We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.
Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.
Atherosclerosis is one of the main causes of mortality and morbidity worldwide1. It is characterized by a chronic inflammatory state where blood vessels are infiltrated by lipids and leukocytes that determine the formation of atherosclerotic plaques. The majority of acute cardiac events are associated with thrombotic events due to plaque rupture. Plaques prone to rupture are defined "vulnerable" and are characterized by increased infiltration of pro-inflammatory leukocytes, a necrotic core, and a thin fibrous cap2. In the last decades, clinical and experimental studies have clarified the complex pathophysiology of the disease. Several animal models are used to investigate the molecular mechanisms involved in the induction of atherosclerosis3. Currently, the mouse is the most frequently used species to study atherosclerosis despite some species-specific differences existing in its pathophysiology in comparison with humans. In particular, circulating cholesterol is mainly composed by high-density lipoproteins (with antiatherogenic properties) in mouse models, while in humans circulating cholesterol is mainly transported as low-density lipoproteins (LDL), probably representing the main reason why wild-type mice do not develop spontaneous atherosclerosis4. Furthermore, wild type mice are generally resistant to absorbing cholesterol from the diet, whereas humans absorb around 50% of dietary cholesterol4. To overcome these limitations, several genetically modified mouse models have been generated. Apolipoprotein E–deficient mouse (ApoE−/−) and LDL receptor–deficient mouse (LDLr−/−) are widely used. On a high-fat high cholesterol diet, ApoE−/− mice develop plaque more rapidly compared to LDLr−/−mice, and for this reason, the use of ApoE−/− mice is more widespread than that of LDLr−/−mice5,6. The ApoE−/− mice develop lesions at any stage of atherosclerosis and are comparable to those observed in humans, although mouse plaques do not show an unstable phenotype7. Generally, ApoE−/− mice spontaneously develop atherosclerosis and this process is accelerated with a western diet3. The severity of atherosclerosis can be evaluated at the level of the carotid, pulmonary, femoral and brachiocephalic artery, and the aortic root6. In particular, the aortic root represents an anatomical site prone to develop atherosclerotic lesions in mice. For this reason, it is common practice to evaluate the formation of atherosclerotic plaques in this region.
Several studies have shown that aldosterone is implicated in the development of atherosclerosis8,9,10. Aldosterone infusion in ApoE−/− mice fed an atherogenic diet accelerates the development of aortic root atherosclerotic lesions inducing inflamed and lipid-rich plaque formation10.
In summary, we describe a protocol including aldosterone infusion, atherogenic diet feeding, embedding of heart tissues, quantification and characterization of atherosclerotic lesions in ApoE-/- mice. This procedure promotes an efficient formation of inflamed, lipid-rich atherosclerotic plaques and represents a valuable model for the study of atherogenesis.
The study was approved by the Italian National Institutes of Health Care and Use Committees, authorization number 493/2016-PR. All procedures were conducted per the guidelines of the European Community for the use of experimental animals (European Directive, 2010/63/UE).
Note: Subcutaneous implantation of osmotic minipump containing vehicle (ethanol in saline solution) or aldosterone (240 µg · kg-1 · d-1) in 8-10 week-old male mice deficient for the ApoE gene. In general, 8-10 week-old male ApoE−/− mice weigh around 25-26 g.
1. Dissolving Aldosterone
2. Osmotic Pump Filling and Implantation
Note: Pumps are supplied in two separate parts: the main body of the pump and the flow regulator.
3. Dissection of the Heart
4. Cutting sections of the Aortic Root
5. Immunohistochemistry
Note: All staining steps reported in the following protocols are performed in glass Coplin staining jars.
6. Image Analysis of Atherosclerotic Lesions in Aortic Root Sections
Note: Aortic root sections stained with Oil Red O or MAC3 or an antibody of interest can be analyzed using appropriate software (indicated in Table of Materials). Total pixels staining positive for the component of interest is normalized to the overall plaque area. Images were collected and analyzed by a treatment-blinded investigator.
In 8-10 weeks Apo-/- mice, minipumps were implanted to infuse with vehicle or aldosterone (Figure 1E-1I) and fed an atherogenic diet (adjusted calories diet 42% from fat) for 4 weeks. At the end of treatment, mice were euthanized and perfused with PBS and 10% formalin as described above. The aortic root was separated from the apical portion of the heart and was embedded in OCT (Figure 2). Cross sections of the aortic roots were prepared for different staining by using a cryostat machine (Figure 3).
Atherosclerotic plaque composition can be characterized through the staining for specific components, which define the stage of plaque development. Oil Red O staining is used to determine lipid content of atherosclerotic lesions as described in step 5.1, Picro Sirius Red staining is used to determine collagen content as described in step 5.2, and Mac3 staining is employed to measure macrophage content described in step 5.3. After 4 weeks of treatment with aldosterone, mice displayed a significant increase in lipid (Figure 4A-4C) and macrophage content (Figure 4D–4F) in atherosclerotic plaques at the aortic root level, but no differences were observed in collagen content (Figure 4G-4I) compared to mice treated with the vehicle. Indeed, aldosterone accelerated atherogenesis in this model, inducing vascular inflammation but not fibrosis (Figure 4).
