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.

<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>

The authors have nothing to disclose.

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 blood from tissue. However, this phenomenon can be advantageous when used to create blood signal enhanced images by applying the quadratic averaging procedure described previously18 and modified for axolotl echocardiography in Protocol Section 6. Since blood speckles are much more dynamic than those found in soft tissue, quadratic averaging will generate pronounced contrast between these two compartments which facilitates image segmentation in two and three dimensions.
This protocol describes three different anesthetics for the axolotl that have been thoroughly tested previously21. Both benzocaine and MS-222 stimulate an increase in heart rate, which can be desirable when testing cardiac function under stress conditions. Propofol induces less stress to the heart during anesthesia and may be used as a substitution for unanesthetized echocardiography in situations where acquisition time exceeds the limits of sedentary behavior in unanesthetized axolotls.
2D echocardiography describing the 3D heart is affected by subjectivity. Therefore, it is imperative to conduct and intra/inter-operator/observer analysis before conducting an actual experiment as described in Protocol Section 5. Likewise, echocardiographic measurements should be viewed more as index values that can be applied to investigate potential differences in cardiac function under different circumstances rather than absolute values. The stroke volume determined by the geometric equation (Equation 2) rarely yields the same absolute value as the pulse wave Doppler equation (Equation 4; Figure 7), and it should be decided which measure to adhere to throughout a series of experiments. The SV(geo) can be obtained more rapidly than the SV(pw), however the spherical assumption of the ventricular shape only applies to healthy uniformly contracting hearts, and in disease and regeneration models, SV(pw) should be considered for a better reflection of the true stroke volume.
The correlation and quadratic averaging procedure of Protocol Section 6 can be implemented in several different imaging and mathematical packages. Since programming skills and access to software packages vary greatly within life science researchers, we have strived toward providing representative scripts for the methods in software packages that most researchers are familiar with (e.g., Excel) that are easily approached and freely available (ImageJ: https://imagej.nih.gov/ij/index.html). Supplementary Files 16-20 provide annotated exemplary scripts written in IJ1 macro language and as .xlsm macros that should be comprehensible even with minimum experience in coding.
Intrinsic heart regeneration is a phenomenon exclusively found in the hearts of small species (relative to human), and thus measurements and imaging of baseline cardiac function and functional progress during regeneration is challenged by the size of the heart and the spatial resolution of the imaging modality applied. High frequency ultrasound imaging provides a desirable trade-off between a high in-plane spatial resolution (~ 30 x 30 &#181;m2 at 50 MHz) that is comparable to in vivo &#181;CT imaging and much higher than in vivo &#181;MRI, which has a depth of penetration (~ 1 cm at 50 MHz) several fold larger than confocal microscopy, and a very high temporal resolution (50-300 frames/s at 50 MHz, 1 cm depth). Coupled with manual or automated z dimensional movement of the transducer, ultrasound enables unmatched reconstruction of cardiac function and anatomical modeling in four dimensions. Additionally, the non-invasive nature of the technique allows for longitudinal experimentation. To our knowledge there are currently no matrix array transducers available for high frequency micro ultrasound imaging. The development of this technology would greatly aid the acquisitions of 3D data of small hearts such as that of the axolotl in a faster procedure than mechanically moving the transducer.

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><tbody><tr><td>Axolotl (Ambystoma mexicanum)</td><td>Exoterra GmbH</td><td>N/A</td><td>All strains (wildtype, melanoid, white, albino, transgenic white with GFP) can be applied for echocardiography</td></tr><tr><td>Vevo 2100</td><td>Fujifilm, Visualsonics</td><td>Vevo 2100</td><td>High frequency ultrasound system</td></tr><tr><td>MS700</td><td>Fujifilm, Visualsonics</td><td>MS700</td><td>50 MHz center frequency, transducer</td></tr><tr><td>MS550s</td><td>Fujifilm, Visualsonics</td><td>MS550s</td><td>40 MHz center frequency, transducer</td></tr><tr><td>Micromanipulator</td><td>Zeiss</td><td>NA</td><td></td></tr><tr><td>Benzocain</td><td>Sigma-Aldrich</td><td>94-09-7</td><td>ethyl 4-aminobenzoate</td></tr><tr><td>MS-222</td><td>Sigma-Aldrich</td><td>886-86-2</td><td>ethyl 3-aminobenzoate methanesulfonic acid</td></tr><tr><td>Propofol</td><td>B. Braun Medical A/S</td><td>NA</td><td>2,6-diisopropylphenol</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>&amp;nbsp;7647-14-5&amp;nbsp;</td><td>NaCl</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>10035-04-8</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Magnesium sulfate heptahydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>&amp;nbsp;10034-99-8&amp;nbsp;</td><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>&amp;nbsp;7447-40-7</td><td>KCl</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>&amp;nbsp;67-64-1&amp;nbsp;</td><td>Propanone</td></tr><tr><td>Soft cloth</td><td>N/A</td><td>N/A</td><td>Any piece of soft cloth measuring appromixately 70 x 55 cm^2 e.g. a dish towel</td></tr><tr><td>Styrofoam block</td><td>N/A</td><td>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><td>Plastic wrap</td><td>N/A</td><td>N/A</td><td>Any piece of plastic wrap e.g. food wrap</td></tr><tr><td>Tape</td><td>BSN Medical</td><td>72359-02</td><td>Leukoplast sleek</td></tr><tr><td>Kimwipes</td><td>Sigma-Aldrich</td><td>Z188956&amp;nbsp;</td><td>Kimwipes, disposable wipers&amp;nbsp;</td></tr><tr><td>Excel 2010</td><td>Microsoft</td><td>N/A</td><td>Excel 2010 or newer</td></tr><tr><td>ImageJ</td><td>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.&amp;nbsp;</td><td></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.

