Name: measuring the stiffness of ex vivo mouse aortas using atomic force microscopy
Uploaded: 2016-10-19T14:00:00.000+00:00
Duration: 10 min 35 s
Description: Watch this Scientific Journal Video about Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy at JoVE.com

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.

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The authors have nothing to disclose.

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 얇은 자연 주어진 마우스 대동맥의 분석에 발생할 수 있습니다. 세정 동맥 길이 방향으로 개방되는 경우, 치료는 탄성률 데이터 수집 내막 (내강 표면)을 방해하지 않도록주의해야한다. 최종 샘플 접시 과잉 PBS는 접시에 동맥의 적절한 접착을 방지하고,이 접착 전에 동맥을 건드리지 않고 종이 티슈를 이용하여 조심스럽게 제거해야한다. 단리 동맥 종이 티슈 WHI 스틱 수 있다는 점에 유의제작 과도한 PBS를 제거하려고하고,이 샘플이 손상 될 수 있습니다. 배양 접시에 접착 동맥 동안, 동맥 연신되지 않는 것이 매우 중요에만 접착제를 소량 동맥의 양쪽 끝 비트 별 적용된다. 강자성 들여 쓰기를 수행 할 때, 접착제가 도포 된 동맥의 끝에서 프로빙을 방지하는 것이 중요하다. 갓 절연 동맥 탄성률의 측정에 사용되기 때문에 마지막 시간은 최소화되어야 측정 수확 경과. 본 연구에서 사용 된 구형 팁은 500 nm의 반경을 가지므로, 최대 500 nm 인 압입 깊이를 정확하게 할 헤르츠 모델을 사용하여 분석 할 수있다. 이 압입 깊이 내막 (21)에있는 내피 세포의 상층과 상관된다. 사용될 수 - 동맥 샘플들의 서브 - 내피 층 역학 큰 콜로이드 프로브 (직경 20 ~ 10 ㎛)를 프로빙들여 쓰기 및 압입 깊이와 계산 된 강성의 변화를 22, 23을 판단 할 수 있습니다. 생물학적 시료는 보통 점탄성보다는 순수 탄성 때문에, (일정한 압입 깊이에 가해진 힘의 감쇠를 모니터링) 응력 완화 측정은 동맥 기계적 특성 (24, 25)의 점성 성분을 결정하기 위해 사용될 수있다. AFM 분석의 전위 한계는 조직의 작은 영역을 측정 스캔 크기이다. 혈관 세포를 포함한 대부분의 생체 시료가 아닌 지형적 및 생체 역학적으로 균질하며, 이러한 이질성은 잠재적 압입 사이트 압입 깊이 및 힘의 크기의 평활도에 따라 탄성률 artifactually 큰 변화가 발생할 수 시료에인가 표면. 전체적인 탄성 계수의 대표 평균 값을 얻기 위해, 반복 AFM-압입 A를 수행하는 것이 중요이전의 연구는 날카로운 피라미드 팁 AFM-덴트 탄성률이 높은 추정 결과 및 소프트 손상된 것을 보여준 바와 같이도 4에 도시 된 바와 같이, 동맥을 통한 t 복수의 임의의 위치가있다. 또한, 우리는 적절한 크기의 구형 팁을 사용하여 생물학적 시료 26, 27. 동맥 경화 우리 생체 AFM 측정 2 보강 평활근 세포의 분자 표지와 잘 일치 나타날지라도, 이들 동맥 샘플을 기계적으로 로딩되지 않는 것을 인식하는 것이 중요하며, 기계적 부하 전체에 중요한 역할을하는 것 같다 동맥 역학. AFM, myography (기계적 하중과 호환 생체 외 방법) (28)와 맥파 속도 (동맥 경화의 생체 측정) (29)에 의해 결정되는 동맥 경화의 비교 가능성이 arter의 가장 포괄적 인 이해를 제공합니다IAL 역학. 우리는 마우스 동맥 생체 강성을 측정하는 접촉 모드의 AFM을 설명 하였다. 이 방법은 임의로 대동맥의 복수의 영역에 압입하여 조직의 평균 강성을 추정한다. 조직의 큰 영역에서 강도의 공간 분포를 조사하기 위해, 최근 논문은 강제 볼륨 모드 AFM (30)에 사용하고있다. 이 방식은 동시에 강성도 변화의 맵과 조직의 표면 지형의 고 해상도 이미지를 획득. 조직 샘플의 강성을 측정하는 이외에, 포스 볼륨 모드 AFM은 세포 10,31 강도의 공간 분포를 모니터링하기 위해 셀에 사용될 수있다.

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.

