Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Summary

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

Abstract

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.

Introduction

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....

Protocol

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) solution.
   CAUTION: Glutaraldehyde is toxic, wear gloves and work in a fume hood.
   1. Make a 0.2 M stock solution of PB, pH 7.4 using 21.8....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Calcium Chloride	Electron Microscopy Sciences	12340	100 g
Carbon Adhesive	Electron Microscopy Sciences	12664	30 g
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	450 ml
Gluteraldehyde, 50% Solution	Electron Microscopy Sciences	16310	EM grade, distillation purified
Hydrochloric Acid	Electron Microscopy Sciences	16760 or 16770	100 ml
Monosodium Phosphate NaH2PO4	Sigma-Aldrich	S9251-250G	250 g
Osmium Tetroxide	Electron Microscopy Sciences	19100	1 g
Silver Conductive Adhesive	Electron Microscopy Sciences	12686-15	15 g
Sodium hydroxide (NaOH)	Sigma-Aldrich	S8045-1KG	1 kg
Sodium Phosphate Dibasic (Na2HPO4)	Sigma-Aldrich	S3264-500G	500 g
Tannic Acid, 5% Aqueous	Electron Microscopy Sciences	21702-5	500 ml
Trihydrate Sodium Cacodylate	Electron Microscopy Sciences	12300	100 g
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