Real-time <i>in vivo</i> imaging of the lung microcirculation in mouse _West China Medical Journal

Authors：

YANG Zhen ¹ , SU Bo ¹ , ZHONG Kangying ² , LI Yansong ³ ,  ZHANG Jie ¹

1. Core Facility, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
2. Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
3. Department of Anesthesiology, the First Affiliated Hospital of Xi'an Jiaotong University, Xi’an, Shaanxi 710000, P. R. China;

Corresponding?author：

ZHANG Jie, Email: zjie@scu.edu.cn

Keywords：

Lung; In vivo; Laser confocal scanning microscope

DOI：

10.7507/1002-0179.201810029

Video：

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ObjectiveTo design a method for observing pulmonary microcirculation in vivo in a native tissue environment, due to the high incidence of lung diseases and the advantages of animal experiments in vivo.MethodsTen BALB/c male mice were randomly divided into group A and group B, with five mice in each group. A self-made apparatus was used to control the movement towards local lung tissues in order to get a stabilized observation plane, and then a 5-minute video was shot with laser confocal scanning microscope. During the filming, the mice in group A were injected with fluorescein isothiocyanate-dextran via the tail vein, and the mice in group B were injected with green fluorescent protein-platelets (extracted from the blood of tie2-cre&rosa26-tomato-EGFP transgenic black C57 male mice). The data of group A was used to observe the lungs perfusion and the damage to tissue by this method, and the data of group B was used to observe the movement of platelets.ResultsImage of lung structure obtained by this method was clear and stable. Mean areas of alveolus in an instant and at the 30th, 60th, 120th, 180th, and 300th second were (1 603±181), (1 588±183), (1 528±363), (1 506±353), (1 437±369), (1 549±307) μm², respectively, and there were no significant differences between each time point (P>0.05). The video was smooth, the rapid movement of platelets was recorded and the particles were clear and without tailing; after the observation, hematoxylin-eosin staining showed no obvious damage to the lung tissue.ConclusionThe method can be used for the observation and research of the lung microcirculatory system in the living state of the mouse, and provides a methodological basis for studies of other lung diseases in vivo.

Citation： YANG Zhen, SU Bo, ZHONG Kangying, LI Yansong, ZHANG Jie. Real-time in vivo imaging of the lung microcirculation in mouse . West China Medical Journal, 2019, 34(1): 43-49. doi: 10.7507/1002-0179.201810029 Copy

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6.	陸月明, 顧秀蓮. 急性呼吸窘迫綜合征研究進展. 中華流行病學雜志, 2005, 26(6): 463-465.
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9.	Kreisel D, Nava RG, Li W, et al. In vivo two-photon imaging reveals monocyte-dependent neutro-phil extravasation during pulmonary inflammation. Proc Natl Acad Sci USA, 2010, 107(42): 18073-18078.
10.	劉斌. 飛秒激光雙光子熒光顯微成像技術研究. 哈爾濱: 哈爾濱工業大學: 2012.
11.	Terry RJ. A thoracic window for observation of the lung in a living animal. Science, 1939, 90(2324): 43-44.
12.	Wearn JT, Ernstene AC, Bromer AW, et al. The normal behavior of the pulmonary blood vessels with observations on the intermittence of the flow of blood in the arterioles and capillaries. Am J Physiol, 1934, 109(2): 236-256.
13.	Tabuchi A, Mertens M, Kuppe H, et al. Intravital microscopy of the murine pulmonary microcir-culation. J App Physiol (1985), 2008, 104(2): 338-346.
14.	夏偉強, 周源, 石明. 雙光子顯微成像技術的新進展. 中國醫療器械雜志, 2011, 35(3): 204-208.
15.	Headley MB, Bins A, Nip A, et al. Visualization of immediate immune responses to pioneer metastatic cells in the lung. Nature, 2016, 531(7595): 513-517.
16.	Vinegoni C, Aguirre AD, Lee S, et al. Imaging the beating heart in the mouse using intravital microscopy techniques. Nat Protoc, 2015, 10(11): 1802-1819.
17.	林巧雅, 黃松林, 駱清銘, 等. 肝臟免疫的活體顯微光學成像研究進展. 生物化學與生物物理進展, 2017, 44(12): 1056-1065.

1. 張文豪, 李建軍, 楊德剛, 等. 雙光子顯微鏡在小動物活體光學成像中的研究進展. 中國康復理論與實踐, 2017, 23(1): 37-41.
2. Chen X, Tang S, Zheng JS, et al. Chemical synthesis of a two-photon-activatable chemokine and photon-guided lymphocyte migration in vivo. Nat Commun, 2015, 6: 7220.
3. Li JL, Goh CC, Keeble JL, et al. Intravital multiphoton imaging of immune responses in the mouse ear skin. Nat Protoc, 2012, 7(2): 221-234.
4. Smith RA, Cokkinides V, Brooks D, et al. Cancer screening in the United States, 2010: a review of current American Cancer Society guidelines and issues in cancer screening. CA Cancer J Clin, 2010, 60(2): 99-119.
5. 包鶴齡, 方利文, 王臨虹. 1990-2014 年中國 40 歲及以上人群慢性阻塞性肺疾病患病率 Meta 分析. 中華流行病學雜志, 2016, 37(1): 119-124.
6. 陸月明, 顧秀蓮. 急性呼吸窘迫綜合征研究進展. 中華流行病學雜志, 2005, 26(6): 463-465.
7. 成令忠, 鐘翠平, 蔡文琴. 現代組織學. 上海: 上海科學技術文獻出版社, 2003.
8. Lefran?ais E, Ortiz-Mu?oz G, Caudrillier A, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature, 2017, 544(7648): 105-109.
9. Kreisel D, Nava RG, Li W, et al. In vivo two-photon imaging reveals monocyte-dependent neutro-phil extravasation during pulmonary inflammation. Proc Natl Acad Sci USA, 2010, 107(42): 18073-18078.
10. 劉斌. 飛秒激光雙光子熒光顯微成像技術研究. 哈爾濱: 哈爾濱工業大學: 2012.
11. Terry RJ. A thoracic window for observation of the lung in a living animal. Science, 1939, 90(2324): 43-44.
12. Wearn JT, Ernstene AC, Bromer AW, et al. The normal behavior of the pulmonary blood vessels with observations on the intermittence of the flow of blood in the arterioles and capillaries. Am J Physiol, 1934, 109(2): 236-256.
13. Tabuchi A, Mertens M, Kuppe H, et al. Intravital microscopy of the murine pulmonary microcir-culation. J App Physiol (1985), 2008, 104(2): 338-346.
14. 夏偉強, 周源, 石明. 雙光子顯微成像技術的新進展. 中國醫療器械雜志, 2011, 35(3): 204-208.
15. Headley MB, Bins A, Nip A, et al. Visualization of immediate immune responses to pioneer metastatic cells in the lung. Nature, 2016, 531(7595): 513-517.
16. Vinegoni C, Aguirre AD, Lee S, et al. Imaging the beating heart in the mouse using intravital microscopy techniques. Nat Protoc, 2015, 10(11): 1802-1819.
17. 林巧雅, 黃松林, 駱清銘, 等. 肝臟免疫的活體顯微光學成像研究進展. 生物化學與生物物理進展, 2017, 44(12): 1056-1065.

West China Medical Journal

Real-time in vivo imaging of the lung microcirculation in mouse

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