Funding for this research includes NIH-T32 (T.H.), R01HL133286 (TT.H.), R01HL094414 (R.M.S.), R01HL138577 (R.M.S.), R01HL159983 and R21AG074593 (R.M.S. and TT.H.). We extend our gratitude to all members of the research group, adjunct investigators, and staff in the Nora Eccles Harrison Cardiovascular Research and Training Institute and Comparative Medicine at the University of Utah. We would also like to extend our appreciation to Dr. Joseph Palatinus MD PhD for his valuable echocardiography training and assistance.

The building and use of the echocardiography cart were conducted in compliance with the animal handling and training standards of the University of Utah&#39;s Institutional Animal Care and Use Committee.
1. Considerations for building the echocardiography cart
<ol>
	<li>Build a contraption that allows access to lateral and ventral standing echocardiography imaging windows.</li>
	<li>Use a cart with tall sides, front, and back to prevent the pigs from climbing or jumping out during echocardiography.</li>
	<li>Use a length- and width-adjustable cart to accommodate pigs of various sizes and ages; however, in research studies in which all pigs are of the same size, breed, and age, this may not be necessary.</li>
	<li>Procure a non-slip ramp for mounting and dismounting the cart. Pigs prefer to walk forward, so a backward dismount is undesirable. In this example, a detachable ramp was utilized, such that it could be disconnected from one end after mounting the cart and then moved to the other end of the cart for dismounting.</li>
	<li>Use a wheeled cart with locking wheels as this allows the contraption to be moved from storage to the animal room.</li>
	<li>To comply with vivarium cleaning and disinfection standards, use plastic, metal, and rubber material.</li>
	<li>Finally, include a detachable feeding trough to the front of the cart to provide the pigs with a distraction during the echocardiography.</li>
</ol>
2. Cart-building specifications
NOTE: The pigs utilized in our study were Yucatan and Gottingen miniature pigs aged 5-10 months old, and, therefore, our cart was constructed with this size in mind.
<ol>
	<li>While a similar structure could be built from scratch, to decrease some of the building work, start from a premade heavy-duty utility cart (Figure 1). Locking wheels are recommended.</li>
	<li>Saw off and remove the front and back of the utility cart, and replace with gates constructed of PVC pipe and chain link. Use the chain link material for hanging a food trough using hooks or carabiners (Figure 2). With the removal of the front and back, the integrity and holding strength of the plastic are reduced, so for larger and heavier pigs, reinforce with metal bars on the undersides of the cart shelves.</li>
	<li>Create an aperture on the top floor of the cart that is large enough to pass a hand holding an ultrasound probe. Retain a plastic cover piece fitted to the top of the aperture, which can be removed for subxiphoid echocardiography access once the pigs are safely standing in the cart (Figure 3).</li>
	<li>As a custom fit, use&#160;aluminum stock (e.g., square tube, bar, and sheet metal)&#160;to build a metal mount/dismount ramp with reinforcements to attach to the modified cart at certain attachment points. Add removable rubber padding for grip using bolts and grommets (Figure 4).</li>
	<li>Create a hinge mechanism for the side gates, with simple pins used to narrow the pig standing area for improved restraint (Figure 5). This provides a snug fit for the pig within the cart and ensures the pigs are restrained in a forward-facing direction without the ability to turn around.</li>
</ol>
3. Training the minipigs to stand in the cart
<ol>
	<li>Pigs must be trained to eat from the trough for a prolonged period of time, walk up the ramp, and walk down the ramp.</li>
	<li>Filling the trough with a frozen treat prolongs the pigs&#39; standing time. Use combinations such as juice, meal replacer drink, or yogurt with cereal and standard chow, biscuits, and/or fruit bars. Freeze these combinations together to create long-lasting frozen treat troughs (Figure 6). Another option to consider is to withhold the animal&#39;s normal meal and instead feed it in the trough in the cart during the echocardiography period.</li>
	<li>Prior to teaching the pigs to eat the frozen treats in the contraption, introduce the frozen treat troughs on the ground, encouraging the recognition of the trough as providing a high-value treat.</li>
	<li>Train the pigs to accept standing echocardiograms for 5-7 days, with one training session per day. Perform the following steps to do this.
	<ol>
		<li>Introduce the pigs to the cart by surrounding the cart with high-value treats (biscuits or cereal) for 1-2 days.</li>
		<li>Introduce the pigs to the ramp for 1-2 days by placing high-value treats along the ramp and providing additional rewards when the pigs walk up the ramp.</li>
		<li>Allow the pigs to stand on the cart without engaging the side restraints or gates (which can induce fear), and provide frozen treat troughs at the front gate for 2-3 days. During the last 1-2 days, while the pigs eat from the troughs, place the ultrasound probe with ultrasound gel on the pigs to accustom them to the sensation of contact with the probe.</li>
		<li>After this training regimen, the pigs will easily allow the restraints and gates to be closed and ultrasound to be performed for the acquisition of echocardiography imaging.</li>
	</ol>
	</li>
</ol>
4. Image acquisition
<ol>
	<li>Obtain images in the echocardiography cart, which allows probe positioning in the following positions, as described below.
	<ol>
		<li>Take images through the sides of the cart to the right and left axillae. These positions are used to obtain the right and left parasternal imaging planes.</li>
		<li>Take images through the floor of the cart to the subxiphoid region to obtain apical views.</li>
	</ol>
	</li>
	<li>Take images for both B-mode and M-mode imaging from these standing positions.</li>
	<li>For a mock awake echo recording procedure, see Video 1. In the video, the animal subject is represented by a real-sized minipig doll, constrained like a real minipig with limited moving room in the cart. The best access window and positioning of the recording echocardiography probe are also shown.</li>
</ol>

<ol>
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I., 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=Heart+disease+and+stroke+statistics+-+2022+Update:+A+report+from+the+American+Heart+Association.">Heart disease and stroke statistics - 2022 Update: A report from the American Heart Association.</a> Circulation. 145 (5), e153-e639 (2022).</li><li>Billig, 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=Transesophageal+echocardiography+in+swine:+evaluation+of+left+and+right+ventricular+structure,+function,+and+myocardial+work.">Transesophageal echocardiography in swine: evaluation of left and right ventricular structure, function, and myocardial work.</a> International Journal of Cardiovascular Imaging. 37 (3), 835-846 (2021).</li><li>Boon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Veterinary Echocardiography., 2nd edition. , (2011).</li><li>Silva, K. A. S., Emter, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+animal+models+of+heart+failure:+A+translational+bridge+to+clinical+success.">Large animal models of heart failure: A translational bridge to clinical success.</a> Journal of the American College of Cardiology: Basic to Translational Science. 5 (8), 840-856 (2020).</li><li>Pilz, P. M., 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=Large+and+small+animal+models+of+heart+failure+with+reduced+ejection+fraction.">Large and small animal models of heart failure with reduced ejection fraction.</a> Circulation Research. 130 (12), 1888-1905 (2022).</li><li>Paslawska, U., 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=Normal+electrocardiographic+and+echocardiographic+(M-mode+and+two-dimensional)+values+in+Polish+Landrace+pigs.">Normal electrocardiographic and echocardiographic (M-mode and two-dimensional) values in Polish Landrace pigs.</a> Acta Veterinaria Scandinavica. 56 (1), 54 (2014).</li><li>Sharp, T. E., 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=Novel+Gottingen+miniswine+model+of+heart+failure+with+preserved+ejection+fraction+integrating+multiple+comorbidities.">Novel Gottingen miniswine model of heart failure with preserved ejection fraction integrating multiple comorbidities.</a> Journal of the American College of Cardiology: Basic to Translational Science. 6 (2), 154-170 (2021).</li><li>Olver, T. D., 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=Western+diet-fed,+aortic-banded+Ossabaw+swine:+A+preclinical+model+of+cardio-metabolic+heart+failure.">Western diet-fed, aortic-banded Ossabaw swine: A preclinical model of cardio-metabolic heart failure.</a> Journal of the American College of Cardiology: Basic to Translational Science. 4 (3), 404-421 (2019).</li><li>Merin, R. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+propofol,+dexmedetomidine,+or+ketofol+on+respiratory+and+hemodynamic+profiles+in+cardiac+patients+undergoing+transesophageal+echocardiography:+A+prospective+randomized+study.">Effects of propofol, dexmedetomidine, or ketofol on respiratory and hemodynamic profiles in cardiac patients undergoing transesophageal echocardiography: A prospective randomized study.</a> Journal of Cardiothoracic and Vascular Anesthesia. 35 (9), 2743-2750 (2021).</li><li>Stoelting, R. K., Hillier, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Pharmacology &amp; Physiology in Anesthetic Practice., 2nd edition. , (2006).</li><li>Kristensen, S. D., 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=ESC/ESA+Guidelines+on+non-cardiac+surgery:+Cardiovascular+assessment+and+management:+The+Joint+Task+Force+on+non-cardiac+surgery:+Cardiovascular+assessment+and+management+of+the+European+Society+of+Cardiology+(ESC)+and+the+European+Society+of+Anaesthesiology+(ESA).">ESC/ESA Guidelines on non-cardiac surgery: Cardiovascular assessment and management: The Joint Task Force on non-cardiac surgery: Cardiovascular assessment and management of the European Society of Cardiology (ESC) and the European Society of Anaesthesiology (ESA).</a> European Heart Journal. 35 (35), 2383-2431 (2014).</li><li>Roth, D. M., Swaney, J. S., Dalton, N. D., Gilpin, E. A., Ross Jr, J. <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> American Journal of Physiology - Heart and Circulatory Physiology. 282 (6), H2134-H2140 (2002).</li><li>Chengode, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+global+systolic+function+assessment+by+echocardiography.">Left ventricular global systolic function assessment by echocardiography.</a> Annals of Cardiac Anaesthesia. 19 (Suppl 1), S26-S34 (2016).</li><li>Cacciapuoti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+evaluation+of+ejection+fraction:+3DE+versus+2DE+and+M-Mode.">Echocardiographic evaluation of ejection fraction: 3DE versus 2DE and M-Mode.</a> Heart Views. 9 (2), 71-79 (2008).</li><li>Yang, H., Galang, K. 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The authors have no financial conflicts of interest to disclose.

The echocardiography cart represents an easily replicable method for monitoring the heart structure and function in an important cardiac research model, the minipig. The cart's novelty lies in the ability to capture echocardiographic images without its biggest caveat: the necessity of using anesthetics or sedatives that change the animals' cardiac function and alter the very measurements used to assess the effects of cardiac therapeutics. Moreover, the cart is safe, inexpensive, and an easy training target for pigs. 
The authors first identified the desired features of the cart and then worked closely with a carpenter to design the product. Standard positive reinforcement training techniques were easy and rapid for teaching the pigs to fearlessly accept the cart and utilize it. With ultrasonography practice, the authors were able to rapidly find and record standard two-dimensional echocardiography imaging planes for later processing. During these standing echocardiograms, sedatives or anesthetics were never administered, and, therefore, the videos and images were representative of awake cardiac function. 
The building of the echocardiography cart is relatively simple for an experienced carpenter or handyman after identifying the key features important to the research group (for example, size adjustability, height, or ultrasound probe access points). During the building process, the features of the cart can be altered to suit individual laboratories' needs. The materials are largely inexpensive, and building the cart may save on the cost of performing echocardiograms with the sedatives and anesthetics typically used.
The limitations of the technique included the motion and the limited timeframe to obtain the images. While the cart could be adjusted to varying sizes to restrain the pigs, and while the animals could not turn around and could only move a few inches in each direction, the animals were still capable of motion within the confines of the cart. A headgate, squeeze chute, or stanchion, such as those used with farm animals, could potentially provide better restraint with additional training. Similarly, successful imaging relied on the animals being distracted by their feed or frozen treats during the echocardiograms. Typically, this allowed approximately 15 min of imaging, which was not always sufficient for obtaining all the desired images. The ability to easily replace the food trough or add feed while the animal remained restrained may have prolonged imaging duration. Finally, due to both of the above limitations, more sensitive imaging techniques, such as tissue Doppler, proved difficult to perform in the standing echocardiography cart.
Other porcine experimental models often utilize non-anesthetic handling techniques, such as the commercially available Panepinto sling17. However, the authors found the sling technique more cumbersome for training pigs, and the sling did not provide the ultrasonographer access to the imaging planes required for echocardiography. Other potential applications for the echocardiography cart could include other non-painful procedures such as abdominal ultrasonography, skin lesion observation, or obtaining blood samples from a vascular access port. The authors often utilize the cart to easily restrain pigs to perform electrocardiograms and program pacemakers, for example. 
In conclusion, the described awake echocardiography technique is easy to perform and valuable for obtaining basic ultrasound imaging of the heart without the cardiovascular depression typical of anesthetic or sedative use. This technique can be utilized to compare anesthetized images to awake images in large animals or for the everyday monitoring of cardiac disease progression in the valuable porcine preclinical translational model of heart disease and failure.

Heart failure is an increasing burden to medical institutions in the United States and abroad, with a worldwide prevalence of 38 million patients1. Approximately 19 million deaths globally were attributed to cardiovascular disease in 2020, demonstrating an 18.7% increase from 20102. New therapy development is slow to catch up with this alarming trend. Heart failure is, therefore, a critical area of research, and the importance of high-fidelity tools for capturing disease development and progression cannot be overstated.
Echocardiography is currently the most clinically important tool for non-invasively measuring the progression of cardiac disease, but in large animal research models, it can be challenging to implement3. Echocardiography uses ultrasonic waves to assess cardiac structure and function and is the standard of care in the clinical setting for cardiac assessment and monitoring4. Preclinical large animal models of cardiac diseases, such as pigs, play a critical role in translating basic science to the development of cardiovascular therapeutics5. It follows, then, that translating echocardiography to large animal models in developing these therapeutics is an important part of this critical endeavor.
Pigs are one of several species commonly used as large animal models of ischemic, pressure overload, and rapid-pacing simulations of heart failure5,6. Pigs are especially important in preclinical studies, as neurohormonal compensatory mechanisms and cardiac remodeling closely mirror human pathophysiology6,7. More recently, miniature pigs, or minipigs, have demonstrated promise as a multiple comorbidities&#160;model of cardiac disease, with obesity, hypertension, hypercholesterolemia, and diabetes reliably resulting in cardiac dysfunction and remodeling8,9.
Safely performing echocardiography in most large animals requires heavy sedation or general anesthesia. However, all anesthetic and sedative drugs depress cardiac function in a dose-dependent manner10,11. Anesthetics and sedatives may cause the depression of cardiac output and blood pressure, increases or decreases in heart rate and systemic vascular resistance, changes in the electrical rhythm, and altered coronary blood flow12. In most cases, anesthetics reduce sympathetic tone, decreasing venous return and lowering blood pressure13. Importantly, anesthetics also affect echocardiographic parameters, complicating the interpretation of this examination in monitoring cardiac disease in animal models14. Awake echocardiography is the closest representation of the native cardiac function.
A porcine restraint device, readily accepted by awake minipigs, is described here that can be used for basic echocardiographic monitoring without requiring the administration of hemodynamic-altering anesthetics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Access Ramp</td>
			<td>N/A - shop built</td>
			<td>58&#34; L x 18&#34; W. Rise of 19&#34; not to exceed 22.5 degree angle.</td>
			<td>Any removable aluminum ramp with capacity to hold weight of pigs</td>
		</tr>
		<tr>
			<td>Fence Feeder with Clips</td>
			<td>DuraFlex&#160;</td>
			<td>E011772</td>
			<td>Feed trough with clips for hanging on chain link, used for frozen treats or feed to distract pigs during echocardiography</td>
		</tr>
		<tr>
			<td>Heavy Duty Utility Cart</td>
			<td>Baxter Medical Equipment &#38; Supplies</td>
			<td>Cart # unk / 45x25x33&#34;; Pipes, sch 40 PVC&#160;</td>
			<td>Made of heavy plastic, with three shelves</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>Image J FIJI&#160;</td>
			<td>https://imagej.net/software/fiji/</td>
			<td>Free scientific image analysis software</td>
		</tr>
		<tr>
			<td>Lumify Ultrasound with S4-1 Phased Array Transducer</td>
			<td>Philips</td>
			<td>FUS6884</td>
			<td>Handheld bedside ultrasound with cardiac probe, used with a tablet device and proprietary software</td>
		</tr>
		<tr>
			<td>Video Editing Software</td>
			<td>Adobe Premiere Pro 2022</td>
			<td>https://www.adobe.com/products/premiere.html</td>
			<td>Commen software part of Adobe Creative Cloud.</td>
		</tr>
	</tbody>
</table>

echocardiography recording in awake miniature pigs

Echocardiography uses ultrasonic waves to non-invasively assess cardiac structure and function and is the standard of care for cardiac assessment and monitoring. The miniature pig, or minipig, is increasingly being used as a model of cardiac disease in medical research. Pigs are notoriously difficult to restrain and handle safely, and, therefore, research echocardiography in this species is almost always performed under anesthesia or heavy sedation. Anesthetics and sedatives universally affect cardiovascular function and may cause the depression of cardiac output and blood pressure, increases or decreases in heart rate and systemic vascular resistance, changes in the electrical rhythm, and altered coronary blood flow. Therefore, sedated or anesthetized echocardiography may not accurately depict the progression of cardiac disease in large animal models, thereby limiting the translational value of these important studies. This paper describes a novel device that allows for standing awake echocardiography in minipigs. In addition, training techniques used to teach pigs to tolerate this painless and non-invasive procedure without the need for hemodynamic-altering anesthetics are described. Standing awake echocardiography represents a safe and feasible way to perform the most common cardiac monitoring test in minipigs for cardiovascular research.

The representative images acquired in a Yucatan minipig at approximately 8 months of age are presented here. The animal was never sedated and was enjoying feed or frozen treat troughs during the image acquisition.
The echocardiography cart is primarily useful for obtaining simple images for calculating left ventricular chamber volumes and ejection fraction (EF) from B-mode or M-mode images and videos. More sensitive imaging, such as vascular imaging or tissue Doppler, may prove too challenging with this technique, as the awake pigs retain limited mobility, and the imaging time frame is limited by the feeding duration.
The laboratory uses a bedside ultrasound machine without post-hoc image analysis capabilities. Therefore, the videos and still images require processing using editing software and measurement using a scientific image analysis software.
Short-axis transverse-view M-mode images (Figure 7) were obtained through the sides of the cart, which were used for the analysis of the left ventricular internal diameter in systole and diastole (LVIDs and LVIDd, respectively) and the subsequent calculation of the ejection fraction (EF), where EF = (EDV - ESV)/EDV &#215; 100% (EDV: end diastolic volume; ESV: end systolic volume) based on volumes calculated with the Teichholz formula (volume = 7D3/[2.4 + D]) (D: linear LV diameter)15. Representative data generated from four M-mode scans obtained from two minipigs are included in Table 1, as well as data generated from M-mode scans recorded from the same animals during sedated echo sessions. As expected, the EF generated from sedated echo sessions tended to be lower than the EF from the conscious echo session.
Parasternal long-axis view B-mode images were also obtained from the sides of the cart (Figure 8). The area-length method for calculating the EF was utilized with these B-mode images16. First, the left ventricular EDV was calculated from the end-diastolic left ventricular major axis length and chamber area using the formula EDV = (0.85 &#215; area2)/length. The left ventricular ESV was calculated in the same manner using the systolic measurements. The ejection fraction was then calculated as EF = (EDV - ESV)/EDV &#215; 100%. Representative data generated from eight B-mode scans obtained from two minipigs are included in Table 2. For comparison, data generated from B-mode scans recorded from the same animals during sedated echo sessions are also included. Using the B-mode images, the EF generated from sedated and conscious echo sessions were closely matched with each other (Table 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64943/64943fig01.jpg" /> 
Figure 1: Side view of the echocardiography cart. The echocardiography cart is built using a pre-made heavy-duty utility cart. <a href="https://www.jove.com/files/ftp_upload/64943/64943fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64943/64943fig02.jpg" /> 
Figure 2: Head view of the echocardiography cart. The front and back of the pre-made cart are replaced with hinged gates made of PVC pipe and chain link (A), which also accommodates a hanging food trough (B). <a href="https://www.jove.com/files/ftp_upload/64943/64943fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64943/64943fig03.jpg" /> 
Figure 3: Top view of the echocardiography cart. An aperture is created on the top floor of the cart for passing a hand holding an ultrasound probe. A plastic over-piece is fitted for safe cart mounting and dismounting. <a href="https://www.jove.com/files/ftp_upload/64943/64943fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64943/64943fig04.jpg" /> 
Figure 4: Aluminum ramp. An aluminum ramp is attached to the front or back of the cart, and removable rubber padding is added for grip using bolts and grommets. <a href="https://www.jove.com/files/ftp_upload/64943/64943fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64943/64943fig05.jpg" /> 
Figure 5: Side gate. Hinges are created for the side gates, with pins to allow for more accurate sizing and restraint. <a href="https://www.jove.com/files/ftp_upload/64943/64943fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64943/64943fig06.jpg" /> 
Figure 6: Treats for conscious echo sessions. Combinations of juice, meal replacer drink, or yogurt combined with cereal and standard chow, biscuits, and/or fruit bars are frozen to create long-lasting frozen treat troughs. <a href="https://www.jove.com/files/ftp_upload/64943/64943fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64943/64943fig07.jpg" /> 
Figure 7: A representative M-mode scan obtained from a conscious animal. Sample image analysis for calculating the left ventricular ejection fraction from M-mode imaging. <a href="https://www.jove.com/files/ftp_upload/64943/64943fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64943/64943fig08.jpg" /> 
Figure 8: A representative B-mode scan obtained from a conscious animal. Sample image analysis for calculating the left ventricular ejection fraction from B-mode imaging. <a href="https://www.jove.com/files/ftp_upload/64943/64943fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mean &#177; SD</td>
			<td>LVIDd (cm)</td>
			<td>LVIDs (cm)</td>
			<td>EF (%)</td>
		</tr>
		<tr>
			<td>Conscious Echo (N=4/2)</td>
			<td>3.8 &#177; 0.5</td>
			<td>2.5 &#177; 0.5</td>
			<td>64.3 &#177; 5.4</td>
		</tr>
		<tr>
			<td>Sedated Echo (N=4/2)</td>
			<td>3.9 &#177; 0.2</td>
			<td>3.0 &#177; 0.0</td>
			<td>48.5 &#177; 7.9</td>
		</tr>
		<tr>
			<td colspan="4">EF, ejection fraction; SD, standard deviation.</td>
		</tr>
		<tr>
			<td colspan="4">N = 4 echo scans obtained from two animals</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of the parameters generated from M-mode images recorded in sedated minipigs versus conscious minipigs constrained in the cart.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>LV-MALd (cm)</td>
			<td>LV-MALs (cm)</td>
			<td>LV-CAd (cm2)</td>
			<td>LV-CAs (cm2)</td>
			<td>EF (%)</td>
		</tr>
		<tr>
			<td>Conscious Echo (N=8/2)</td>
			<td>5.8 &#177; 0.8</td>
			<td>4.5 &#177; 0.6</td>
			<td>18.6 &#177; 5.0</td>
			<td>10.7 &#177; 2.8</td>
			<td>57.3 &#177; 5.2</td>
		</tr>
		<tr>
			<td>Sedated Echo (N=8/2)</td>
			<td>5.9 &#177; 0.5</td>
			<td>4.8 &#177; 0.4</td>
			<td>21.8 &#177; 2.7</td>
			<td>13.1 &#177; 2.4</td>
			<td>55.3 &#177; 9.0</td>
		</tr>
		<tr>
			<td colspan="6">LV-MAL, left ventricular major axis length; LV-CA, left ventricular chamber area;</td>
		</tr>
		<tr>
			<td colspan="6">EF, ejection fraction; SD, standard deviation. N=8 echo scans obtained from 2 animals</td>
		</tr>
	</tbody>
</table>
Table 2: Comparison of the parameters generated from B-mode images recorded in sedated minipigs versus conscious minipigs constrained in the cart.
Video 1: A mock awake echocardiogram recording procedure performed on a real-sized minipig doll using the echocardiography cart. <a href="https://www.jove.com/files/ftp_upload/64943/Vedio 1.mov" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Echocardiography Recording in Awake Miniature Pigs at JoVE.com

Echocardiography Recording in Awake Miniature Pigs

A simple cart construct, built to perform research echocardiography in standing awake minipigs, is described, along with building considerations, training techniques, and representative ultrasound images.

Echocardiography uses ultrasonic waves to non-invasively assess cardiac structure and function and is the standard of care for cardiac assessment and ...

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950 Views.  University of Utah. A simple cart construct, built to perform research echocardiography in standing awake minipigs, is described, along with building considerations, training techniques, and representative ultrasound images. 

Video: Echocardiography Recording in Awake Miniature Pigs

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  ...

Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through implanted electrodes. Deep brain stimulation was first implemented as a therapeutic modality by Benabid et al. in the late 1980s, when he used this technique to stimulate the ventral intermediate nucleus of the thalamus for the treatment of tremor 1. Currently, the procedure is used to treat patients who fail to respond adequately to medical management for diseases such as Parkinson's, dystonia, and essential tremor. The efficacy of this procedure for the treatment of Parkinson's disease has been demonstrated in well-powered, randomized controlled trials 2. Presently, the U.S. Food and Drug Administration has approved DBS as a treatment for patients with medically refractory essential tremor, Parkinson's disease, and dystonia. Additionally, DBS is currently being evaluated for the treatment of other psychiatric and neurological disorders, such as obsessive compulsive disorder, major depressive disorder, and epilepsy. 
DBS has not only been shown to help people by improving their quality of life, it also provides researchers with the unique opportunity to study and understand the human brain. Microelectrode recordings are routinely performed during DBS surgery in order to enhance the precision of anatomical targeting. Firing patterns of individual neurons can therefore be recorded while the subject performs a behavioral task. Early studies using these data focused on descriptive aspects, including firing and burst rates, and frequency modulation 3. More recent studies have focused on cognitive aspects of behavior in relation to neuronal activity 4,5. This article will provide a description of the intra-operative methods used to perform behavioral tasks and record neuronal data with awake patients during DBS cases. Our exposition of the process of acquiring electrophysiological data will illuminate the current scope and limitations of intra-operative human experiments.

Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through ...

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 ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

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 ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

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 ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

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 ...