Skip to main content
SpringerLink
Log in

Live Cell Multiphoton Microscopy of Atherosclerotic Plaques in Mouse Aortas

  • Chapter
  • First Online:
Cardiovascular Imaging

  • 1777 Accesses

Abstract

Atherosclerosis is a chronic inflammatory disease with both innate and adaptive immune components. Various static methods have been applied to investigate mechanisms of atherosclerosis development. However, they did not allow for monitoring dynamic changes in leukocyte behavior in normal and atherosclerotic aortas. Live cell imaging is necessary to study dynamic or transient leukocyte functions relevant to disease pathology, such as antigen presentation, cell migration, and cell-cell interaction. We developed a protocol for ex vivo multiphoton microscopy of atherosclerotic aortas and used it to demonstrate that antigen presentation may occur within the arterial wall. Aortas are harvested from transgenic reporter mice with fluorescent myeloid cells and then incubated with labeled T cells. The cells are imaged with a multiphoton microscope, while a superfusion system maintains the explant in physiologic condition. Cells are tracked from the videos, and their motion is quantified. This system was used to demonstrate antigen presentation in the arterial wall in the context of atherosclerosis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. World Health Organization. Global burden of disease, http://www.who.int/healthinfo/global_burden_disease/estimates_country/en/index.html (2013). Accessed 7 May 2013.

  2. Galkina E, Ley K. Immune and inflammatory mechanisms of atherosclerosis. Annu Rev Immunol. 2009;27:165–97. doi:10.1146/annurev.immunol.021908.132620.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ, Ley K. Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent. The Journal of experimental medicine. 2006;203(5):1273–82. doi:10.1084/jem.20052205.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Schulte S, Sukhova GK, Libby P. Genetically programmed biases in Th1 and Th2 immune responses modulate atherogenesis. Am J Pathol. 2008;172(6):1500–8. doi:10.2353/ajpath.2008.070776.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Egen JG, Rothfuchs AG, Feng CG, Horwitz MA, Sher A, Germain RN. Intravital imaging reveals limited antigen presentation and T cell effector function in mycobacterial granulomas. Immunity. 2011;34(5):807–19. doi:10.1016/j.immuni.2011.03.022.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Han JW, Shimada K, Ma-Krupa W, Johnson TL, Nerem RM, Goronzy JJ, Weyand CM. Vessel wall-embedded dendritic cells induce T-cell autoreactivity and initiate vascular inflammation. Circ Res. 2008;102(5):546–53. doi:10.1161/CIRCRESAHA.107.161653.

    Article  CAS  PubMed  Google Scholar 

  7. Choi JH, Do Y, Cheong C, Koh H, Boscardin SB, Oh YS, Bozzacco L, Trumpfheller C, Park CG, Steinman RM. Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves. J Exp Med. 2009;206(3):497–505. doi:10.1084/jem.20082129.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Miller MJ, Wei SH, Parker I, Cahalan MD. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science. 2002;296(5574):1869–73. doi:10.1126/science.1070051.

    Article  CAS  PubMed  Google Scholar 

  9. Mempel TR, Henrickson SE, Von Andrian UH. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature. 2004;427(6970):154–9. doi:10.1038/nature02238.

    Article  CAS  PubMed  Google Scholar 

  10. Koltsova EK, Garcia Z, Chodaczek G, Landau M, McArdle S, Scott SR, von Vietinghoff S, Galkina E, Miller YI, Acton ST, Ley K. Dynamic T cell-APC interactions sustain chronic inflammation in atherosclerosis. J Clin Invest. 2012;122(9):3114–26. doi:10.1172/JCI61758.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Frigault MM, Lacoste J, Swift JL, Brown CM. Live-cell microscopy - tips and tools. J Cell Sci. 2009;122(Pt 6):753–67. doi:10.1242/jcs.033837.

    Article  CAS  PubMed  Google Scholar 

  12. Stephens DJ, Allan VJ. Light microscopy techniques for live cell imaging. Science. 2003;300(5616):82–6. doi:10.1126/science.1082160.

    Article  CAS  PubMed  Google Scholar 

  13. Nikon. MicroscopyU-introduction to live-cell imaging techniques. http://www.microscopyu.com/articles/livecellimaging/index.html (2013). Accessed 26 Apr 2013.

  14. Zeiss. Live-cell imaging. http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/index.html (2013). Accessed 26 Apr 2013.

  15. Andor Technology. iXon Ultra 897. http://www.andor.com/pdfs/specifications/Andor_iXon_Ultra_897_Specifications.pdf (2013). Accessed 3 May 2013.

  16. McNally JG, Karpova T, Cooper J, Conchello JA. Three-dimensional imaging by deconvolution microscopy. Methods. 1999;19(3):373–85. doi:10.1006/meth.1999.0873.

    Article  CAS  PubMed  Google Scholar 

  17. Leica. Leica SD AF – the integrated confocal spinning disk solution. http://www.leica-microsystems.com/news-media/news/news-details/article/leica-sd-af-the-integrated-confocalspinning-disk-solution/.

  18. Ntziachristos V. Going deeper than microscopy: the optical imaging frontier in biology. Nat Methods. 2010;7(8):603–14. doi:10.1038/nmeth.1483.

    Article  CAS  PubMed  Google Scholar 

  19. Zipfel WR, Williams RM, Webb WW. Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol. 2003;21(11):1369–77. doi:10.1038/nbt899.

