Mouse Models of Atherosclerosis

E. Maganto‐Garcia1, M. Tarrio1, A.H. Lichtman1

1 Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.24
DOI:  10.1002/0471142735.im1524s96
Online Posting Date:  February, 2012
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Genetically altered mice carrying mutations of genes encoding crucial components of the immune system and lipid metabolism have been widely used to study the role of immune responses and inflammation in atherosclerosis. These mice are often fed a diet, with a high content of cholesterol and saturated fat in order to induce hypercholesterolemia and arterial lesions. We review the different mouse models of atherosclerosis, type of diets, and techniques to measure lipid deposition and lesion size in the arterial walls. Moreover, the methods used to determine the presence of the immune cells in atherosclerotic lesions are also described here. Curr. Protoc. Immunol. 96:15.24.1‐15.24.23. © 2012 by John Wiley & Sons, Inc.

Keywords: atherosclerosis; mice; diet; lipids; staining

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Table of Contents

  • Introduction
  • Choice of Mouse Models to Study Atherosclerosis
  • Sex and Age of Mice
  • Basic Protocol 1: Induction of Atherosclerotic Disease in Genetically Susceptible Mice
  • Support Protocol 1: Measurement of Blood Lipids in Mice
  • Basic Protocol 2: Quantitative Analysis of Mouse Aortic Atherosclerotic Lesions
  • Alternate Protocol 1: Preparation of Aortic Arch Sections for Histologic Analysis
  • Basic Protocol 3: Lipid Staining of Aortic Lesions
  • Alternate Protocol 2: Sudan IV Staining of Whole Aortas for En Face Analysis
  • Basic Protocol 4: Quantification of Atherosclerotic Lesions
  • Support Protocol 2: Collagen Staining of Mouse Aortas
  • Support Protocol 3: Immune Cells Detection in Aortic Tissue
  • Support Protocol 4: Immunofluorescence Microscopy
  • Support Protocol 5: Flow Cytometric Analysis of Aortic Digests
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Induction of Atherosclerotic Disease in Genetically Susceptible Mice

  • Chow‐ or cholesterol‐containing diets (see Table 15.24.3; e.g., Research Diets, Harlan Laboratories, or Purina Diets)
  • Mice genetically altered (6‐ to 8‐week‐old ldlr− /− mice)
  • Additional reagents and equipment for euthanizing the animal (unit 1.8)

Support Protocol 1: Measurement of Blood Lipids in Mice

  • Mice under control or cholesterol diet
  • Total cholesterol analysis kit (Raichem)
  • ELISA kits
  • EDTA (American Bioanalytical)
  • Isoflurane (Webster Veterinary)
  • Capillary blood collection tubes (Fisher)
  • Additional reagents and equipment for blood collection from mice (unit 1.7)

Basic Protocol 2: Quantitative Analysis of Mouse Aortic Atherosclerotic Lesions

  • Mice with atherosclerotic disease ( protocol 1)
  • Phosphate‐buffered saline (Invitrogen, cat. no. 10010‐023)
  • Heparin (Sigma)
  • OCT (optimal cutting temperature compound; Tissue‐Tek)
  • Isopentane
  • Mini Vanna scissors and forceps
  • 10‐ml syringes
  • 27‐G needles
  • Dissecting light microscope
  • Cryosection plastic mold
  • −80°C freezer
  • Cryostat microtome
  • Camel hair brush, No3
  • Glass slides (FisherBrand)
  • Plastic wrap
  • Additional reagents and equipment for euthanizing the mouse (unit 1.8) and immunohistochemistry ( protocol 9)

Alternate Protocol 1: Preparation of Aortic Arch Sections for Histologic Analysis

  • Frozen sections (see protocol 3)
  • 10% neutral buffered formalin (Fisher)
  • Distilled water
  • 100% and 85% propylene glycol (Fisher)
  • Oil Red O (ORO; Solvent Red 27, Sudan Red 5B, C.I. 26125, C 26H 24N 4O) solution (see recipe)
  • Mayer's Hematoxylin solution (Newcomer Supply)
  • Glycerol/gelatin mounting medium (Sigma)
  • 4% (v/v) paraformaldehyde/PBS (Fisher)
  • Phosphate‐buffered saline (PBS; Invitrogen cat. no. 10010‐023)
  • 0.1% (v/v) Triton X‐100/PBS (MP Biomedicals)
  • 36% (v/v) triethylphosphate/H 2O (Fisher)
  • Filipin (Sigma)
  • Dimethyl sulfoxide (DMSO)
  • Mounting medium with DAPI (Vectashield, Vector Laboratories)
  • 37° or 60°C incubator
  • Coverslips (Fisherfinest)
  • Digital camera
  • Light microscope
  • Fluorescence microscope
  • 4°C shaking incubator
  • Silicon‐elastomere (Factor II) plates
  • 60‐mm dishes
  • Stainless steel minutien pins (Fine Science Tools)
  • 25°C incubator

