Abstract
Lymphatic malformations and other conditions where lymphatic function is disturbed in the respiratory tract present diagnostic and therapeutic challenges. Advances in lymphatic development, growth regulation, function, and imaging have increased the understanding of lymphatics, but the airways and lungs have not received as much attentions as many other organs. The lung presents challenges for studies of lymphatics because of the complex, densely packed three-dimensional architecture of the airways and vasculature, and because it cannot readily be examined in its entirety. To address this problem, we developed methods for immunohistochemical examination of the lymphatics in mouse lungs, based on approaches we devised for lymphatic vessels and blood vessels in whole mounts of the mouse trachea. This report provides a practical guide for visualizing by fluorescence and confocal microscopy the lymphatics in mouse airways and lungs under normal conditions and in models of disease. Materials and methods are described for immunohistochemical staining of lymphatics in whole mounts of the mouse trachea and 200-μm sections of mouse lung. Also described are mouse models in which lymphatics proliferate in the lung, blocking antibodies for preventing lymphatic growth, methods for fixing mouse lungs by vascular perfusion, and techniques for staining, visualizing, and analyzing lymphatic endothelial cells and other cells in the lung. These methods provide the opportunity to learn as much about lymphatics in the lung as in other organs.
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Stump B, Cui Y, Kidambi P, Lamattina AM, El-Chemaly S (2017) Lymphatic changes in respiratory diseases: more than just remodeling of the lung? Am J Respir Cell Mol Biol 57:272–279
Baluk P, Fuxe J, Hashizume H, Romano T, Lashnits E, Butz S, Vestweber D, Corada M, Molendini C, Dejana E et al (2007) Functionally specialized junctions between endothelial cells of lymphatic vessels. J Exp Med 204:2349–2362
Meinecke AK, Nagy N, Lago GD, Kirmse S, Klose R, Schrodter K, Zimmermann A, Helfrich I, Rundqvist H, Theegarten D et al (2012) Aberrant mural cell recruitment to lymphatic vessels and impaired lymphatic drainage in a murine model of pulmonary fibrosis. Blood 119:5931–5942
Kretschmer S, Dethlefsen I, Hagner-Benes S, Marsh LM, Garn H, Konig P (2013) Visualization of intrapulmonary lymph vessels in healthy and inflamed murine lung using CD90/Thy-1 as a marker. PLoS One 8:e55201
Baluk P, Tammela T, Ator E, Lyubynska N, Achen MG, Hicklin DJ, Jeltsch M, Petrova TV, Pytowski B, Stacker SA et al (2005) Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation. J Clin Invest 115:247–257
Baluk P, Yao LC, Feng J, Romano T, Jung SS, Schreiter JL, Yan L, Shealy DJ, McDonald DM (2009) TNF-alpha drives remodeling of blood vessels and lymphatics in sustained airway inflammation in mice. J Clin Invest 119:2954–2964
Baluk P, Adams A, Phillips K, Feng J, Hong YK, Brown MB, McDonald DM (2014) Preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation. Am J Pathol 184:1577–1592
Baluk P, Phillips K, Yao LC, Adams A, Nitschke M, McDonald DM (2014) Neutrophil dependence of vascular remodeling after mycoplasma infection of mouse airways. Am J Pathol 184:1877–1889
Yao LC, Baluk P, Feng J, McDonald DM (2010) Steroid-resistant lymphatic remodeling in chronically inflamed mouse airways. Am J Pathol 176:1525–1541
Yao LC, Baluk P, Srinivasan RS, Oliver G, McDonald DM (2012) Plasticity of button-like junctions in the endothelium of airway lymphatics in development and inflammation. Am J Pathol 180:2561–2575
Harding J, Ritter A, Rayasam A, Fabry Z, Sandor M (2015) Lymphangiogenesis is induced by mycobacterial granulomas via vascular endothelial growth factor receptor-3 and supports systemic T-cell responses against mycobacterial antigen. Am J Pathol 185:432–445
Baluk P, Hogmalm A, Bry M, Alitalo K, Bry K, McDonald DM (2013) Transgenic overexpression of interleukin-1beta induces persistent lymphangiogenesis but not angiogenesis in mouse airways. Am J Pathol 182:1434–1447
Baluk P, Yao LC, Flores JC, Choi D, Hong YK, McDonald DM (2017) Rapamycin reversal of VEGF-C-driven lymphatic anomalies in the respiratory tract. JCI Insight 2(16):pii: 90103
Yao LC, Testini C, Tvorogov D, Anisimov A, Vargas SO, Baluk P, Pytowski B, Claesson-Welsh L, Alitalo K, McDonald DM (2014) Pulmonary lymphangiectasia resulting from vascular endothelial growth factor-C overexpression during a critical period. Circ Res 114:806–822
Cui Y, Liu K, Monzon-Medina ME, Padera RF, Wang H, George G, Toprak D, Abdelnour E, D'Agostino E, Goldberg HJ et al (2015) Therapeutic lymphangiogenesis ameliorates established acute lung allograft rejection. J Clin Invest 125:4255–4268
Choi I, Chung HK, Ramu S, Lee HN, Kim KE, Lee S, Yoo J, Choi D, Lee YS, Aguilar B et al (2011) Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse. Blood 117:362–365
Lohela M, Helotera H, Haiko P, Dumont DJ, Alitalo K (2008) Transgenic induction of vascular endothelial growth factor-C is strongly angiogenic in mouse embryos but leads to persistent lymphatic hyperplasia in adult tissues. Am J Pathol 173:1891–1901
Baluk P, McDonald DM (2008) Markers for microscopic imaging of lymphangiogenesis and angiogenesis. Ann N Y Acad Sci 1131:1–12
Gotts JE, Abbott J, Matthay MA (2014) Influenza causes prolonged disruption of the alveolar-capillary barrier in mice unresponsive to mesenchymal stem cell therapy. Am J Physiol Lung Cell Mol Physiol 307:L395–L406
Ortiz-Munoz G, Looney MR (2015) Non-invasive intratracheal instillation in mice. Bio Protoc 5(12):pii: e1504
Acknowledgments
This work was supported in part by grants R01 HL127402, R01 HL059157, and P01 HL024136 from the NIH National Heart, Lung, and Blood Institute and 11CVD03 from the Leducq Foundation.
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Baluk, P., McDonald, D.M. (2018). Imaging Lymphatics in Mouse Lungs. In: Oliver, G., Kahn, M. (eds) Lymphangiogenesis. Methods in Molecular Biology, vol 1846. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8712-2_11
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DOI: https://doi.org/10.1007/978-1-4939-8712-2_11
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