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Histochemical analyses and quantum dot imaging of microvascular blood flow with pulmonary edema in living mouse lungs by “in vivo cryotechnique”

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Abstract

Light microscopic imaging of blood vessels and distribution of serum proteins is essential to analyze hemodynamics in living animal lungs under normal respiration or respiratory diseases. In this study, to demonstrate dynamically changing morphology and immunohistochemical images of their living states, “in vivo cryotechnique” (IVCT) combined with freeze-substitution fixation was applied to anesthetized mouse lungs. By hematoxylin–eosin staining, morphological features, such as shapes of alveolar septum and sizes of alveolar lumen, reflected their respiratory conditions in vivo, and alveolar capillaries were filled with variously shaped erythrocytes. Albumin was usually immunolocalized in the capillaries, which was confirmed by double-immunostaining for aquaporin-1 of endothelium. To capture accurate time-courses of blood flow in peripheral pulmonary alveoli, glutathione-coated quantum dots (QDs) were injected into right ventricles, and then IVCT was performed at different time-points after the QD injection. QDs were localized in most arterioles and some alveolar capillaries at 1 s, and later in venules at 2 s, reflecting a typical blood flow direction in vivo. Three-dimensional QD images of microvascular networks were reconstructed by confocal laser scanning microscopy. It was also applied to lungs of acute pulmonary hypertension mouse model. Erythrocytes were crammed in blood vessels, and some serum components leaked into alveolar lumens, as confirmed by mouse albumin immunostaining. Some separated collagen fibers and connecting elastic fibers were still detected in edematous tunica adventitia near terminal bronchioles. Thus, IVCT combined with histochemical approaches enabled us to capture native images of dynamically changing structures and microvascular hemodynamics of living mouse lungs.

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Acknowledgments

The authors thank Mr. Y. Kitahara at the Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, for his technical assistance. This study was partly supported by a grant for young investigators to Y. Saitoh from the University of Yamanashi.

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Authors and Affiliations

  1. Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo City, Yamanashi, 409-3898, Japan

    Yurika Saitoh, Nobuo Terada, Sei Saitoh, Nobuhiko Ohno & Shinichi Ohno

  2. Laboratory for Nano-Bio Pobes, RIKEN Quantitative Biology Center, Suita, Osaka, 565-0874, Japan

    Takashi Jin

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  1. Yurika Saitoh
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  2. Nobuo Terada
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  4. Nobuhiko Ohno
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  5. Takashi Jin
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  6. Shinichi Ohno
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Correspondence to Shinichi Ohno.

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Saitoh, Y., Terada, N., Saitoh, S. et al. Histochemical analyses and quantum dot imaging of microvascular blood flow with pulmonary edema in living mouse lungs by “in vivo cryotechnique”. Histochem Cell Biol 137, 137–151 (2012). https://doi.org/10.1007/s00418-011-0892-1

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  • DOI: https://doi.org/10.1007/s00418-011-0892-1

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