We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.
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Method Article
Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy
In This Article
Summary
Abstract
Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.
Introduction
The biomechanical properties of arteries are a critical determinant in cardiovascular disease (CVD) and aging. Arterial stiffness, a major cholesterol independent risk factor and an indicator for the progression of CVD, increases with vascular injury, atherosclerosis, age, and diabetes1-8. Arterial wall stiffening is associated with increased dedifferentiation, migration, and proliferation of vascular smooth muscle cells9-12. In addition, increased arterial stiffness has been linked to enhanced macrophage adhesion1, endothelial permeability and leukocyte transmigration13, and vessel wall remodeling14,15. Thus, the....
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Protocol
Animal work in this study was approved by the Institutional Animal Care and Use Committees of the University of Pennsylvania. The methods were carried out in accordance with the approved guidelines.
1. Preparing the Mouse and Isolation of the Aorta
- Anesthetize a mouse with ketamine (80 - 100 mg/kg), xylazine (8 - 10 mg/kg) and acepromazine (1 - 2 mg/kg) intraperitoneally. Confirm anesthesia with a tail pinch test. Once the mouse is fully anesthetized, euthanize the mouse by cervical dislocation.
- Place the mouse on its back and pin the mouse to a dissection board. Clean the abdomen area with 70% (v/v) ethanol wipes.
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Results
Figure 5A shows a phase contrast image of the descending (thoracic) aorta from a 6-month old, male C57BL/6 mouse. The AFM cantilever is in place directly above the tissue and ready for indentation. Figures 5B and 5C demonstrate representative force curves obtained by AFM indentation in contact mode. Green lines shown in Figures 5B and 5C represent the best fit curves obtained using the Hertzian model for a sphere. In Figure 5D
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Discussion
AFM indentation can be used to characterize the stiffness (elastic modulus) of cells and tissues. In this paper, we provide detailed step-by-step protocols to isolate the descending aorta and aortic arch in the mouse and determine the elastic moduli of these arterial regions ex vivo. We now summarize and discuss the technical issues and limitations of the method described in this paper.
Several technical issues can arise in the isolation and analysis of mouse aortas given their small .......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
AFM analysis was performed on instrumentation supported by the Pennsylvania Muscle Institute and the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, the University of Pennsylvania. This work was supported by NIH grants HL62250 and AG047373. YHB was supported by post-doctoral fellowship from the American Heart Association.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| BioScope Catalyst AFM system | Bruker | ||
| Nikon Eclipse TE 200 inverted microscope | Nikon Instruments | ||
| Silicon nitride AFM probe | Novascan Technologies | PT.SI02.SN.1 | 0.06 N/m cantilever; 1 µm SiO2 particle |
| Dumont #5 forceps | Fine Science Tools | 11251-10 | See section 1.4 |
| Dumont #5SF forceps | Fine Science Tools | 11252-00 | See section 1.8 |
| Fine Scissors-ToughCut | Fine Science Tools | 14058-11 | See section 1.4 (medium sized) |
| Vannas-Tübingen spring scissors | Fine Science Tools | 15008-08 | See section 1.6 (small sized) |
| 60 mm TC-treated cell culture dish | Corning | 353004 | |
| Dulbecco's Phosphate-Buffered Saline, 1x | Corning | 21-031-CM | Without calcium and magnesium |
| Krazy Glue instant all purpose liquid | Krazy Glue | KG58548R | See section 2.2 |
| Gel-loading tips, 1 - 200 µl | Fisher | 02-707-139 | See section 2.2 |
| Tip Tweezers | Electron Microscopy Sciences | 78092-CP | See section 3.2 |
| 50-mm, clear wall glass bottom dishes | TED PELLA | 14027-20 | See section 4.4 |
References
- Kothapalli, D., et al. Cardiovascular Protection by ApoE and ApoE-HDL Linked to Suppression of ECM Gene Expression and Arterial Stiffening. Cell Rep. 2, 1259-1271 (2012).
- Liu, S. -L., et al. Matrix me....
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