    Article  CAS  PubMed  Google Scholar 

  20. Drobizhev M, Makarov NS, Tillo SE, Hughes TE, Rebane A. Two-photon absorption properties of fluorescent proteins. Nat Methods. 2011;8(5):393–9. doi:10.1038/nmeth.1596.

    Article  CAS  PubMed  Google Scholar 

  21. Taatjes DJ, Wadsworth MP, Schneider DJ, Sobel BE. Improved quantitative characterization of atherosclerotic plaque composition with immunohistochemistry, confocal fluorescence microscopy, and computer-assisted image analysis. Histochem Cell Biol. 2000;113(3):161–73.

    Article  CAS  PubMed  Google Scholar 

  22. Zoumi A, Yeh A, Tromberg BJ. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc Natl Acad Sci USA. 2002;99(17):11014–9. doi:10.1073/pnas.172368799.

  23. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992;258(5081):468–47.

    Article  CAS  PubMed  Google Scholar 

  24. Lindquist RL, Shakhar G, Dudziak D, Wardemann H, Eisenreich T, Dustin ML, Nussenzweig MC. Visualizing dendritic cell networks in vivo. Nat Immunol. 2004;5(12):1243–50. doi:10.1038/ni1139.

    Article  CAS  PubMed  Google Scholar 

  25. Barnden MJ, Allison J, Heath WR, Carbone FR. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol Cell Biol. 1998;76(1):34–40. doi:10.1046/j.1440-1711.1998.00709.x.

    Article  CAS  PubMed  Google Scholar 

  26. Oxenius A, Bachmann MF, Zinkernagel RM, Hengartner H. Virus-specific MHC-class II-restricted TCR-transgenic mice: effects on humoral and cellular immune responses after viral infection. Eur J Immunol. 1998;28(1):390–400.

    Article  CAS  PubMed  Google Scholar 

  27. Hermansson A, Ketelhuth DF, Strodthoff D, Wurm M, Hansson EM, Nicoletti A, Paulsson-Berne G, Hansson GK. Inhibition of T cell response to native low-density lipoprotein reduces atherosclerosis. J Exp Med. 2010;207(5):1081–93. doi:10.1084/jem.20092243.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Palinski W, Horkko S, Miller E, Steinbrecher UP, Powell HC, Curtiss LK, Witztum JL. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma. J Clin Invest. 1996;98(3):800–14. doi:10.1172/JCI118853.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Schett G, Xu Q, Amberger A, Van der Zee R, Recheis H, Willeit J, Wick G. Autoantibodies against heat shock protein 60 mediate endothelial cytotoxicity. J Clin Invest. 1995;96(6):2569–77. doi:10.1172/JCI118320.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Zoumi A, Lu X, Kassab GS, Tromberg BJ. Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy. Biophys J. 2004;87(4):2778–86. doi:10.1529/biophysj.104.042887.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Cinamon G, Shinder V, Alon R. Shear forces promote lymphocyte migration across vascular endothelium bearing apical chemokines. Nat Immunol. 2001;2(6):515–22. doi:10.1038/88710.

    Article  CAS  PubMed  Google Scholar 

  32. Megens RT, Reitsma S, Schiffers PH, Hilgers RH, De Mey JG, Slaaf DW, oude Egbrink MG, van Zandvoort MA. Two-photon microscopy of vital murine elastic and muscular arteries. Combined structural and functional imaging with subcellular resolution. J Vasc Res. 2007;44(2):87–98. doi:10.1159/000098259.

    Article  CAS  PubMed  Google Scholar 

  33. Sorokin L. The impact of the extracellular matrix on inflammation. Nat Rev Immunol. 2010;10(10):712–23. doi:10.1038/nri2852.

    Article  CAS  PubMed  Google Scholar 

  34. Caldwell CC, Kojima H, Lukashev D, Armstrong J, Farber M, Apasov SG, Sitkovsky MV. Differential effects of physiologically relevant hypoxic conditions on T lymphocyte development and effector functions. J Immunol. 2001;167(11):6140–9.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA, 92037, USA

    Sara McArdle, Ekaterina Koltsova, Grzegorz Chodaczek & Klaus Ley MD

  2. Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA

    Sara McArdle

Authors
  1. Sara McArdle
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ekaterina Koltsova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Grzegorz Chodaczek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Klaus Ley MD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Klaus Ley MD .

Editor information

Editors and Affiliations

  1. Cardiovascular Medicine, Brigham and Women’s Hospital Harvard Medical School, Boston, Massachusetts, USA

    Elena Aikawa

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer International Publishing Switzerland

About this chapter

Cite this chapter

McArdle, S., Koltsova, E., Chodaczek, G., Ley, K. (2015). Live Cell Multiphoton Microscopy of Atherosclerotic Plaques in Mouse Aortas. In: Aikawa, E. (eds) Cardiovascular Imaging. Springer, Cham. https://doi.org/10.1007/978-3-319-09268-3_7

Download citation

  • DOI: https://doi.org/10.1007/978-3-319-09268-3_7

  • Published:

  • Publisher Name: Springer, Cham

  • Print ISBN: 978-3-319-09267-6

  • Online ISBN: 978-3-319-09268-3

  • eBook Packages: MedicineMedicine (R0)

Publish with us

Policies and ethics

Search

Navigation