Name: transthoracic echocardiography in mice
Uploaded: 2010-05-28T17:00:00
Duration: 8 min 9 s
Description: Watch this Scientific Journal Video about Transthoracic Echocardiography in Mice at JoVE.com

We would like to thank Dr. Corey Reynolds and the BCM Mouse Phenotyping Core for their expert help with this project and kind access to the facilities. X.H.T.W. is a W.M. Keck Foundation Distinguished Young Scholar in Medical Research, and is also supported by NIH/NHLBI grants R01-HL089598 and R01-HL091947, and Muscular Dystrophy Association grant #69238. This work is also supported in part by the Foundation Leducq Alliance for CaMKII Signaling in Heart.

1. Preparing Mice for Imaging Studies
<ol>
 <li>Prior to the imaging studies, anesthetize the mouse (2% isoflurane mixed with 0.5 L/min 100% O2)in the induction chamber. Be sure to filter waste gas for the safety of the operator. Remove the animal from the induction chamber and use hair clippers to shave the fur from the neckline to mid chest level. Then remove the remaining body hair with hair removal cream. This preparation may be performed a day prior to imaging studies to minimize potential undesired stress responses in the mice.</li>
 <li>Apply duralube gel to both eyes to prevent drying of the sclera (avoid contacting the cornea with the gel applicator). Place the anesthetized mouse in a supine position atop a heating pad with embedded ECG leads in order to maintain body temperature.</li>
 <li>Place the snout within a nose cone connected to the anesthesia system to maintain a steady-state sedation level throughout the procedure (1.0% to 1.5% isoflurane mixed with 0.5 L/min 100% O2). Perform a toe or tail pinch to confirm sedation. If necessary, the level of anesthesia can be adjusted to obtain a target heart rate of 450 &plusmn; 50 beats per minute (bpm). </li>
 <li>Gently insert a rectal probe (after lubricating) to continuously monitor and adjust body temperature via the heating pad. It is important to maintain the body temperature within a narrow range (37.0 &deg;C &plusmn; 0.5 &deg;C), as even moderate changes in temperature and heart rate affect cardiac function in mice. </li>
 <li>Apply electrode gel to the four paws and tape them to the ECG electrodes. </li>
</ol>
2. Evaluating Systolic Function of the Heart
<ol>
 <li>To begin this procedure, apply a layer of preheated ultrasound gel to the chest, primarily the area overlying the heart. Gel should be warmed to approximate body temperature. A volume of cold gel may rapidly add to the rate of body temperature loss. Avoid air bubbles in the gel, which can interfere with ultrasonic imaging.</li>
 <li>Immobilize the mouse in a slight upward position (head up) under a slight angle. Using a micromanipulator, immobilize the ultrasound probe with a 90&deg; angle between the probe and the heart. </li>
 <li>To begin making short-axis echocardiographic measurements, lower the probe onto the thorax parasternally. Avoid placing the probe directly over the sternum or onto the skin, as this will distort the signal.</li>
 <li>First, we will perform two-dimensional (2D) imaging ('B-mode') to obtain a view along the parasternal short axis. Adjust the viewing area of the probe to obtain a complete image depending on the size of the animal's heart. </li>
 <li>A proper image in this orientation will include the left ventricle and a slight portion of the right ventricular wall. The arrow (shown in video) indicates the anterolateral and posteromedial papillary muscles, the posterior and anterior walls of the LV, the intraventricular septal wall, and a slight portion of the right ventricular wall. During these measurements, pay special attention to potential wall motion abnormalities (e.g., hypokinesia, akinasia, asynchrony) or aneurysms. </li>
 <li>Next, we will use M-mode echocardiography, which provides a 1-dimensional (1D) view, to obtain fine measurements of cardiac dimensions and contractility. Store acquired images for later evaluation of parameters of systolic left ventricular function. </li>
</ol>
3. Evaluating Diastolic Function of the Heart
<ol>
	<li>While still viewing the parasternal short axis, we can perform tissue Doppler imaging (TDI) to measure velocity of myocardial motion. The region of interest is marked to include a posterior left ventricular wall for radial axis evaluation. The waveform obtained will have four peaks to evaluate diastolic function: IVRT, E', A', and IVCT. S' represents systolic velocity. (Figure 3B) These parameters will be described later in the representative results section. </li>