Basic Protocol 3: Lipid Staining of Aortic Lesions

  • Sudan IV (Sigma)
  • 70% (v/v) ethanol (Fisher)
  • Acetone (Fisher)
  • Distilled water
  • Light microscope

Alternate Protocol 2: Sudan IV Staining of Whole Aortas for En Face Analysis

  • ORO‐stained sections of aortic root and aortic valve (see protocol 5)
  • Sudan IV‐stained sections (see protocol 6)
  • IMAGEPRO PLUS software (Media Cybernetics)

Basic Protocol 4: Quantification of Atherosclerotic Lesions

  • Mice with atherosclerotic disease ( protocol 1)
  • Tap water
  • Distilled water
  • Sirius Red F3BA (Polysciences)
  • Picric Acid (VWR)
  • HCl (Sigma)
  • Xylene (Fisher)
  • Appropriate aqueous mounting medium
  • Slide rack
  • Light microscope
  • Additional reagents and equipment for removing aortas ( protocol 3) and fixing frozen sections in formalin ( protocol 5)

Support Protocol 2: Collagen Staining of Mouse Aortas

  • Sections of aorta (see protocol 3)
  • Acetone
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (CMF‐PBS, Invitrogen, cat. no. 10010‐023)
  • Normal serum (serum of the species that the secondary biotinylated antibody is raised in; use heat‐inactivated serum)
  • Wash fluid (see recipe)
  • Avidin/Biotin Blocking kit (Vector) containing:
    • Avidin solution
    • Biotin solution
  • Primary antibody (e.g., rat anti‐mouse CDY, clone RM4‐5; Pharmingen)
  • Secondary antibody (e.g., biotinylated goat anti‐rat Ig; Invitrogen)
  • Hydrogen peroxide
  • ABC complexes (Vector)
  • 0.1 M acetate buffer, pH 5.2 (see recipe)
  • AEC solution (see recipe)
  • Gill's Hematoxilin No. 2 (Fisher)
  • Aqueous mounting medium
  • Coverslips
  • Light microscope

Support Protocol 3: Immune Cells Detection in Aortic Tissue

  • 4% paraformaldehyde
  • En face sections of the aortic arch (see protocol 3)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010‐023)
  • Bovine serum albumin (BSA; American Bioanalytical)
  • Primary antibodies (e.g., rat anti‐mouse CD4; Biolegend)
  • Secondary antibodies (e.g., Alexa 555‐labeled goat anti‐rat Ig; Invitrogen)
  • Immunofluorescence microscope

Support Protocol 4: Immunofluorescence Microscopy

  • Mouse aortas (see protocol 3)
  • Digestion buffer (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010‐023)
  • Fluorescently labeled antibodies (e.g., APC‐labeled rat anti‐mouse CD3; Pharmingen)
  • Surgical scissors
  • 70‐µm strainer (BD Bioscience)
  • 3‐ml syringes
  • Centrifuge
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Literature Cited

