Meten van de stijfheid van<em> Ex Vivo</em> Mouse aorta's met behulp van 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
Arteriële verstijving is een belangrijke risicofactor en biomarker voor cardiovasculaire ziekte en een kenmerk van veroudering. Atomic force microscopie (AFM) is een veelzijdig analytisch hulpmiddel voor het karakteriseren viscoelastische mechanische eigenschappen voor verschillende materialen, variërend van harde (plastic, glas, metaal, enz.) Oppervlakken van cellen op een substraat. Het is wijd gebruikt om de stijfheid van de cellen te meten, maar minder vaak gebruikt om de stijfheid van de aorta meten. In dit artikel zullen we de procedures voor het gebruik van AFM in contact modus om de ex vivo elasticiteitsmodulus van onbelaste muis slagaders te meten te beschrijven. We beschrijven onze procedure voor het isoleren van de muis aorta, en vervolgens gedetailleerde informatie voor de AFM analyse. Dit omvat stap-voor-stap instructies voor het richten van de laserbundel, kalibratie van de veerconstante en de doorbuiging gevoeligheid van de AFM sonde, en verkrijging van kracht curves. We bieden ook een gedetailleerd protocol voor data analysis van de kracht 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, 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 inspringing kan worden gebruikt om de stijfheid (elasticiteitsmodulus) van cellen en weefsels te karakteriseren. In dit document, bieden we uitvoerige stap-voor-stap protocollen de afdalende aorta en de aortaboog in de muis isoleren en bepalen de elastische moduli van deze slagaderlijke gebieden ex vivo. We hebben nu samen te vatten en bespreken de technische problemen en beperkingen van de in dit artikel beschreven methode.
Verschillende technische problemen kunnen voordoen in …
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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