Advertisement

Live Cell Multiphoton Microscopy of Atherosclerotic Plaques in Mouse Aortas

  • Sara McArdle
  • Ekaterina Koltsova
  • Grzegorz Chodaczek
  • Klaus LeyEmail author
Chapter

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.

Keywords

Multiphoton Microscopy Atherosclerotic Aorta Atherosclerotic Mouse Laser Scanning Confocal System Multiphoton Imaging 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 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. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  3. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  4. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  5. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  6. 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.PubMedCrossRefGoogle Scholar
  7. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. 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.PubMedCrossRefGoogle Scholar
  10. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 12.
    Stephens DJ, Allan VJ. Light microscopy techniques for live cell imaging. Science. 2003;300(5616):82–6. doi: 10.1126/science.1082160.PubMedCrossRefGoogle Scholar
  13. 13.
    Nikon. MicroscopyU-introduction to live-cell imaging techniques. http://www.microscopyu.com/articles/livecellimaging/index.html (2013). Accessed 26 Apr 2013.
  14. 14.
    Zeiss. Live-cell imaging. http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/index.html (2013). Accessed 26 Apr 2013.
  15. 15.
    Andor Technology. iXon Ultra 897. http://www.andor.com/pdfs/specifications/Andor_iXon_Ultra_897_Specifications.pdf (2013). Accessed 3 May 2013.
  16. 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.PubMedCrossRefGoogle Scholar
  17. 17.
  18. 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.PubMedCrossRefGoogle Scholar
  19. 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.PubMedCrossRefGoogle Scholar
  20. 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.PubMedCrossRefGoogle Scholar
  21. 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.PubMedCrossRefGoogle Scholar
  22. 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. 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.PubMedCrossRefGoogle Scholar
  24. 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.PubMedCrossRefGoogle Scholar
  25. 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.PubMedCrossRefGoogle Scholar
  26. 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.PubMedCrossRefGoogle Scholar
  27. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  28. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  29. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  30. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  31. 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.PubMedCrossRefGoogle Scholar
  32. 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.PubMedCrossRefGoogle Scholar
  33. 33.
    Sorokin L. The impact of the extracellular matrix on inflammation. Nat Rev Immunol. 2010;10(10):712–23. doi: 10.1038/nri2852.PubMedCrossRefGoogle Scholar
  34. 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.PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Sara McArdle
    • 1
    • 2
  • Ekaterina Koltsova
    • 1
  • Grzegorz Chodaczek
    • 1
  • Klaus Ley
    • 1
    Email author
  1. 1.Division of Inflammation BiologyLa Jolla Institute for Allergy and ImmunologyLa JollaUSA
  2. 2.Department of BioengineeringUniversity of CaliforniaSan Diego, La JollaUSA

Personalised recommendations