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 therapies^2,3,4,5. 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. ....

Protocol

mT/mG (stock no. 007676), and Apoe^-/- (stock no. 002052) mice were purchased from the Jackson Laboratory. Myh11-CreER^T2 mice were a gift from Stefan Offermanns (available from the Jackson Laboratory as stock no. 019079). Tgfbr2^fl/fl mice were obtained from Harold L. Moses (Vanderbilt University). All animal procedures were performed using protocols approved by the Yale University Institutional Animal Care and Use Committee.

1. Mice

1. Produce MYH11-CreER^T2;mT/mG^f/f;Apoe^-/- and MYH11-CreER

Results

In this protocol, atherosclerotic lesions in TGFβR2^iSMC-Apoe mice were analyzed after 4 months on a HCHF diet⁷. In addition to extensive atherosclerosis, these mice developed both thoracic and abdominal aortic aneurysms, as previously reported. Compared to Apoe^-/- mice, TGFβR2^iSMC-Apoe mice aortic walls showed severe atherosclerosis, making it difficult to dissect the lesions (Figure 2C,D,E). In addition, the aneurysms.......

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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