Published: September 26th, 2018
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 o....
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 Apo.......
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 section.......
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 .......
|Adjusted calories diet (42 % from fat)
|Osmotic minipump with filling tube
|for continous realase
|Alchol Preps saturated with 70 % Isopropyl Alcohol
|Surgery wire (Vicryl 6.0)
|10% Povidone/iodine ointment
|to fix vascolature
|instrument for sectioning
|Dulbecco's Phosphat Buffered Saline
|Adhesion Slides Polysine
|Oil Red O solution (0.5 % in isopropanol)
|Acqueous Mounting Medium
|Phosphomolybdic acid solution
|Direct Red 80 (Picrosirius Red)
|Bio Clear (clearing agent of terpene origin)
|product for the preparation of histological samples
|Eukitt Quick Hardening mounting medium (Poly(butyl methacrylate-co-methyl methacrylate)
|Sodium Dodecyl Sulfate
|Hydrogen Peroxide solution (30 %)
|Vectorstain ABC KIT including: anti-rabbit IgG ABC, normal rabbit serum, Secondary-biotinylated Anti-Rat IgG ,
|ImagePro Premier 9
|software to analyze images
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