Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.
Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model 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.
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.
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
2. Anesthetize Axolotls
3. 2D Echocardiography on Anesthetized Axolotl
4. 2D Echocardiography on Unanesthetized Axolotl
5. Evaluate 2D Echocardiography Data and Minimize Subjectivity
6. 3D Echocardiography on Anesthetized Axolotl
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 (> 20 g, > 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 applied for Figure 3, Figure 4, Figure 5, and Figure 9.
The long axis view generally provides a good overview of cardiac anatomy in the axolotl. Entering at the midline plane with the sinus venosus, atria, and part of the ventricle in plane (Figure 3A, B, Supplementary File 2), either the ventricular plane (Figure 3C–H) or the atrial plane (Figure 4A–D) can be reached by translating the transducer to the right or left of the animal, respectively. The ventricle will appear spherical and highly trabeculated (Figure 3C, Supplementary Files 3–5), whereas the atria have a more irregular shape and almost no trabeculation (Figure 4A, Supplementary File 6, Supplementary File 7). The short axis view (Figure 5A, B, Supplementary File 10) provides a less easily interpretable overview of the cardiac anatomy of the axolotl heart, however it contributes to the evaluation of correct cardiac contraction (e.g., infarcted or non-contracting zones of the circular ventricle are clearly visualized in this view plane). In the long axis view plane, the center of the outflow tract is positioned closely to the center of the ventricle (Figure 2A, and compare Figure 3C with Figure 4E and Supplementary File 3 with Supplementary File 8). Since the soft tissue of the outflow tract will be moving upon blood ejection, the high intensity blood signal during a cardiac cycle measured by pulse wave Doppler in both the long axis and the oblique paragill plane will be adjoined by low intensity noise from the movements of the surrounding soft tissue (gray area surrounding white area in the velocity/time curve in Figure 4G and Figure 5E). Generally, the contrast between blood signal and soft tissue noise should be large enough to segment out only the blood signal when measuring the velocity time integral (Figure 4G (g1 magnification) and Figure 5E (e1 magnification)).
For qualitative evaluation of blood flow patterns, color Doppler and power Doppler imaging provide visualizations of flow patterns in different cardiac chambers (ventricle: Figure 3E–H, Supplementary File 4, Supplementary File 5; atria: Figure 4C, D, Supplementary File 7; outflow tract: Figure 4F, Figure 5D, Supplementary File 9, Supplementary File 12).
Axolotls used for laboratory experimentation vary in size from the early post larval stage of 2-4 g to full maturity at 10-30 g and larger animals weighing > 100 g. Likewise, cardiac function and some absolute values of functional parameters described here depend on the size of the animals. Generally, fractional area change is constant in different size groups with values ranging at 40-50% (skewed toward lower values for larger animals). Stroke volume is highly dependent on the size of the animal, i.e., the size of the heart, ranging from e.g., 20-30 µL in 5 g axolotls, 50-70 µL in 10 g axolotls, and 250-300 µL in 50 g axolotls. Heart rate and to some degree stroke volume are highly dependent on the applied anesthetic and the level of anesthesia (Figure 6A–F, Figure 7).
Traditional intra/inter-operator/observer analysis involves graphical representations (Q-Q Plots and Bland-Altman plots) and testing for equal mean (t-test) and variance (F-test) to evaluate normal distribution of data and to compare accuracy and precision between two persons (Figure 6G).
3D echocardiography adds an additional dimension (z or depth) to the more traditional 2D acquisition. This allows for multi-planar visualization of data (Figure 9A), reslicing (Figure 9B), surface and volume reconstructions (Figure 9C, Supplementary File 13, and Supplementary File 14), and segmentation and generation of 3D models (Figure 9C, Supplementary File 15).
Figure 1. Preparation of bed and container for echocardiography of anesthetized and unanesthetized axolotl. (A) A soft piece of cloth is folded once and rolled into "burrito" shape. (B) The ends are bent back and taped to form a lip shaped bed for the axolotl during underwater scanning. (C) For 2D and 3D echocardiography of an anesthetized axolotl, the animal is gently placed in a supine position in the crevice of the lip shaped bed and fixed with rubber bands over the mid-mandibular and sacral region. (D, E) A hammock is prepared by carving out a square hole in a piece of polystyrene foam and taping plastic wrap to the upper surface. (F) For 2D echocardiography of an unanesthetized axolotl, the animal is placed in a natural prone position in the hammock and approached with a gel covered transducer tip from underneath. Please click here to view a larger version of this figure.
