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.

Déclaration d&#39; éthique: Les protocoles pour la manipulation des animaux ont été approuvés par le Vanderbilt Institutional Animal Care et utilisation Commission (IACUC, protocoles nombre M / 10/117 (porcine) et M / 10/219 (souris) et menées conformément aux normes AAALAC-International. l&#39;utilisation de tissus cardiaques humaines en banque a été approuvé par l&#39;Université Vanderbilt Medical Center IRB (numéro de protocole 100887). 1. Exemp...

Cross préparation section de surface est l&#39;étape la plus critique au cours du protocole. Afin de préserver la structure fine, les échantillons déshydratés doivent rester en éthanol à 100% en tout temps jusqu&#39;à introduit dans le processus de séchage au point critique. Par conséquent, le découpage des échantillons pour réaliser des surfaces d&#39;examen EM doivent être effectués alors que les échantillons sont immergés dans de l&#39;éthanol dans un plat peu profond. Il est également essentiel q...

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.

<table border="0" cellpadding="0" cellspacing="0" width="791">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Calcium Chloride</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">12340</td>
			<td style="width:167px;">100g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Carbon Adhesive</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">12664</td>
			<td>30g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Carbon Adhesive Tabs</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">77825</td>
			<td>order to fit stubs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Double edge razor blades stainless steel</td>
			<td style="width:207px;">Ted Pella, Inc</td>
			<td style="width:119px;">121-6</td>
			<td>250/pkg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ethanol</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15055</td>
			<td>450mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Gluteraldehyde, 50% solution</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">16310</td>
			<td>EM grade, distillation purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Hydrochloric Acid</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">16760 or 16770</td>
			<td>100mL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:299px;">Monosodium phosphate NaH2PO4</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9251-250G</td>
			<td>250g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Osmium Tetroxide</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">19100</td>
			<td>1g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Silver Conductive Adhesive</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">12686-15</td>
			<td>15g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sodium hydroxide (NaOH)</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8045-1KG</td>
			<td>1KG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:299px;">Sodium phosphate dibasic (Na2HPO4)</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:119px;">S3264-500G</td>
			<td>500g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Tannic acid, 5% aqueous&#160;</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">21702-5</td>
			<td>500mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trihydrate Sodium Cacodylate</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">12300</td>
			<td>100g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Gold-palladium Alloy or Gold</td>
			<td style="width:207px;">Refining Systems, Inc.&#160;</td>
			<td style="width:119px;">varies</td>
			<td>&#160;specific to the sputter coater make and model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Critical Point Dryer</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Plain Wooden Applicators</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:119px;">23-400-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Quanta 250 Environmental SEM</td>
			<td style="width:207px;">FEI</td>
			<td style="width:119px;">Q250 SEM</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Sputter coater</td>
			<td style="width:207px;">Cressington Scientific Instruments Ltd.</td>
			<td style="width:119px;">Model 108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Alluminum SEM Sample Stubs</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">75220-12</td>
			<td>specific to the miscroscope</td>
		</tr>
	</tbody>
</table>

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.

La technique mise en évidence a été appliquée aux tissus cardiaques à partir d&#39; un donneur de greffe de cœur humain utilisé (Figure 1), les tissus explantées de receveurs de greffe, des cœurs de type sauvage et souris dystrophique (Figure 3), et post-myocardique échantillons cardiaques infarctus d&#39;un porcine modèle de lésion cardiaque (Figure 2). Comme on le voit sur ​​la figure 1, la matrice card...

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.

Microscopie électronique à balayage du tissu macéré pour visualiser la matrice extracellulaire

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 ...

Research

JoVE Journal

Biology

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

Lumière corrélative et microscopie électronique (CLEM) comme un outil pour visualiser des molécules et de leurs microinjecté eucaryotes Sous-cellulaires cibles


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Recherche

Biologie

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Une méthode standardisée pour l&#39;analyse des cellules endothéliales et sinusoïdale du foie Leurs fenestrations par microscopie électronique à balayage

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

L&#39;étude de l&#39;Organisation supramoléculaire de photosynthétiques Membranes dans les tissus foliaires Gel fracturés par Cryo-microscopie électronique à balayage

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Microscopie électronique à balayage (MEB) Protocoles pour Problematic plantes, Oomycètes et échantillons Fungal

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

Préparation des organismes procaryotes et eucaryotes à l’aide de séchage chimique pour l’analyse morphologique en microscopie électronique (MEB)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

Préparation de l’échantillon biologique par congélation à haute pression, amélioration du contraste assistée par micro-ondes et minime résine enrobage pour l’imagerie de Volume

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Flux de travail de tomographie en réseau pour l’acquisition ciblée d’informations de volume à l’aide de la microscopie électronique à balayage

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

Microscopie électronique à balayage par blocs série (SBF-SEM) d’échantillons de tissus biologiques

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

Préparation de grille cryo-microscopique électronique pour des études résolues dans le temps à l’aide d’un nouveau système robotique, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

Microscopie confocale à balayage laser de la dynamique du calcium dans les tranches de tissu pancréatique aigu de souris

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...