 <li>Pulse-wave Doppler (PWD) can be used to measure blood flow velocity within a small area at a specific depth in the myocardial tissue. To image the trans-mitral flow pattern, tilt the animal backwards in the Trendelenburg position. Tilt the probe upward, such that the probe is orthogonal with the apex of the heart. </li>
 <li>Now increase the isoflurane level to lower the heart rate to 300-350 bpm, which will slow the movement of the mitral valve. For comparison studies, it is important to maintain similar heart rates between animals. </li>
 <li>Visualize the movements of the mitral valve as a reference point to measure the flow across the valve. Place the sample volume at the tip of the mitral valve. </li>
 <li>Measure the flow patterns across the mitral valve using PWD. Two waves are characteristically seen, one representing passive filling of the ventricle (early [E] wave), and one consistent with active filling with atrial systole (atrial [A] wave). In a healthy heart, the E-wave velocity is slightly greater than that of the A wave (Figure 3C). </li>
 <li>Once all measurements are completed, remove the electrode gel (by gently wiping with standard gauze pads) and restraints from the animal. Allow the unrestrained animal to recover atop the heated ECG pad in an upright position and turn off the anesthesia. When the mouse awakens, return it to its cage. </li>
</ol>
4. Representative Transthoracic Echocardiography Results
Noninvasive echocardiographic studies can be used to assess left ventricular morphology and function in murine models of cardiac disease.4,5 This technique is widely used and provides an alternative to terminal procedures to assess LV function, such as intracardiac pressure-volume loop measurements.8 or the ex vivo working heart model.9 Assessment of LV chamber dimensions by transthoracic M-mode echocardiography allows for detection of abnormal LV chamber enlargement or increased wall thickness in various models of heart disease.5 Thus, transthoracic echocardiography, combined with pulse wave Doppler, constitutes a comprehensive avenue for the non-invasive evaluation of systolic and diastolic cardiac function in mice.
<img src="/files/ftp_upload/1738/1738fig1.jpg" alt="Figure 1" /> Figure 1. Overview of Vevo 770 High Resolution In Vivo Micro-Imaging System for Echocardiography and Doppler Imaging. (A) We use the VisualSonics integrated rail system with physiological monitoring unit, EKG board, and RMV 30mHz scan head. (B) The mouse is positioned and properly restraint on the heating board with integrated ECG electrodes (gold color). The four limbs are taped into the ECG electrodes. (C-D) Photographs showing proper angles for pulse-wave Doppler used to obtain diastolic function measurements, as described in section 4 of the protocol. Probe orientation for systolic function measurements are show in the video protocol.
<img src="/files/ftp_upload/1738/1738fig2.jpg" alt="Figure 2" /> Figure 2. Representative 2D Echocardiography Images (B-mode). (A) Para-sternal short-axis view showing the left ventricular anterior (AW) and posterior (PW) walls, the intraventricular septal wall (SW), and lateral wall (LW). The diameter of the left ventricular lumen can be measured as the left ventricular internal diameter (LVID). The asterisk indicates the posteromedial papillary muscle. (B) Apical four-chamber view with a viewing area over the mitral valve for determination of E/A peak velocities. MVAL, mitral valve anterior leaflet; MVPL, mitral valve posterior leaflet; RV, right ventricle; LV, left ventricle.
<img src="/files/ftp_upload/1738/1738fig3.jpg" alt="Figure 3" /> Figure 3. Acquisition and Quantification of Echocardiography and Doppler Data. This figure contains representative images of acquired data corresponding to the 2D echocardiography images presented in Figure 2. (A) M-mode tracing with lines indicating end-systolic (ESD) and end-diastolic diameters (EDD). LVAWD/S, left ventricular anterior wall thickness (diastole/ systole); LVPWD/S, left ventricular posterior wall thickness (diastole/ systole). (B) Representative tracings for tissue Doppler imaging of the LV posterior wall. IVRT, isovolumic relaxation time; IVCT, isovolumic contraction time. The E' wave corresponds to the motion of the mitral annulus during early diastolic filling of the LV, and the A' wave originates from atrial systole during late filling of the LV. S' represent systolic velocity. (C) Pulse wave Doppler recording of mitral valve leaflet tips provides mitral inflow velocity patterns from which early diastolic velocity (E), late diastolic velocity with diastolic contraction (A), and the E/A ratio can be derived. The IVRT is also a useful variable to characterize diastolic function and filling pressures. ET indicates ejection time.

