Models and Analysis of Atherosclerosis, Restenosis, and Aneurysm Formation in the Mouse
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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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PDF or HTML at Wiley Online LibraryTable 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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Basic Protocol 1: Setup of an Experiment for Diet‐Induced Atherosclerosis
Materials
Basic Protocol 2: Carotid Artery Ligation to Study Restenosis
Materials
Basic Protocol 3: Angiotensin II–Induced Aneurysm Formation
Materials
Support Protocol 1: Excision of the Aortic Root for Embedding for Cryosections
Materials
Support Protocol 2: Excision of the Arterial Tree (Including Aortic Root, Aortic Arch, and Branch Points) for Formaldehyde Fixation
Materials
Support Protocol 3: Atherosclerosis Quantification in Cryosections of the Aortic Root
Materials
Support Protocol 4: Paraffin Embedding of the Aortic Arch and Its Main Branch Points After Formaldehyde Fixation
Materials
Support Protocol 5: Preparation of Slides for Analysis of Atherosclerosis in the Aortic Arch and Its Main Branch Points
Materials
Support Protocol 6: Immunohistochemistry on Cryosections
Materials
Support Protocol 7: Immunohistochemistry on Formalin‐Fixed, Paraffin‐Embedded Sections
Materials
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Figure 1. Carotid artery ligation and processing of the carotid artery after harvest. (A ) In the left panel, the positions of the trachea and the salivary glands are indicated when the skin is opened at the onset of the surgery to ligate the carotid artery. In the right panel, the branching of the right and left carotid artery from the aortic arch are schematically indicated, as well as the bifurcation of the left carotid artery and the position of the ligation. (B ) Schematic overview of the carotid artery after ligation and lesion formation. The reference point is indicated (see text for further details), and on the right the corresponding sections are shown. The remnants of suture and the internal and external elastic laminae are indicated. (C ) Once the ligated left carotid artery and the non‐ligated right carotid artery are harvested, they need to be embedded in paraffin. To illustrate the embedding procedure, two small red rods are used in the pictures (the carotids are white and much more difficult to see in solid paraffin). In panel 1, it is shown that the carotids are lined up with the suture of the left carotid at the bottom. A thin layer of paraffin is poured in and allowed to solidify. With a warm tweezer, a scratch is made in the paraffin (panel 2), and the piece of paraffin with the carotid is folded upward (Panel 3). The mold is filled with paraffin (panel 4) and solidified with the lid on top (panel 5). Sectioning is this way started from the ligation into the lesion. View Image -
Figure 2. Aneurysms. (A ) A saccular aneurysm in the ascending aorta is shown macroscopic on the left with an arrow pointing at the lesion. A cross‐section of the aortic arch is shown in overview in the middle and the saccular aneurysm is enlarged in the right panel. (B ) A thoracic aneurysm is shown on the left within the total aorta and in more detail on the right. (C ) An aorta with an AAA is shown and on the right an AAA is presented in more detail. (D ) Cross‐sections of the abdominal aorta are stained with Masson's Trichrome stain, showing adventitia (collagen) in blue and the medial SMCs and foam cell macrophages (cytoplasm) in red/pink. In the left panel, the control aorta is shown with a healthy ratio between media and adventitia. In the middle panel, the media is disrupted (arrow at medial dissection), and in the right panel, extensive remodelling in an atherosclerotic aneurysm is shown, where the media is disintegrated (arrows). View Image -
Figure 3. Schematic overview of the heart. To illustrate how the heart needs to be processed to obtain sections from the aortic root at the aortic valves, a schematic of the heart is given and the red dotted lines indicate where the heart needs to be cut under the tips of the atria at the bottom and through the aorta at the top. The heart is sectioned in the direction of the arrow (see also Fig. 4). View Image -
Figure 4. Work flow of lesion assessment in the aortic root. (A ) Indicated are the different locations encountered in the aortic root when sectioning. Sections are collected once the aortic wall thickens and the valve cusps are present; sectioning is stopped when the valve cusps have disappeared. (B ) The sections from the area of A are collected on 24 slides, where the first proper section is put on slide 1, the second section on slide 2, etc. The 25th section is put on slide 1 again, next to the first section. Sections are collected in this way until the valve cusps have disappeared, as indicated in A. (C ) Representative example of an aortic root section. In the right panel, the atherosclerotic lesions in the areas under the three valves are filled in; this is the area that needs to be determined to assess total lesion area in this section. Note that the lesions attached to the cusps are only partially included for the measurement. View Image -
Figure 5. Aortic root lesions. (A ) Healthy vessel wall of the aortic root. (B ‐F ) Atherosclerotic lesions at different stages of the disease. (B ) early fatty streak; (C ) regular fatty streak; (D ) mild plaque; (E ) moderate plaque; and (F ) severe plaque, as described in , step 19 (semiquantitative evaluation of the lesion by simplified classification). View Image -
Figure 6. Aortic arch with lesions. (A ) Macroscopic view of an aortic arch with the branching arteries indicated, stained whole mount with Oil‐Red‐O to visualize atherosclerosis at the various sites in the arch. (B ‐F ) Atherosclerotic lesions at different stages of the disease. (B) Unaffected artery; (C) intimal xanthoma (fatty streak); (D) mild plaque with fibrous cap; (E) advanced fibrous cap atheroma with cholesterol clefts; and (F), advanced fibrous cap atheroma with intraplaque hemorrhage (star). View Image -
Figure 7. En face staining of lipids in aorta. The entire aorta including the aortic arch was harvested and stained for lipids with Sudan IV. Afterwards, the aorta was cut open longitudinally and pinned down for quantitative analysis. View Image
Videos
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