sertliği ölçme<em> Ex Vivo</emAtomik Kuvvet Mikroskobu Kullanma> Fare aorta
Summary
We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.
Abstract
Arteriyel sertleşme önemli bir risk faktörüdür ve kardiyovasküler hastalık için biomarker ve yaşlanma bir özelliğidir. Atomik kuvvet mikroskopisi (AFM), sert (plastik, cam, metal, vs.) herhangi bir alt-tabaka üzerinde hücrelere yüzeyleri arasında değişen çeşitli malzemeler için viskoelastik mekanik özelliklerini karakterize etmek için çok yönlü bir analiz aracı. Yaygın hücre sertliğini ölçmek için kullanılan, daha az sıklıkla aorta sertliğini ölçmek için kullanılmıştır. Bu yazıda, yüksüz fare arterlerin ex vivo elastik modülü ölçmek için temas modunda AFM kullanarak işlemleri anlatacağım. Biz fare aorta izolasyonu için prosedürü tarif ve sonra AFM analizi için ayrıntılı bilgi sağlar. Bu lazer ışını uyumu, yay sabiti ve AFM prob sapma duyarlılığı kalibrasyonu ve kuvvet eğrileri elde edilmesi için adım adım yönergeler içerir. Biz aynı zamanda veri analy için ayrıntılı bir protokol sağlarkuvvet eğrilerinin sis.
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 girinti hücre ve dokuların sertliği (esneklik katsayısı) karakterize etmek için kullanılabilir. Bu yazıda fare azalan aort ve aort arkı izole etmek ve bu arter bölgelerin ex vivo elastik modüle belirlemek için ayrıntılı adım adım protokolleri sağlar. Biz şimdi özetlemek ve bu yazıda anlatılan yöntemin teknik konuları ve sınırlamaları tartışmak.
Çeşitli teknik konular izolasyonu ve bunların küçük ve ince yapısı verilen fare aorta analizinde or…
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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