Name: echocardiographic and histological examination of cardiac morphology in the mouse
Uploaded: 2017-10-26T12:30:00
Description: Watch this Scientific Journal Video about Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse at JoVE.com

The work was supported by the French Government (National Research Agency, ANR) through the &#34;Investments for the Future&#34; LABEX SIGNALIFE program (reference ANR-11-LABX-0028-01) and by grants to K. D. W. from the Association pour la Recherche sur le Cancer, Fondation de France, and Plan Cancer Inserm. D. B. and A. V. received fellowships from the Fondation pour la Recherche M&#233;dicale and from the City of Nice, respectively. The echocardiograph and the transducer were kindly provided by Philips. We thank A. Borderie, S. Destree, M. Cutajar-Bossert, A. Landouar, A. Martres, A. Biancardini, and S. M. Wagner for their skilled technical assistance.

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 (Comit&#233; Institutionnel d&#39;Ethique Pour l&#39;Animal de Laboratoire; number NCE/2012-106).
1. Echocardiography
<ol>
	<li>Determine the body weight of the mouse using a standard laboratory balance while holding it lightly by the tail to ensure proper positioning.</li>
	<li>Anestheti...

<ol>
	<li>Ormandy, E. H., Dale, J., Griffin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+animals:+ethical+issues,+including+welfare+concerns.">Genetic engineering of animals: ethical issues, including welfare concerns.</a> Can Vet J. 52 (5), 544-550 (2011).</li><li>Karl, T., Pabst, R., von H&#246;rsten, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+of+mice+in+pharmacological+and+toxicological+research.">Behavioral phenotyping of mice in pharmacological and toxicological research.</a> Exp Toxicol Pathol. 55 (1), 69-83 (2003).</li><li>Phoon, C. K., Turnbull, D. 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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 &#62;10 MHz should be used, although successful measurements have been repo...

A large variety of genetically modified mouse models are available, and the number of pharmacological studies in mice is high1,2. 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 mice4,5,6,7,8,9,10,11,12. Pachon et al.11 identified 205 studies published in Circulation, Circulation Research, American Journal of Physiology - Heart and Circulatory Physiology, and Cardiovascular Research between 2012 and 2015 that used echocardiographic examination in animals.
Echocardiography is used to identify cardiac phenotypes in genetically modified mice5,6,13,14,15,16,17,18,19,20,21,22, as well as to analyze cardiac function in chronic overload-induced hypertrophy, myocardial ischemia, and cardiomyopathy models in mice (reviewed in12). Improved echocardiography equipment allows for the the standard measure of left-ventricular (LV) systolic and diastolic dimensions, tissue Doppler imaging, myocardial contrast echography, and the assessment of LV regional function and coronary reserve12. Ideally, echocardiographic examination should be performed in conscious mice to avoid the negative effects of anesthesia on contractile function, autonomic reflex control, and heart rate11. Nevertheless, this approach is limited by the requirement to train the animals; difficulties in keeping the body temperature stable; movement artifacts; stress; very high cardiac frequencies; and the requirement for at least two investigators to perform the experiment, especially if a large number of animals are under investigation. Interestingly, a recent study reported no differences in echocardiographic parameters in trained and untrained animals19. We perform echocardiographic measurements in anesthetized mice. Different anesthesia protocols will be discussed below.
Although standard resolution echocardiography (&#62;10 MHz) is sufficient to measure LV systolic and diastolic dimensions and cardiac function in adult mice, the method is limited in its description of underlying structural phenomena. Thus, we combine the in vivo measurements with histological and immunohistological analyses to measure, for example, cardiomyocyte diameter and vessel density. Other histological and immunohistological investigations, such as the determination of proliferation, examination of apoptosis, infarct size measurements, determination of fibrosis, and specific marker expression, can also be performed on the same type of processed tissue but are not the subject of this protocol. The combination of in vivo echocardiographic examination with histological analyses provides additional insights into underlying structural alterations. In an additional step, we can complete these measurements with molecular and ultra-structural investigations. Histological analyses not only complete the echocardiographic examination but also become indispensible when the resolution of echocardiography is not sufficient. This is especially the case in models of genetically modified mice that are embryonic lethal23,24.

