Pulmonary hypertension (PH) is a life-threatening disease characterized by pulmonary arterial remodeling, including the progressive obliteration of arterioles, which can lead to increased pulmonary vascular resistance and fatal respiratory and circulatory failure. The histological analysis of PH vessels by 2-dimensional imaging methods has revealed constrictive inward remodeling with the proliferation of smooth muscle cells (SMCs) and endothelial cells (ECs). Although 3-dimensional (3D) tissue visualization systems that use confocal and multiphoton microscopy allow imaging of complex microstructures in several organs, imaging of changes in vascular architecture has remained challenging because factors, such as tissue opacity, light scattering, and absorbance, 1 limit the depth of light penetration to≈ 100 to 200 µm. Recent remarkable progress in tissue-clearing techniques has been able to overcome these technical barriers to visualize whole organs without thin sectioning. Specifically, the clear unobstructed brain/body imaging cocktails and computational analysis (CUBIC) technique is a newly developed tissue-clearing method that enables 3D imaging of whole organs. 1 To analyze vascular remodeling in PH, we developed a new 3D visualization system of murine pulmonary vasculature using the CUBIC tissue-clearing method and multiphoton microscopy. All experiments were approved by the Ethics Committee for Animal Experiments of the University of Tokyo and strictly adhered to stipulated guidelines of the University of Tokyo for animal experiments. The data that support the findings described here are available from the corresponding authors on reasonable request.

For sample preparation, mice were transcardially and sequentially perfused with 4%(wt/vol) paraformaldehyde in phosphate-buffered saline and 50%(v/v) CUBIC-1 reagent. Postcaval lobes of the right lung were excised and continuously immersed in the CUBIC-1 reagent at 37 C, either for 5 days for whole-mount staining or for 1 day for fluorescent protein labeling (Figure [A]). Next, for whole-mount immunostaining, lungs were immersed in 20%(wt/vol) sucrose in phosphate-buffered saline, frozen in optimal cutting temperature compound at− 80 C overnight to increase antibody penetration, 2 thawed at room temperature, and then subjected to whole-mount immunostaining with primary antibodies diluted in 2%(v/v) Triton X-100–phosphate-buffered saline for 3 days. The fluorescent protein–labeled and–stained samples were finally immersed in CUBIC-2 reagent for 1 day before imaging by multiphoton excitation fluorescence microscopy with tile scanning. The lung tissue was successfully transparentized (Figure [B]). Fluorescence attributable to ECs expressing tdTomato or fluorescein isothiocyanate–conjugated anti–α smooth muscle actin antibody staining enabled 3D visualization of whole-lung vasculature, at full depth and at a single-cell resolution, in pulmonary EC-specific tdTomato-expressing mice