Histological Quantification of Chronic Myocardial Infarct in Rats

Summary

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

Abstract

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

Introduction

Myocardial infarction (MI) is a leading cause of death and disability worldwide. Coronary heart disease is the main cause; MI results from ischemia consecutive to coronary events such as occlusion. When reperfusion is not performed within the first 6 hr, ischemia induces irreversible myocardial necrosis. In patients, the characterization of MI relies on different diagnostic tools, including clinical signs, electrocardiography, assessment of plasma levels of biomarkers, echocardiography, MRI imaging, and histological analyses¹. Acute and chronic MI are classified as two different phases of injury according to the timing of the myocardial necrosis relative to....

Protocol

All animals received humane care in compliance with the European Convention on Animal Care. Surgical procedures were performed in accordance with the Swiss Animal Protection Law after obtaining permission of the State Veterinary Office, Fribourg, approved by the Swiss Federal Veterinary Office, Switzerland.

1. Heart Harvesting

NOTE: All surgical interventions were performed under isoflurane anesthesia. Efforts were made to diminish animal suffering. In particular, all animals received a subcutaneous injection of 0.1 mg/kg buprenorphine pre-anesthesia. The surgical protocol for inducting myocardial infarct....

Results

Six weeks post-LAD ligation, hearts were harvested from Lewis rats. 2-mm tissue sections were obtained from apex to base. A TTC staining procedure was performed to visualize the infarct area, which appears in white, and the healthy myocardium, which appears in red (Figure 2). Depending on the site of ligation of the LAD, the infarct size varies. For large MI, transmural infarcts were observed from apex to base (Figure 2A). Smaller infarcts presented white.......

Discussion

Critical Steps within the Protocol

Fibrotic tissue can be accurately assessed in a chronic MI rat model using systematic sampling of the harvested heart and image analyses of trichromatic-stained histological sections obtained from base to apex. Two steps are particularly important for successful protocol implementation. First, the use of KCl for heart harvesting allows the cardiac muscle to be maintained in a relaxed state. This step is important for comparisons of infarct di.......

Materials

Name	Company	Catalog Number	Comments
Acrylic rat heart matrix 2mm	72-5015	Harvard Appartus
INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR	HARVARD APPARATUS	557058
CATHETER INSYTE 14G	BD	381267
O.C.T	BDHA361603E	VWR
TTC	T8877-10G	Sigma Aldrich
Mayer hematoxylin	MHS32-1L	Sigma Aldrich
Acid Fuchsin CI 42685	F8129-50G	Sigma Aldrich
Ponceau Xylidin CI 16150	P2395-25G	Sigma Aldrich
Orange G CI 16230	O3756-100G	Sigma Aldrich
Light green CI 42095	L5382-25G	Sigma Aldrich
KCl	P9333-500G	Sigma Aldrich
Xylol	10315083	HoneyWell
Ethanol absolute	10303990	HoneyWell
2-methylbutane	M32631-1L	Sigma Aldrich
Stereogical microscope	SM2800	Nikon
Formaldehyde	99340	Reactolab
Embedding cassette	K113.1	Carl Roth
Bersoft Image measurement Software		Bersoft.com	Licensed software