<table>
	<tbody>
		<tr>
			<td>Wheat germ agglutinin (WGA) conjugated tetramethylrhodamine</td>
			<td>Life Technologies, Molecular Probes</td>
			<td>W849</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated Goat Anti-Rabbit IgG Antibody</td>
			<td>Vectorlabs</td>
			<td>BA-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Avidin/Biotin Blocking Kit</td>
			<td>Vectorlabs</td>
			<td>SP-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)</td>
			<td>Vectorlabs</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td>Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>SIGMAFAST 3,3&#39;-Diaminobenzidine tablets</td>
			<td>Sigma</td>
			<td>D4168</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Pecam-1 (CD31) antibody</td>
			<td>Abcam</td>
			<td>ab28364</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound transmission gel, Gel Aquasonic 100</td>
			<td>Parker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Linear ultrasound probe, L15-7io</td>
			<td>Philips Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Echocardiograph, IE33 xMATRIX</td>
			<td>Philips Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope, Leica DMi8</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Filterset DAPI</td>
			<td>Leica</td>
			<td>11525304</td>
			<td></td>
		</tr>
		<tr>
			<td>Filterset TxR</td>
			<td>Leica</td>
			<td>11525310</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera, SPOT RT3 Color Slider</td>
			<td>Spot Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Software, SPOT 5.2 Advanced and Basic Software</td>
			<td>Spot Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Computer</td>
			<td>Dell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Scissors</td>
			<td>Fine Science Tools</td>
			<td>14501-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Fine Science Tools</td>
			<td>10023-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11650-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent shaver</td>
			<td>Harvard Apparatus</td>
			<td>34-0243</td>
			<td></td>
		</tr>
		<tr>
			<td>cassettes for paraffin embedding</td>
			<td>Sakura</td>
			<td>4155F</td>
			<td></td>
		</tr>
		<tr>
			<td>neutral buffered Formalin</td>
			<td>Sakura</td>
			<td>8727</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sakura</td>
			<td>8733</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffine TEK III</td>
			<td>Sakura</td>
			<td>4511</td>
			<td></td>
		</tr>
		<tr>
			<td>automated embedding apparatus, Tissue-Tek VIP</td>
			<td>Sakura</td>
			<td>6032</td>
			<td></td>
		</tr>
		<tr>
			<td>paraffin-embedding station Tissue-Tek TEC 5</td>
			<td>Sakura</td>
			<td>5229</td>
			<td></td>
		</tr>
		<tr>
			<td>microtome blades,Accu-Edge S35</td>
			<td>Sakura</td>
			<td>4685</td>
			<td></td>
		</tr>
		<tr>
			<td>microscopy slides, Tissue-Tek</td>
			<td>Sakura</td>
			<td>9533</td>
			<td></td>
		</tr>
		<tr>
			<td>cover slips, Tissue-Tek</td>
			<td>Sakura</td>
			<td>9582</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium Tissue-Tek</td>
			<td>Sakura</td>
			<td>1408</td>
			<td></td>
		</tr>
		<tr>
			<td>slide boxes</td>
			<td>Sakura</td>
			<td>3958</td>
			<td></td>
		</tr>
		<tr>
			<td>eosine solution</td>
			<td>Sakura</td>
			<td>8703</td>
			<td></td>
		</tr>
		<tr>
			<td>hematoxyline solution</td>
			<td>Sakura</td>
			<td>8711</td>
			<td></td>
		</tr>
		<tr>
			<td>microtome, RM2125RT</td>
			<td>Leica</td>
			<td>720-1880 (VWR)</td>
			<td></td>
		</tr>
		<tr>
			<td>water bath, Leica HI1210</td>
			<td>Leica</td>
			<td>720-0113(VWR)</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>ACRO444220050</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>VWR</td>
			<td>734-0451</td>
			<td></td>
		</tr>
		<tr>
			<td>staining glass dish</td>
			<td>VWR</td>
			<td>MARI4220004</td>
			<td></td>
		</tr>
		<tr>
			<td>staining jars</td>
			<td>VWR</td>
			<td>MARI4200005</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Binder</td>
			<td>9010-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB and urea hydrogen peroxide tablets, SIGMAFAST 3,3&#8242;-Diaminobenzidine tablets</td>
			<td>Sigma</td>
			<td>D4293</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011044</td>
			<td></td>
		</tr>
	</tbody>
</table>

echocardiographic and histological examination of cardiac morphology in the mouse

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&#160;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.

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 ...

