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 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 arteries^12-14. 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

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 needle puncture into the left ventricle. To permit drainage, a small incision is made in the right atrium. After rinsing the vasculature, tissue samples of liver, muscle and adipose tissue can be collected, flash frozen in liquid nitrogen, and stored at -80 ºC for future analysis.
4. The vasculature is fixed by gravity perfusion into the left ventricle with 10% neutral buffered formalin.
   Note: Once the perfusion is started, the flow of 10% neutral buffered formalin is adjusted to a slow drip so that the process takes 1-2 min. Organs and tissues that have been perfusion fixed can be used to the study of protein expression through immunofluorescence or histochemistry, but they generally cannot be used as samples for western blotting or to the study of gene expression.
5. Carefully extract the heart and approximately 2 mm of proximal aorta from the carcass. Other tissues including the descending aorta can be collected and stored for subsequent analysis.
   Note: The descending aorta (to the iliac bifurcation) can also be used to examine atherosclerosis.
6. Make a transversal cut with a scalpel on the removed heart in a manner that is perpendicular to the ascending aorta.
   Note: This is facilitated by the use of a dissecting microscope and can be effectively done by cutting along a straight line joining the lower tips of the right and left atria (Figure 1).
7. Store the apical (top) part of the heart in an embedding cassette and submerge it in neutral buffered formalin for processing.

2. Preparation of the Aorta

1. Place the cassette with the dissected tissue into the tissue processor.
2. Process tissue samples.
   Note: Generally, tissue processors are programmed to work overnight because of the length of the operation and the solutions involved in this process. Samples are processed following an established protocol:
   1. 10% neutral buffered formalin for 1 hr at 42 ºC.
   2. 10% neutral buffered formalin for 1 hr at 42 ºC.
   3. 70% ethanol for 30 min at 40 ºC.
   4. 85% ethanol for 30 min at 40 ºC.
   5. 100% ethanol for 45 min at 40 ºC.
   6. 100% ethanol  for 45 min at 40 ºC.
   7. 100% ethanol for 45 min at 40 ºC.
   8. Xylene for 1 hr at 40 ºC.
   9. Xylene for 1 hr at 40 ºC.
   10. Xylene for 1 hr at 40 ºC.
   11. Paraffin wax for 45 min at 63 ºC.
   12. Paraffin wax for 45 min at 63 ºC.
   13. Paraffin wax for 45 min at 63 ºC.
   14. Paraffin wax for 45 min at 63 ºC.
3. Melt the paraffin wax overnight at 62 ºC (Figures 1D-E). Estimate the total amount of paraffin wax to be used base on the grams of paraffin wax needed to fill a histology mold and the number of blocks to be prepared.
   Note: A deep histology mold is preferred to shallow mold because it provides a wider potential angle of rotation when sectioning. A single deep histology mold requires 11 g of paraffin wax.
4. Place each processed tissue into a deep histology mold and then pour melted paraffin wax to completely fill the mold (Figure 1F). Cover the mold with a labeled plastic cassette (Figure 1G).
5. Cool the filled mold on ice, or on a cold surface (Figure 1H), in order to create the solid block and separate the block from the mold (Figures 1I-J).
   Note: When placing the dissected heart in the mold, ensure that the inner face of the heart is touching the base of the mold, and then proceed to fill the block with melted paraffin. A labeled plastic cassette covers the mold before it solidifies in order to provide a base to handle the block. Once cooled, the mold is removed and the paraffin blocks can be stored indefinitely at room temperature.

3. Sectioning of the Aorta

1. Position the block with the heart in the specimen holder of the microtome so that it can be sectioned from the inside toward the top of the heart (Figure 2)
2. Adjust the microtome to cut a section thickness of 10 µm and proceed to section the heart.
3. Collect sections on a glass slide and examine under a light microscope to determine position and orientation.
4. When 1 or 2 valves of the aortic sinus become evident adjust the angle of the block as necessary (Figure 3A).
   Note: The sinus should appear as three bipartite valve bases with attached leaflets. If only one or two valve leaflets are observed then the cutting angle should be adjusted accordingly.
5. Adjust the microtome to cut at 4-5 µm and collect the sections on coated glass slides.
   Note: Although the procedure can be varied, a systematic approach is recommended, by which the first 10 sections, from the aortic sinus advancing up the ascending aorta, are collected onto the top portion of a labeled glass slides (slides labeled 1-10). Subsequent sections are collected following the same pattern: #11-20, #21-30, #31-40 until the atherosclerotic lesions are no longer observed (Figure 3B). In this way the atherosclerotic lesions can be examined and characterized at precise locations along the ascending aorta. For example, Slide 1 holds sections 1, 11, 21, 31, 41, and 51, representing atherosclerotic lesions from the aortic sinus up the ascending aorta. Additional slides, labeled 11-20, can be included if the lesion is large, to collect more distal sections following the same pattern.
6. Continue to collect sections until no atherosclerotic lesion is observed.
   Note: Slides can be stored at room temperature for months, before further analysis.

4. Staining and Quantification of Atherosclerotic Lesions

Atherosclerotic lesions can be analyzed in many different ways using a variety of techniques. The most basic analysis involves determination of lesion cross sectional area. In order to accurately compared lesion area, it is important that the starting point for lesion measurement is consistent from sample to sample. This is facilitated by using the valve leaflets for orientation. To facilitate the accurate identification and quantification of the lesion area, select cross sections are stained with hematoxylin and eosin. This involves immersing the slides in a series of solutions for specified times, that will deparaffinize/stain the tissue.

