GHK is supported by NIH/NHLBI K08-HL098565 and the Institute for Cardiovascular Research at the University of Chicago. All experimental methods described are approved by the Institutional Animal Care and Use Committee at the University of Chicago.

1. Preparing Mice for Imaging
<ol>
	<li>Prior to the imaging study, anesthetize the dam (2-3% isoflurane) in the induction chamber. Remove the animal from the induction chamber and immediately place the snout within a nose cone connected to the anesthesia system. Remove fur from the mid chest level to lower limbs (See Figure 1) with hair clippers. Remove the remaining body hair with depilatory cream. Depilatory cream may also be used without hair clippers, and should be thoroughly rinsed off of the skin after use to prevent irritation.</li>
	<li>Place the anesthetized mouse in a supine position on a heating pad with embedded ECG leads in order to maintain body temperature (Figure 1). Apply electrode gel to the four paws and tape them to the ECG electrodes.</li>
	<li>Obtain a steady-state sedation level throughout the procedure (1.0% to 1.5% isoflurane mixed with 100% O2). The level of anesthesia can be adjusted to maintain a target heart rate of 450 &plusmn; 50 beats per min (bpm). Careful attention should be paid to minimize dosage of isoflurane and duration of sedation to less than one hour.</li>
	<li>Gently insert a rectal probe (after lubricating) to monitor body temperature via the heating pad. It is important to maintain the body temperature within a narrow range (37.0 &deg;C &plusmn; 0.5 &deg;C). Controlled anesthetic and constant body temperature is essential for hemodynamic stability of the mother and fetuses.</li>
</ol>
Technical Considerations
The acquisition of fetal echocardiograms can be challenging. Data obtained from these studies can confounded due to the response to stress from both the dam and the fetus. Ideally, the animal temperature should be maintained using a heated imaging platform, circulating warming pad, heating lamps, or autoregulated heating blankets. In addition, routine use of a warmed acoustic gel is recommended. Although the predominant concern regarding body temperature control is to avoid hypothermia, development of hyperthermia should be of equal concern. An unmonitored heating apparatus such as a simple heating pad or the presence of close proximity to halogen illumination can result in fast and dangerous elevation of body temperature. Since substantial body temperature fluctuations in either direction places the animal at risk, every attempt should be made to maintain normal body temperatures.
The sonographer acquiring the images needs to avoid placing excessive pressure on the cavity with the transducer, since the weight of the transducer alone may result in altered cardiac function. The duration of image acquisition must also be kept to a minimum (ideally less than one hour) in order to reduce the physiologic and hemodynamic changes resulting from prolonged sedation. Additionally, length of time and exposure to isoflurane for each study must be kept to a minimum due to potential teratogenic effects of isoflurane 12.
2. Identification of Embryos
<ol>
	<li>Imaging is started using the mother's bladder as a landmark, with fetuses on the left and right uterine horns labeled as L1, 2,3, et cetera (left side) and R1, 2,3, et cetera (right side) (see Figure 1C). Notation of fetus location is useful for retrieval of specimens after imaging.</li>
	<li>Specimens situated too deep in the abdomen are scanned to document their presence but are excluded from data analysis because of poor resolution. The ability to scan adjacent embryos may help in tracking fetuses (Figure 2A).</li>
