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 distributio...
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| Format: | Article |
| Language: | English |
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Wiley
2020-01-01
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| Series: | International Journal of Biomedical Imaging |
| Online Access: | http://dx.doi.org/10.1155/2020/8815231 |
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| author | Kohei Togami Hiroaki Ozaki Yuki Yumita Anri Kitayama Hitoshi Tada Sumio Chono |
| author_facet | Kohei Togami Hiroaki Ozaki Yuki Yumita Anri Kitayama Hitoshi Tada Sumio Chono |
| author_sort | Kohei Togami |
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| description | 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 ClearT2 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 ClearT2 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. |
| format | Article |
| id | doaj-art-159c751f520b4b7e9c06c1af992917a5 |
| institution | Kabale University |
| issn | 1687-4188 1687-4196 |
| language | English |
| publishDate | 2020-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | International Journal of Biomedical Imaging |
| spelling | doaj-art-159c751f520b4b7e9c06c1af992917a52025-08-20T03:54:25ZengWileyInternational Journal of Biomedical Imaging1687-41881687-41962020-01-01202010.1155/2020/88152318815231Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular MatrixKohei Togami0Hiroaki Ozaki1Yuki Yumita2Anri Kitayama3Hitoshi Tada4Sumio Chono5Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8585, JapanDivision of Pharmaceutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8585, JapanDivision of Pharmaceutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8585, JapanDivision of Pharmaceutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8585, JapanDepartment of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8585, JapanDepartment of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8585, JapanIdiopathic 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 ClearT2 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 ClearT2 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.http://dx.doi.org/10.1155/2020/8815231 |
| spellingShingle | Kohei Togami Hiroaki Ozaki Yuki Yumita Anri Kitayama Hitoshi Tada Sumio Chono Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix International Journal of Biomedical Imaging |
| title | Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix |
| title_full | Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix |
| title_fullStr | Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix |
| title_full_unstemmed | Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix |
| title_short | Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix |
| title_sort | three dimensional imaging of pulmonary fibrotic foci at the alveolar scale using tissue clearing treatment with staining techniques of extracellular matrix |
| url | http://dx.doi.org/10.1155/2020/8815231 |
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