Models and Analysis of Atherosclerosis, Restenosis, and Aneurysm Formation in the Mouse

Vivian de Waard1, Marion J.J. Gijbels2, Esther Lutgens3, Menno P.J. de Winther1, Carlie J.M. de Vries1

1 Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 2 Department of Pathology and Department of Molecular Genetics, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands, 3 Institute for Cardiovascular Prevention (IPEK), Ludwig Maximilians University (LMU), Munich, Germany
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo120069
Online Posting Date:  December, 2012
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Abstract

Atherosclerosis is considered a chronic inflammatory condition of the vessel wall and involves a high chronic concentration of low‐density lipoprotein (LDL) in blood. In humans, restenosis develops after intravascular interventions such as angioplasty and stent placement to treat atherosclerosis, and this process is characterized by excessive smooth muscle cell proliferation that re‐occludes the vessel lumen. Aortic aneurysm formation is caused by severe degradation and thus dilatation of the vessel wall, in part due to atherosclerosis. Each of these vascular pathologies has its specific characteristics at onset and during development of the disease, and to study the involvement of specific genes in detail, various (transgenic) mice have been generated. Here, we aim to provide detailed insight in considerations to choose and set up the appropriate mouse model for specific vascular research questions. Additionally, we provide technical details to execute experiments with these animal models. Curr. Protoc. Mouse Biol. 2:317‐345 © 2012 by John Wiley & Sons, Inc.

Keywords: atherosclerosis; restenosis; aneurysm; high‐fat/cholesterol diet; LDLR‐knockout mice; ApoE‐knockout mice

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Setup of an Experiment for Diet‐Induced Atherosclerosis
  • Basic Protocol 2: Carotid Artery Ligation to Study Restenosis
  • Basic Protocol 3: Angiotensin II–Induced Aneurysm Formation
  • Support Protocol 1: Excision of the Aortic Root for Embedding for Cryosections
  • Support Protocol 2: Excision of the Arterial Tree (Including Aortic Root, Aortic Arch, and Branch Points) for Formaldehyde Fixation
  • Support Protocol 3: Atherosclerosis Quantification in Cryosections of the Aortic Root
  • Support Protocol 4: Paraffin Embedding of the Aortic Arch and Its Main Branch Points After Formaldehyde Fixation
  • Support Protocol 5: Preparation of Slides for Analysis of Atherosclerosis in the Aortic Arch and Its Main Branch Points
  • Support Protocol 6: Immunohistochemistry on Cryosections
  • Support Protocol 7: Immunohistochemistry on Formalin‐Fixed, Paraffin‐Embedded Sections
  • Support Protocol 8: En Face Aorta Preparation and Sudan IV Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Setup of an Experiment for Diet‐Induced Atherosclerosis

  Materials
  • Mice (age‐and sex‐matched, ≥10 weeks old)
  • Anesthetic mix (see recipe)
  • Eye lubricant
  • 70% ethanol
  • Antagonizing mix (see recipe)
  • Paraffin
  • Hematoxylin and eosin (e.g., Sigma)
  • Operation tablet (12 × 15 cm Perspex sheet)
  • Sensitive Fixation Tape (white tape; Hansaplast, http://int.hansaplast.com/)
  • Hair removal cream (Veet)
  • Gauze
  • Dissecting microscope
  • Surgical instruments:
    • General‐purpose stainless steel forceps (blunt, curved)
    • Stainless steel microtweezers (sharp, straight and curved)
    • General‐purpose stainless steel surgical scissors
    • Microscissors straight
  • Surgical silk sutures (nonabsorbable): natural silk, black, P6/0, metric 0.7
  • Surgical vicryl sutures (absorbable): vicryl P4/0
  • Heating pad
  • Metal molds for paraffin embedding
  • Lids to paraffin blocks
  • Additional reagents and equipment for injection of mice (Donovan and Brown, ) and harvesting of blood vessels for fixation (Basic Protocols protocol 11 and protocol 22)

