The authors have nothing to disclose.

The protocol follows the guidelines of the Institutional Animal Care and Use Committee of University of Washington.
1. Surgical Procedure and Preparation for Imaging
<ol>
	<li>Subject C57BL/6 mice to TAC or sham surgery as previously described10.</li>
	<li>One week after TAC or sham surgery, anesthetize the mouse in the induction chamber with 2% isoflurane mixed with 1 L/min O2. Confirm proper anesthetization by unresponsiveness to toe or tail pinching. Use veterinary ointment on eyes to prevent dryness while under anesthesia.&#160;Remove the chest hair by applying hair removal cream. Disinfect mouse skin with 70% ethanol.</li>
	<li>Secure the mouse to an animal-handling platform in the supine position. To maintain a steady level of anesthesia, use a nosecone to deliver 0.5 - 1% isoflurane mixed with 1 L/min O2.</li>
	<li>Apply electrode gel to the paws of the mouse and tape them to the electrode pad.</li>
	<li>Insert a rectal probe to monitor body temperature. Maintain the body temperature at 37 &#176;C via a heating pad or lamp.</li>
	<li>Apply a layer of pre-warmed ultrasound gel to the mouse chest, mainly the area overlying the heart.&#160;&#160;Note: remove ultrasound gel and dry the mouse with sterile gauze after the imaging procedure.</li>
</ol>
2. In the Aortic Arch View, Use B-mode and Doppler Imaging to Evaluate Transverse Aortic Constriction
<ol>
	<li>Use the B-mode setting to obtain the aortic arch view in order to visualize the aorta, major arterial branches, and the constriction site.
	<ol>
		<li>Tilt the left side of the platform up as far as possible to rotate the mouse into left decubitus position. Hold the ultrasound transducer by stand in vertical position and place it on the chest along the right parasternal line, with the notch pointing towards the chin of the mouse.&#160;Note: Do not compress the mouse thorax when lowering the transducer; minimal amount of pressure is required.</li>
		<li>Tilt the transducer up at the level of scapula and rotate slightly clockwise until aortic arch comes into view. Observe the transverse aortic constriction site, which is located between the branching of innominate artery (IA) and left common carotid artery (LCCA) (Figure 1). 
		Note: No constriction is detected in sham-operated mouse.</li>
	</ol>
	</li>
	<li>Click the &#34;color Doppler&#34; button on the workstation to switch to color Doppler mode to monitor directionality and velocity of the blood flow across the constriction site. Acquire and store images by clicking the &#34;cine store&#34; button.</li>
	<li>Click the &#34;PW Doppler&#34; button to switch to pulse wave Doppler mode, and place sample volume (the dashed cursor box) immediately distal to the constriction site to search for the stenotic jet with the highest velocity, then click the &#34;PW Doppler&#34; button to obtain waveforms of aortic flow and measure peak velocity (Figure 2).</li>
	<li>Calculate pressure gradient across the constriction site using the modified Bernoulli&#39;s equation: pressure gradient = 4 x Vmax2. Only include mice with a pressure gradient ranging from 40 to 80 mmHg for further analysis.</li>
</ol>
3. In the Parasternal Long Axis View, Use B-mode and M-mode Imaging to Assess Cardiac Dimensions and Contractility
<ol>
	<li>With the mouse lying in the supine position on the platform, hold the transducer in vertical fashion with the notch pointing to the mouse&#39;s head. Lower the transducer on the thorax parallel to the left parasternal line and rotate 30&#176; counter-clockwise.</li>
	<li>Use B-mode imaging to obtain a full long axis &#34;sagittal&#34; view of the heart. Adjust the angle of the transducer and focus depth to visualize left ventricle, the intraventricular septal wall, and a slight portion of the right ventricular wall. Save the images for later measurements of cardiac wall thickness and chamber dimension. Using &#34;cardiac package&#34;, select parameters such as IVS or LVAW, LVID, and LVPW, and then click on the image to draw corresponding lines for each parameter to obtain the measurements.</li>
	<li>Observe cardiac wall movement patterns and check for possible motion abnormalities, including akinesia, hypokinesia, and asynchrony. 
	Note: Akinesia and hypokinesia denote complete and partial loss of movement of the cardiac wall, respectively. Asynchrony denotes irregular, uncoordinated cardiac wall movement.</li>
	<li>Switch to M-mode, place M-mode cursor perpendicular to the LV walls at the level of the papillary muscle, and acquire images for later measurement of cardiac dimensions and fractional shortening (Figure 3).</li>
</ol>
4. In the Parasternal Short Axis View, Use B-mode and M-mode Imaging to Assess Cardiac Morphology and Function
<ol>
	<li>From the parasternal long axis view, obtain parasternal short axis view by rotating the transducer 90&#176; clockwise. Adjust the transducer to give a horizontal cross-sectional &#34;transverse&#34; view of the heart in B-mode, with both papillary muscles clearly visible and located to the right (the 2 and 4 o&#39;clock position).</li>
	<li>Switch to M-mode and place the M-mode axis at the mid-level of the left ventricle. Acquire and store images for later measurements of cardiac wall thickness, chamber dimension, and fractional shortening (Figure 4). Using &#34;cardiac package&#34;, select parameters in SAX (short axis) including IVS or LVAW, LVID, and LVPW, and click on the image to draw corresponding lines for each parameter to obtain the measurements. 
	Note: Measurements obtained here should correlate closely to those obtained in the parasternal long axis view (Figure 5).</li>
</ol>
5. In the Apical Four-chamber View, Use Doppler Imaging to Assess Systolic and Diastolic Function
<ol>
	<li>Obtain the apical four-chamber view to visualize both left and right ventricles with the atria at the bottom of the screen. In B-mode, from the short axis view, tilt the upper left corner of the platform to angle the mouse&#39;s head down and orient the transducer towards the right shoulder of the mouse. This is essentially to achieve a &#34;coronal&#34; view of the heart looking up towards the apex.</li>
	<li>Visualize the mitral valve in B-mode, and switch to color Doppler mode, placing the sample volume (the dashed cursor box) at the tip of the mitral valve.</li>
	<li>Switch to PW Doppler mode to assess flow patterns across the mitral valve. Align the Doppler probe cursor parallel to the direction of mitral blood flow. Use a probe angle less than 20&#176; to determine peak velocity (Figure 6).</li>
	<li>Save the images for later measurements. Use &#34;cardiac package&#34; and select &#34;MV flow&#34;. Click each parameter and draw corresponding lines to obtain the measurements. Available measurements include: peak E velocity (early filling with active ventricular relaxation), peak A velocity (late filling with atrial contraction), mitral isovolumic relaxation and contraction times (IVRT and IVCT respectively), and ejection time (ET).</li>
	<li>Calculate myocardial performance index (MPI) by MPI = (IVCT + IVRT)/ET.</li>
</ol>
6. Post-procedural Treatment of Animal
<ol>
	<li>Give analgesia and/or sterile saline intraperitoneally to surgical animals when necessary.</li>
	<li>Allow the animal to recover on a heating pad in the prone position. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone the procedure to the company of other animals until fully recovered.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+models+of+heart+failure:+development+of+novel+therapies,+past+and+present.">Small animal models of heart failure: development of novel therapies, past and present.</a> Circ Heart Fail. 2 (2), 138-144 (2009).</li><li>Heineke, J., Molkentin, J. 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D., Aronovitz, M. J., Bridgman, P., Pandian, N. G. <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), 708-714 (2002).</li><li>deAlmeida, A. C., van Oort, R. J., Wehrens, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transverse+aortic+constriction+in+mice.">Transverse aortic constriction in mice.</a> J Vis Exp. (38), e1729 (2010).</li><li>Pacher, P., Nagayama, T., Mukhopadhyay, P., B&#225;tkai, S., Kass, D. A. <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), 1422-1434 (2008).</li><li>Larsen, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+working+mouse+heart:+methodological+considerations.">The isolated working mouse heart: methodological considerations.</a> Pflugers Arch. 437 (6), 979-985 (1999).</li><li>Roth, D. M., Swaney, J. S., Dalton, N. D., Gilpin, E. 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This work was supported in part by NIH/NHLBI grants R00HL0908076 and R01HL116507 (to Q.L.).

