Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix

Idiopathic pulmonary fibrosis is a progressive, chronic lung disease characterized by the accumulation of extracellular matrix proteins, including collagen and elastin. Imaging of extracellular matrix in fibrotic lungs is important for evaluating its pathological condition as well as the distribution of drugs to pulmonary focus sites and their therapeutic effects. In this study, we compared techniques of staining the extracellular matrix with optical tissue-clearing treatment for developing three-dimensional imaging methods for focus sites in pulmonary fibrosis. Mouse models of pulmonary fibrosis were prepared via the intrapulmonary administration of bleomycin. Fluorescent-labeled tomato lectin, collagen I antibody, and Col-F, which is a fluorescent probe for collagen and elastin, were used to compare the imaging of fibrotic foci in intact fibrotic lungs. These lung samples were cleared using the Clear(T2) tissue-clearing technique. The cleared lungs were two dimensionally observed using laser-scanning confocal microscopy, and the images were compared with those of the lung tissue sections. Moreover, three-dimensional images were reconstructed from serial two-dimensional images. Fluorescent-labeled tomato lectin did not enable the visualization of fibrotic foci in cleared fibrotic lungs. Although collagen I in fibrotic lungs could be visualized via immunofluorescence staining, collagen I was clearly visible only until 40 μm from the lung surface. Col-F staining facilitated the visualization of collagen and elastin to a depth of 120 μm in cleared lung tissues. Furthermore, we visualized the three-dimensional extracellular matrix in cleared fibrotic lungs using Col-F, and the images provided better visualization than immunofluorescence staining. These results suggest that Clear(T2) tissue-clearing treatment combined with Col-F staining represents a simple and rapid technique for imaging fibrotic foci in intact fibrotic lungs. This study provides important information for imaging various organs with extracellular matrix-related diseases.