<table><tbody><tr><td>BioScope Catalyst AFM system</td><td>Bruker</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE 200 inverted microscope</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Silicon nitride AFM probe</td><td>Novascan Technologies</td><td>PT.SI02.SN.1</td><td>0.06 N/m cantilever; 1 &amp;micro;m SiO&lt;sub&gt;2&lt;/sub&gt; particle</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-10</td><td>See section 1.4</td></tr><tr><td>Dumont #5SF forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>See section 1.8</td></tr><tr><td>Fine Scissors-ToughCut</td><td>Fine Science Tools</td><td>14058-11</td><td>See section 1.4 (medium sized)</td></tr><tr><td>Vannas-T&amp;uuml;bingen spring scissors</td><td>Fine Science Tools</td><td>15008-08</td><td>See section 1.6 (small sized)</td></tr><tr><td>60 mm TC-treated cell culture dish</td><td>Corning</td><td>353004</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, 1x</td><td>Corning</td><td>21-031-CM</td><td>Without calcium and magnesium</td></tr><tr><td>Krazy Glue instant all purpose liquid</td><td>Krazy Glue</td><td>KG58548R</td><td>See section 2.2</td></tr><tr><td>Gel-loading tips, 1 - 200 &amp;micro;l</td><td>Fisher</td><td>02-707-139</td><td>See section 2.2</td></tr><tr><td>Tip Tweezers</td><td>Electron Microscopy Sciences</td><td>78092-CP</td><td>See section 3.2</td></tr><tr><td>50-mm, clear wall glass bottom dishes</td><td>TED PELLA</td><td>14027-20</td><td>See section 4.4</td></tr></tbody></table>

measuring the stiffness of ex vivo mouse aortas using atomic force microscopy

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.

<html> 도 5a는 6 개월, 남성 C57BL / 6 마우스에서 하강 (흉부) 대동맥의 위상 콘트라스트 이미지를 보여줍니다. AFM 캔틸레버 직접 조직 위의 자리에 들여 쓰기를위한 준비.도 5b 및도 5c는 접촉 모드 AFM 들여 쓰기에 의해 얻어진 대표 힘 곡선을 보여줍니다. 도 5b 및도 5c에 도시 된 그린 선 구체 대한 헤르츠 모델을 이용하여 얻어지는 최적 곡선을 나타낸다. 본문에 설명 된도 5d에서, 하행 대동맥 대동맥 아치의 평균 강성 세 개별 쥐에서 측정 하였다. 하행 대동맥과 대동맥 궁은 ​​각각 층류 및 방해 흐름의 영역을 표현합니다. 그럼에도 불구하고, 자신의 (기계적으로 언로드) 탄성 계수는 ​​AFM의 결정에 따라 유사하다. 0 / 54630fig5.jpg &quot;/&gt; 그림 5 : 분석 포스 들여 쓰기 곡선과 하강 마우스 대동맥 생체 통해 AFM 캔틸레버를 나타내는 강성 데이터 (A) 위상차 현미경 사진.. 스케일 바, 100 μm의. (BC) (B) 하행 대동맥과 (C) 대동맥 궁 취득이 대표 강제 압입 곡선의 집합입니다. (D) 평균 마우스 하행 대동맥의 강성과 대동맥 아치로 하였다 위의 설명. 오차 막대는 평균 ± SEM을 보여; N = 3 독립적 인 생물 복제. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54630/54630fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy at JoVE.com

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

의 강성 측정<em&gt; 전의 VIVO</em원자 힘 현미경을 사용하여&gt; 마우스 대동맥

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

measuring-stiffness-ex-vivo-mouse-aortas-using-atomic-force

Universidad San Sebastian

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Biology

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

10.5K 조회수.  University of Pennsylvania. We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

비디오: Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

예 VIVO 바이러스성 전달을 사용하여 헤어 대과에 GFP 타겟팅 표현

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

연구

생물학

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

원자 힘 현미경을 사용하여 살아있는 세포의 기계적 특성을 측정

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

생체공학

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

오름차순 대동맥 강성 측정<em&gt; 생체</em&gt; 초음파를 사용하여 마우스의

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

의학

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

광학 트랩을 사용하여 박테리아 세포의 굽힘 강성을 측정

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

기계 자극에 라이브 세포 응답 통합 광학과 원자 힘 현미경에 의해 연구

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

원자 힘 현미경을 사용하여 폐 조직의 미세 기계적 특성

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

절연, 문화, 그리고 성인 마우스 된 심근의 기능적 특성

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level.
To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young&#39;s (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice.
A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

We present a protocol to measure the elastic moduli of collagen-rich areas in normal and diseased liver using atomic force microscopy. The simultaneous use of polarization microscopy provides high spatial precision for localizing collagen-rich areas in the liver sections.

편광 현미경과 결합된 원자력 현미경을 사용한 간 경화도 측정

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell ...

Measurement of Stiffness in mouse retinal capillaries and subendothelial matrix(마우스 망막 모세혈관 및 내피하 기질의 강성 측정)

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced retinal capillary stiffening plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. The increase in retinal capillary stiffness results from the overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix, and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and subendothelial matrix using atomic force microscopy.

저자 스포트라이트: 초기 당뇨병성 망막병증의 혈관 경직 및 염증 조사

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.

의 강성 측정

요약

더 많은 비디오 탐색

이 비디오의 챕터

예 VIVO 바이러스성 전달을 사용하여 헤어 대과에 GFP 타겟팅 표현

원자 힘 현미경을 사용하여 살아있는 세포의 기계적 특성을 측정

오름차순 대동맥 강성 측정

광학 트랩을 사용하여 박테리아 세포의 굽힘 강성을 측정

기계 자극에 라이브 세포 응답 통합 광학과 원자 힘 현미경에 의해 연구

원자 힘 현미경을 사용하여 폐 조직의 미세 기계적 특성

절연, 문화, 그리고 성인 마우스 된 심근의 기능적 특성

편광 현미경과 결합된 원자력 현미경을 사용한 간 경화도 측정

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy