Сканирующая электронная микроскопия из размоченной ткани для визуализации внеклеточный матрикс

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 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-1β, 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.

Protocol

Заявление по этике: протоколы для обработки животных были одобрены Вандербильта Институциональные уходу и использованию животных комитета (IACUC, протоколы число М / 10/117 (свиной) и М / 10/219 (мышей) и проводится в соответствии с AAALAC-Международными стандартами. Использовани…

Representative Results

Выделенное методика была применена к сердечной ткани от неиспользованного человеческого трансплантата сердца донора (рис 1), эксплантированных тканей от реципиентов, сердца от дикого типа и дистрофических мышей (рисунок 3), и в пост-инфарктов образц?…

Discussion

Подготовка Сечение поверхности является наиболее важным шагом в процессе протокола. Чтобы сохранить тонкую структуру, обезвоженные образцы должны оставаться в 100% этаноле в любое время до тех пор, пока вводить в процесс критической точки сушки. Поэтому нарезка образцов для достижения …

Materials

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