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. 2017 Aug 17;2(16):e90103. doi: 10.1172/jci.insight.90103

Figure 1. Lymphatics in tracheas of CCSP/VEGF-C mice before and after doxycycline.

Figure 1

(A) VEGF-C mRNA expression (relative to β-actin) in trachea increased on doxycycline and returned almost to baseline after 14-day doxycycline washout period. (B) Number of Prox1-positive nuclei before and during doxycycline administration. (C–E) Overview of tracheal whole mounts stained for LYVE-1 (green) and Prox1 (red) from mice given 0.01 mg/ml doxycycline for 7 days (P21–P28) and perfused at P28 (C and D) or after 14-day washout period (P42) (E). (C) Trachea from a control (single-transgenic) mouse, with normal lymphatics restricted to intercartilage spaces and over the trachealis muscle at the center. (D) Trachea from a double-transgenic CCSP/VEGF-C mouse after doxycycline for 7 days, where lymphatics cover a much larger area and increase in abundance toward the caudal end. (E) Trachea from a P42 double-transgenic mouse 14 days after doxycycline withdrawal; lymphatics are less numerous than at P28, especially in central muscular region, but most of the lymphatic abnormality persists. Boxed regions are shown at higher magnification below. The middle row shows the boxed region stained for LYVE-1 showing normal lymphatics (C), lymphangiectasia at P28 (D), and spontaneous regression between P28 and P42 (E). Some lymphatics are narrowed regions (arrows). The lower row shows Prox1-positive nuclei at the beginning (C) and end of doxycycline treatment (D) and 14 days after withdrawal (E). Scale bar: 1 millimeter (top row); 50 μm (middle and bottom rows). (F) Extent of LYVE-1 lymphatics in trachea before doxycycline (blue box), after doxycycline (red box), and after doxycycline withdrawal (gray boxes). Values show the lymphatic expansion during doxycycline and some regression during the 2-week withdrawal period (P28–P42) but none thereafter throughout the 9-month study period (gray boxes were not significantly different from each other). n = 3 to 10 mice/group. *P < 0.05 vs. baseline group, †P < 0.05 vs. P28, ANOVA. Box and whisker plots show the median, first and third quartiles, and maximum and minimum.