	<li>Scan planes are modified by changing the orientation of the mouse with respect to the scan plane. Images are obtained in 2 orthogonal planes for each fetus (Figure 2). An effort is made to obtain views approximating the transverse, frontal, or sagittal planes but sometimes is limited to oblique planes by the position of the uterus in the abdomen. Rotation of the scanning head will also allow for modification of orientation without moving the dam.</li>
</ol>
Technical Considerations
While handheld operation of the probe is feasible in adult mouse echocardiography, handheld operation in fetal imaging in not recommended. Identification of fetuses is complicated by the variable nature of uterine location, tortuosity, and movement. To minimize difficulties with fetus localization, stationary transducer use (Figure 1) with minimal movement beyond the horizontal plane of the dam is essential.
3. Evaluation of Structure and Function
<ol>
	<li>Scanning b-mode images are used to identify basic cardiac structures such as the atria, interventricular septum, ventricular chambers, and left and right outflow tracts (Figure 2).</li>
	<li>M-mode images are obtained from the short axis view and are used to measure ventricular wall thickness and chamber dimensions (Figure 3). If correct alignment is not obtainable due to fetal lie, measurements from b-mode images can be used to quantify % fractional shortening (FS). Temporal changes between LV end-systolic dimension (LVESD) and LV end-diastolic dimension (LVEDD) throughout the cardiac cycle are used for the calculation of shortening fraction (FS), as follows: 
	%FS = [(LVEDD - LVESD) / LVEDD] x 100</li>
	<li>The left and right ventricles are identified by scanning from head to tail. Left and right sides should be annotated. Visible flow streams generated by echogenic fetal blood facilitates accurate placement of the Doppler sample volume within the mitral orifice. Left ventricular inflow velocity is obtained from the mitral valves in apical four-chamber and LV long axis views (Figure 4,C). Aortic outflow measurements can be used to measure systolic ejection time (Figure 4,D). Heart rate may be calculated from the measurement of one flow cycle to the following flow cycle (Figure 4, C and D). Care should be taken to align blood flow and the Doppler beam to minimize the Doppler angle. Values taken beyond an angle of 60 degrees are inaccurate and should be avoided.</li>
	<li>Scanning b-mode images are used to identify common structural congenital abnormalities such as ventricular septal defects (Figure 5). Doppler sample volume within the ventricles may be used to identify flow across the ventricular septum. Additional parameters that are easily monitored include fetal size, heart rates, flow velocities, pericardial effusions, and fetal hydrops. The definitive diagnosis of specific cardiac defects requires additional evaluation by necropsy and histopathology.</li>
</ol>
Technical Considerations
Identification of left and right chambers can be difficult in fetal cardiac imaging due to the similar dimensions of ventricular chambers during development. One strategy is to establish right and left fetal orientation in real time by moving the imaging platform in the horizontal plane. Identification of snout, limb buds, and spine will help identify the let/right orientation of the fetus. If possible, tracking of the outflow track to the arch or the visualization of the main bifurcation of the pulmonary artery will allow for identification of the left ventricular outflow tract or right outflow tract respectively. For each fetus studied, it is important to note the determined left-right orientation on saved images.
 
Post-imaging Animal Monitoring and Care
 
Following the completion of imaging, the dam is returned to the appropriate housing and monitored according to standard institutional post-procedure protocol. Analgesia following this imaging procedure is not required. Full resumption of normal activity can be anticipated within five minutes.
4. Representative Results of Fetal Echocardiography
The development of high frequency probes (above 8 MHz), have allowed commercial echocardiographic equipment to have an axial resolution of approximately 0.2 mm with a lateral resolution of 0.3 mm when the image is zoomed and acquired at a depth of 1 cm. Most of the recently developed transducers are linear which have the advantage of avoiding near field artifacts. High frequency (30-50 MHz) mechanical probes have been recently developed which are adequate for the murine chest and heart rate, allowing an axial resolution of approximately 50 &mu;m at a depth of 5-12 mm. Most recently, these high frequency mechanical probes have added color Doppler capabilities enabling a complete evaluation of ventricular and valvular function and identification of shunt lesions in the fetal heart. The methods described here are performed on a VisualSonics Vevo 770 system and can be applied to nearly all equivalent systems. Current commercially available ultra-high frequency ultrasound system can operate at 40 Hz with maximal imaging depth of 7 to 14 mm, with up to 60 mm lateral and 50 to 100 mm axial resolution (Vevo770, VisualSonics, Inc.). This compares with 60 Hz and 20 mm imaging depth, with 50 to 100 mm axial and 200 to 500 mm lateral resolution with the clinical Acuson Sequoia ultrasound system.
Given the small size of the fetal mouse heart, fetal echocardiography studies in mice are technically challenging. Unlike echocardiography in adult mice, the sonographer must use nonconventional ultrasound imaging planes defined by the fetus' body axes. Tortuosity of the uterus also affects the orientation of the fetus and must be taken into consideration. Additionally, the inherent limitation in penetration depth of ultra-high frequency ultrasound can make it difficult to image all specimens in pregnancies with a large number of fetuses.
An ultrasound imaging strategy allows for high throughput screening for congenital cardiovascular and extracardiac defects 7. Beyond the study of genetic alterations, this technique may be used to screen for defects in pharmacologic and toxicology studies. This methodology can also be used as a guidance tool for interventional procedures such as injections or measurement of ventricular pressures 13.
The noninvasive nature of the fetal ultrasound is advantageous, not only because it allows cardiovascular function to be assessed under physiological conditions, but also because this provides critical phenotypic information in real time. Longitudinal examination of embryonic hearts, though technically possible, remains challenging for several reasons. Serial examination of the same fetus and identification of the same fetus at each exam is challenging in the absence of an evident structural defect. Movement of the uterus and the fetus may completely change the orientation of the specimen and thus make longitudinal tracking and follow-up measurements difficult 14.
Though echocardiography is a powerful technique for identification of cardiac abnormalities, the specific diagnosis of structural heart defects requires further detailed phenotyping by necropsy and histopathology 15. Correlation of genotype and a specific fetus requires harvesting fetuses by hysterotomy, preferably immediately after an echo study and while the dam is still anesthetized to minimize changes in orientation and embryo location.
Normal values for chamber dimensions and function have been reported for embryonic mice and users of this technique are advised to review the references cited for these values 6-10. Assessment of valvular morphology is limited by image resolution; however, annular dimension measurements and velocity measurements through the major vessels is feasible even as early as ED 9.5. Care must be taken to obtain adequate alignment with blood flow and the transducer 10, 16.