Protocol for hematoxylin and eosin staining:

1. Put the slides in the air drier for 8 min then deparaffinize the sections by washing 4× in xylene, for 3 min per wash.
2. Wash slides in 100% ethanol 3× for 3 min each.
3. Wash 3× in 70% ethanol for 3 min each and then 2× in 50% ethanol for 3 min each.
4. Rinse with distilled water and then immerse in Mayer’s Hematoxylin for 15 min.
5. Wash in running tap water for 5 min then wash in distilled water for 5 min.
6. Immerse in Eosin Y for 1-5 min.
7. Wash in distilled water 3× for 5 min each, wash in 50% ethanol 2× for 2 min each, then 70% ethanol 3× for 2 min each.
8. Wash in 100% ethanol 3× for 2 min each then wash in xylene 4× for 2 min each.
9. Mount sections using xylene based mounting medium and capture images using a digital camera mounted on a light microscope.
10. Identify and quantify lesion area using imaging software.
    Note: The lesion area can be determined at specific distances from the aortic sinus at 40-50 μm intervals (depending upon the thickness of each section). A plot of these area measurements versus distance gives a profile of the atherosclerotic lesion (Figure 4). The area under the curve represents an estimate of the total volume of the atherosclerotic lesion. Additional characterization of the lesion can include the determination of necrotic area. The necrotic core (nc) of an advanced lesion is formed by apoptotic foam cells. With practice, this can be determined by examination of the acellular regions of the hematoxylin and eosin stained sections (Figure 4A). Early lesions tend to have very little necrosis while more advanced lesions have increased necrotic areas that may contribute to plaque instability. Serial sections of the same aorta should be examined to confirm the presence of a necrotic region.

5. Further Characterization of the Atherosclerotic Lesion

Specific details regarding the stage of atherosclerotic development can be determined by staining with antibodies directed against specific cell types or markers. Immunohistochemistry on paraffin sections is a standard technique that is used to identify specific cell types or markers within the context of the surrounding unstained tissue. Antigen detection is generally achieved using a horseradish peroxidase substrate.

Immunofluorescence staining involves the use of fluorophore-conjugated antibodies and requires a fluorescence microscope and appropriate filters for detection. This technique permits simultaneous staining of two (or more) antigens.

General Protocol for Immunohistochemical staining:

1. Place slides in the air drier for 8 min.
2. Deparaffinize sections in 4 changes of xylene, 3 min each.
3. Wash in 100% ethanol 3× for 3 min each to remove xylene.
4. Wash 3× in 70% ethanol for 3 min each, then 2× in 50% ethanol for 3 min each.
5. Wash in distilled water 5 min each then wash 3× in PBS for 5 min each.
6. Perform antigen retrieval. This can be accomplished using the Heat-Induced Epitope Retrieval (HIER) method in a microwave pressure cooker and antigen retrieval solution (10 mM citrate buffer, pH 6.0).
   Note: The process of formalin fixation may mask the antigens of interest. In our experience, the HIER significantly improves the performance of some antibodies, but is not required for others. In addition, there are other available alternatives to the HIER method of antigen retrieval.
7. When the pressure cooker has cooled, place slides in PBS for 15 min.
8. Incubate sections in normal serum (from the same species that the secondary antibody was derived from) for 30 min. DO NOT WASH SLIDES - DRAIN SERUM OFF.
9. Incubate with primary antibody diluted in normal blocking serum for 2 hr at room temperature or overnight at 4 °C.
10. Wash with PBS 3× for 5 min each.
11. Incubate sections with the biotinylated secondary antibody which has been diluted in PBS for 1 hr at room temperature.
12. Incubate slides with horseradish peroxidase streptavidin for 30 min then wash with PBS 3× for 5 min each.
13. Chromogenic reaction- incubate slides in fresh DAB (3,3'-diaminobenzidine) for 5-30 min. This reaction should be monitored using a microscope. Stop the reaction by washing slides in tap water.
14. Counter stain with hematoxylin for 1 min. Wash with tap water for 5 min then wash in distilled water 3× for 5 min each.
15. Repeat steps 4.7-4.9 for mounting of slides.

6. General Protocol for Immunofluorescence Staining

A similar protocol, with minor changes, can be followed for immunofluorescence staining.

1. Place slides in the air drier for 8 min then deparaffinize sections in 4 changes of xylene, 3 min each.
2. Wash in 100% ethanol 3× for 3 min each to remove xylene.
3. Wash 3× in 70% ethanol for 3 min each, then 2× in 50% ethanol for 3 min each.
4. Wash in distilled water for 5 min each then wash in PBS 3× for 5 min each.
5. Perform antigen retrieval. This can be accomplished using the Heat-Induced Epitope Retrieval (HIER) method in a microwave pressure cooker and antigen retrieval solution (10 mM citrate buffer, pH 6.0).
   Note: The process of formalin fixation may mask the antigens of interest. In our experience, the HIER significantly improves the performance of some antibodies, but is not required for others. In addition, there are other available alternatives to the HIER method of antigen retrieval.
6. When the pressure cooker has cooled, place slides in PBS for 15 min.
7. Incubate sections in normal serum (from the same species that the secondary antibody was derived from) for 30 min. DO NOT WASH SLIDES - DRAIN SERUM OFF.
8. Incubate with primary antibody diluted in normal blocking serum for 2 hr at room temperature or overnight at 4 °C.
9. Wash with PBS 3× for 5 min each.
10. Add secondary fluorophore-conjugated antibody that is diluted in normal blocking serum (or PBS). Incubate for 1.5 hr at room temperature.
11. Wash with PBS 3× for 5 min each.
12. Counter stain with DAPI.
13. Wash in PBS 3× for 5 min each.
14. Apply a coverslip using antifade mounting media.

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