Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Summary

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Abstract

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

Introduction

In the last two decades the development and use of atherosclerosis-prone mouse models through dietary and/or genetic manipulation have significantly increased our understanding of the molecular and cellular mechanisms involved in atherosclerotic lesion development^1-3. A great deal of our knowledge and understanding of atherogenesis comes from studies carried out in apolipoprotein (Apo)-E deficient⁴ mice, in which atherosclerotic lesions develop spontaneously and low density lipoprotein receptor (LDLR)-deficient⁵ mice, in which ....

Protocol

The McMaster University Animal Research Ethics Board has preapproved all procedures described herein.

1. Harvesting Heart and Aorta

1. Anesthetize the mouse and carefully open the chest cavity to reveal the heart and proximal aorta.
   1. Extract the blood from the mouse by direct punctuation of the right ventricle of the heart.
      Note: The blood can be stored and used for the analysis of plasma lipids and other blood borne factors.
2. Euthanize the mouse by cervical dislocation.
3. Rinse the vasculature with 5 ml of saline.
   Note: This is accomplished by gravity perfusion through a nee....

Results

 Five week old LDLR-/- mice were fed a standard diet or a high fat diet for ten weeks. Mice were sacrificed and perfused with formalin as described above. Cross sections of the aortic sinus were prepared and stained with hematoxylin and eosin to determine lesion area and volume (Figure 4). When mice are fed a standard chow diet, atherosclerotic development is very limited and may not be detectable at 15 weeks of age. High fat diet significantly accelerates atherogenesis in this model and induces the form.......

Discussion

Atherosclerosis is a complex chronic disease of the large muscle arteries that is a major underlying cause of myocardial infarction and stroke. Disease progression involves the interplay of many different cell types within the artery wall, with circulating blood cells, lipoprotein particles and other blood-borne factors that we are just beginning to understand. Much of our current knowledge regarding the development and progression of atherosclerosis has come from studies carried out in specially designed atherosclerosis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LDLR-deficient mice (D2.129S7(B6)-Ldlrtm1Her/J)	The Jackson Laboratory	stock #002207
Formalin	Sigma-Aldrich	HT5011
Histology Molds	Ted Pella Inc	27195
Paraffin	Paramat	19286-10
Coated slides	Fisher	12-550-15
Solvent containers	Tissue Tek II	4457
Microtome	Leica	RM22S5
Water bath	VWR	80086-982
Microscope	Olympus	BX41TF
Camera	Olympus	DP72
ImageJ	NIH	www.rsbweb.nih.gov/ij/
Processor	Sakura Finetek	VIP6-A1
Processing cassettes	VWR	18000-134
Preassure Cooker	Nordic Ware
Coverslip	Fisher	12-545-M
PBS	Sigma-Aldrich	P3813
Microtome blade	Thermo Scientific	30-518-35
Antifade mounting medium	Sigma-Aldrich	F-4680
Xylene mounting medium	Sigma-Aldrich	44581
Aquous mounting medium	Sigma-Aldrich	I1161
Mac-3 anbody	BD Pharmingen	553322
DAPI	Sigma-Aldrich	D9542
F4/80 antibody	Abcam	Ab6640
Alpha actin antibody	Santa Cruz Biotechnology	SC-32251
Mayer’s Hematoxylin	Sigma-Aldrich	E4382
Eosin Y	Sigma-Aldrich	MHS16
DAB (3,3'-Diaminobenzidine)	Dako	K3468