<ol>
	<li>Zhang, T. M. a. i. e. r., S, L., Dalton, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deltaC+isoform+of+CaMKII+is+activated+in+cardiac+hypertrophy+and+induces+dilated+cardiomyopathy+and+heart+failure.">The deltaC isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure.</a> Circ Res. 92 (8), 912-912 (2003).</li><li>Srivastava, D., Thomas, T., Lin, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cardiac+mesodermal+and+neural+crest+development+by+the+bHLH+transcription+factor,+dHAND.">Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND.</a> Nat Genet. 16 (2), 154-154 (1997).</li><li>Sood, S., Chelu, M. G., van Oort, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+leak+due+to+FKBP12.6+deficiency+in+mice+facilitates+the+inducibility+of+atrial+fibrillation.">Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation.</a> Heart Rhythm. 5 (7), 1047-1047 (2008).</li><li>Gardin, J. M., Siri, F. M., Kitsis, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+assessment+of+left+ventricular+mass+and+systolic+function+in+mice.">Echocardiographic assessment of left ventricular mass and systolic function in mice.</a> Circ Res. 76 (5), 907-907 (1995).</li><li>Tanaka, N., Dalton, N., Mao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transthoracic+echocardiography+in+models+of+cardiac+disease+in+the+mouse.">Transthoracic echocardiography in models of cardiac disease in the mouse.</a> Circulation. 94 (5), 1109-1109 (1996).</li><li>Patten, R. D., Aronovitz, M. J., Bridgman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+pulse+wave+and+color+flow+Doppler+echocardiography+in+mouse+models+of+human+disease.">Use of pulse wave and color flow Doppler echocardiography in mouse models of human disease.</a> J Am Soc Echocardiogr. 15 (7), 708-708 (2002).</li><li>Garcia-Fernandez, M. A., Azevedo, J., Moreno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+Left+Ventricular+Diastolic+Dysfunction+Evaluated+by+Pulsed-Tissue+Doppler+Echocardiography.">Regional Left Ventricular Diastolic Dysfunction Evaluated by Pulsed-Tissue Doppler Echocardiography.</a> Echocardiography. 16 (5), 491-491 (1999).</li><li>Pacher, P., Nagayama, T., Mukhopadhyay, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cardiac+function+using+pressure-volume+conductance+catheter+technique+in+mice+and+rats.">Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats.</a> Nat Protoc. 3 (9), 1422-1422 (2008).</li><li>Larsen, T. S., Belke, D. D., Sas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pflugers Arch. 437 (6), 979-979 (1999).</li><li>Roth, D. M., Swaney, J. S., Dalton, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+anesthesia+on+cardiac+function+during+echocardiography+in+mice.">Impact of anesthesia on cardiac function during echocardiography in mice.</a> Am J Physiol Heart Circ Physiol. 282 (6), H2134-H2134 (2002).</li></ol>