It should be emphasized that cardiac dimensions vary according to mice strains, gender and age, and rapidly change at different embryonic time points and heart rates. It is important to verify that groups of mice are matched for these parameters. Fetal imaging varies by mouse strain as well. For example, the pregnant CD-1 dam routinely contains more embryos compared to the C57/BL6 strain and thus may be more difficult to visualize all specimens. For these reasons, use of age and strain matched controls for each experiment should be used instead of reference values. In addition, measurements of individual parameters such as left ventricular end-diastolic dimension and posterior wall thickness may vary in normal mice up to 25% 8.
<img alt="Figure 1" src="/files/ftp_upload/4416/4416fig1.jpg" /> 
Figure 1. Overview of set-up using VisualSonics Vevo 770 system. (A) VisualSonics integrated rail system with physiological monitoring unit. (B) The mouse is positioned and properly restraint on the heating board. The four limbs are taped into the ECG electrodes. (C) Schematic of pregnant mouse and layout of embryos. The number of embryos within each horn of the uterus can vary significantly in addition to the orientation of the fetus. (D) A positioned dam on the imaging platform with arrows noting the planes of manipulation (X-axis and Y-axis) to move the dam for imaging. The Z-axis refers to movement of the transducer upward and downward (as indicated by the arrow in panel B). B, bladder; L, left; R, right.
<img alt="Figure 2" src="/files/ftp_upload/4416/4416fig2.jpg" /> 
Figure 2. Representative b-mode images. This figure contains representative b-mode images of embryonic day 14.5 fetus. (A) Visualization of two neighboring fetuses. Boxes indicate location of fetal heart. (B) Anatomic landmarks in a fetus to guide orientation. Embryonic day 14.5 heart in a four-chambered view (C), short-axis view of the left and right ventricles (D), right ventricular outflow tract and pulmonary artery (PA) (E), and left ventricular outflow tact (LVOT) and aorta (F).
<img alt="Figure 3" src="/files/ftp_upload/4416/4416fig3.jpg" /> 
Figure 3. Representative assessment of ventricular function. This figure contains representative images of 2D echocardiography of the long axis view of the heart at embryonic day 14.5 (A), and a four-chambered view (B). (C) M-mode tracing with lines indicating left and right ventricular internal diameter at diastole (R/LVIDd) and systole (R/LVIDs) from the four chamber image plane. Interventricular septum (IVS) is also visualized.
<img alt="Figure 4" src="/files/ftp_upload/4416/4416fig4.jpg" /> 
Figure 4. Representative Doppler assessment. This figure contains representative images of 2D echocardiography of embryonic day 14.5 heart in an apical four-chamber view (A). The left atrium and left ventricular cavity have been outlined. (B) Representative placement of Pulse wave Doppler sample volume for recording of mitral inflow. (C) Mitral inflow Doppler patterns from which early diastolic velocity (denoted "E") and atrial contraction (denoted "A") velocities can be measured. (D) Representative aortic Doppler waveform. Aortic Doppler jet can be used to measure ejection time (ET). Heart rate (HR) may be calculated from the measurement of one flow cycle to the following flow cycle. <a href="https://www.jove.com/files/ftp_upload/4416/4416fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 5" src="/files/ftp_upload/4416/4416fig5.jpg" /> 
Figure 5. Representative detection of ventricular septal defect. This figure contains representative b-mode images of embryonic day 14.5 heart in an apical four-chamber view (A) with the right and left ventricular cavities outlined in (B). Note the presence of the interventricular septum. (C) Transverse section of imaged heart stained with hematoxylin and eosin. (D) B-mode image of embryonic day 14.5 heart with a ventricular sepal defect (VSD) indicated by the arrow. (E) Right and left ventricular cavities are outlined with superimposed placement of Pulse wave Doppler sample volume for recording of flow across the interventricular septum. (F) Transverse section stained with hematoxylin and eosin of imaged heart after specimen retrieval. (G) Superimposed placement of Pulse wave Doppler sample volume for recording of flow across the interventricular septum. (H) Representative Doppler tracing from (G) demonstrating flow from the left to right ventricle. <a href="https://www.jove.com/files/ftp_upload/4416/4416fig5large.jpg" target="_blank"> Click here to view larger figure</a>.

