LNT, mice underwent FITC painting of the ipsilateral distal hindlimb. Eighteen hours later, the number of DCs migrating to the inguinal lymph node was analyzed using flow cytometry. B) Left panel: Representative flow cytometry plots of inguinal lymph node cells gated for side scatter analysis (SSA) and FITC. The red boxes indicate the gating for FITC+ DCs. Right panel: Quantification of FITC+ DCs in the inguinal lymph nodes of mice treated with or without LNT.
Fig 6.
LNT improves T cell-mediated immune responses.
A) Schematic diagram of experimental protocol. Ten weeks after surgery with or without LNT, mice were sensitized with topical 0.5% DNFB to the ipsilateral hindlimb once daily for three days. T cell response was elicited by challenging the contralateral ear with topical 0.3% DNFB 5 days later. The ears were harvested for analysis 3 days later. B) Left panel: Representative H&E stain of the ear skin from mice with and without LNT. Note the increased inflammatory reaction in mice treated with LNT (+LNT). Right panel: Quantification of epidermal and dermal thickness of ear skin. Note significant increase in both epidermal and dermal thickness in mice treated with LNT (+LNT). C) Left panel: Representative images demonstrating immunofluorescent localization of CD45+ cells (red) in ear skin of mice with and without LNT. Right panel: Quantification of CD45+ cells per HPF (80x) in mice treated with and without LNT. Note the more robust inflammatory response as indicated by the greater amount of CD45+ cells in mice treated with LNT (+LNT).