Name: scanning electron microscopy of macerated tissue to visualize the extracellular matrix
Uploaded: 2016-06-14T15:00:00
Description: Watch this Scientific Journal Video about Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix at JoVE.com

This study was funded by grants from the National Institutes of Health (NIH), Heart, Lung, and Blood Institute (NIHLB): K01-HL-121045, K08-HL-094703, 5T32HL007411-35, P20 HL101425, U01 HL100398. 
Imaging and tissue processing (after NaOH maceration) were performed through the use of the Vanderbilt University Medical Center (VUMC) Cell Imaging Shared Resource (CISR) (supported by NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126). We are especially grateful to the VUMC CISR core directors (Dr. Sam Wells and Dr. W. Gray (Jay) Jerome) for valuable technical advice and also for providing core space and resources for the purposes of filming the technique highlighted in this paper. 
We would like to extend our deepest appreciation to Dr. Yan Ru Su and Ms. Kelsey Tomasek in the Cardiology Core Lab for Translational and Clinical Research at Vanderbilt University for providing technical expertise and for collecting human tissue samples used in this study.

윤리 문 : 동물 처리를위한 프로토콜 밴더빌트 기관 동물 관리 및 사용위원회 (IACUC에 의해 승인 된, AAALAC-국제 표준에 따라 수 M / 1백17분의 10 (돼지) 및 M / 219분의 10 (마우스) 및 실시를 프로토콜. 뱅크 인간의 심장 조직의 사용법은 밴더빌트 대학 의료 센터 IRB (프로토콜 번호 100887)에 의해 승인되었다. 1. 시료 채취 및 보관 <ol><li> 0.1 M 인산 완충액 (PB) 용액에 신?...

단면 표면 처리 프로토콜 중 가장 중요한 단계입니다. 임계점 건조 공정에 도입 될 때까지 미세 구조를 보존하기 위해 탈수 시험편은 항상 100 % 에탄올에 남아 있어야한다. 따라서 표본의 슬라이스는 표본이 얕은 접시에 에탄올에 잠겨있는 동안 EM 시험을위한 표면을 수행해야합니다 달성했다. 노출 된 표면이 접촉 또는 후속 처리 중에 프로브되지 않는 것이 중요하다. 프로토콜의 SEM 부분에 관계?...

Fibrosis disrupts the normal myocardial collagen network, which is critical for normal mechanistic functions of cardiomyocytes 1,2 as well as for inter-cellular communication, intracellular signaling, and cell survival 3. The development of fibrosis is a major determinant of cardiac function, and fibrotic remodeling of the cardiac interstitium arising from a variety of etiologies leads to increased left ventricular stiffness and diastolic dysfunction 4. Myocardial fibrosis may also lead to arrhythmias, and whether the progression of fibrotic remodeling is a general or local phenomenon, it is highly associated with a poor prognosis in patients with ischemic and non-ischemic cardiomyopathy 5. Likewise, the absence of myocardial fibrosis is a strong predictor of ventricular functional recovery and long-term survival 6. 
The hallmark of fibrosis is the deposition of excess collagen, which has the tensile strength of steel 7 and can adversely affect cardiomyocyte function on multiple levels. Mechanical forces resulting from an excessively collagenous matrix can lead to cardiomyocyte atrophy 8,9, passive tissue stiffness 10, tonic contraction-induced myocardial stiffness 11-13, and reduced delivery of oxygen to the remaining population of cardiomyocytes. Gap junction coupling of cardiomyocytes and myoFbs can also compromise the heart's electrical characteristics, creating a greater risk for the development of arrhythmias 14-16. Perivascular fibrosis alters vasomotor reactivity of intramural coronary arteries and arterioles 17 and contributes to luminal narrowing that reduces the supply of oxygen and thus the survival of cardiomyocytes 17-22. Pathogenic fibrotic and electrical remodeling, emanating from an initial site of ischemic injury or energy imbalance, inevitably progresses to heart failure. 
Cardiomyocyte necrosis initiates the fibrotic response, and subsequent adverse fibrotic remodeling can occur irrespective of etiology. Finding a way to control cardiac fibrosis would be clinically beneficial for the treatment of ischemic and idiopathic cardiomyopathies, hypertensive heart disease, hypertrophic cardiomyopathy, valvular heart disease and dystrophinopathies 23-42. Regardless of how the fibrotic disease process begins, soluble, profibrotic factors can cross the interstitial space and provoke activation of interstitial and adventitial fibroblasts at sites remote to the initial fibrotic scar, creating a cascade effect that ultimately leads to heart failure. The optimum scenario would be to exploit the fibrillogenic process using a targeted therapeutic that can be applied during the compensative hypertrophic stage of cardiomyopathy before it progresses to systolic pump failure, diastolic heart failure, or other end-stage outcomes. The ultimate goal would be to reverse fibrosis so that dead cardiomyocytes can be replaced and heart function restored completely. 
The importance of the matrix is widely understood, yet methods to study the matrix are limited mainly to quantitative measurements of major structural components, particularly collagen, and relative levels of different matrix and matricellular proteins. This protocol highlights a rarely used technique that is useful for assessing qualitative differences in the cardiac matrix. This technique has been recently used to compare and contrast fundamental differences in heart matrices from different etiologies of heart disease (in human explants), to examine hearts from post-infarcted pigs treated with the glial growth factor (GGF) isoform of neuregulin-1&#946;, relative to untreated animals 43, and to probe for differences in the matrices of cardiac tissues from mdx mice (a commonly used animal model of Duchenne Muscular Dystrophy) at different ages and compared to wild-type controls. This technique was first introduced by Drs. Caulfield and Borg in 1979 44, but few studies have since employed this powerful technique 45-47, re-introduced here with only slight modification. This methodology is a valuable research tool, because it provides qualitative information about extracellular matrix ultrastructure that might otherwise be overlooked when simply measuring matrix component content and/or level of fibrosis.

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scanning electron microscopy of macerated tissue to visualize the extracellular matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

강조 표시된 기술은 사용하지 않는 인간의 심장 이식 기증자 (그림 1), 이식, 야생형과 이영 마우스 (그림 3)에서 마음에서 외식 조직에서 심장 조직에, 그리고 돼지에서 후 심근 경색 심장 샘플에 적용되었다 심장 손상 모델 (그림 2). 도 1에 도시 된 바와 같이, 인간의 심장 행렬은 단면에서 보았을 때, 벌집 형 패턴을 표시 ...

Watch this Scientific Journal Video about Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix at JoVE.com

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

침연 (macerated) 조직의 전자 현미경을 스캔하면 세포 외 매트릭스를 시각화하기

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

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