Figure 2. Transducer placement. (A, B) Model of the arterial network in the axolotl with the approximate position of the transducer for long axis and short axis view (A) and oblique paragill view (B). (C) Transillumination with a powerful cold light source can aid in finding the exact location of the cardiac chambers before applying the transducer (see Supplementary File 1). Anatomical abbreviations: A, atria; OFT, outflow tract; SinV, sinus venosus; V, ventricle. Please click here to view a larger version of this figure.
Figure 3. Representative long axis echocardiographic views of the ventricle. (A, B) Typical long axis midline view in B-mode (yellow line in Figure 2A) in the ventricular end-diastolic (A) and end-systolic (B) phases (see Supplementary File 2 for video representation). (C, D) Long axis view of the ventricle in B-mode (black line in Figure 2A) in the ventricular end-diastolic (C) and end-systolic (D) phases (see Supplementary File 3 for video representation). (E–H) Similar view plane as in (A) and (B) in color Doppler (CD) and power Doppler (PD) mode demonstrating blood flow (see Supplementary File 4 and Supplementary File 5 for video representation of CD- and PD-mode, respectively). Red colors in CD-mode images indicate blood flowing toward the transducer and blue colors indicate the opposite. Cardiac chambers and blood flow have been highlighted with dotted lines. Inserted cartoons in (A) and (C) show placement of transducer and translation relative to the long axis midline view. Anatomical abbreviations: A, atria; DC(L), left duct of Cuvier; OFT, outflow tract; SinV, sinus venosus; V, ventricle. Please click here to view a larger version of this figure.
Figure 4. Representative long axis echocardiographic views of the atria and outflow tract. (A, B) Long axis view of the atria in B-mode (green line in Figure 2A) in the atrial end-diastolic (A) and end-systolic (B) phases (see Supplementary File 6 for video representation). (C, D) Similar view plane as in (A) and (B) in color Doppler (CD) mode demonstrating blood flow (see Supplementary File 7 for video representation). (E) Long axis view of the outflow tract in B-mode (blue line in Figure 2A) in the mid-ejection phase (see Supplementary File 8 for video representation). F: Similar view plane as in (E) in CD-mode demonstrating blood flow (see Supplementary File 9 for video representation). (G) Similar view plane as in (E) and (F) in pulse wave Doppler (PW) mode allowing for heat rate detection and velocity time integral (VTI) measurement for stroke volume calculation. Red colors in CD-mode images indicate blood flowing toward the transducer and blue colors indicate the opposite. Cardiac chambers and blood flow have been highlighted with dotted lines. Yellow and red arrow heads indicate semilunar valves at the root of the outflow tract and the spiral valve in the outflow tract, respectively. Inserted cartoons in (A) and (E) show placement of transducer and translation relative to the long axis midline view. Anatomical abbreviations: A(R), right atrium; A(L), left atrium; OFT, outflow tract; SinV, sinus venosus; V, ventricle; VC, vena cava. Please click here to view a larger version of this figure.
Figure 5. Representative short axis and oblique paragill echocardiographic views of the ventricle and outflow tract. (A, B) Short axis view of the ventricle in B-mode (grey line in Figure 2A) in the ventricular end-diastolic (A) and end-systolic (B) phases (see Supplementary File 10 for video representation). (C) Oblique paragill view of the outflow tract in B-mode (purple line in Figure 2B) in the mid-ejection phase (see Supplementary File 11 for video representation). (D) Similar view plane as in (C) in CD-mode demonstrating blood flow (see Supplementary File 12 for video representation). (E) Similar view plane as in (C) and (D) in pulse wave Doppler (PW) mode allowing for heat rate detection and velocity time integral (VTI) measurement for stroke volume calculation. Red colors in CD-mode images indicate blood flowing toward the transducer and blue colors indicate the opposite. Cardiac chambers and blood flow have been highlighted with dotted lines. Inserted cartoons in (A) and (C) show placement of transducer and translation relative to the long axis midline view. Anatomical abbreviations: A, atria; OFT, outflow tract; SinV, sinus venosus; V, ventricle. Please click here to view a larger version of this figure.
Figure 6. Representative results of heart rate and stroke volume measurements, the effect of anesthesia, and representative intra/inter-operator/observer analysis. (A–C) Heart rate (HR) relative to unanesthetized baseline plotted over time (0 h is at full anesthesia) for six axolotls anesthetized in benzocaine (A), MS-222 (B), and propofol (C). (D–F) Stroke volume (SV) relative to unanesthetized baseline plotted over time (0 h is at full anesthesia) for six axolotls anesthetized in benzocaine (D), MS-222 (E), and propofol (F). (G) Intra/inter-operator/observer analysis of stroke volume. Bland-Altman plots [difference (Dif) between operators (Op)/observers (Obs) plotted against average (Avg)] should reveal no systematic bias in the normally distributed measurements (Q-Q Plots) obtained by different operators and observers. Testing for equal mean (t-test) and equal variance (F-test) should reveal no significant differences between operators/observers (table in lower right). A–F was modified from material available under the Creative Commons Attribution License (Figure 1 of Thygesen et al.21). Please click here to view a larger version of this figure.