Echocardiography has been widely used to assess cardiac function in rodent models of heart disease2,6. Compared to invasive or terminal methodologies such as pressure-volume loop measurement11 and ex vivo working heart12, echocardiography provides a powerful, noninvasive tool to assess ongoing cardiac structural and functional changes in living animals. To obtain reliable data, it is important to maintain body temperature and heart rate within physiological range13 by careful adjusting the heating apparatus and the anesthesia level. All images should be captured and analyzed consistently according to the standardized imaging procedures, to facilitate the comparison between mice of different strain or genotype.
TAC is commonly used to induce cardiac hypertrophy and heart failure in mice1. Noninvasive measurement of pressure gradient across the constriction site by Doppler imaging represents a reliable assessment of the degree of pressure overload in mice. Successful TAC typically produces a pressure gradient &#8805;40 mmHg. Only mice subjected to similar degree of pressure overload should be included for further analysis, while mice with a pressure gradient too low or too high should be excluded. Following TAC, mice are expected to develop cardiac hypertrophy within 1-2 weeks, and cardiac dilatation after 4 weeks, depending the degree of pressure overload and the genetic background of mice tested. The dynamic cardiac remodeling and functional changes following TAC can be reliably assessed by echocardiographic imaging as described above.
In contrast to its frequent usage in humans14, color/PW Doppler has only been recently available in rodent ultrasound imaging9. Here also we described the applications of Doppler imaging in measuring pressure gradient as well as systolic and diastolic performance. Measurement of mitral and tricuspid blood flow directionality and velocity (i.e., E/A ratio, IVRT, IVCT, ET, and MPI) provides important information on cardiac function. Thus echocardiographic imaging represents an important tool to study cardiac physiology and pathophysiology in small animals.
The limitation of cardiac ultrasound imaging is related to measurement variability and reproducibility. To reduce inter- and intra- operator variability, it is important to standardize how images are acquired and analyzed. Measurements should be performed from multiple acoustic windows and modes (B-mode, M-mode, and PW/color Doppler) and at least 3 separate measurements should be averaged to ensure accuracy and reliability. In addition, there are limited acoustic windows and sometimes low quality images are obtained in small rodents subjected to surgical procedures such as TAC, due to tissue swelling, surgical scars, and lung edema that interfere with the ultrasound beams. For Doppler imaging, sometimes it is challenging to separate E and A waves and obtain a complete waveform of the mitral flow, due to a relatively high heart rate in small rodents, especially in mice subjected to TAC or MI surgery. Lowering the heart rate may be helpful to get measurements, but this will affect values obtained by Doppler imaging and hence the data interpretation.
With recent technical advances, newly released ultrasound systems provide high image resolution and frame/sampling rates to ensure accurate quantitative measurement in small animals. New echocardiography technologies will also improve the sensitivity of echocardiographic evaluation of cardiac function and permit early detection of cardiac pathology. For example, speckle-tracking strain imaging15 has been used to precisely measure regional myocardial function. New transducer technologies currently under development will provide the potential for real-time, 3D or 4D imaging. Contrast echocardiography that is in advanced development will allow for volumetric measurements, tissue perfusion assessments, molecular imaging of cardiovascular disease, and delivery of therapeutic agents.

