Heterogenous Intrapulmonary Distribution of Aerosolized Model Compounds in Mice with Bleomycin-Induced Pulmonary Fibrosis.

Background: A distinctive pathological feature of idiopathic pulmonary fibrosis (IPF) is the aberrant accumulation of extracellular matrix components in the alveoli in abnormal remodeling and reconstruction following scarring of the alveolar structure. The current antifibrotic agents used for IPF therapy frequently result in systemic side effects because these agents are distributed, through the blood, to many different tissues after oral administration. In contrast to oral administration, the intrapulmonary administration of aerosolized drugs is believed to be an efficient method for their direct delivery to the focus sites in the lungs. However, how fibrotic lesions alter the distribution of aerosolized drugs following intrapulmonary administration remains largely unknown. In this study, we evaluate the intrapulmonary distribution characteristics of aerosolized model compounds in mice with bleomycin-induced pulmonary fibrosis through imaging the organs and alveoli. Methods: Aerosolized model compounds were administered to mice with bleomycin-induced pulmonary fibrosis using a Liquid MicroSprayer®. The intrapulmonary distribution characteristics of aerosolized model compounds were evaluated through several imaging techniques, including noninvasive lung imaging using X-ray computed tomography, ex vivo imaging using zoom fluorescence microscopy, frozen tissue section observation, and three-dimensional imaging with tissue-clearing treatment using confocal laser microscopy. Results: In fibrotic lungs, the aerosolized model compounds were heterogeneously distributed. In observations of frozen tissue sections, model compounds were observed only in the fibrotic foci near airless spaces called honeycombs. In three-dimensional imaging of cleared tissue from fibrotic lungs, the area of the model compound in the alveolar space was smaller than in healthy lungs. Conclusion: The intrapulmonary deposition of extracellular matrix associated with pulmonary fibrosis limits the intrapulmonary distribution of aerosolized drugs. The development of delivery systems for antifibrotic agents to improve the distribution characteristics in fibrotic foci is necessary for effective IPF therapy.

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 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.

Evaluation of various tissue-clearing techniques for the three-dimensional visualization of liposome distribution in mouse lungs at the alveolar scale.

PURPOSE: To develop a three-dimensional visualization method for evaluating the distribution of pulmonary drug delivery systems and compare four tissue-clearing techniques (ClearT2, CUBIC, ScaleS, and SeeDB2) using intrapulmonary liposomes as drug carriers. METHODS: Rhodamine B-labeled liposomes were administered intrapulmonarily to mice using a MicroSprayer, and then fluorescent-labeled tomato lectin was administered intravenously to visualize the general lung structure. Tissue-clearing treatment of the mouse lungs was performed using the standard protocols of the ClearT2, CUBIC, ScaleS, and SeeDB2 techniques. Lung clearing was clarified using laser-scanning confocal microscopy, and three-dimensional images were reconstructed. RESULTS: Fluorescent-labeled tomato lectin was preserved using ClearT2 and SeeDB2 but not using CUBIC and ScaleS. In addition, the liposomes were stable in ClearT2 reagent, but they were mostly degraded in other reagents by surface-active agents. ClearT2 treatment enabled the three-dimensional visualization of intrapulmonary rhodamine B-labeled liposomes at the alveolar scale. CONCLUSIONS: These results suggest that the ClearT2 tissue-clearing technique was appropriate for the three-dimensional visualization of intrapulmonary liposomes at the alveolar scale. This study provides important information for selecting and optimizing suitable optical tissue-clearing techniques in lungs for evaluating the distribution of pulmonary drug delivery systems.