Mesure de la rigidité de<em> Ex Vivo</em> Souris aortes Utiliser Atomic Force Microscopy
Summary
We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.
Abstract
raidissement artérielle est un facteur de risque significatif et biomarqueur pour une maladie cardiovasculaire et une caractéristique de vieillissement. La microscopie à force atomique (AFM) est un outil d' analyse polyvalent pour la caractérisation des propriétés mécaniques viscoélastiques pour une variété de matériaux allant du disque (plastique, verre, métal, etc.) des surfaces à des cellules sur tout substrat. Il a été largement utilisé pour mesurer la rigidité des cellules, mais moins fréquemment utilisé pour mesurer la rigidité des aortes. Dans cet article, nous allons décrire les procédures d'utilisation de l' AFM en mode contact pour mesurer l'ex vivo module d' élasticité des artères de souris non chargées. Nous décrivons notre procédure d'isolement des aortes de souris, puis des informations détaillées pour l'analyse de l'AFM. Cela inclut des instructions étape par étape pour l'alignement du faisceau laser, l'étalonnage de la constante du ressort et de la sensibilité de déviation de la sonde AFM, et l'acquisition de courbes de force. Nous fournissons également un protocole détaillé pour analy de donnéessis des courbes de force.
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 indentation peut être utilisée pour caractériser la rigidité (module d'élasticité), des cellules et des tissus. Dans cet article, nous fournissons des protocoles étape par étape détaillées pour isoler l'aorte descendante et l' arc aortique chez la souris et déterminer les modules élastiques de ces régions artérielles ex vivo. Nous résumons maintenant et discuter les problèmes techniques et les limites de la méthode décrite dans le présent document.
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Declarações
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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