의 강성 측정<em> 전의 VIVO</em원자 힘 현미경을 사용하여> 마우스 대동맥
Summary
We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.
Abstract
동맥 경직은 중요한 위험 인자 및 심혈관 질환 바이오 마커 및 노화의 특징이다. 원자력 현미경 (AFM)은 하드 (플라스틱, 유리, 금속 등)의 임의의 기판 위에 세포 표면에 이르기까지 다양한 재료에 대한 점탄성 기계적 성질을 특성화하기위한 다용도 분석 도구이다. 널리 세포의 강성을 측정하는 데 사용되지만 자주 대동맥의 강성을 측정하는 데 사용되었다. 본 논문에서는 무부하 마우스 동맥의 생체 탄성 계수를 측정하기 위해 접촉 모드 AFM을 사용하기위한 절차를 설명합니다. 우리는 마우스 대동맥의 분리를 위해 우리의 절차를 설명하고 AFM 분석에 대한 자세한 정보를 제공합니다. 이것은 레이저 빔의 배향, 스프링 상수 및 AFM 프로브의 편향 감도를 교정 한 힘 곡선 취득 단계가 설명되어있다. 우리는 또한 데이터 analy에 대한 자세한 프로토콜을 제공힘 곡선의 동생.
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 압입은 세포 및 조직의 강성 (탄성 계수)을 특성화하는데 사용될 수있다. 본 논문에서는 마우스의 하행 대동맥과 대동맥 궁을 분리하고이 동맥 영역 생체의 탄성 계수를 결정하는 자세한 단계별 프로토콜을 제공합니다. 우리는 지금 요약하고이 문서에 설명 된 방법의 기술적 인 문제 및 제한 사항에 대해 설명합니다.
몇 가지 기술적 인 문제는 고립과 자신의 작고 ?…
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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