Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model
Summary
This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals.
Abstract
Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for atherosclerosis research. They are used to study the impact of a various genetic factors and different cell types on atherosclerotic lesion formation and as well as test the development of new therapies. Isolation, excision of the whole aorta, and quantification of Oil Red O-stained atherosclerotic lesions are basic morphometric methods used to evaluate atherosclerotic burden. The goal of this protocol is to describe an optimized, step-by-step surgical method to dissect, perfuse-fix, isolate, stain, image and analyze atherosclerotic lesions in mouse aortas with Oil Red O. Because atherosclerotic lesions can form anywhere in the entire aortic tree, this whole aorta Oil Red O staining method has the advantage of evaluating lipid-laden plaques in the entire aorta and all branches in a single mouse. In addition to Oil Red O staining, fresh isolated whole aortas can be used for variety of in vitro and in vivo experiments and cell isolations.
Introduction
Coronary artery disease, a leading cause of mortality in the US, is usually caused by atherosclerosis, a process that leads to the buildup of plaque inside arterial walls1. Hyperlipidemia-prone Apoe- and Ldlr-deficient mice are central to investigations of atherosclerosis and its complications and development of therapies2,3,4,5. Quantification of atherosclerotic lesions from an en face aorta is an important endpoint analysis for evaluating the impact of genetic manipulation in different cell types. It also helps to study novel therapies designed to affect atherosclerotic disease initiation, progression, and regression. Atherosclerotic lesions can form anywhere in the aorta and its branches (i.e., brachiocephalic, carotid and subclavian arteries in the chest, as well as renal, common iliac and femoral arteries below the diaphragm)6. A comprehensive evaluation of atherosclerosis burden and appropriate therapy requires assessment of disease burden in different locations, a challenge that is often overlooked.
This protocol describes how to perform a comprehensive analysis of atherosclerotic lesions, starting with an unopened whole aorta and proceeding to en face preparation, in a single mouse. Unopened whole aorta Oil Red O staining allows rapid, qualitative assessment of lipid-laden plaques in the entire aorta and its branches, while en face preparation provides a quantitative assessment of atherosclerotic lesion distribution in the mouse aorta.
The technique uses 8 week-old mice with a smooth muscle cell-specific TGFβR2 deletion on the Apoe-/- hyperlipidemic background (MYH11-CreERT2;Tgfbr2f/f;mT/mGf/f;Apoe-/-; hereafter referred to as TGFβR2iSMC-Apoe mice) and littermate Apoe-/- controls (MYH11-CreERT2;mT/mGf/f;Apoe-/-; hereafter referred to as Apoe-/- mice). The animals are kept for 16 weeks on a high cholesterol high fat diet (HCHFD) as study materials7. At study termination, the unopened whole aortas are stained and imaged (including all major branches) with Oil Red O for qualitative assessment of lipid-laden plaques. The aortas are cut open via en face preparation, and all atherosclerotic lesions are imaged and quantified. This protocol can be used to study atherosclerotic lesion development in Apoe-/- or Ldlr-/- hyperlipidemia mice models and extended to general aorta-related vascular biology applications.
Protocol
Representative Results
Discussion
Apolipoprotein E (Apoe) and low density lipoprotein receptor (Ldlr) deficient mice are useful for studying development and treatment of atherosclerosis. Investigators can evaluate the impact of genetics and therapeutic manipulations on atherosclerosis-related diseases initiation, progression, and regression using Oil Red O staining of the whole aorta9. Aorta Oil Red O staining and lesion quantification is the gold standard endpoint for atherosclerosis research. This technique is …
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported, in part, by a Joint Biology Consortium Microgrant provided under NIH grant P30AR070253 (P.-Y.C.), and HL135582 (M.S.). We are grateful to R. Webber and L. Coon for maintaining the mice used in this study.
Materials
| 1.5 mL Eppendorf tube | DENVILLE | C2170 | |
| 10 mL syringe | BD | 302995 | |
| 16% Formaldehyde | Polysciences | 18814-10 | |
| 70% ethanol | VWR | RC2546.70-5 | To clean the dissection tools |
| Black dissection wax | CR Scientific | C3541 | |
| Corn oil | Sigma | C8267 | Solvent for Tamoxifen |
| DNeasy Blood & Tissue kit | QIAGEN | 69506 | To isolate DNA from mouse ear |
| Dulbecco’s Phosphate-buffered saline (1X DPBS), pH 7.4 | Gibco | 14190-144 | |
| Fine scissors | Fine Science Tools | 14059-11 | To cut the mouse skin and open the ribcage |
| Fisherbrand Economy Plain Glass Microscope Slides | Fisher Scientific | 12-550-A3 | |
| High cholesterol high fat diet | Research Diets | D12108 | To induce atherosclerosis |
| Imaging software | National Institutes of Health | Image J | Aortic lesion quantification |
| Isopropanol | VWR | JT9079-5 | |
| Kimwipes | Fisher Scientific | 06-666A | To clean the glass microscope slides |
| McPherson-Vannas Micro Dissecting Spring Scissors | ROBOZ | RS-5602 | To separate the heart and the aorta and to cut open the aorta and aorta branches |
| Microscope control software | Olympus | DP Controller | For aorta imaging |
| Minutien pins | Fine Science Tools | 26002-10 | |
| Needle-25G | BD | 305124 | |
| NonWoven Sponge | McKesson | 94442000 | |
| Oil Red O | Sigma | O-0625 | To stain the atherosclerosis lesions |
| Pall Acrodisc Sterile Syringe Filters with Super Membrane | VWR | 28143-312 | To filter working Oil Red O solution |
| Spring Scissors | Fine Science Tools | 15021-15 | To dissect and clean the aorta |
| Statistical software | GraphPad | Prism 8 | Statical analysis |
| Stereomicroscope | Nikon | SMZ1000 | For aorta dissection |
| Stereomicroscope | Olympus | SZX16 | For aorta imaging |
| Tamoxifen | Sigma | T5648 | To induce Cre-loxP recombination |
| Tissue-Tek O.C.T Compound, Sakura Finetek | VWR | 25608-930 | |
| Tweezer Style 4 | Electron Microscopy Sciences | 0302-4-PO | To cut the mouse skin and open the ribcage |
| Tweezer Style 5 | Electron Microscopy Sciences | 0302-5-PO | To dissect and clean the aorta |
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