Figure 7. Comparison of stroke volume estimated by the geometric and the pulse wave Doppler method. Comparison of stroke volume (SV) estimated by either two-dimensional B-mode geometric (geo) measurements or pulse wave Doppler measurements on the velocity of blood exiting the outflow tract. SV(geo) and SV(pw) is recorded in the same six animals with seconds in between the two measurement types and using three different anesthetics, benzocaine (blue tilted squares), MS-222 (red squares), and propofol (green triangles) with one week of recovery between applying the different anesthetics. Please click here to view a larger version of this figure.
Figure 8. Representative spatiotemporal image correlation for 3D echocardiography. (A) Curve representation of yielded correlation values of a correlation operation in a 1,000 frame cine dataset with 75 frames per cardiac cycle. Two frames with only small differences, indicating matching cardiac phases, will yield a high correlation value. Subsequently a local maxima searching algorithm can be applied on the data to detect all matching frames. (B) Graphical representation of the same data as in (A). When correlation values are obtained by comparing the first cardiac cycle with the entire cine stack, diagonal lines of maximum correlation indicate matching cardiac phases. Please click here to view a larger version of this figure.
Figure 9. Representative 3D echocardiography. (A) Multi-planar view of 3D reconstructed axolotl heart. The spatiotemporal image correlation procedure allows for the reconstruction of a full cardiac cycle with several distinct phases (here 70 phases) in three spatial dimensions that can then be sliced as ones for desired investigation of spatiotemporal phenomena in the beating heart. (B) Three transversal slices of the reconstructed 115 slices 3D data. The quadratic averaging procedure enhances the blood-to-tissue contrast and lowers the signal-to-noise ratio allowing for a better appreciation of the trabeculated nature of the axolotl ventricle and a clear visualization of the interatrial septum and the valves in the outflow tract. (C) Surface and volume representations of the beating heart at three phases along a color coded segmented model (see Supplementary File 13 and Supplementary File 14 for video representations of the surface and volume rendered beating heart, and Supplementary File 15 for a three-phase segmented interactive 3D model). Anatomical abbreviations: A, atria; Cau, caudal; Cra, cranial; Dex, dexter (to the animal right); Dor, dorsal; OFT, outflow tract; Sin, sinister (to the animals left); SinV, sinus venosus; V, ventricle; Ven, ventral. Please click here to view a larger version of this figure.
Supplementary File 1. Transillumination to locate cardiac chambers in theaxolotl. See Figure 2C. Please click here to download this file.
Supplementary File 2. Long axis, midline view, B-mode. See Figure 3A, B. Please click here to download this file.
Supplementary File 3. Long axis, ventricular view, B-mode. See Figure 3C, D. Please click here to download this file.
Supplementary File 4. Long axis, ventricular view, Color Doppler mode. See Figure 3E, F. Please click here to download this file.
Supplementary File 5. Long axis, ventricular view, Power Doppler mode. See Figure 3G, H. Please click here to download this file.
Supplementary File 6. Long axis, atrial view, B-mode. See Figure 4A, B. Please click here to download this file.
Supplementary File 7. Long axis, atrial view, Color Doppler mode. See Figure 4C, D. Please click here to download this file.
Supplementary File 8. Long axis, outflow tract view, B-mode. See Figure 4E. Please click here to download this file.
Supplementary File 9. Long axis, outflow tract view, Color Doppler mode. See Figure 4F. Please click here to download this file.
Supplementary File 10. Short axis, ventricular view, B-mode. See Figure 5A, B. Please click here to download this file.
Supplementary File 11. Oblique paragill, outflow tract view, B-mode. See Figure 5C. Please click here to download this file.
Supplementary File 12. Oblique paragill, outflow tract view, Color Doppler mode. See Figure 5D. Please click here to download this file.
Supplementary File 13. Three-dimensional surface rendering of beating heart in 70 phases (19.6 ms temporal resolution). See Figure 9C. Please click here to download this file.
Supplementary File 14. Three-dimensional volume rendering of beating heart in 70 phases (19.6 ms temporal resolution). See Figure 9C. Please click here to download this file.