   An, G., Miwa, T., Song, W.L., Lawson, J.A., Rader, D.J., Zhang, Y., and Song, W.C. 2009. CD59 but not DAF deficiency accelerates atherosclerosis in female ApoE knockout mice. Mol. Immunol. 46:1702‐1709.
   Basso, F., Amar, M.J., Wagner, E.M., Vaisman, B., Paigen, B., Santamarina‐Fojo, S., and Remaley, A.T. 2006. Enhanced ABCG1 expression increases atherosclerosis in LDLr‐KO mice on a western diet. Biochem. Biophys. Res. Comm. 351:398‐404.
   Buono, C., Come, C.E., Stavrakis, G., Maguire, G.F., Connelly, P.W., and Lichtman, A.H. 2003. Influence of interferon‐gamma on the extent and phenotype of diet‐induced atherosclerosis in the LDLR‐deficient mouse. Arterioscler. Thromb. Vasc. Biol. 23:454‐460.
   Buono, C., Binder, C.J., Stavrakis, G., Witztum, J.L., Glimcher, L.H., and Lichtman, A.H. 2005. T‐bet deficiency reduces atherosclerosis and alters plaque antigen‐specific immune responses. Proc. Natl. Acad. Sci. U.S.A. 102:1596‐1601.
   Cybulsky, M.I., Lichtman, A.H., Hajra, L., and Iiyama, K. 1999. Leukocyte adhesion molecules in atherogenesis. Clin. Chim. Acta 286:207‐218.
   Dansky, H.M., Charlton, S.A., Harper, M.M., and Smith, J.D. 1997. T and B lymphocytes play a minor role in atherosclerotic plaque formation in the apolipoprotein E‐deficient mouse. Proc. Natl. Acad. Sci. U.S.A. 94:4642‐4646.
   Fuster, V., Ross, R., and Topol, E.J. 1996. Atherosclerosis and Coronary artery disease, vol. 1. Lippincott‐Raven, Philadelphia.
   Galkina, E. and Ley, K. 2009. Immune and inflammatory mechanisms of atherosclerosis. Ann. Rev. Immunol. 27:165‐197.
   Galkina, E., Kadl, A., Sanders, J., Varughese, D., Sarembock, I.J., and Ley, K. 2006. Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L‐selectin dependent. J. Exp. Med. 203:1273‐1282.
   Gotsman, I., Grabie, N., Dacosta, R., Sukhova, G., Sharpe, A., and Lichtman, A.H. 2007. Proatherogenic immune responses are regulated by the PD‐1/PD‐L pathway in mice. J. Clin. Invest. 117:2974‐2982.
   Hansson, G.K. and Libby, P. 2006. The immune response in atherosclerosis: A double‐edged sword. Nat. Rev. 6:508‐519.
   Iiyama, K., Hajra, L., Iiyama, M., Li, H., DiChiara, M., Medoff, B.D., and Cybulsky, M.I. 1999. Patterns of vascular cell adhesion molecule‐1 and intercellular adhesion molecule‐1 expression in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation. Circ. Res. 85:199‐207.
   Ishibashi, S., Brown, M.S., Goldstein, J.L., Gerard, R.D., Hammer, R.E., and Herz, J. 1993. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus‐mediated gene delivery. J. Clin. Invest. 92:883‐893.
   Ishibashi, S., Goldstein, J.L., Brown, M.S., Herz, J., and Burns, D.K. 1994. Massive xanthomatosis and atherosclerosis in cholesterol‐fed low density lipoprotein receptor‐negative mice. J. Clin. Invest. 93:1885‐1893.
   Junqueira, L.C., Bignolas, G., and Brentani, R.R. 1979. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem. J. 11:447‐455.
   Koopman, R., Schaart, G., and Hesselink, M.K. 2001. Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids. Histochem. Cell Bio. 116:63‐68.
   Libby, P. 2004. What happens inside an atherosclerotic plaque? International Congress Series Atherosclerosis XIII Proceedings of the 13th International Atherosclerosis Symposium 1262:253‐256.
   Lichtman, A.H., Clinton, S.K., Iiyama, K., Connelly, P.W., Libby, P., and Cybulsky, M.I. 1999. Hyperlipidemia and atherosclerotic lesion development in LDL receptor‐deficient mice fed defined semipurified diets with and without cholate. Arterioscler. Thromb. Vasc. Biol. 19:1938‐1944.
   Mach, F., Schonbeck, U., Sukhova, G.K., Atkinson, E., and Libby, P. 1998. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature 394:200‐203.
   Mallat, Z., Taleb, S., Ait‐Oufella, H., and Tedgui, A. 2009. The role of adaptive T cell immunity in atherosclerosis. J. Lipid Res. 50:S364‐S369.
   Merat, S., Casanada, F., Sutphin, M., Palinski, W., and Reaven, P.D. 1999. Western‐type diets induce insulin resistance and hyperinsulinemia in LDL receptor‐deficient mice but do not increase aortic atherosclerosis compared with normoinsulinemic mice in which similar plasma cholesterol levels are achieved by a fructose‐rich diet. Arterioscler. Thromb. Vasc. Biol. 19:1223‐1230.