Figure 1. Preparation and implantation of osmotic minipump with vehicle or aldosterone. A-B) Steps to fill osmotic minipumps with vehicle or aldosterone. C) Steps to close the body of pump with the flow regulator. D-F) Steps to create an incision for the implantation of pump on the back of the mouse. G-I) Implantation of osmotic minipump and suturing of incision. Please click here to view a larger version of this figure.
Figure 2. Preparing the heart and the aortic root for embedding and sectioning. A-B) The heart is cut transversely along a straight line that joins the lower points of the right and left atrium. The apical part of the heart is processed to quantify atherosclerotic lesions composition in the aortic root. C) The aortic root from inside the heart. D-E) Filling of the heart cavity with OCT. F) Orientation of the dissected heart in a mold pre-filled with OCT (with the heart cavity in direction of the bottom of mold) G) Freezing of the dissected heart with OCT in the mold. Please click here to view a larger version of this figure.
Figure 3. Sectioning of aortic root using a cryostat microtome. A) Pre-cool the cryostat and set the right temperature for the machine and chuck. B-D) The solid OCT block is separated from the mold and the excess OCT around the heart is eliminated using a blade. E-I)The block of OCT is fixed, using liquid OCT, on the support. J-K) The support is inserted in the chuck and oriented with the ventricular facing outward. L-M) Section the block until the dissected heart is completely exposed. N-O) Set the thickness to 10 and/or 6 µm and collect the slices on the glass. P-Q) Check the presence of all three valves under the microscope. R) Subsequent slices are collected, as shown, until one or more valves are not observed anymore. Please click here to view a larger version of this figure.
Figure 4. Quantification of atherosclerotic plaque composition in aldosterone- or vehicle-treated ApoE-/-. A-B) Representative Red Oil O stained cross sections of aortic root in 8-10 week-old ApoE-/- mice treated with vehicle (A) or aldosterone (B) and fed an atherogenic diet for 4 weeks. C) Quantification of lipid content as percentage of positive staining/total plaque area. D-E) Representative Mac3 stained cross sections of aortic root. F) Quantification of macrophage content as percentage of positive staining/total plaque area. G-H) Representative Picro Sirius Red stained cross sections of aortic root. I) Quantification of collagen content as a percentage of positive staining/total plaque area. Values are expressed as mean ± Standard Error Mean (SEM; n=6 for each group). Please click here to view a larger version of this figure.
Atherosclerosis is a chronic inflammatory disorder associated with large and medium vessels involving interactions between multiple cell types, such as macrophages, T-lymphocytes, endothelial cells and smooth muscle cells1. Despite the limitations of murine atherogenic models, a large body of evidence on the atherosclerotic process is available. These models have the advantage of rapidly generating experimental cohorts of a specific age and gender. Mice also show a defined and homogeneous genetic background.
The development of atherosclerotic plaques, observed in mouse models prone to atherosclerosis, partially resembles that observed in human subjects, except for unstable plaques formation4. Plaque rupture and subsequent thrombosis, the main cause of myocardial infarction in humans, is not observed in murine atherogenic models. In our recent papers, we showed that infusion of aldosterone is able to induce, after 4 weeks, an unstable plaque phenotype in ApoE-/-mice fed an HFD10. In fact, this treatment is able to increase plaque area, lipid content, and inflammatory area at the aortic root level, with no significant difference in collagen content compared to untreated mice10. Of note, these mice show increased development of hypertension-independent atherosclerosis, given that blood pressure was not significantly altered by aldosterone treatment10.
In the present manuscript, we illustrate in detail the technical procedure to embed the heart tissue in OCT as well as the sequential steps, i.e., aortic root sectioning and plaque analysis. Compared to paraffin, OCT allows section preparation with a high range of thicknesses (1-100 µm) with a cryostat microtome and minimal tissue processing. Limitations include the quality of frozen tissue, which is potentially affected by the formation of ice crystals and can alter subcellular details and generate artifacts detectable by immunohistological staining. Indeed, cryopreservation is thought to better preserve antigen and antigenicity. In fact, frozen tissues preserve the structure of antigens. Furthermore, frozen tissue processing for immunohistochemistry allows to avoid the use of formalin, which is a toxic and carcinogenic compound.