Mouse models of heart disease, such as TAC and myocardial infarction (MI), have been proven to be valuable to study disease mechanisms as well as to develop novel therapeutic strategies3. TAC initially induces compensatory hypertrophy, but prolonged pressure overload leads to cardiac dilatation and heart failure4. The tightness of the aortic constriction directly determines the degree of cardiac hypertrophy and its transition to heart failure. Noninvasive and reliable measurement of pressure gradient across the aortic constriction is essential for the success of these studies. Doppler imaging has been used to assess pressure gradient produced by TAC5, which is a noninvasive alternative for catheter-based pressure measurement.
Echocardiography has been widely used to noninvasively measure cardiac morphology as well as systolic and diastolic function in mice6-8. Two-dimensional B-mode imaging is used to detect abnormal movements or structural changes of the heart. One-dimensional M-mode imaging is used for quantification of cardiac dimensions and contractility. Color and PW Doppler imaging has recently been used on rodent ultrasound, which has broad applications for echocardiography, including measurement of flow directionality and velocity, as well as systolic and diastolic performance9. 
Longitudinal real-time monitoring of cardiac function using echocardiography in B-mode, M-mode, color and PW Doppler mode provides comprehensive assessment of cardiac structure and function in mice under physiological and pathological conditions. Here we provide a detailed description of the use of echocardiographic imaging to monitor dynamic cardiac morphological and functional changes in mice following TAC or sham surgery.

<table><tbody><tr><td>Anesthesia equipment</td><td>Harvard Apparatus, 84 October Hill Road&lt;br/&gt; Holliston, MA</td><td>723015</td><td></td></tr><tr><td>Vevo 2100 Imaging System</td><td>VisualSonics Inc., 3080 Yonge Street Suite 6100, Box 66, Toronto, Ontario, Canada</td><td>Vevo 2100</td><td></td></tr><tr><td>Aquasonic ultrasound gel</td><td>Parker Laboratories, 286 Eldridge Rd, Fairfield, NJ&amp;nbsp;</td><td>03-50</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Healthcare, Inc, 3950 Schelden Circle&lt;br/&gt; Bethlehem, PA&amp;nbsp;</td><td>NDC 66794-017-25</td><td></td></tr><tr><td>F/air anesthesia gas filter unit</td><td>A.M. Bickford, Inc, 12318 Big Tree Rd, Wales Center, NY&amp;nbsp;</td><td>80120</td><td></td></tr></tbody></table>

assessment of cardiac morphological and functional changes in mouse model of transverse aortic constriction by echocardiographic imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

