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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Abstract

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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Materials

Basic Protocol 1: Induction of Atherosclerotic Disease in Genetically Susceptible Mice

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

Videos

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