Basic Protocol 2: Carotid Artery Ligation to Study Restenosis

  Materials
  • 12‐week‐old male ApoE‐/‐ mice
  • Cholesterol‐ and triglyceride‐rich diet (Arieblok Diet W, Hope Farms, cat. no. 4021.06, http://www.hopefarms.nl)
  • Angiotensin II (Sigma‐Aldrich)
  • Sterile saline (0.9% NaCl)
  • Carprofen (Pfizer, 50 mg/ml)
  • Isoflurane
  • 70% ethanol
  • 4% (w/v) paraformaldehyde
  • Osmotic minipumps (Alzet, model 2004: 4 week pump)
  • Standard equipment for isoflurane anesthesia
  • Sensitive Fixation Tape (white tape; Hansaplast)
  • Electric razor
  • Surgical instruments:
    • General‐purpose stainless steel forceps (blunt, curved)
    • General‐purpose stainless steel surgical scissors
    • Wound clips
    • Wound clip applicator
  • Additional reagents and equipment for injection of mice (Donovan and Brown, ) and harvesting of blood vessels for fixation (Basic Protocols protocol 11 and protocol 22)

Basic Protocol 3: Angiotensin II–Induced Aneurysm Formation

  Materials
  • Mouse with induced lesion (Basic Protocol protocol 22 or protocol 33)
  • Tissue‐Tek (Sakura)
  • Isopentane (also called 2‐methylbutane)
  • Dry ice
  • Dissecting board
  • Dissecting equipment including forceps and scissors
  • Dissecting microscope
  • Plastic cryomolds (Sakura)
  • Additional reagents and equipment for euthanasia of the mouse by CO 2 asphyxiation (Donovan and Brown, )

Support Protocol 1: Excision of the Aortic Root for Embedding for Cryosections

  Materials
  • Paraformaldehyde (Merck)
  • Phosphate‐buffered saline (PBS; PAA Laboratories, cat. no. H15‐002; http://www.paa.com/)
  • Sodium nitroprusside dihydrate (MP Biomedicals, Inc.)
  • Normal saline (0.9% NaCl)
  • Mouse with induced lesion (Basic Protocol protocol 11 or protocol 33)
  • Sodium pentobarbital (Nembutal, 60 mg/ml)
  • 0.5 M tetrasodium EDTA
  • Aluminum foil
  • 23‐G, 1‐in. and 30‐G, 0.5‐in. needles
  • 1‐, 10‐, and 50‐ml syringes
  • Tape
  • Scissors, spring scissors, forceps
  • Dissecting microscope
  • Cork
  • Pins
  • Small and large tubes to store tissues
NOTE: You will need 1% (w/v) paraformaldehyde in PBS to perfuse mice (approximately 10 ml/mouse) as well as to store arteries. 4% (w/v) paraformaldehyde is needed to perfuse organs once they are removed (approximately 90 ml/mouse). PBS and nitroprusside are only needed for perfusion (approximately 10 ml/mouse).

Support Protocol 2: Excision of the Arterial Tree (Including Aortic Root, Aortic Arch, and Branch Points) for Formaldehyde Fixation

  Materials
  • Tissue‐Tek (Sakura)
  • 0.2% toluidine blue staining solution (Sigma)
  • ‘Dry’ acetone: 100% analytical‐grade acetone (Merck; dried overnight by adding 0.5 nm molecular sieves, also from Merck)
  • Entellan mounting medium (Merck)
  • Cryotome
  • Glass slides (Starfrost, Knittel)
  • Vacuum desiccator containing small tray of silica gel (Merck)
  • Plastic slide racks containing a small amount of silica gel (Merck)
  • Sealing tape
  • Fan
  • Software capable of morphometric analyses (e.g., Adobe Photoshop, Leica LAS, QWin)
  • Additional reagents and equipment for isolating the mouse heart ( protocol 4)

Support Protocol 3: Atherosclerosis Quantification in Cryosections of the Aortic Root

  Materials
  • Formaldehyde‐fixed tissue sections ( protocol 5)
  • Eosin solution (IHC World; http://www.IHCWorld.com)
  • Paraffin block
  • Dissecting microscope
  • Dissecting instruments including forceps and scissors
  • Filter paper for embedding
  • Paraffin mold
  • Additional reagents and equipment for paraffin embedding (Zeller, )

Support Protocol 4: Paraffin Embedding of the Aortic Arch and Its Main Branch Points After Formaldehyde Fixation

  Materials
  • Sections, either frozen or fixed (Support Protocols protocol 41 or protocol 52, respectively)
  • Hematoxylin and eosin (e.g., Sigma)
  • Microscope equipped with camera/computer and software for quantitative analyses (see protocol 6)

