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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Published: June 14th, 2016

DOI:

10.3791/54005

1Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, 2Department of Medicine, Vanderbilt University Medical Center, 3Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 4Cardiovascular Institute, Maine Medical Center

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

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.

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 p....

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Ethics Statement: The protocols for animal handling were approved by the Vanderbilt Institutional Animal Care and Use Committee (IACUC, protocols number M/10/117 (swine) and M/10/219 (mice) and conducted according to AAALAC-International standards. Usage of banked human cardiac tissues was approved by the Vanderbilt University Medical Center IRB (protocol number 100887).

1. Sample Collection and Storage

  1. Make fresh 4% glutaraldehyde in 0.1 M phosphate buffer (PB) so.......

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The highlighted technique was applied to cardiac tissues from an unused human heart transplant donor (Figure 1), explanted tissues from transplant recipients, hearts from wild-type and dystrophic mice (Figure 3), and in post-myocardial infarction heart samples from a swine model of heart injury (Figure 2). As shown in Figure 1, the human cardiac matrix is an intricate weave of cross-linked proteins that display a honeycom.......

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Cross section surface preparation is the most critical step during the protocol. To preserve fine structure, dehydrated specimens must remain in 100% ethanol at all times until introduced to the critical point drying process. Therefore the slicing of the specimens to achieve surfaces for EM examination must be done while specimens are submersed in ethanol in a shallow dish. It is also critical that the exposed surface is not touched or probed during subsequent handling. No major modifications are anticipated for applicat.......

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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 fil....

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Name Company Catalog Number Comments
Calcium Chloride Electron Microscopy Sciences 12340 100g
Carbon Adhesive Electron Microscopy Sciences 12664 30g
Carbon Adhesive Tabs Electron Microscopy Sciences 77825 order to fit stubs
Double edge razor blades stainless steel Ted Pella, Inc 121-6 250/pkg
Ethanol Electron Microscopy Sciences 15055 450mL
Gluteraldehyde, 50% solution Electron Microscopy Sciences 16310 EM grade, distillation purified
Hydrochloric Acid Electron Microscopy Sciences 16760 or 16770 100mL
Monosodium phosphate NaH2PO4 Sigma-Aldrich S9251-250G 250g
Osmium Tetroxide Electron Microscopy Sciences 19100 1g
Silver Conductive Adhesive Electron Microscopy Sciences 12686-15 15g
Sodium hydroxide (NaOH) Sigma-Aldrich S8045-1KG 1KG
Sodium phosphate dibasic (Na2HPO4) Sigma-Aldrich S3264-500G 500g
Tannic acid, 5% aqueous  Electron Microscopy Sciences 21702-5 500mL
Trihydrate Sodium Cacodylate Electron Microscopy Sciences 12300 100g
Gold-palladium Alloy or Gold Refining Systems, Inc.  varies  specific to the sputter coater make and model
Critical Point Dryer Electron Microscopy Sciences 850
Plain Wooden Applicators Fisher Scientific 23-400-102
Quanta 250 Environmental SEM FEI Q250 SEM
Sputter coater Cressington Scientific Instruments Ltd. Model 108
Alluminum SEM Sample Stubs Electron Microscopy Sciences 75220-12 specific to the miscroscope

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