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 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 ...

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

Research

JoVE Journal

Medicine

14.6K 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 ...

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 functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

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 

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

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

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

Murine Fetal Echocardiography

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

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

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

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 ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

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 traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

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

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 ...

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 mexicanum). Most clinical spinal cord injuries occur as high energy blunt traumas, inducing contusion injuries. However, most studies in the axolotl spinal cord have been conducted with sharp traumas. Hence, this study aims to produce a more clinically relevant regenerative model. Due to their impressive ability to regenerate almost any tissue, axolotls are widely used as models in regenerative studies and have been used extensively in spinal cord injury (SCI) studies. In this protocol, the axolotls are anesthetized by submersion in a benzocaine solution. Under the microscope, an angular incision is made bilaterally at a level just caudal to the hind limbs. From this incision, it is possible to dissect and expose the spinous processes. Using forceps and scissors, a two-level laminectomy is performed, exposing the spinal cord. A custom trauma device consisting of a falling rod in a cylinder is constructed, and this device is used to induce a contusion injury to the spinal cord. The incisions are then sutured, and the animal recovers from anesthesia. The surgical approach is successful in exposing the spinal cord. The trauma mechanism can produce contusion injuries to the spinal cord, as confirmed by histology, MRI, and neurological examination. Finally, the spinal cord regenerates from the injury. The critical step of the protocol is removing the spinous processes without inflicting damage to the spinal cord. This step requires training to ensure a safe procedure. Furthermore, wound closure is highly dependent on not inflicting unnecessary damage to the skin during incision. The protocol was performed in a randomized study of 12 animals.

This manuscript presents protocols for surgically inflicting controlled blunt and sharp spinal cord injuries to a regenerative axolotl (Ambystoma mexicanum).

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

Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. The model can be used for planning and simulating critical steps of a surgical approach. Therefore, it is important that anatomical structures such as blood vessels inside a tumor can be printed to be colored not only on their surface, but throughout their whole volume. During simulation this allows for the removal of certain parts (e.g., with a high-speed drill) and revealing internally located structures of a different color. Thus, diagnostic information from various imaging modalities (e.g., CT, MRI) can be combined in a single compact and tangible object.
However, preparation and printing of such a fully colored anatomical model remains a difficult task. Therefore, a step-by-step guide is provided, demonstrating the fusion of different cross-sectional imaging data sets, segmentation of anatomical structures, and creation of a virtual model. In a second step the virtual model is printed with volumetrically colored anatomical structures using a plaster-based color 3D binder jetting technique. This method allows highly accurate reproduction of patient-specific anatomy as shown in a series of 3D-printed petrous apex chondrosarcomas. Furthermore, the models created can be cut and drilled, revealing internal structures that allow for simulation of surgical procedures.

The protocol describes the fabrication of fully colored three-dimensional prints of patient-specific, anatomical skull models to be used for surgical simulation. The crucial steps of combining different imaging modalities, image segmentation, three-dimensional model extraction, and production of the prints are explained.

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. ...

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 for cancer cell receptors. After injection, the radiotracer is distributed throughout the entire body, with a higher uptake in tissues where targeted receptors are overexpressed. The use of beta/gamma radionuclide emitters enables therapy imaging (beta-emission) and post-therapy imaging (gamma-emission) to be performed at the same time. Post-treatment sequential images permit absorbed dose calculation based on local uptake and wash-in/wash-out kinetics. We implemented a hybrid method that combines information derived from both 2D and 3D images. Serial whole-body images and blood samples are acquired to estimate the absorbed dose to different organs at risk and to lesions disseminated throughout the body. A single 3D-SPECT/CT image, limited to the abdominal region, overcomes projection overlap on planar images of different structures such as the intestines and kidneys. The hybrid 2D+3D-SPECT/CT method combines the effective half-life information derived from 2D planar images with the local uptake distribution derived from 3D images. We implemented this methodology to estimate the absorbed dose for patients undergoing PRRT with 177Lu-PSMA-617. The methodology could, however, be implemented with other beta-gamma radiotracers. To date, 10 patients have been enrolled into the dosimetry study with 177Lu-PSMA-617 combined with drug protectors for kidneys and salivary glands (mannitol and glutamate tablets, respectively). The median ratio between kidney uptake at 24 h evaluated on planar images and 3D-SPECT/CT is 0.45 (range:0.32-1.23). The comparison between hybrid and full 3D approach has been tested on one patient, resulting in a 1.6% underestimation with respect to full 3D (2D: 0.829 mGy/MBq, hybrid: 0.315 mGy/MBq, 3D: 0.320 mGy/MBq). Treatment safety has been confirmed, with a mean absorbed dose of 0.73 mGy/MBq (range:0.26-1.07) for kidneys, 0.56 mGy/MBq (0.33-2.63) for the parotid glands and 0.63 mGy/MBq (0.23-1.20) for submandibular glands, values in accordance with previously published data.

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

This method estimates the absorbed dose of different structures for peptide-receptor-radionuclide-therapy (PRRT) with the possibility of avoiding organ overlap on 2D-projections. Serial whole-body planar images permit estimation of mean absorbed doses along the whole body, while the hybrid approach, combining planar images and 3D-SPECT/CT image, overcomes the limitations of structure overlapping.

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