Support Protocol 5: Preparation of Slides for Analysis of Atherosclerosis in the Aortic Arch and Its Main Branch Points

  Materials
  • Isolated heart, frozen at –20°C ( protocol 4)
  • ‘Dry’ acetone: 100% analytical‐grade acetone (Merck; dried overnight by adding 0.5 nm molecular sieves, also from Merck)
  • Phosphate‐buffered saline (PBS; PAA Laboratories, cat. no. H15‐002; http://www.paa.com/)
  • Fetal bovine serum (FBS)
  • Avidin‐biotin complex (ABC) kit (Vector Laboratories)
  • Primary antibody (Table 12.0.6900)
  • Secondary antibody: horseradish peroxidase (HRP)–conjugated antibody appropriately recognizing primary antibody
  • Normal mouse serum (NMS, Sigma)
  • AEC kit (Vector Laboratories)
  • Hematoxylin (Sigma)
  • Aqueous mounting medium (Faramount, Dako)
  • Coplin jars
  • Fan
  • Paraffin pen (Dako)
  • Horizontal staining container: a box with a lid in which the slides can be placed horizontally; to create a humid environment, some wet tissue is placed in the box (for commercial examples, see http://www.IHCWorld.com)
    Table 2.0.1   MaterialsAntibodies for Immunohistochemistry on Mouse Tissue

    Cells Marker Antibody Application Supplier a
    Macrophages CD68 Rat FA‐11 Cryo HBT, Serotec
    Moma2 Rat Moma2 Cryo Serotec
    Mac3 Rat M3/84 Paraffin Pharmingen
    Leukocytes CD45 Rat 30F11 Cryo/paraffin Pharmingen
    T cells CD3 SP‐7 Rabbit SP7 Cryo/paraffin Labvision
    Neutrophils Ly‐6G Rat 1A8 Cryo/Paraffin BD
    SMC αSM‐actin Rat 1A4 Cryo/Paraffin Sigma

     aAbbreviations: HBT, Hycult Biotech; BD, Becton Dickinson.

Support Protocol 6: Immunohistochemistry on Cryosections

  Materials
  • Xylenes
  • Ethanol
  • Hydrogen peroxide (H 2O 2)
  • Methanol
  • Tris‐buffered saline (TBS; 10 mM Tris⋅Cl, pH 7.5 containing 150 mM NaCl)
  • Fetal bovine serum (FBS)
  • Primary antibody (Table 12.0.6900)
  • Bovine serum albumin (BSA;
  • Tween 20
  • Avidin‐biotin complex (ABC) kit (Dako)
  • 3,3′‐diaminobenzidine (Bright DAB solution; ImmunoLogic, cat. no. BS04‐500A/B; http://www.immunologic.nl/)
  • Vector Red (Vector Laboratories, cat. no. SK4800)
  • Hematoxylin (Sigma)
  • Imsol (Klinipath, http://www.klinipath.nl/)
  • Entellan mounting medium (Merck)
  • Coplin jars
  • Paraffin pen (Dako)

Support Protocol 7: Immunohistochemistry on Formalin‐Fixed, Paraffin‐Embedded Sections

  Materials
  • 1% (w/v) paraformaldehyde
  • Phosphate‐buffered saline (PBS; PAA Laboratories, cat. no. H15‐002; http://www.paa.com/)
  • 70% ethanol
  • Sudan IV solution (Klinipath, http://www.klinipath.nl/)
  • 80% ethanol
  • Coplin jars
  • Spring scissors
  • Standard black wax dissection pan
  • 0.15‐mm black anodized pins (Fine Science Tools)
  • Camera‐equipped dissecting microscope
  • Leica Quantimet software or ImageJ
  • Additional reagents and equipment for excision of the arterial tree ( protocol 5)
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Figures

Videos

Literature Cited

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Internet Resources
   http://www.hbt.nl
  Web site of Hycult Biotech.
  http://www.ab‐direct.com/index
  Web site of AbD Serotec.
   http://www.bdbeurope.com/
  Web site of Becton Dickinson.
   http://www.sigmaaldrich.com
  Sigma‐Aldrich Web site.
   http://www.labvision.com
  Web site of Thermo Scientific Lab Vision.
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