Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

Summary

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

Abstract

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Introduction

A large variety of genetically modified mouse models are available, and the number of pharmacological studies in mice is high^1,2. Echocardiography and MRI are commonly used approaches for the phenotypic characterization of cardiac function and morphology in these mouse models³. The aim of the presented protocol is to analyze cardiac function and morphology in adult mice. It combines echocardiographic, histological, and immunohistochemical measurements. Echocardiographic examination is widely used in mice^4,5,

Protocol

The experiments described here were carried out in compliance with the relevant institutional and French animal welfare laws, guidelines, and policies. They have been approved by the French ethics committee (Comité Institutionnel d'Ethique Pour l'Animal de Laboratoire; number NCE/2012-106).

1. Echocardiography

1. Determine the body weight of the mouse using a standard laboratory balance while holding it lightly by the tail to ensure proper positioning.
2. Anesthetize the animal by the intraperitoneal (i.p.) injection of 50 mg/kg pentobarbital^25,26.
   ....

Results

In Figure 1, representative echocardiographic recordings demonstrate the usefulness of echocardiography to identify cardiac phenotypes in genetically modified mice. The difference between a mouse with normal cardiac function (Figure 1A) and an animal with a dilated left ventricle and reduced LV function (Figure 1B) can easily be identified. Figure 2 shows the comparison .......

Discussion

Different methods have been developed to evaluate cardiac structure and function in mice, including echocardiography, contrast-enhanced MRI, micro CT, and PET scan. Due to its cost-effectiveness and simplicity, echocardiography is the most widely used technique for functional analysis in mice¹¹. In general, because of the small size of the heart and the high frequency of the heart rate in mice, transducers with a frequency >10 MHz should be used, although successful measurements have been repo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Wheat germ agglutinin (WGA) conjugated tetramethylrhodamine	Life Technologies, Molecular Probes	W849
Biotinylated Goat Anti-Rabbit IgG Antibody	Vectorlabs	BA-1000
Avidin/Biotin Blocking Kit	Vectorlabs	SP-2001
VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)	Vectorlabs	PK-6100
VECTASHIELD Antifade Mounting Medium with DAPI	Vectorlabs	H-1200
SIGMAFAST 3,3'-Diaminobenzidine tablets	Sigma	D4168
Hydrogen peroxide solution	Sigma	H1009
Anti-Pecam-1 (CD31) antibody	Abcam	ab28364
Ultrasound transmission gel, Gel Aquasonic 100	Parker
Linear ultrasound probe, L15-7io	Philips Healthcare
Echocardiograph, IE33 xMATRIX	Philips Healthcare
Microscope, Leica DMi8	Leica
Fluorescence Filterset DAPI	Leica	11525304
Filterset TxR	Leica	11525310
Digital Camera, SPOT RT3 Color Slider	Spot Imaging
Imaging Software, SPOT 5.2 Advanced and Basic Software	Spot Imaging
Imaging Computer	Dell
Fine Scissors	Fine Science Tools	14028-10
Large Scissors	Fine Science Tools	14501-14
Scalpel blades	Fine Science Tools	10023-00
Graefe Forceps	Fine Science Tools	11650-10
Rodent shaver	Harvard Apparatus	34-0243
cassettes for paraffin embedding	Sakura	4155F
neutral buffered Formalin	Sakura	8727
Xylene	Sakura	8733
Paraffine TEK III	Sakura	4511
automated embedding apparatus, Tissue-Tek VIP	Sakura	6032
paraffin-embedding station Tissue-Tek TEC 5	Sakura	5229
microtome blades,Accu-Edge S35	Sakura	4685
microscopy slides, Tissue-Tek	Sakura	9533
cover slips, Tissue-Tek	Sakura	9582
Mounting medium Tissue-Tek	Sakura	1408
slide boxes	Sakura	3958
eosine solution	Sakura	8703
hematoxyline solution	Sakura	8711
microtome, RM2125RT	Leica	720-1880 (VWR)
water bath, Leica HI1210	Leica	720-0113(VWR)
Ethanol	VWR	ACRO444220050
15 ml tubes	VWR	734-0451
staining glass dish	VWR	MARI4220004
staining jars	VWR	MARI4200005
Incubator	Binder	9010-0012
DAB and urea hydrogen peroxide tablets, SIGMAFAST 3,3′-Diaminobenzidine tablets	Sigma	D4293
PBS (10X)	Thermo Fisher Scientific	70011044