Fig. 6: Analysis of lymphatic vessels in the pulmonary fibrosis model using TDA.

a Induction of pulmonary fibrosis using bleomycin. Mice were instilled with bleomycin intratracheally (1.25–2.5 mg/kg) in Prox1-GFP mice (male, 4–16 months). Saline was instilled in the control group. The lungs were subjected to hematoxylin-eosin (HE) and Azan staining. Representative images from three independent experiments are shown. b The 3D whole-lung images of lymphatic vessels in the fibrosis model. Prox1-GFP mice were instilled intratracheally with bleomycin or saline. The mice were then sacrificed, and the lungs were subjected to CUBIC procedures. Representative 3D whole-lung images of lymphatic vessels are shown (Z = 10 μm step, digital zoom: 1.25). The enlarged 3D images (white insets) are shown in the bottom. c Quantification of lymphatic vessels (volume, branching/end points, and lengths and radii of branches). All the classified signals as lymphatic vessels are counted as a volume at the whole lung level. Using TubeMap, the number of branching/end points (vertex), and lengths and radii of branches were calculated. Data are shown with mean ± SD. Representative data from two independent experiments are shown (control (no aspiration); n = 3 mice, saline; n = 6 mice, bleomycin; n = 6 mice). One-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test were used. Source data are provided as a Source Data file. d The classified signals as lymphatic vessels in the lung fibrosis model. Classified signals as lymphatic vessels after training with ilastik software are shown (left). Persistent diagram of mouse lung lymphatic vessels in the fibrosis model. PH was applied to extracted signals. Persistent diagrams are shown in each sample (right). The red points (dimention1) and blue triangles (dimention2) represent planar feature points (loop) and spatial feature points (void), respectively, observed by the persistent homology method. Source data are provided as a Source Data file. e The geometric features of lymphatic vessels in the lung fibrosis model. To calculate the distance of each features in these persistent diagrams, the Sliced Wasserstein kernel is applied⁸¹. The results of (d) are shown in MDS. Representative data from two independent experiments are shown (control (no aspiration); n = 3 mice, saline; n = 6 mice, bleomycin; n = 6 mice). Source data are provided as a Source Data file.