We thank the animal physiology core, division of molecular medicine at UCLA for providing&#160;technical support&#160;and open access to the Vevo 2100 ultrasound biomicroscopy (UBM) system. This study was supported by the NIH/Child Health Research Center (5K12HD034610/K12), the UCLA-Children&#39;s Discovery Institute and Today and Tomorrow Children&#39;s Fund, and David Geffen School of Medicine Research Innovation award to M. Touma.

The University of California, Los Angeles, Animal Care and Use Committee has approved all procedures shown in this protocol. The experiments were conducted as part of an ongoing study under active animal protocols approved by the institutional Animal Care and Use Committee of University of California, Los Angeles, California, USA. Animal handling and care followed the standards of the Guide for the Care and Use of Laboratory Animals.
1. Preparing the High Frequency Ultrasound Imaging System
...

<ol>
	<li>Touma, M., Reemtsen, B., Halnon, N., Alejos, J., Finn, J. P., Nelson, S. F., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Path+to+Implement+Precision+Child+Health+Cardiovascular+Medicine.">A Path to Implement Precision Child Health Cardiovascular Medicine.</a> Front Cardiovasc Med. 4, 36 (2017).</li><li>Triedman, J. K., Newburger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+Congenital+Heart+Disease.+The+Next+Decade.">Trends in Congenital Heart Disease. The Next Decade.</a> Circulation. 133, 2716-2733 (2016).</li><li>Gilboa, S. 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=Congenital+Heart+Defects+in+the+United+States+Estimating+the+Magnitude+of+the+Affected+Population+in+2010.">Congenital Heart Defects in the United States Estimating the Magnitude of the Affected Population in 2010.</a> Circulation. 134, 101-109 (2016).</li><li>Pruetz, J. D., 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=Outcomes+of+critical+congenital+heart+disease+requiring+emergent+neonatal+cardiac+intervention.">Outcomes of critical congenital heart disease requiring emergent neonatal cardiac intervention.</a> Prenat Diagn. 34, 1127-1132 (2014).</li><li>Peterson, C., 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=Mortality+among+Infants+with+Critical+Congenital+Heart+Disease:+How+Important+Is+Timely+Detection?.">Mortality among Infants with Critical Congenital Heart Disease: How Important Is Timely Detection?.</a> Birth Defects Res A Clin Mol Teratol. 97 (10), 664-672 (2013).</li><li>Atz, A. 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=Prenatal+Diagnosis+and+Risk+Factors+for+Preoperative+Death+in+Neonates+with+Single+Right+Ventricle+and+Systemic+Outflow+Obstruction:+Screening+Data+from+the+Pediatric+Heart+Network+Single+Ventricle+Reconstruction+Trial.">Prenatal Diagnosis and Risk Factors for Preoperative Death in Neonates with Single Right Ventricle and Systemic Outflow Obstruction: Screening Data from the Pediatric Heart Network Single Ventricle Reconstruction Trial.</a> J Thorac Cardiovasc Surg. 140 (6), 1245-1250 (2010).</li><li>Lalani, S. R., Belmont, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Basis+of+Congenital+Cardiovascular+Malformations.">Genetic Basis of Congenital Cardiovascular Malformations.</a> Eur J Med Genet. 57 (8), 402-413 (2014).</li><li>Hanchard, N. A., Swaminathan, S., Bucasas, K., Furthner, D., Fernbach, S., Azamian, M. 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=A+genome-wide+association+study+of+congenital+cardiovascular+left-sided+lesions+shows+association+with+a+locus+on+chromosome+20.">A genome-wide association study of congenital cardiovascular left-sided lesions shows association with a locus on chromosome 20.</a> Hum Mol. 11, 2331-2341 (2016).</li><li>Arsenijevic, V., Davis-Dusenbery, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible,+Scalable+Fusion+Gene+Detection+from+RNA-Seq.">Reproducible, Scalable Fusion Gene Detection from RNA-Seq.</a> Methods Mol Biol. 1381, 223-237 (2016).</li><li>LaHaye, S., Corsmeier, D., Basu, M., Bowman, J. L., Fitzgerald-Butt, S., Zender, G., 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=Utilization+of+Whole+Exome+Sequencing+to+Identify+Causative+Mutations+in+Familial+Congenital+Heart+Disease.">Utilization of Whole Exome Sequencing to Identify Causative Mutations in Familial Congenital Heart Disease.