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 development1-3. 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 atherosclerosis is diet induced. An important advantage of these models is that they permit the study of relatively large numbers of genetically defined animals under controlled dietary and environmental conditions. In addition, the advanced atherosclerotic lesions that develop in some of these mouse strains appear to be very similar to those observed in human subjects, containing lipid filled necrotic cores and fibrous caps. Atherosclerosis develops rapidly in these mouse models making it very feasible to study lesion development, from fatty streak to advanced plaque, over a matter of weeks. Furthermore, known risk factors for cardiovascular disease in humans, including diabetes6, dyslipidemia7, obesity8, hypertension9, cigarette smoke10, and a sedentary environment11 have been shown to further accelerate lesion development.
In most, if not all atherosclerosis-prone mouse models, lesion development can be first detected at the aortic sinus. The time of onset depends on mouse strain and diet. As lesions increase in size they tend to grow up the ascending aorta. In ApoE-/- and LDLR-/- mice, subsequent lesion development can be detected at aortic bifurcations in the aortic arch, in the descending aorta and in other larger arteries12-14. Dr Beverly Paigen and colleagues, in addition to developing some of the earliest models of diet-induced atherosclerosis, also established an assay for the quantification of atherosclerotic lesions at the aortic sinus that has become the standard of lesion measurement in mouse models15. Over the years this technique has been refined and described in detail16. Here we present a modified version of the "Paigen method" for the quantification and characterization of atherosclerotic lesions in a mouse. The analysis of serial aortic cross sections from a specific vascular region and in a defined and fixed orientation, facilitates precise data collection and permits the accurate detection of variations in lesion development in different treatment groups. The methods presented here builds upon the previous techniques. Specifically, we describe how the characterization of lesion in terms of area, volume, necrotic, and cellular content is possible through the examination of serial sections using a combination of histochemistry and immunohistochemistry.
Protocol
Representative Results
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This research was funded by an operating grant from the Canadian Institutes of Health Sciences and the Canadian Diabetes Association. DEV is supported by a scholarship from the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT, Chile).
Materials
| 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 |
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