Figure 1 shows B-mode images of the aortic arch view of mouse heart subjected to sham (Figure 1A) or TAC surgery (Figure 1B). The aortic arch, innominate artery, left common carotid artery, and left subclavian artery are shown. Note that aortic constriction is clearly visible in TAC but not sham heart. Color Doppler images from aortic view are shown in Figure 2A. The waveforms of aortic flow across the constriction site were captured by PW Doppler imaging (Figure 2B). Successful TAC will lead to a significantly increased flow velocity downstream the constriction site (typically ~4 m/sec in TAC mice). Pressure gradient across the constriction was calculated based on peak flow velocity, according to the modified Bernoulli&#39;s equation (Figure 2C).
Figure 3 shows B- and M-mode images of the parasternal long axis view of sham (Figure 3A) or TAC heart (Figure 3B). The upper panel shows the B-mode images of the left ventricle, the interventricular septum, and a portion of the right ventricle from sham or TAC mice. The lower panel shows the M-mode tracings of several cardiac cycles from sham or TAC mice. The measurements of cardiac dimensions are shown, including the left ventricular anterior wall thickness (LVAW), left ventricular internal diameter (LVID), left ventricular posterior wall thickness (LVPW) in diastole and systole. Note significantly increased wall thickness in mouse heart subjected to TAC compared with sham surgery.
Figure 4 shows images of the parasternal short axis view of sham (Figure 4A) or TAC heart (Figure 4B). The upper portion of each panel shows the M-mode axis (the dotted line) placed in the center of the left ventricle. The lower portion of each panel is the M-mode tracing with lines indicating cardiac dimensions as described above. As a marker of hypertrophy, ventricular and septal wall thickness can be accurately determined. Mice subjected to TAC showed increased wall thickness as assessed by LVAWd and LVPWd, ventricular dilatation as assessed by LVISd and LVISs, decreased contractility as assessed by LVFS and LVEF, and increased LV mass (Figure 5).
Figure 6 shows B-mode apical four-chamber view (Figure 6A,B) and PW Doppler images of transmitral flow patterns (Figure 6C,D). Measurements of peak E and A velocity, IVCT, IVRT, and ET are shown. The E/A ratio and MPI are calculated (Figure 6E - I). A healthy mouse heart has an E/A ratio &#8805;1 and a MPI value &#8804;0.5. In pathological conditions with diastolic or systolic cardiac dysfunction, such as in mice subjected to TAC, a decreased E/A ratio and/or an increased MPI value are typically observed.
<img alt="Figure 1" src="/files/ftp_upload/54101/54101fig1.jpg" /> 
Figure 1. B-mode Image of the Aortic Arch View of Mouse Heart Subjected to Sham Surgery (A) or TAC (B). Major aortic branches including innominate artery (IA), left common carotid artery (LCCA), and left subclavian artery (LSA) are shown. Note that transverse aortic constriction (indicated by the white arrow) can be visualized in TAC but not sham heart. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54101/54101fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54101/54101fig2.jpg" /> 
Figure 2. Color/PW Doppler Imaging of the Transverse Aorta Blood Fow from the Aortic Arch View. Color (A) and PW (B) Doppler images from sham and TAC hearts are shown. Peak aortic velocity obtained from PW Doppler imaging is used to calculate pressure gradient according to the modified Bernoulli equation (C). These data confirm a successful TAC surgery with the pressure gradient of ~70 mmHg. *P&#160;&#60;0.05 vs. Sham. Data are expressed as mean &#177; s.e.m. n = 15 for Sham and n = 13 for TAC. Student&#39;s t-test was used to determine statistical significance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54101/54101fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54101/54101fig3.jpg" /> 
Figure 3. Parasternal Long Axis (PLAX) View of Mouse Heart Subjected to Sham Surgery (A) or TAC (B). M-mode images indicate the placement of the sample volume (dotted yellow line in the upper panel) and measurement of cardiac dimensions in diastole and systole (blue lines in the lower panel). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54101/54101fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54101/54101fig4.jpg" /> 
Figure 4. Parasternal Short Axis (PSAX) View of Mouse Heart Subjected to Sham Surgery (A) or TAC (B). M-mode images indicate the placement of the sample volume (dotted yellow line in the upper panel) and measurement of cardiac dimensions in diastole and systole (blue lines in the lower panel). The asterisks indicate papillary muscles. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54101/54101fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54101/54101fig5.jpg" /> 
Figure 5. Echocardiographic Assessment of Cardiac Morphological and Functional Changes Following TAC. M-mode imaging in short axis view was performed as in Figure 4. (A) LVAWd, left ventricular anterior wall thickness in diastole. (B) LVPWd, left ventricular posterior wall thickness in diastole. (C) LVIDd, left ventricular internal diameter in diastole. (D) LVIDs, left ventricular internal diameter in systole. (E) LVFS, left ventricular fractional shortening. LVFS (%) = (LVIDd-LVIDs)/LVIDd x100%. (F) LVEF, left ventricular ejection fraction. LVEF (%) = (LVEDV-LVESV)/LVEDV x100%. LVEDV and LVESV denote left ventricular end-diastolic and end-systolic volume, respectively. LV volume and ejection fraction are precisely assessed by Simpson&#39;s method. LV volume is estimated by fitting numerous disks into the ventricle: Simpson volume = [area(1) + area(2) + &#8230; + area(n)] x length. Simpson area and length are obtained by tracing the endocardial border of the LV in the long axis and short axis view. (G) LV (left ventricular) mass. LV mass (mg) = 1.05 x [(LVIDd + LVPWd + IVSd)3&#160;- (LVIDd)3]. The factor 1.05 represents the specific density of the myocardium. (H)&#160;HR, heart rate. *P&#160;&#60;0.05 vs. Sham. The number of mice analyzed is shown in the bars of each panel. Data are expressed as mean &#177; s.e.m. Student&#39;s t-test was used to determine statistical significance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54101/54101fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54101/54101fig6.jpg" /> 
Figure 6. Assessment of Transmitral Blood Flow by Doppler Imaging. (A and B) B-mode apical four-chamber view of sham (A) or TAC (B) heart. LV, left ventricle; RV, right ventricle; MV, mitral valve; TV, tricuspid valve; LA, left atrium; RA, right atrium.&#160;(C and D)&#160;PW Doppler waveform of trans-mitral blood flow in sham (C) or TAC (D) heart.&#160;Relevant measurements are shown. (E) E/A, peak E and A velocity ratio. (F) IVCT, isovolumic contraction time. (G) IVRT, isovolumic relaxation time. (H) ET, ejection time. (I) MPI, myocardial performance index. *P&#160;&#60;0.05 vs. Sham. The number of mice analyzed is shown in the bars of each panel. Data are expressed as mean &#177; s.e.m. Student&#39;s t-test was used to determine statistical significance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54101/54101fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

