Figure 4

Lung lymphatic dysfunction after CS exposure in mice. Fluorescence (A) and brightfield (B) microscopy of mediastinal lymph nodes (mLN) from mice after 4 weeks CS exposure or room air. mLNs were harvested 60 min after intra-tracheal administration of dextran-555 (red). (C) Quantification of dextran-555 uptake to mediastinal lymph nodes by mean fluorescence intensity (AU). (D,E) Whole mount microscopy of lung lymphatics from Prox1-EGFP mice exposed to CS (E) or room air (D) for 8 weeks. (F,G) Quantification of nuclear orientation (angle from vessel axis) and nuclear roundness (length:width ratio) in lung lymphatics after 8 weeks CS or room air. (H) Cell trafficking assay to assess lymphatic leukocyte migration from lungs to draining lymph nodes was performed using intra-tracheal administration of CTV-labeled leukocytes followed by harvest of mLNs for flow cytometry. Quantification of CTV⁺ leukocytes in mLNs is shown as percent of total cells by flow cytometry. (I) Expression of CCL21b by quantitative PCR using whole lung tissue from mice after 4 months of CS exposure compared to room air, normalized to GAPDH. (J,K) Immunohistochemistry for CCL21 (green) using lung tissue from mice after 4 month CS or room air. Whole mount microscopy images are representative of lung tissue from at least 4 mice in each group. All values are means ± SEM. P value calculated by Student’s t test. *P < 0.05. ns not significant. Images in (A,B) shown at 5 × magnification. Scale bars in (D,E,J,K) = 50 μm.