We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.
enrijecimento arterial é um fator de risco significativo e biomarcador para a doença cardiovascular e uma característica do envelhecimento. Microscopia de força atómica (AFM), é uma ferramenta analítica versátil para a caracterização de propriedades mecânicas viscoelásticas para uma variedade de materiais que vão desde a dura (de plástico, vidro, metal, etc.) superfícies de células em qualquer substrato. Tem sido largamente utilizado para medir a rigidez das células, mas menos frequentemente utilizados para medir a rigidez das aortas. Neste artigo, vamos descrever os procedimentos para a utilização AFM no modo de contato para medir o módulo de elasticidade ex vivo das artérias descarregados do mouse. Nós descrevemos o nosso procedimento para isolamento de aortas do rato e, em seguida, forneça informações detalhadas para a análise AFM. Isso inclui instruções passo-a-passo para o alinhamento do feixe de laser, a calibração da constante da mola e sensibilidade deflexão da sonda de AFM, e aquisição de curvas força. Nós também fornecemos um protocolo detalhado para Analy dadossis das curvas de força.
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.
AFM recuo pode ser utilizado para caracterizar a rigidez (módulo de elasticidade) de células e tecidos. Neste artigo, nós fornecemos protocolos detalhados passo-a-passo para isolar a aorta descendente e arco aórtico em ratos e determinar o módulo de elasticidade dessas regiões arteriais ex vivo. Vamos agora resumir e discutir as questões técnicas e limitações do método descrito neste artigo.
Várias questões técnicas podem surgir no isolamento e análise de aortas de ra…
The authors have nothing to disclose.
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.
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 |