Supplementary File 15. Three-dimensional interactive model of beating heart in 3 phases: Ventricular end-systole, ventricular mid-ejection, and ventricular end-systole. See Figure 7C. The interactive PDF file should be viewed in Adobe Acrobat Reader 9 or higher. To activate the 3D feature, click the model. Using the cursor, it is now possible to rotate, zoom, pan the model, and in the model tree all segments of the model can be turned on/off or made transparent. The model tree is a hierarchy containing several sub layers that can be opened (+). Please click here to download this file.
Supplementary File 16. Representative annotated script for calculating the correlation value of a 1,000 frames acquisition with an upper estimation of 75 frames/cardiac cycle. The script is written in IJ1 macro language and can be implemented as a batch macro in ImageJ to calculate correlation values (75,000 per acquisition) across an entire z-stack of 3D data. Please click here to download this file.
Supplementary File 17. Representative script for automatic peak detection in a series of correlation values from a 1,000 frames acquisition with an upper estimation of 75 frames/cardiac cycle. The series of correlation values (Column B, marked in yellow) can be replaced and after activation of the macro (Ctrl + r) the list of commands to select matching cardiac phases and perform quadratic averaging will be displayed (Column Q, marked in green). Please click here to download this file.
Supplementary File 18. Representative annotated script to select matching cardiac phases and perform quadratic averaging of a 1,000 frames acquisition with an upper estimation of 75 frames/cardiac cycle (Column Q in Supplementary File 17). The script is written in IJ1 macro language and can be implemented as a macro in ImageJ to create an ensemble averaged one cycle (75 phases) 2D slice. Please click here to download this file.
Supplementary File 19. Representative annotated script for calculating the correlation value between a 70 frames reference slice and an adjacent 75 frames test slice. The script is written in IJ1 macro language and can be implemented as a macro in ImageJ to calculate correlation values (5,250). Please click here to download this file.
Supplementary File 20. Representative Excel script for automatic peak detection in a series of correlation values from a comparison between a 70 frames reference slice and an adjacent 75 frames test slice. The series of correlation values (Column C, marked in yellow) can be replaced and after activation of the macro (Ctrl + t) the list of slices to be selected as a substack in the test slice will be displayed (Column L, Row 2, marked in green). The test slice substack will have spatially matching frames to the reference slice. Please click here to download this file.
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 µm2 at 50 MHz) that is comparable to in vivo µCT imaging and much higher than in vivo µMRI, 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.
The authors have nothing to disclose.
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.
Axolotl (Ambystoma mexicanum) | Exoterra GmbH | N/A | All strains (wildtype, melanoid, white, albino, transgenic white with GFP) can be applied for echocardiography |
Vevo 2100 | Fujifilm, Visualsonics | Vevo 2100 | High frequency ultrasound system |
MS700 | Fujifilm, Visualsonics | MS700 | 50 MHz center frequency, transducer |
MS550s | Fujifilm, Visualsonics | MS550s | 40 MHz center frequency, transducer |
Micromanipulator | Zeiss | NA | |
Benzocain | Sigma-Aldrich | 94-09-7 | ethyl 4-aminobenzoate |
MS-222 | Sigma-Aldrich | 886-86-2 | ethyl 3-aminobenzoate methanesulfonic acid |
Propofol | B. Braun Medical A/S | NA | 2,6-diisopropylphenol |
Sodium chloride | Sigma-Aldrich | 7647-14-5 | NaCl |
Calcium chloride dihydrate | Sigma-Aldrich | 10035-04-8 | CaCl2·2H2O |
Magnesium sulfate heptahydrate | Sigma-Aldrich | 10034-99-8 | MgSO4·7H2O |
Potassium chloride | Sigma-Aldrich | 7447-40-7 | KCl |
Acetone | Sigma-Aldrich | 67-64-1 | Propanone |
Soft cloth | N/A | N/A | Any piece of soft cloth measuring appromixately 70 x 55 cm^2 e.g. a dish towel |
Styrofoam block | N/A | N/A | Any piece of Styrofoam block measuring approximately 33 x 27 x 5 cm^3 e.g. a medium sized Styrofoam cooler lid |
Plastic wrap | N/A | N/A | Any piece of plastic wrap e.g. food wrap |
Tape | BSN Medical | 72359-02 | Leukoplast sleek |
Kimwipes | Sigma-Aldrich | Z188956 | Kimwipes, disposable wipers |
Excel 2010 | Microsoft | N/A | Excel 2010 or newer |
ImageJ | National Institutes of Health | 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. |