Figure 1.
Representative intravital color image of the mouse foot showing lymphatic trafficking of EBD 5 mins after i.d. injection of EBD (A).
Arrow, EBD injection site. After a small incision of the skin, the blue PLN under fascia (black dotted circle) was observed (B). Arrowhead, surgical incision site. Scale, 1 mm. The EBD-filled PLN was excised (C). Double arrow, dissected PLN. Scale, 1 mm. Ankle thickness (D) and cross-sectional leg area (E) from axial CT slices were measured from stereoscopic imaging and CT scans, respectively, and the operated side was normalized to the contralateral right side.
Figure 2.
Representative intravital fluorescence images of mice 5 mins after i.d. injection of ICG (green) and Alexa680-BSA (red) on the dorsal aspect of left and right paw, respectively, in control mice prior to surgery and 2 days after surgical removal of the left PLN.
Double arrow, thoracic duct. Arrowhead, RLN. Arrow, LLN.
Figure 3.
White light and fluorescent images in mice prior to and 2, 6, 13, 20, and 27 days after PLN removal.
Images were acquired 10 mins after i.d. injection of ICG. Magnified fluorescent images of the red rectangles (a, b, c, and d) were also acquired. Arrow, ICG injection site. Double arrow, PLN. Arrowhead, ILN. Asterisk, bright spots due to leaked-out ICG. Open arrow, newly detected fluorescent lymphatic vessels after PLN surgery. Inset, fluorescent image in the ventral view of the mouse showing lymphatic drainage to the ILN. Scale bar, 1 mm.
Figure 4.
White light and fluorescent images in mice prior to and 2, 6, 13, 20, and 27 days after PLN removal.
Images were acquired 10 mins after i.d. injection of ICG. Magnified fluorescent images of the red rectangles (a, b, c, and d) were also acquired. Arrow, ICG injection site. Double arrow, PLN. Open arrowhead, popliteal efferent collecting lymphatic vessel. Arrowhead, ILN. Open arrow, lymphatic vessel branch points from previously observed lymphatic vessels after PLN surgery. Double open arrow, IsLN. Inset, Intravital color image after i.d. injection of EBD. Scale bar, 1 mm.
Figure 5.
White light and fluorescent images in mice prior to and 2, 6, and 20 days after PLN removal.
Images were acquired 10 mins after i.d. injection of ICG. Magnified fluorescent images of the red rectangles (a, b, and c) were also acquired. Arrow, ICG injection site. Double arrow, PLN. Open arrowhead, popliteal efferent collecting lymphatic vessel. Arrowhead, ILN. Open arrow, newly detected fluorescent lymphatic vessels after PLN removal. Scale bar, 1 mm.
Figure 6.
White light and fluorescent images in mice prior to and 2, 13, and 20 days after PLN removal.
Images were acquired 10 mins after i.d. injection of ICG. Magnified fluorescent images of the red rectangles (a and b) were also acquired. Arrow, ICG injection site. Double arrow, PLN. Open arrowhead, popliteal efferent collecting lymphatic vessel. Arrowhead, ILN. Open arrow, lymphatic vessels branching off from collecting lymphatic vessels. Scale bar, 1 mm.
Figure 7.
IHC staining for LYVE-1 in skin tissues from (A) the wound region, (B) the left ankle distal to the wound area, and (C) the contralateral right ankle in mice at 2 days post-surgery.
Computer-assisted image analysis shows significantly increased number of lymphatic vessels (D) and relative area occupied by lymphatics in the wound area (E), whereas there is no significant difference between tissues from the left ankle in the distal wound site and the contralateral right ankle. Scale: 100 µm. Whole mount staining of lymphatic vessels of (F) the left ankle distal to the wound area, and (G) the contralateral right ankle and (H) fluorescence analysis of vessel area fraction n = 4 mice. Scale bar, 200 µm. * p<0.05 vs. wound.
Figure 8.
Fluorescent images prior to the popliteal lymphadenectomy (A, G), at 27 days post-surgery (B, H), and 21 days after B16F10 cell inoculation (C, I).
At 3 weeks post inoculation, intravital color images showed B16F10 metastasis to the ILN (D), IsLN (E), and LLN (F) in a mouse with lymphatic drainage to ILN and IsLN across the popliteal wound site as demonstrated by the presence of black melanin pigment in LNs. In a mouse with lymphatic drainage to the ILN (I), ILN metastasis (J) was observed, but not in the mouse IsLN which was not involved in the lymphatic drainage pathway (K). In addition, in-transit metastasis was detected proximal to the wound site (L). White arrow, ICG injection site. Double arrow, PLN. Open arrowhead, popliteal efferent collecting lymphatic vessel. Arrowhead, ILN. Open arrow, B16F10 metastasis. Black arrow, in-transit metastasis. Scale bar, 1 mm.
Figure 9.
The quantification of lymphatic contractility in the popliteal afferent (A, C) and efferent (B, D) lymphatic vessels in mice after PLN removal (A; n = 19, B; n = 15) and in control mice (C, D; n = 10).
* p<0.05 vs. baseline.