It is important to standardize the level of anesthesia,10 body temperature, and heart rate within a cohort of mice to facilitate the comparison between different groups or genotypes of mice when using M-mode echocardiography. For PWD assessment of diastolic function, heart rates must be lowered in order to evaluate the diastolic properties of the murine heart. The vital functions of the animal must be closely observed in order to prevent hemodynamic failure and death. On the other hand, for systolic function measurements using M-model echocardiography, heart rates below 400 beats per minute can cause end-diastolic diameters to increase due to increased filling of the LV.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Vevo 770 high-resolution in vivo micro-imaging system *</td>
<td>&nbsp;</td>
<td>VisualSonics</td>
<td>&nbsp;</td>
<td>www.visualsonics.com/products</td>
</tr>
<tr>
<td>RMV (Real-time MicroVisualization) 707B 30MHz ccanhead with encapsulated transducer</td>
<td>&nbsp;</td>
<td>VisualSonics</td>
<td>&nbsp;</td>
<td>www.visualsonics.com/products</td>
</tr>
<tr>
<td>Vevo integrated rail system including physiological monitoring unit</td>
<td>&nbsp;</td>
<td>VisualSonics</td>
<td>&nbsp;</td>
<td>www.visualsonics.com/products</td>
</tr>
<tr>
<td>Vevo compact anesthesia system</td>
<td>&nbsp;</td>
<td>VisualSonics</td>
<td>&nbsp;</td>
<td>www.visualsonics.com/products</td>
</tr>
<tr>
<td>Heating lamp </td>
<td>&nbsp;</td>
<td>General Supply</td>
<td>&nbsp;</td>
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<td>Hair removal lotion (Nair)</td>
<td>&nbsp;</td>
<td>General Supply</td>
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<td>Sigma cr&eacute;me (electrode cream)</td>
<td>&nbsp;</td>
<td>Parker Labs</td>
<td>&nbsp;</td>
<td>Part#17-05; www.cardioogyshop.com</td>
</tr>
<tr>
<td>Ecogel 100 ultrasound gel</td>
<td>&nbsp;</td>
<td>Eco-Med Pharmaceuticals Inc.</td>
<td>&nbsp;</td>
<td>Code 30GB;
www.eco-med.com</td>
</tr>
<tr>
<td>Standard gauze pads</td>
<td>&nbsp;</td>
<td>Safe Cross First Aid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Tape/ cotton swabs/ heating pads (x2)/ Clidox/ ddH2O</td>
<td>&nbsp;</td>
<td>General Supply </td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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			* Required software is Vevo software including B-Mode application, pulse wave Doppler application, and cardiovascular analysis tools package.		</div>

transthoracic echocardiography in mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Watch this Scientific Journal Video about Transthoracic Echocardiography in Mice at JoVE.com

Transthoracic Echocardiography in Mice

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

Research

JoVE Journal

Medicine

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the examiners&#39; expertise. Speckle tracking Echocardiography (STE) is a promising but technically challenging method that can be used to quantitatively assess regional and global systolic and diastolic myocardial performance. Myocardial strain and strain rate can be measured in all three dimensions&#160;&#8212; radial, circumferential, longitudinal &#8212; of myocardial deformation. Standard cross-sectional two-dimensional B-mode images are recorded and subsequently postprocessed by automated continuous frame-by-frame tracking and motion analysis of speckles within the myocardium. Images are recorded as digital loops and synchronized to a 3-lead EKG for timing purposes. Longitudinal deformation is assessed in the apical 4-, 3-, and 2-chamber views. Circumferential and radial deformation are measured in the parasternal short axis plane.
Optimal image quality and accurate tissue tracking are paramount for the correct determination of myocardial performance parameters. Utilizing transthoracic STE in a healthy volunteer, the present article is a detailed outline of the essential steps and potential pitfalls of quantitative echocardiographic myocardial deformation analysis.

Speckle tracking echocardiography is an emerging diagnostic imaging technique for the quantitative assessment of global and regional myocardial performance. Standard view echocardiographic motion images are recorded and deformation parameters are subsequently measured by automated continuous frame-by-frame tracking and motion analysis of speckles within the B-mode images of the myocardium.

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model species such as zebrafish and salamanders that are capable of intrinsic heart regeneration hold promise for future regenerative therapies for human patients. To evaluate the outcome of cardioregenerative experiments it is imperative that heart function can be monitored. The axolotl salamander (A. mexicanum) represents a well-established model species in regenerative biology attaining sizes that allows for evaluation of cardiac function. The purpose of this protocol is to establish methods to reproducibly measure cardiac function in the axolotl using echocardiography. The application of different anesthetics (benzocaine, MS-222, and propofol) is demonstrated, and the acquisition of two-dimensional (2D) echocardiographic data in both anesthetized and unanesthetized axolotls is described. 2D echocardiography of the three-dimensional (3D) heart can suffer from imprecision and subjectivity of measurements, and to alleviate this phenomenon a solid method, namely intra/inter-operator/observer analysis, to measure and minimize this bias is demonstrated. Finally, a method to acquire 3D echocardiographic data of the beating axolotl heart at a very high spatiotemporal resolution and with pronounced blood-to-tissue contrast is described. Overall, this protocol should provide the necessary methods to evaluate cardiac function and model anatomy, and flow dynamics in the axolotl using ultrasound imaging with applications in both regenerative biology and general physiological experiments.

2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum

Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...