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18.1K Views.  University of Washington. The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

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

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.

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 ...

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  ...

Ultrasonic Assessment of Myocardial Microstructure

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. Ultrasonic assessments of cardiac tissue can include analyses of backscatter signal intensity within a given region of interest. Previously established techniques have relied predominantly on the integrated or mean value of backscatter signal intensities, which may be susceptible to variability from aliased data from low frame rates and time delays for algorithms based on cyclic variation. Herein, we describe an ultrasound-based imaging algorithm that extends from previous methods, can be applied to a single image frame&#160;and accounts for the full distribution of signal intensity values derived from a given myocardial sample. When applied to representative mouse and human imaging data, the algorithm distinguishes between subjects with and without exposure to chronic afterload resistance. The algorithm offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and ...

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, ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Biology

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

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.

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 ...

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic dobutamine stress echocardiography in humans, a reductionist investigation approach to unmask subtle changes in cardiac function has become possible. Here, we provide a protocol for using this technique in mice to facilitate expanded analysis of LV architecture and function in physiology and pathology enabling the observation of alterations in models of cardiac disease hidden in unstressed hearts. This investigation can be performed in one and the same animal and allows both, basal and pharmacologically stress-induced measurements. We outline detailed criteria for appropriate anesthesia, imaging-based LV analysis, consideration of intra- and interobserver variability, and obtaining positive inotrope response that can be attained in mice after intraperitoneal injection of dobutamine under near physiological conditions. To recapitulate the characteristics of human physiology and disease in small animal models, we highlight critical pitfalls in evaluation, e.g., a pronounced Bowditch effect in mice. To further meet translational objectives, we compare stress-induced effects in humans and mice. When used in translational studies, attention must be paid to physiological differences between mice and human. Experimental rigor dictates that some parameters assessed in patients can only be used with caution due to restrictions in spatial and temporal resolution in mouse models.

Left ventricular dysfunction constitutes the final common pathway for a host of cardiac disorders. We here provide a detailed protocol of transthoracic dobutamine stress echocardiography approach for comprehensive evaluation of the left ventricular function of mouse models of cardiac disease as well as cardiac phenotyping.

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...