Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix.
{"title":"Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix.","authors":"Kohei Togami, Hiroaki Ozaki, Yuki Yumita, Anri Kitayama, Hitoshi Tada, Sumio Chono","doi":"10.1155/2020/8815231","DOIUrl":null,"url":null,"abstract":"<p><p>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<sup>T2</sup> 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 <i>μ</i>m from the lung surface. Col-F staining facilitated the visualization of collagen and elastin to a depth of 120 <i>μ</i>m 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<sup>T2</sup> 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.</p>","PeriodicalId":47063,"journal":{"name":"International Journal of Biomedical Imaging","volume":null,"pages":null},"PeriodicalIF":3.3000,"publicationDate":"2020-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787752/pdf/","citationCount":"3","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"International Journal of Biomedical Imaging","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1155/2020/8815231","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2020/1/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"ENGINEERING, BIOMEDICAL","Score":null,"Total":0}
引用次数: 3
Abstract
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.
期刊介绍:
The International Journal of Biomedical Imaging is managed by a board of editors comprising internationally renowned active researchers. The journal is freely accessible online and also offered for purchase in print format. It employs a web-based review system to ensure swift turnaround times while maintaining high standards. In addition to regular issues, special issues are organized by guest editors. The subject areas covered include (but are not limited to):
Digital radiography and tomosynthesis
X-ray computed tomography (CT)
Magnetic resonance imaging (MRI)
Single photon emission computed tomography (SPECT)
Positron emission tomography (PET)
Ultrasound imaging
Diffuse optical tomography, coherence, fluorescence, bioluminescence tomography, impedance tomography
Neutron imaging for biomedical applications
Magnetic and optical spectroscopy, and optical biopsy
Optical, electron, scanning tunneling/atomic force microscopy
Small animal imaging
Functional, cellular, and molecular imaging
Imaging assays for screening and molecular analysis
Microarray image analysis and bioinformatics
Emerging biomedical imaging techniques
Imaging modality fusion
Biomedical imaging instrumentation
Biomedical image processing, pattern recognition, and analysis
Biomedical image visualization, compression, transmission, and storage
Imaging and modeling related to systems biology and systems biomedicine
Applied mathematics, applied physics, and chemistry related to biomedical imaging
Grid-enabling technology for biomedical imaging and informatics