   Nakashima, Y., Plump, A.S., Raines, E.W., Breslow, J.L., and Ross, R. 1994. ApoE‐deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler. Thromb. Vasc. Biol. 14:133‐140.
   Packard, R.R., Maganto‐Garcia, E., Gotsman, I., Tabas, I., Libby, P., and Lichtman, A.H. 2008. CD11c(+) dendritic cells maintain antigen processing, presentation capabilities, and CD4(+) T‐cell priming efficacy under hypercholesterolemic conditions associated with atherosclerosis. Circ. Res. 103:965‐973.
   Paigen, B., Morrow, A., Brandon, C., Mitchell, D., and Holmes, P. 1985. Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis 57:65‐73.
   Plump, A.S., Smith, J.D., Hayek, T., Aalto‐Setala, K., Walsh, A., Verstuyft, J.G., Rubin, E.M., and Breslow, J.L. 1992. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E‐deficient mice created by homologous recombination in ES cells. Cell 71:343‐353.
   Powell‐Braxton, L., Veniant, M., Latvala, R.D., Hirano, K.I., Won, W.B., Ross, J., Dybdal, N., Zlot, C.H., Young, S.G., and Davidson, N.O. 1998. A mouse model of human familial hypercholesterolemia: Markedly elevated low density lipoprotein cholesterol levels and severe atherosclerosis on a low‐fat chow diet. Nat. Med. 4:934‐938.
   Prophet, E.B., Mills, B., Arrington, J.B., and Sobin, L.H. 1992. Laboratory Methods in Histotechnology, pp. 66‐67. Armed Forces Institute of Pathology, Washington, D.C.
   Puchtler, H., Waldrop, F.S., and Valentine, L.S. 1973. Polarization microscopic studies of connective tissue stained with picro‐sirius red FBA. Beitr. Pathol. 150:174‐187.
   Purcell‐Huynh, D.A., Farese, R.V. Jr., Johnson, D.F., Flynn, L.M., Pierotti, V., Newland, D.L., Linton, M.F., Sanan, D.A., and Young, S.G. 1995. Transgenic mice expressing high levels of human apolipoprotein B develop severe atherosclerotic lesions in response to a high‐fat diet. J. Clin. Invest. 95:2246‐2257.
   Sanan, D.A., Newland, D.L., Tao, R., Marcovina, S., Wang, J., Mooser, V., Hammer, R.E., and Hobbs, H.H. 1998. Low density lipoprotein receptor‐negative mice expressing human apolipoprotein B‐100 develop complex atherosclerotic lesions on a chow diet: No accentuation by apolipoprotein(a). Proc. Natl. Acad. Sci. U.S.A. 95:4544‐4549.
   Schiller, N.K., Kubo, N., Boisvert, W.A., and Curtiss, L.K. 2001. Effect of gamma‐irradiation and bone marrow transplantation on atherosclerosis in LDL receptor‐deficient mice. Arterioscler. Thromb. Vasc. Biol. 21:1674‐1680.
   Taatjes, D.J., Wadsworth, M.P., Schneider, D.J., and Sobel, B.E. 2000. Improved quantitative characterization of atherosclerotic plaque composition with immunohistochemistry, confocal fluorescence microscopy, and computer‐assisted image analysis. Histochem. Cell Biol. 113:161‐173.
   van Ree, J.H., van den Broek, W.J., Dahlmans, V.E., Groot, P.H., Vidgeon‐Hart, M., Frants, R.R., Wieringa, B., Havekes, L.M., and Hofker, M.H. 1994. Diet‐induced hypercholesterolemia and atherosclerosis in heterozygous apolipoprotein E‐deficient mice. Atherosclerosis 111:25‐37.
   van Vlijmen, B.J., van den Maagdenberg, A.M., Gijbels, M.J., van der Boom, H., HogenEsch, H., Frants, R.R., Hofker, M.H., and Havekes, L.M. 1994. Diet‐induced hyperlipoproteinemia and atherosclerosis in apolipoprotein E3‐Leiden transgenic mice. J. Clin. Invest. 93:1403‐1410.
   VanderLaan, P.A., Reardon, C.A., and Getz, G.S. 2004. Site specificity of atherosclerosis: Site‐selective responses to atherosclerotic modulators. Arterioscler. Thromb. Vasc. Biol. 24:12‐22.
   Whitfield, G.B., Brock, T.D., Ammann, A., Gottlieb, D., and Carter, H.E. 1955. Filipin, an Antifungal Antibiotic: Isolation and Properties. J. Am. Chem. Soc. 77:4799‐4801.
   Whitman, S.C., Ravisankar, P., and Daugherty, A. 2002. IFN‐gamma deficiency exerts gender‐specific effects on atherogenesis in apolipoprotein E‐/− mice. J. Interferon. Cytokine Res. 22:661‐670.
   Whittaker, P., Kloner, R.A., Boughner, D.R., and Pickering, J.G. 1994. Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light. Basic Res. Cardiol. 89:397‐410.
   Wilson, L.B. 1905. A method for the rapid preparation of fresh tissues for the microscope. J. Am. Med. Assoc. 45:1737.
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