<ol>
	<li>Wessels, A., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+anatomy+of+the+heart:+a+tale+of+mice+and+man.">Developmental anatomy of the heart: a tale of mice and man.</a> Physiol. Genomics. 15, 165 (2003).</li><li>Snider, P., Conway, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+human+cardiovascular+congenital+disease+using+transgenic+mouse+models.">Probing human cardiovascular congenital disease using transgenic mouse models.</a> Prog. Mol. Biol. Transl. Sci. 100, 83 (2011).</li><li>Clark, K. L., Yutzey, K. E., Benson, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+and+congenital+heart+defects.">Transcription factors and congenital heart defects.</a> Annu. Rev. Physiol. 68, 97 (2006).</li><li>Leatherbury, L., Yu, Q., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+phenotypic+analysis+of+cardiovascular+structure+and+function+in+fetal+mice+using+ultrasound.">Noninvasive phenotypic analysis of cardiovascular structure and function in fetal mice using ultrasound.</a> Birth Defects Res C Embryo Today. 69, 83 (2003).</li><li>Spurney, C. F., Lo, C. W., Leatherbury, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+mouse+imaging+using+echocardiography:+a+review+of+current+technology.">Fetal mouse imaging using echocardiography: a review of current technology.</a> Echocardiography. 23, 891 (2006).</li><li>Spurney, C. F., Leatherbury, L., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+ultrasound+database+profiling+growth,+development,+and+cardiovascular+function+in+C57BL/6J+mouse+fetuses.">High-frequency ultrasound database profiling growth, development, and cardiovascular function in C57BL/6J mouse fetuses.</a> J. Am. Soc. Echocardiogr. 17, 893 (2004).</li><li>Shen, 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=Cardiovascular+phenotyping+of+fetal+mice+by+noninvasive+high-frequency+ultrasound+facilitates+recovery+of+ENU-induced+mutations+causing+congenital+cardiac+and+extracardiac+defects.">Cardiovascular phenotyping of fetal mice by noninvasive high-frequency ultrasound facilitates recovery of ENU-induced mutations causing congenital cardiac and extracardiac defects.</a> Physiol. Genomics. 24, 23 (2005).</li><li>Yu, Q., Leatherbury, L., Tian, X., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+assessment+of+fetal+mice+by+in+utero+echocardiography.">Cardiovascular assessment of fetal mice by in utero echocardiography.</a> Ultrasound Med. Biol. 34, 741 (2008).</li><li>Linask, K. K., Huhta, 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=Use+of+Doppler+echocardiography+to+monitor+embryonic+mouse+heart+function.">Use of Doppler echocardiography to monitor embryonic mouse heart function.</a> Methods Mol. Biol. 135, 245 (2000).</li><li>Hinton, R. B., 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=Mouse+heart+valve+structure+and+function:+echocardiographic+and+morphometric+analyses+from+the+fetus+through+the+aged+adult.">Mouse heart valve structure and function: echocardiographic and morphometric analyses from the fetus through the aged adult.</a> Am. J. Physiol Heart Circ. Physiol. 294, H2480 (2008).</li><li>Gui, Y. H., Linask, K. K., Khowsathit, P., Huhta, 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=Doppler+echocardiography+of+normal+and+abnormal+embryonic+mouse+heart.">Doppler echocardiography of normal and abnormal embryonic mouse heart.</a> Pediatr. Res. 40, 633 (1996).</li><li>Purssell, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+high-resolution+ultrasound+reveals+structural+and+functional+deficits+in+dimethadione-exposed+fetal+rat+hearts+in+utero.">Noninvasive high-resolution ultrasound reveals structural and functional deficits in dimethadione-exposed fetal rat hearts in utero.</a> Birth Defects Res. B Dev. Reprod. Toxicol. , (2011).</li><li>Le, V. P., Kovacs, A., Wagenseil, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Left+Ventricular+Pressure+in+Late+Embryonic+and+Neonatal+Mice.">Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice.</a> J. Vis. Exp. (60), e3756 (2012).</li><li>Ji, R. P., Phoon, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+localization+of+nuclear+factor+of+activated+T+cells+c1-/-+mouse+embryos+by+ultrasound+biomicroscopy-Doppler+allows+genotype-phenotype+correlation.">Noninvasive localization of nuclear factor of activated T cells c1-/- mouse embryos by ultrasound biomicroscopy-Doppler allows genotype-phenotype correlation.</a> J. Am. Soc. Echocardiogr. 18, 1415 (2005).</li><li>Kim, G. H., Samant, S. A., Earley, J. U., Svensson, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+of+FOG-2+expression+in+cardiomyocytes+by+microRNA-130a.">Translational control of FOG-2 expression in cardiomyocytes by microRNA-130a.</a> PLoS One. 4, e6161 (2009).</li><li>Momoi, N., 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=Modest+maternal+caffeine+exposure+affects+developing+embryonic+cardiovascular+function+and+growth.">Modest maternal caffeine exposure affects developing embryonic cardiovascular function and growth.</a> Am. J. Physiol. Heart Circ. Physiol. 294, H2248 (2008).</li><li>Tobita, K., Liu, X., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+modalities+to+assess+structural+birth+defects+in+mutant+mouse+models.">Imaging modalities to assess structural birth defects in mutant mouse models.</a> Birth Defects Res. C Embryo Today. 90, 176 (2010).</li></ol>