The aortic root is a site relatively straightforward to identify for histological processing. This anatomical site is a gold standard to study atherosclerotic lesion development in mice prone to atherosclerosis. Data obtained by the analysis of this specific region allow comparisons among mice belonging to the same group or different experimental groups. In fact, during sectioning of the aortic root, it is easy to recognize a specific region characterized by the presence of all three valves. In order to obtain such a specific region, it is strictly recommended to pay particular attention to several critical steps during embedding and sectioning. It is important to fill the cavity of the upper part of the sectioned heart with OCT to avoid the formation of air bubbles in the tissue that can compromise the integrity of the sections. Furthermore, to ensure that the cutting angle is exactly perpendicular to the ascending aorta during sectioning, it is necessary to properly orient the upper sectioned heart perpendicular to the bottom surface of the tissue mold. In order to obtain intact slices, the temperature of the cryostat should be exactly at -20 °C. In fact, a lower temperature could compromise the quality of sections (i.e., the blade could crumble the sections making them unusable).
Of note, when the three valves appear, it is recommended to cut the tissue with different thicknesses, depending on the type of the analysis that will be performed. In fact, some slides will be used for the determination of plaque progression, whereas the remaining slides could be used for different stainings (i.e., Picro Sirius, Mac3, etc.).
Atherosclerotic lesions in mice treated with aldosterone appear rich in lipid and macrophages characterized by the use of Oil Red O (for lipid staining), Mac3 (for macrophage staining), Sirius red (for the staining of collagen), or other specific antibodies for proteins of interest. It is important to highlight that OCT embedding, unlike paraffin embedding, avoids the use of organic solvents that remove the lipid content of the lesions. Therefore, lipid staining with Oil Red O is feasible in OCT-embedded sections and lipid content of atherosclerotic plaques can be precisely quantified.
In the present manuscript we showed that aldosterone infusion is a valuable method to promote atherosclerosis in ApoE-/- mice. In fact, aldosterone infusion in ApoE-/- mice is able to: 1) increase the activation of endothelial cells, as shown by the increased expression of molecules involved in the adhesion and migration of monocytes at the early stages of atherosclerotic plaque formation12,13; and 2) increase lipid and pro-inflammatory macrophages content at aortic root level10,13. These effects are not due to hemodynamic effects of aldosterone10. In conclusion, the proposed protocol represents a valid method to generate and characterize atherosclerosis in mice; this could help monitor early stages of disease and outcomes of therapeutic intervention and rehabilitation in atherosclerosis.
The authors have nothing to disclose.
This work was supported by grants from the Italian Ministry of Health (Ricerca Corrente, GR-2009-1594563 and PE-2011-02347070 to M.C.)
Adjusted calories diet (42 % from fat) | Envigo | TD.88137 | atherogenic diet |
Osmotic minipump with filling tube | Alzet | model 1004 | for continous realase |
Aldosterone | SIGMA | A9477 | hormone |
Ethanol | SIGMA | 34852-M | solvent |
Alchol Preps saturated with 70 % Isopropyl Alcohol | Kendal Webcol | 6818 | Disinfectant |
Surgery wire (Vicryl 6.0) | Demas | 500004 | surgery |
10% Povidone/iodine ointment | Aplicare-Meriden | 52380-0026-1 | Antiseptic |
Formalin 10% | SIGMA | HT5012 | to fix vascolature |
Cryomold | Bio-Optica | 07-MP1515 | for embedding |
O.C.T. Compound | Sakura Finetek | 4583 | for embedding |
Cryostat | Leica | CM1900 | instrument for sectioning |
Dulbecco's Phosphat Buffered Saline | Aurogene | AU-L0615-500 | buffer solution |
Adhesion Slides Polysine | VWR | 631-0107 | microscope glasses |
Cover Glasses | Bio-Optica | 72015 | cover glasses |
Formaldehyde 37% | SIGMA | 252549 | solvent |
Oil Red O solution (0.5 % in isopropanol) | SIGMA | O1391 | staining solution |
Mayer’s Hematoxylin | SIGMA | MHS32 | staining solution |
Lithium Carbonate | SIGMA | 62470 | washing buffer |
Acqueous Mounting Medium | Thermo Scientific | TA-125-AM | mounting solution |
Acetone | SIGMA | 179124 | solvent |
Phosphomolybdic acid solution | SIGMA | HT153 | for hystology |
Direct Red 80 (Picrosirius Red) | SIGMA | 365548 | staining solution |
Bio Clear (clearing agent of terpene origin) | Bio-Optica | 06-1782D | product for the preparation of histological samples |
Eukitt Quick Hardening mounting medium (Poly(butyl methacrylate-co-methyl methacrylate) | SIGMA | 3989 | mounting solution |
Sodium Dodecyl Sulfate | Fluka | 71725 | powder |
Hydrogen Peroxide solution (30 %) | SIGMA | H1009 | solution |
Vectorstain ABC KIT including: anti-rabbit IgG ABC, normal rabbit serum, Secondary-biotinylated Anti-Rat IgG , | Vector Laboratories | PK-6100 | staining solution |
3-amino-9-ethylcarbazole | Vector Laboratories | SK-4200 | staining solution |
Mac3 antibody | BD Biosciences | 553322 | antibody |
ImagePro Premier 9 | Media Cybernetics | 050910000-2534 | software to analyze images |