Figure 2. Standard protocol and comparison of effects of six treatment strategies.

(A) Standard protocol for studying effects of treatment on lymphangiectasia in CCSP/VEGF-C mice on doxycycline (0.01 mg/ml) for 7 days, off doxycycline for 14 days, and then treated for 14 days (n = 4–6 mice/group). (B) Approach for measuring line density of tracheal lymphatics stained for LYVE-1 by overlaying 3 parallel lines and assessing the fraction of pixels with a brightness of 100 or greater (on a scale of 0–255) with ImageJ. (C) Density of tracheal lymphatics in CCSP/VEGF-C mice (P42–P56) after treatment with one of several vehicles used for the active therapeutics. (D) Lymphatic density in tracheas of CCSP/VEGF-C mice treated in the standard protocol with doxycycline (P21–P28), off doxycycline (P28–P42), and treated with one of six therapeutics for 14 days (P42–P56). Only the high dose of rapamycin (green box, 20 mg/kg) induced significant regression compared with the vehicle-treated group (red box). Treatment with lower doses of candidate therapeutics had no significant effect. (E) Effects of treatment with rapamycin in combination with other agents (P42–P56). The amount of lymphatic regression was not significantly greater (†) than that found after rapamycin alone (green box) but was less than that after treatment with vehicle (*P < 0.05 vs. vehicle, ANOVA). Box and whisker plots show the median, first and third quartiles, and maximum and minimum.