Fig 1.
Schematic illustrating the surgical procedure.
(A) Located LNs, arrowheads indicate popliteal LN (left, black) and inguinal LN (right, white) after injection at hind footpad and base of tail, respectively. (B) Incision line (dashed line) was located above the location of the LNs for operated limb. LN, lymph node.
Fig 2.
Customized camera to visualize lymphatic pattern (left) and classification of lymphatic pattern (right). We classified and scored ICG lymphography images as follows: i) linear, clear lines appear (score 1); ii) splash with linear, the linear lymphatic vessels lie and several linear lymphatic vessels have a winding appearance (score 2); iii) stardust, shining dots are dispersed together with a faint luminous lymphatic vessel (score 3); iv) diffuse, blurry without a clear shape (score 4); and v) none, there is no pattern (score 5).
Fig 3.
Comparison of changes in swelling, lymphatic pattern and pathophysiologic analysis between exercise and control groups.
(A) Thickness difference of both ankles in the exercise, control and subgroup measured with ImageJ. The p-values for each time point between exercise and control groups are as follows: week 1, p = .15; week 2, p = .631; week 3, p = .055; week 4, p = .016; and week 5, p = .423 (n = 6 per group; * p < .05, Mann–Whitney U test). Subgroup represents a group of rats that underwent removal of only popliteal LN (n = 3). EG, exercise group; CG, control group; LN, lymph node. (B) Comparison between EG and CG of ICG lymphography patterns on ventroproximal. Data are expressed as mean ± standard deviation. (left) Time course of score on the severity of the patterns in the EG and CG. In the EG, there were more linear and splash patterns than in the CG. The Wilcoxon signed rank test was performed to compare severity over time in each group. (right) Representative ICG images of the hindlimb at weeks 1, 3, and 5. Arrow indicates a linear signal. EG, exercise group; CG, control group. (C-I) Comparison of skin thickness, fibrosis and the number of LV between both groups. (C) Representative images of H&E-stained skin thickness of the operated leg. (D, E) Control group showed significantly thicker epidermis and dermis compared with the exercise group (n = 6, respectively; p < .05, Mann–Whitney U test). (F) MT staining of operated limbs showed the extent of skin fibrosis. (G) Collagen area fraction (%) in the control group was significantly higher than in the exercise group. (H, I) The number of lymphatic vessels per HPF significantly increased after exercise. Arrows indicate positive podoplanin lymphatic vessels. Scale bar: C = 50 μm and 200 μm, respectively; F and I = 50 μm. Original magnification: C = × 200 and × 40, respectively; F and I = × 400. Epi, epidermis; De, dermis; LV, lymphatic vessel; H&E, hematoxylin and eosin; MT, Masson’s trichrome; HPF, high-power field. * < .05, ** < .01.
Table 1.
Thickness difference between both ankles for each week.