Watch this Scientific Journal Video about Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse at JoVE.com

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

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.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

The overall goal of this echocardiographic examination combined with histological investigations is to examine cardiac function and morphology in different transgenic mouse models under baseline and pathophysiological conditions. To begin, while lightly holding a mouse by it's tail, use a standard laboratory balance to determine the body weight of the animal. After anesthetizing the mouse according to the text protocol lightly squeeze a rear foot and observe if the leg retracts.Use a commercial rodent shaver to shave the left side of the thorax and the left armpit. Position the animal on a warm pad set at 40 to 42 degrees Celsius in a shallow left sided position, with the head at 12 o'clock and the tail at 6 o'clock. Then use tape to fix the left arm, left leg and tail.Apply pre-warmed echocardiography gel onto the shaved chest and the head of the transducer. Next place the echocardiograph transducer parasternal left, directing it to the right side of the neck to obtain a two-dimensional parasternal long access view on the level of the papillary muscle. Then turn the transducer 90 degrees clockwise to obtain a short access view.Establish a minimal depth setting and a zoom to maximize image quality and frame rate. Set the sweep speed to the maximum. Record 2-D guided M-mode images in short access view.Record at least three series of three heartbeat semiloops for each animal. After obtaining successful recordings, remove the tape from the limbs and tail. Wipe off the echocardiogrpahy gel from the mouse thorax heating pad and transducer.Leave the mouse under observation on the heating pad, covered with tissue to avoid unnecessary light exposure and heat loss until it wakes up. Then put the animal back in the cage. Analyze recorded M-mode images from short access view to determine left ventricular or LV dimensions and function.Using the identification of the tissue blood interface on the stored images, measure the thickness of the LV anterior wall in systole and diastole, the LV internal end-systolic and end-diastolic diameters, and the LV posterior wall thickness in systole and diastole. Measure the diastolic dimensions at the time of the apparent maximal LV diastolic dimensions and the LV end-systolic dimensions at the end of the most anterior systolic excursion of the LV posterior wall. Tap the touchscreen on the analyze icon and then on the LVAWd icon.Position the electronic caliper on the interface between the right ventricular cavity and the LV anterior wall in diastole. Next position the electronic caliper on the interface between the LV anterior wall and the LV cavity to obtain LV diastolic anterior wall thickness. Position the caliper on the interface between the LV cavity and the LV posterior wall to obtain the LV internal and diastolic diameter.Position the caliper on the interface between the LV posterior wall and the pericardium to obtain the LV diastolic posterior wall thickness. For LV systolic dimensions, tap the touchscreen on the LVAWs icon and position the electronic caliper on the interface between the right ventricular cavity and the LV anterior wall in systole. Position the electronic caliper on the interface between the LV anterior wall and the LV cavity to obtain the LV systolic anterior wall thickness.Position the caliper on the interface between the LV cavity and the LV posterior wall to obtain the LV internal and systolic diameter. Position the caliper on the interface between the LV posterior wall and the pericardium to obtain the LV systolic posterior wall thickness. After sacrificing the animals according to the text protocol measure their body weights.Then use a 70%alcohol swab to disinfect the chest and abdomen. Make a transverse incision in the skin, one centimeter distal to the sternum. Then using blunt forceps, remove the skin from the thorax moving in the direction of the head.With fine forceps, hold the sternum lightly and open the diaphragm by inserting the blunt end of fine scissors. Cut the ribcage on both sides parallel to the sternum. Then move the sternum in the direction of the head.Locate the heart in the thorax. With fine forceps, hold the vascular trunk of the heart and use fine scissors to cut below the organ. Then measure the heart weight and establish a heart-to-body weight ratio.Transfer the heart into two milliliters of 10%neutral buffered Formalin solution in a 15 milliliter tube and fix the organ at four degrees Celsius overnight. To perform Wheat Germ Agglutinin or WGA staining, place the previously prepared slides in Tetramethylrhodamine conjugated WGA and incubate the samples at room temperature in a humid chamber for sixty minutes. Use PBS to wash the samples three times for five minutes each.Then use fluorescence mounting medium contained DAPI to mount the sections. Store them at four degrees Celsius in the dark before analysis. These 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 and an animal with a dilated left ventricle or LV and reduced LV function can easily be identified. Shown here is a comparison of cardiomyocyte diameter measurements in animals without and with cardiac hypertrophy using H&E stained paraffin heart sections and measurements of longitudinal sections of heart tissue. This figure illustrates the measurement of cardiomyocyte transverse diameters using WGA stained paraffin sections from hearts of control mice and animals with cardiac hypertrophy.These panels depict the utility of PECAM1 Immunohistochemistry of cardiac sections to determine the vessel density of normal heart tissue and hearts with increased angiogenesis.

Research

JoVE Journal

Medicine

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Technical Aspects of the Mouse Aortocaval Fistula

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Echocardiographic Assessment of the Right Heart in Mice

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Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

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Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

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Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

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Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

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Cardiac Magnetic Resonance Imaging at 7 Tesla

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Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

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