No conflicts of interest declared.

The ability to perform serial measurements and to detect mutant fetuses with cardiac defects highlights the usefulness of echocardiography for investigating normal and abnormal cardiovascular development. Analysis of cardiac structure and function in vivo has become an integral part in the description of genetic and non-genetic modifications to normal fetal development. The availability of 2D-guided Doppler makes it possible to monitor heart rate and blood flow patterns while obtaining real-time images. De...

<table border="1">
<tbody>
 <tr>
 <td>Vevo 770 Imaging System</td>
 <td>VisualSonics</td>
 <td></td>
 <td>(Toronto, Canada)</td>
 </tr>
 <tr>
 <td>RMV707B.15-45 MHz transducer</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Tec 3 Isoflurane Vaporizer</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Murine Fetal Echocardiography at JoVE.com

Murine Fetal Echocardiography

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 ...

Research

JoVE Journal

Medicine

17.1K Views.  University of Chicago. Morte fetale e perinatale è una caratteristica comune quando si studiano le alterazioni genetiche che influenzano lo sviluppo cardiaco. Alta frequenza ecografia ha migliorato 2-D risoluzione e può fornire informazioni excellent precoce sviluppo cardiaco ed è un metodo ideale per rilevare l'impatto sulla struttura e funzione cardiaca prima della morte.

Video: Murine Fetal Echocardiography

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  ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

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 ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

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 ...

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model species such as zebrafish and salamanders that are capable of intrinsic heart regeneration hold promise for future regenerative therapies for human patients. To evaluate the outcome of cardioregenerative experiments it is imperative that heart function can be monitored. The axolotl salamander (A. mexicanum) represents a well-established model species in regenerative biology attaining sizes that allows for evaluation of cardiac function. The purpose of this protocol is to establish methods to reproducibly measure cardiac function in the axolotl using echocardiography. The application of different anesthetics (benzocaine, MS-222, and propofol) is demonstrated, and the acquisition of two-dimensional (2D) echocardiographic data in both anesthetized and unanesthetized axolotls is described. 2D echocardiography of the three-dimensional (3D) heart can suffer from imprecision and subjectivity of measurements, and to alleviate this phenomenon a solid method, namely intra/inter-operator/observer analysis, to measure and minimize this bias is demonstrated. Finally, a method to acquire 3D echocardiographic data of the beating axolotl heart at a very high spatiotemporal resolution and with pronounced blood-to-tissue contrast is described. Overall, this protocol should provide the necessary methods to evaluate cardiac function and model anatomy, and flow dynamics in the axolotl using ultrasound imaging with applications in both regenerative biology and general physiological experiments.

2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum

Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model ...

Model Surgical Training: Skills Acquisition in Fetoscopic Laser Photocoagulation of Monochorionic Diamniotic Twin Placenta Using Realistic Simulators

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome (TTTS), but is technically challenging and can lead to significant complications. Acquiring and maintaining the necessary surgical skills require consistent practice, a critical caseload, and time. Training on realistic surgical simulators can potentially shorten this steep learning curve and enables several proceduralists to acquire procedure-specific skills simultaneously. Here we describe realistic simulators designed to allow the user familiarity with the equipment and specific steps required in the surgical treatment of TTTS, including fetoscopic handling, approaches to anterior and posterior placenta, recognition of anastomoses, and efficient coagulation of vessels. We describe the skills that are especially important in conducting placental laser coagulation that the surgeon can practice on the model and apply in a clinical case. These models can be adapted easily depending on the availability of materials and require standard fetoscopy equipment. Such training systems are complementary to traditional surgical apprenticeships and can be useful aids for fetal medicine units that provide this clinical service.

Practicing the specific skills required for fetoscopic laser coagulation of monochorionic placental anastomoses on realistic models can aid less experienced surgeons in overcoming the steep learning curve associated with this procedure that is now regarded as the standard of care for twin-twin transfusion syndrome.

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome ...