Name: 2d and 3d echocardiography in the axolotl (ambystoma mexicanum)
Uploaded: 2018-11-29T12:30:00
Description: Watch this Scientific Journal Video about 2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum at JoVE.com

We would like to acknowledge Kasper Hansen, Institute for Bioscience, Aarhus University for providing access to and assistance with the electronic micromanipulator for 3D echocardiographic acquisition.

The procedures carried out in this protocol were in accordance with the national Danish legislation for care and use of laboratory animals and the experiments were approved by the Danish National Animal Experiments Inspectorate (protocol# 2015-15-0201-00615).
1. Preparations
<ol>
	<li>Prepare axolotl medium.
	<ol>
		<li>Apply high quality non-chemically treated tap water as axolotl medium. If this is unavailable, apply 40% Holtfreter&#39;s solution.</li>
		<li>Prepare 40% (wt/vol) Holtfreter...

<ol>
	<li>Forouzanfar, M. H., 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=Assessing+the+Global+Burden+of+Ischemic+Heart+Disease.">Assessing the Global Burden of Ischemic Heart Disease.</a> Glob. Heart. 7, 331-342 (2012).</li><li>Go, A. 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=Heart+Disease+and+Stroke+Statistics--2014+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2014 Update: A Report From the American Heart Association.</a> Circulation. 129, e28-e292 (2014).</li><li>Leferovich, J. 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=Heart+regeneration+in+adult+MRL+mice.">Heart regeneration in adult MRL mice.</a> Proc. Natl. Acad. Sci. USA. 98, 9830-9835 (2001).</li><li>Leferovich, J. M., Heber-Katz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+scarless+heart.">The scarless heart.</a> Semin. Cell Dev. Biol. 13, 327-333 (2002).</li><li>Nakada, Y., 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=Hypoxia+induces+heart+regeneration+in+adult+mice.">Hypoxia induces heart regeneration in adult mice.</a> Nature. 541, 222-227 (2017).</li><li>Poss, K. D., Wilson, L. G., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+regeneration+in+zebrafish.">Heart regeneration in zebrafish.</a> Science. 298, 2188-2190 (2002).</li><li>Chablais, F., Veit, J., Rainer, G., Jazwinska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+heart+regenerates+after+cryoinjury+induced+myocardial+infarction.">The zebrafish heart regenerates after cryoinjury induced myocardial infarction.</a> BMC Dev. Biol. 11, 21 (2011).</li><li>Gemberling, M., Bailey, T. J., Hyde, D. R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+complex+tissue+regeneration.">The zebrafish as a model for complex tissue regeneration.</a> Trends. Genet. 29, 611-620 (2013).</li><li>Gonzalez-Rosa, J. M., Martin, V., Peralta, M., Torres, M., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+scar+formation+and+regression+during+heart+regeneration+after+cryoinjury+in+zebrafish.">Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish.</a> Development. 138, 1663-1674 (2011).</li><li>Schnabel, K., Wu, C. C., Kurth, T., Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+cryoinjury+induced+necrotic+heart+lesions+in+zebrafish+is+associated+with+epicardial+activation+and+cardiomyocyte+proliferation.">Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation.</a> PLoS One. 6 (4), e18503 (2011).</li><li>Oberpriller, J. O., Oberpriller, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+the+adult+newt+ventricle+to+injury.">Response of the adult newt ventricle to injury.</a> J. Exp. Zool. 187, 249-260 (1974).</li><li>Witman, N., Murtuza, B., Davis, B., Arner, A., Morrison, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulation+of+developmental+cardiogenesis+governs+the+morphological+and+functional+regeneration+of+adult+newt+hearts+following+injury.">Recapitulation of developmental cardiogenesis governs the morphological and functional regeneration of adult newt hearts following injury.</a> Dev. Biol. 354, 67-76 (2011).</li><li>Gressens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+the+Mexican+axolotl+(Ambystoma+mexicanum).">An introduction to the Mexican axolotl (Ambystoma mexicanum).</a> Lab Animal. 33, 41-47 (2004).</li><li>Cano-Mart&#237;nez, A., Vargas-Gonz&#225;lez, A., Guarner-Lans, V., Prado-Zayago, E., Le&#243;n-Oleda, M., Nieto-Lima, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+regeneration+in+the+axolotl+heart+(Ambystoma+mexicanum)+after+partial+ventricular+amputation.">Functional and structural regeneration in the axolotl heart (Ambystoma mexicanum) after partial ventricular amputation.</a> Arch. Cardiol. Mex. 80, 79-86 (2010).</li><li>McCusker, C., Gardiner, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axolotl+model+for+regeneration+and+aging+research:+a+mini-review.">The axolotl model for regeneration and aging research: a mini-review.</a> Gerontology. 57, 565-571 (2011).</li><li>Khattak, 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=Optimized+axolotl+(Ambystoma+mexicanum)+husbandry,+breeding,+metamorphosis,+transgenesis+and+tamoxifen-mediated+recombination.">Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination.</a> Nat. Protoc. 9, 529-540 (2014).</li><li>Nakamura, R., 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=Expression+analysis+of+Baf60c+during+heart+regeneration+in+axolotls+and+neonatal+mice.">Expression analysis of Baf60c during heart regeneration in axolotls and neonatal mice.</a> Develop. Growth Differ. 58, 367-382 (2016).</li><li>Tan, G. X. Y., Jamil, M., Tee, N. G. Z., Zhong, L., Yap, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Reconstruction+of+Chick+Embryo+Vascular+Geometry+Using+Non-Invasive+High-Frequency+Ultrasound+for+Computational+Fluid+Dynamics.">3D Reconstruction of Chick Embryo Vascular Geometry Using Non-Invasive High-Frequency Ultrasound for Computational Fluid Dynamics.</a> Ann. Biomed. Eng. 43, 2780-2793 (2015).</li><li>Ho, S., Tan, G. X. Y., Foo, T. J., Phan-Thien, N., Yap, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+Dynamics+and+Fluid+Dynamics+of+the+HH25+Chick+Embryonic+Cardiac+Ventricle+as+Revealed+by+a+Novel+4D+High-Frequency+Ultrasound+Imaging+Technique+and+Computational+Flow+Simulations.">Organ Dynamics and Fluid Dynamics of the HH25 Chick Embryonic Cardiac Ventricle as Revealed by a Novel 4D High-Frequency Ultrasound Imaging Technique and Computational Flow Simulations.</a> Ann. Biomed. Eng. , (2017).</li><li>Wasmeier, G. H., 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=Reproducibility+of+transthoracic+echocardiography+in+small+animals+using+clinical+equipment.">Reproducibility of transthoracic echocardiography in small animals using clinical equipment.</a> Coron. Artery. Dis. 18, 283-291 (2007).</li><li>Thygesen, M. M., Rasmussen, M. M., Madsen, J. G., Pedersen, M., Lauridsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propofol+(2,6-diisopropylphenol)+is+an+applicable+immersion+anesthetic+in+the+axolotl+with+potential+uses+in+hemodynamic+and+neurophysiological+experiments.">Propofol (2,6-diisopropylphenol) is an applicable immersion anesthetic in the axolotl with potential uses in hemodynamic and neurophysiological experiments.</a> Regeneration. 4 (3), (2017).</li></ol>

Echocardiography in the axolotl and other non-mammalian species yields fundamentally different data than mammalian echocardiography because of the nucleated nature of red blood cells in all vertebrates except adult mammals. This results in a pronounced blood signal and less blood-to-tissue contrast in axolotl echocardiographic images compared to e.g., mouse or human echocardiography. This can make image segmentation on unprocessed single frame ultrasound images more difficult as it can be hard to distinguish blo...

Ischemic heart disease is a leading cause of death worldwide1,2. Although many survive a myocardial infarction due to rapid and fine-tuned medical intervention, ischemic incidents in humans often lead to fibrotic scarring associated with hypertrophy, electrical malfunction, and a diminished functional capacity of the heart. This lack of regenerative potential of cardiac tissue is shared among mammals and although controversial claims of mammalian cardiac regeneration have been reported, these have been limited to specific murine strains3,4 and hypoxia treated mice5. Thus, the field of cardiac regenerative medicine and biology is generally limited to non-mammalian animal models to study intrinsic heart regenerative phenomena. The zebrafish (Danio rerio) has in the past decade been established as the most well characterized model for intrinsic heart regeneration6,7,8,9,10. Due to easy laboratory maintenance, a short generation time and a wide array of molecular tools available, the zebrafish is well adapted as a model for genetic and molecular mechanisms underlying cardiac development and regeneration. However, the minute dimensions of the zebrafish heart make it less suited for functional evaluation, and complicated surgical procedures and the non-tetrapod phylogeny of the zebrafish limits the sensible extrapolation of findings in this species, thus justifying the use of other larger tetrapod models. One of the earliest models of vertebrate heart regeneration was a caudate amphibian, the Eastern newt (Notophthalmus viridescens)11, a species that remains a valuable model12.
In recent years another caudate amphibian, the Mexican axolotl (A. mexicanum) has entered the scene as a large (up to 100 g of body mass) and highly laboratory adaptable animal model for a wide array of regenerative disciplines spanning limb regeneration, spinal cord injury, and cardiac regeneration13,14,15,16,17. The axolotl is highly amenable to functional measurements on the heart using high frequency echocardiography and the absence of calcified structures on the ventral side of the heart allows for ultrasound imaging with a much lower level of image artifacts (acoustic shadowing and reverberation in particular) than observed in other model animals with calcified sternum and ribs.
The following protocol describes several different methods and preparations (Figure 1, Figure 2) to acquire reproducible echocardiographic measurements on the axolotl heart in both anesthetized (applying three different anesthetics: benzocaine, MS-222, and propofol) and unanesthetized animals in two (Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Supplementary Files 1-12) and three (Figure 8, Figure 9, Supplementary Files 13-14) spatial dimensions. The amphibian heart is three-chambered (two atria and a single ventricle). The atria are supplied by a large sinus venosus and the ventricle empties into the conus arteriosus outflow tract (Figure 2). Since most emphasis is traditionally placed on ventricular regeneration and less on the recovery of atria6,7,8,9,10,11,12,14,17, this protocol mainly focuses on measurements of ventricular function.
Amphibian echocardiography is not well-described in the literature, and the development of the 2D methods described in this paper have been driven by the need to best represent the functionality of the beating axolotl heart at a given time and experimental setting. Thus, the methods described here are applicable in heart regenerative experiments where cardiac function can be repeatedly monitored over the course of a regeneration process. Additionally, the methods can be applied in cardiophysiological experiments on the axolotl in general or modified slightly to span other caudate or anuran amphibian models (e.g.,Xenopus). The axolotl exists in several different strains and color variations (e.g., wildtype, melanoid, white, albino, transgenic white with green fluorescence protein expression), however these characteristics do not affect the compatibility of the axolotl with the described protocol. The method described here to acquire 3D echocardiographic data is a modified version of the spatiotemporal image correlation (STIC) technique developed for clinical ultrasound and the quadratic averaging method described previously in the developing chicken to enhance the signal of blood speckles in soft tissues in species containing nucleated red blood cells18,19. This method allows for advanced modeling of cardiac contraction and computed fluid dynamics in the axolotl heart.

<table border="0" cellpadding="0" cellspacing="0" width="1230">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axolotl (Ambystoma mexicanum)</td>
			<td style="width:147px;">Exoterra GmbH</td>
			<td style="width:103px;">N/A</td>
			<td style="width:765px;">All strains (wildtype, melanoid, white, albino, transgenic white with GFP) can be applied for echocardiography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vevo 2100</td>
			<td style="width:147px;">Fujifilm, Visualsonics</td>
			<td style="width:103px;">Vevo 2100</td>
			<td>High frequency ultrasound system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MS700</td>
			<td style="width:147px;">Fujifilm, Visualsonics</td>
			<td style="width:103px;">MS700</td>
			<td>50 MHz center frequency, transducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS550s</td>
			<td style="width:147px;">Fujifilm, Visualsonics</td>
			<td>MS550s</td>
			<td>40 MHz center frequency, transducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td style="width:147px;">Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzocain</td>
			<td>Sigma-Aldrich</td>
			<td>94-09-7</td>
			<td>ethyl 4-aminobenzoate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS-222</td>
			<td>Sigma-Aldrich</td>
			<td>886-86-2</td>
			<td>ethyl 3-aminobenzoate methanesulfonic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Propofol</td>
			<td>B. Braun Medical A/S</td>
			<td style="width:103px;">NA</td>
			<td>2,6-diisopropylphenol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:103px;">&#160;7647-14-5&#160;</td>
			<td>NaCl</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:103px;">10035-04-8</td>
			<td>CaCl2&#183;2H2O</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Magnesium sulfate heptahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:103px;">&#160;10034-99-8&#160;</td>
			<td>MgSO4&#183;7H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:103px;">&#160;7447-40-7</td>
			<td>KCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td style="width:103px;">&#160;67-64-1&#160;</td>
			<td>Propanone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Soft cloth</td>
			<td style="width:147px;">N/A</td>
			<td style="width:103px;">N/A</td>
			<td>Any piece of soft cloth measuring appromixately 70 x 55 cm^2 e.g. a dish towel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Styrofoam block</td>
			<td style="width:147px;">N/A</td>
			<td style="width:103px;">N/A</td>
			<td>Any piece of Styrofoam block measuring approximately 33 x 27 x 5 cm^3 e.g. a medium sized Styrofoam cooler lid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic wrap</td>
			<td style="width:147px;">N/A</td>
			<td style="width:103px;">N/A</td>
			<td>Any piece of plastic wrap e.g. food wrap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tape</td>
			<td style="width:147px;">BSN Medical</td>
			<td style="width:103px;">72359-02</td>
			<td>Leukoplast sleek</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kimwipes</td>
			<td>Sigma-Aldrich</td>
			<td style="width:103px;">Z188956&#160;</td>
			<td>Kimwipes, disposable wipers&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Excel 2010</td>
			<td style="width:147px;">Microsoft</td>
			<td style="width:103px;">N/A</td>
			<td>Excel 2010 or newer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ</td>
			<td colspan="2">National Institutes of Health</td>
			<td>ImageJ 1.5e or newer. Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997-2016.&#160;</td>
		</tr>
	</tbody>
</table>

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.

Intrapericardial space in the axolotl is dependent on the size of the animal. Smaller animals (2-20 g, 7-15 cm) will have an excess of pericardial fluid (appearing dark in echocardiography) surrounding the cardiac chambers whereas in larger sexually mature animals (&#62; 20 g, &#62; 15 cm) the chambers will occupy most of the intrapericardial space. To provide the best overview for representative results of echocardiographic views of the axolotl heart, a smaller animal (10 g, 10 cm) was a...

Watch this Scientific Journal Video about 2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum at JoVE.com

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.

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 ...

This method can help answer key questions in the regenerative field, such as how cardiac regeneration progresses following myocardial injury. The main advantage of this technique is that it provides high resolution, noninvasive, and repeatable evaluation of cardiac function in the axolotl. Additionally, this method can also be applied to other amphibian model organisms, such as newts and xenopus.Begin with positioning an anesthetized axolotl supine in a lip-shaped animal bed. When the medium gets added, the animal will float, so it must be secured with loose rubber bands. Next, fill the bed with medium containing anesthetics so that the thorax is three to five millimeters deep.If only using B-mode, color-Doppler mode, and pulse-wave Doppler mode data, then the axolotl can be alert for this procedure. In this situation, position the animal prone in a hammock, and let it recover from the stress for 30 to 60 minutes before proceeding. For white or albino animals, have a cold light source ready to help place the transducer.For a transducer, use either a 40-or 50-hertz model depending on the animal's mass. Then, prepare the transducer ultrasound gel, and proceed with collecting data. Begin with positioning the ultrasound transducer over the midline of the animal in the thoracic region, and parallel to its long axis.A small portion of the ventricle, positioned to the right in the thoracic cavity, should appear in-frame at the ventricular diastole. A large portion of both atria should also be visible, as should the sinus venosus. These structures should be identifiable in the diastole or systole view.Next, translate the transducer one to three millimeters to the right, to obtain the ventricular long-axis view. Ultimately, the correct position is attained when the cross-sectional area of the end-systole ventricle is at its maximum. Correct 2D ultrasound measurements are highly dependent on correct positioning of the transducer.Practice the transducer placement meticulously and perform inter-operator analysis to minimize subjectivity. Now, in B-mode, acquire at least three cardiac cycles at a minimum of 50 frames per second. Choose between using the general imaging mode or the cardiology mode.Next, translate the transducer along the long axis of the animal until the center of the ventricle is in the middle of the screen. Then, rotate the transducer 90 degrees clockwise to obtain the midventricular short-axis view. From this position, evaluate the circular shape of the ventricle by translating the transducer along the long axis of the heart.Then, return the transducer to the long axis plane, and translate it back towards the midline to obtain an atrial long-axis two-chamber view. The correct position is achieved when the cross-sectional areas of the end-systole atria are at their maxima, and the two atria combined assume the outline of the number eight when tilted about 45 degrees to the left. Then, collect B-mode images.To proceed, translate the transducer to the right until the outflow tract appears. The ventricular end-systole view is correct when the diameter of the outflow is at its largest, and when, during mid-injection, two of the semiluminar valves, at the entrance of the outflow, are visible. From this view, velocity and flow measurements can be made using Doppler imaging.Apply the color-Doppler mode to map blood flow velocities in the outflow tract during cardiac injection. Next, apply color-Doppler imaging and power-Doppler imaging to visualize the blood flow in the ventricular view, and do the same in the atrial view. Next, use color-Doppler mode directed at the location of maximum blood velocity in the outflow tract running directly towards the transducer.When the outflow is not completely perpendicular to the transducer, apply a beam angle and angular correction to correct the image by up to 45 degrees. Next, collect velocity-time data in the pulse-wave Doppler mode. During no phase of the cardiac cycle should the spiral valve overlap the view of the outflow.Collect data from at least three cardiac cycles. Then, without moving the transducer, acquire data in B-mode from at least three cardiac cycles. Next, for anesthetized animals only, rotate the animal 90 degrees so that the right part of the animal is facing upwards, and move the transducer to the oblique para-gill view, just parallel and posterior to the protruding gills.This view offers an alternative measurement of the blood flow velocity in the outflow tract. The outflow tract should be running downward at about 45 degrees, and the atria should appear under the outflow tract during injection. Finally, switch to pulse-wave Doppler mode and position the transducer to view the maximum blood velocity running away from the transducer in the outflow tract.As needed, use up to 45 degrees of beam angle and angular correction to make the outflow perpendicular to the transducer. Then, collect data in pulse-wave Doppler mode and in B-mode from the same position as before. 3D acquisition takes a while, so the axolotl must be anesthetized.Place it supine in the lip-shaped animal bed. Secure it with rubber bands, and submerge its thoracic surface in three to five millimeters of ultrasound medium containing an anesthetic. Movements during 3D acquisition are detrimental to subsequent reconstruction.If the animal moves during 3D ultrasound acquisition, the procedure must be repeated from the beginning. Next, position the transducer over the midline in the thoracic region, and place it parallel to the long axis for a sagittal 3D recording, or orthogonal to the long axis for transversal 3D recording. Then, translate the transducer to insure that the entire cardiac region gets covered in the subsequent 3D capture.Move it in both the in-plane dimension and in the out-of-plane dimension. For the imaging mode, if the animal's heart rate is between 20 and 60 beats per minute, select the general imaging mode. Otherwise, choose the cardiology mode.Turn off the light. Now, in the raw B-mode image, adjust the 2D gain to a level where the anatomical structures are barely recognizable. This will increase signal-to-noise ratio in the final reconstructions.Then, decide the size of the Z-step or slice thickness. Now, translate the transducer one Z-step at a time, taking a recording containing 1, 000 frames at each Z-step until the entire cardiac region has been covered. A 2D echocardiographic analysis of a 10-gram, 10-centimeter axolotl was performed using the described technique.The long-axis view provided a good starting point. The midline plane showed the sinus venosus, atria, and parts of the ventricle. Viewing the ventricular plane showed the ventricle to be spherical and highly trabeculated.In the atrial plane, the atria appeared more irregular and barely trabeculated. The center of the outflow tract is close to the center of the ventricle. Measurements of the cardiac cycle using pulse-wave Doppler from the long-axis and from the oblique para-gill plane had some background noise.This noise was surmountable when making measurements of the velocity time integral. Color-Doppler and power-Doppler imaging showed the flow pattern through the heart chambers. Views were possible from the ventricle, from the atria, and from the outflow tract.3D echocardiography was also performed. This multiplanar view of the heart has many uses, such as surface and volume reconstructions, or segmentation and generation of 3D models. After watching this video, you should have a good understanding of how to perform 2D and 3D echocardiography in the axolotl.Once mastered, the 2D ultrasound technique can be done in less than five minutes per animal, if it is performed properly, whereas the 3D acquisition can take up to an hour per animal. Following this procedure, other methods like heart extraction and histology can be performed in order to answer additional questions related to cardiac structure and anatomy.

Research

JoVE Journal

Medicine

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 ...

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

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  ...

Murine Fetal Echocardiography

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

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Contusion Spinal Cord Injury via a Microsurgical Laminectomy in the Regenerative Axolotl

The purpose of this study is to establish a standardized and reproducible regenerative blunt spinal cord injury model in the axolotl (Ambystoma ...

A Whole Body Dosimetry Protocol for Peptide-Receptor Radionuclide Therapy (PRRT): 2D Planar Image and Hybrid 2D+3D SPECT/CT Image Methods

A Whole Body Dosimetry Protocol for Peptide-Receptor Radionuclide Therapy PRRT: 2D Planar Image and Hybrid 2D+3D SPECT/CT Image Methods

Peptide-receptor-radionuclide-therapy (PPRT) is a targeted therapy that combines a short-range energy radionuclide with a substrate with high specificity ...

Morphological and Functional Assessment of the Right Ventricle Using 3D Echocardiography

Traditionally, it was believed that the right side of the heart has a minor role in circulation; however, more and more data suggest that right ...

Hemodynamic Precision in the Neonatal Intensive Care Unit using Targeted Neonatal Echocardiography

Targeted neonatal echocardiography (TnECHO) refers to the use of comprehensive echocardiographic evaluation and physiologic data to obtain accurate, ...