测量的刚度<em>离体</em>鼠标主动脉用原子力显微镜
Summary
We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.
Abstract
动脉硬化是一个显著风险因子和生物标志物用于心血管病和老化的标志。原子力显微镜（AFM）是用于多种材料从硬（塑料，玻璃，金属等 ）表面的任何基底上细胞的表征粘弹性的机械性能的多功能的分析工具。它已被广泛地用于测量细胞的刚度，但不经常用于测量主动脉的刚度。在本文中，我们将介绍使用AFM在接触模式下测量空载小鼠动脉离体弹性模量的过程。我们描述程序的鼠标主动脉隔离，然后提供的AFM分析的详细信息。这包括用于激光束的对准，弹簧常数和原子力显微镜探针的偏转灵敏度的校正，并采集力曲线的一步一步的说明。我们还提供了数据ANALY了详细的方案力曲线的妹妹。
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压痕可用于表征细胞和组织的刚度（弹性模量）。在本文中，我们提供详细的一步一步的协议来隔离小鼠降主动脉和主动脉弓，并确定这些地区的动脉离体的弹性模量。我们现在总结和讨论在本文所描述的方法的技术问题和限制。
几个技术问题可以在隔离，并给予他们的小而薄的自然鼠标主动脉的分析产生。当清洗过的动脉被纵向打开，必须小心不要破坏那里的弹?…
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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