We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.
irrigidimento arteriosa è un fattore di rischio significativo e biomarker per malattie cardiovascolari e un segno distintivo di invecchiamento. Microscopia a forza atomica (AFM) è uno strumento analitico versatile per la caratterizzazione viscoelastiche proprietà meccaniche per una varietà di materiali che vanno dal disco (plastica, vetro, metallo, ecc) le superfici di celle su qualsiasi substrato. È stato ampiamente utilizzato per misurare la rigidità delle cellule, ma usato meno frequentemente per misurare la rigidità di aorte. In questo articolo, descriveremo le procedure per l'utilizzo in modalità AFM contatto per misurare la ex vivo modulo elastico delle arterie scaricati mouse. Descriviamo la nostra procedura per l'isolamento di aorte del mouse, e quindi fornendo informazioni dettagliate per l'analisi AFM. Questo include le istruzioni passo-passo per l'allineamento del raggio laser, la taratura della costante della molla e la sensibilità deflessione della sonda AFM, e l'acquisizione di curve di forza. Forniamo anche un protocollo dettagliato per analy datisis delle curve di forza.
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 indentazione può essere utilizzato per caratterizzare la rigidità (modulo elastico) di cellule e tessuti. In questo lavoro, forniamo dettagliate protocolli passo-passo per isolare l'aorta discendente e arco aortico nel topo e determinare il modulo elastico di queste regioni arteriosi ex vivo. Ora riassumere e discutere le questioni tecniche e le limitazioni del metodo descritto in questo articolo.
Diversi problemi tecnici possono sorgere in isolamento e l'analisi di to…
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 |