La medición de la rigidez de<em> Ex Vivo</em> Ratón aortas Usando microscopía de fuerza atómica
Summary
We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.
Abstract
la rigidez arterial es un importante factor de riesgo y biomarcadores para la enfermedad cardiovascular y un sello de envejecimiento. Microscopía de fuerza atómica (AFM) es una herramienta analítica versátil para la caracterización de propiedades mecánicas viscoelásticas para una variedad de materiales que van desde el disco (plástico, vidrio, metal, etc.) las superficies a las células sobre cualquier sustrato. Ha sido ampliamente utilizado para medir la rigidez de las células, sino que se utiliza con menos frecuencia para medir la rigidez de aortas. En este artículo, vamos a describir los procedimientos para el uso de AFM en modo de contacto para medir el módulo elástico ex vivo de las arterias de ratones sin carga. Describimos nuestro procedimiento para el aislamiento de las aortas de ratón y, a continuación ofrecemos información detallada para el análisis AFM. Esto incluye instrucciones paso a paso para la alineación del haz de láser, la calibración de la constante de elasticidad y sensibilidad de desviación de la sonda de AFM, y la adquisición de curvas de fuerza. También proporcionamos un protocolo detallado para Analy datossis de las curvas de fuerza.
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, therapies that could prevent arterial stiffening in CVD or aging might complement currently available pharmacological interventions that treat CVD by reducing high blood cholesterol.
AFM is a powerful analytical tool used for various physical and biological applications. AFM is increasingly used to obtain the high-resolution images and characterize the biomechanical properties of soft biological samples such as tissues and cells1,2,10,16,17 with a great degree of accuracy at nanoscale levels. A major advantage of AFM is the fact that it can be used with living cells.
This paper describes our method for measuring the elastic modulus of mouse arteries ex vivo using AFM. The described method shows how we 1) properly isolate mouse arteries (descending aorta and aortic arch) and 2) measure the elastic modulus of these tissues by AFM. Measurements of unloaded elastic moduli in arteries can help to elucidate changes in the extracellular matrix (ECM) that occur in response to vascular injury, CVD, and aging.
Protocol
Representative Results
Discussion
AFM indentación puede ser utilizado para caracterizar la rigidez (módulo elástico) de las células y tejidos. En este trabajo, ofrecemos protocolos detallados paso a paso para aislar la aorta descendente y arco aórtico en el ratón y determinar los módulos de elasticidad de estas regiones arteriales ex vivo. Ahora tenemos resumir y discutir las cuestiones técnicas y las limitaciones del método descrito en este documento.
Varios problemas técnicos pueden surgir en el aislamie…
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.
Materials
| 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) |
| 60mmTC-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 |
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