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 ...

2d-and-3d-echocardiography-in-the-axolotl-ambystoma-mexicanum

Universidad San Sebastian

Research

JoVE Journal

Medicine

14.7K Views.  Aarhus University. 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.

Video: 2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum

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  ...

Cryo-injury Induced Heart Regeneration in the Axolotl and Echocardiography and Unbiased Quantitative Histology to Evaluate Regenerative Progression

The urodele amphibians, salamanders and newts, represent the phylogenetic group closest to mammals capable of performing complete myocardial regeneration following ventricular resection. The resection model has generated a basic knowledge of the processes involved in cardiac repair. However, the model does not relate well to clinical situations in which tissue damage, apoptosis, necrosis, fibrosis, and hypertrophy are all key detrimental consequences of ischemia-induced myocardial infarctions rather than tissue removal. On the other hand, cryoinjury-induced myocardial infarction resembles ischemia-induced myocardial infarction more closely. Here we provide a detailed description of the cryoinjury procedure in the axolotl (Ambystoma mexicanum), which provides a tool for investigating basic mechanisms in cardiac repair in a tetrapod model. Additionally, we provide quantitative methods for estimating infarction size non-invasively in vivo with echocardiography and for measuring infarction size precisely with unbiased quantitative histology ex vivo.

Here we present a surgery protocol to induce cryoinjury to the ventricular myocardium of the axolotl. Additionally, we present a protocol to non-invasively estimate infarction fraction during the regenerative process with echocardiography and a protocol to precisely measure infarction fraction in the excised heart with unbiased quantitative histology.

The urodele amphibians, salamanders and newts, represent the phylogenetic group closest to mammals capable of performing complete myocardial regeneration ...

Developmental Biology

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.

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 ...

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 ...

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 ...

Evaluation of Left Ventricular Structure and Function using 3D Echocardiography

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk stratification in various cardiac disorders. Recently, 3D echocardiography became available in routine cardiology practice; however, high-quality image acquisition and subsequent analysis have a steep learning curve. The present article aims to guide the reader through a detailed 3D protocol by presenting tips and tricks and also by highlighting the potential pitfalls to facilitate the widespread but technically sound use of this important technique concerning the LV. First and foremost, we show the acquisition of a high-quality 3D dataset with optimal spatial and temporal resolution. Then, we present the analytical steps toward a detailed quantification of the LV by using one of the most widely applied built-in software. We will quantify LV volumes, sphericity, mass and also systolic function by measuring ejection fraction and myocardial deformation (longitudinal and circumferential strain). We will discuss and provide clinical examples about the essential scenarios where the transition from a conventional echocardiographic approach to a 3D-based quantification is highly recommended.

In this article, we provide a step-by-step acquisition and analysis protocol for the volumetric assessment and speckle-tracking analysis of the left ventricle by 3D echocardiography, particularly focusing on practical aspects that maximize the feasibility of this technique.

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk ...

Bioengineering

Transthoracic Echocardiographic Examination in the Rabbit Model

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

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

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

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

Transthoracic Echocardiography to Assess Post-Resuscitation Left Ventricular Dysfunction After Acute Myocardial Infarction and Cardiac Arrest in Pigs

One of the main causes of out-of-hospital cardiac arrest is acute myocardial infarction (AMI). After successful resuscitation from cardiac arrest, approximately 70% of patients die before hospital discharge due to post-resuscitation myocardial and cerebral dysfunction. In experimental models, myocardial dysfunction after cardiac arrest, characterized by an impairment in both left ventricular (LV) systolic and diastolic function, has been described as reversible but very little data are available in cardiac arrest models associated with AMI in pigs. Transthoracic echocardiography is the first-line diagnostic test for the assessment of myocardial dysfunction, structural changes and/or AMI extension. In this pig model of ischemic cardiac arrest, echocardiography was done at baseline and 2-4 and 96 hours after resuscitation. In the acute phase, the examinations are done in anesthetized, mechanically ventilated pigs (weight 39.8 &#177; 0.6 kg) and ECG is recorded continuously. Mono- and bi-dimensional, Doppler and tissue Doppler recordings are acquired. Aortic and left atrium diameter, end-systolic and end-diastolic left ventricular wall thicknesses, end-diastolic and end-systolic diameters and shortening fraction (SF) are measured. Apical 2-, 3-, 4-, and 5-chamber views are acquired, LV volumes and ejection fraction are calculated. Segmental wall motion analysis is done to detect the localization and estimate the extent of myocardial infarction. Pulsed Wave Doppler echocardiography is used to record trans-mitral flow velocities from a 4-apical chamber view and trans-aortic flow from a 5-chamber view to calculate LV cardiac output (CO) and stroke volume (SV). Tissue Doppler Imaging (TDI) of LV lateral and septal mitral anulus is recorded (TDI septal and lateral s&#39;, e&#39;, a&#39; velocities). All the recordings and measurements are done according to the recommendations of the American and European Societies of Echocardiography Guidelines.

Transthoracic echocardiography is the first-line diagnostic test for post-resuscitation left ventricular dysfunction and structural changes in a pig model of cardiac arrest.