</a> Circ Cardiovasc Genet. 9 (4), 320-329 (2016).</li><li>Zaidi, S., Choi, M., Wakimoto, H., Ma, L., Jiang, J., Overton, J. D., 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=De+novo+mutations+in+histone+modifying+genes+in+congenital+heart+disease.">De novo mutations in histone modifying genes in congenital heart disease.</a> Nature. 498 (7453), 220-223 (2016).</li><li>Leirgul, E., Brodwall, K., Greve, G., Vollset, S. E., Holmstrom, H., Tell, G. 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=Maternal+Diabetes,+Birth+Weight,+and+Neonatal+Risk+of+Congenital+Heart+Defects+in+Norway,+1994-2009.">Maternal Diabetes, Birth Weight, and Neonatal Risk of Congenital Heart Defects in Norway, 1994-2009.</a> Obstet Gynecol. 128 (5), 1116-1125 (2016).</li><li>Garry, D. J., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Common+Progenitor+at+the+Heart+of+Development.">A Common Progenitor at the Heart of Development.</a> Cell. 127 (6), 1101-1104 (2006).</li><li>Postma, A. V., Bezzina, C. R., Christoffels, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+congenital+heart+disease:+the+contribution+of+the+noncoding+regulatory+genome.">Genetics of congenital heart disease: the contribution of the noncoding regulatory genome.</a> J Hum Genet. 61, 13-19 (2016).</li><li>Ganguly, A., Touma, M., Thamotharan, S., De Vivo, D. C., Devaskar, S. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Calorie+Restriction+Causing+Uteroplacental+Insufficiency+Differentially+Affects+Mammalian+Placental+Glucose+and+Leucine+Transport+Molecular+Mechanisms.">Maternal Calorie Restriction Causing Uteroplacental Insufficiency Differentially Affects Mammalian Placental Glucose and Leucine Transport Molecular Mechanisms.</a> Endocrinology. Oct. 157 (10), 4041-4054 (2016).</li><li>Lluri, G., Huang, V., Touma, M., Liu, X., Harmon, A. W., Nakano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+progenitors+are+required+for+proper+development+of+coronary+vasculature.">Hematopoietic progenitors are required for proper development of coronary vasculature.</a> J Mol Cell Cardiol. 86, 199-207 (2015).</li><li>Bishop, K. C., Kuller, J. A., Boyd, B. K., Rhee, E. H., Miller, S., Barker, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+Examination+of+the+Fetal+Heart.">Ultrasound Examination of the Fetal Heart.</a> Obstet Gynecol Surv. 72 (1), 54-61 (2017).</li><li>He, H., Gan, J., Qi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+extensive+cardiac+echography+examination+for+detecting+foetal+congenital+heart+defects+during+early+and+late+gestation:+a+systematic+review+and+meta-analysis.">Assessing extensive cardiac echography examination for detecting foetal congenital heart defects during early and late gestation: a systematic review and meta-analysis.</a> Acta Cardiol. 71 (6), 699-708 (2016).</li><li>Hobbs, C. A., Cleves, M. A., Karim, M. A., Zhao, W., MacLeod, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Folate-Related+Gene+Environment+Interactions+and+Congenital+Heart+Defects.">Maternal Folate-Related Gene Environment Interactions and Congenital Heart Defects.</a> Obstet Gynecol. 116 (2 Pt 1), 316-322 (2016).</li><li>Gabbay-Benziv, 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=A+step-wise+approach+for+analysis+of+the+mouse+embryonic+heart+using+17.6+Tesla+MRI.">A step-wise approach for analysis of the mouse embryonic heart using 17.6 Tesla MRI.</a> Magn Reson Imaging. 35, 46-53 (2017).</li><li>Kim, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+fetal+echocardiography.">Murine fetal echocardiography.</a> J Vis Exp. (72), e4416 (2013).</li><li>Zhou, Y. Q., Cahill, L. S., Wong, M. D., Seed, M., Macgowan, C. K., Sled, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+flow+distribution+in+the+mouse+fetal+circulation+at+late+gestation+by+high-frequency+Doppler+ultrasound.">Assessment of flow distribution in the mouse fetal circulation at late gestation by high-frequency Doppler ultrasound.</a> Physiol Genomics. 46 (16), 602-614 (2014).</li><li>Greco, A., Coda, A. R., Albanese, S., Ragucci, M., Liuzzi, R., Auletta, L., Gargiulo, S., Lamagna, F., Salvatore, M., Mancini, M. <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+for+the+Study+of+Early+Mouse+Embryonic+Cardiovascular+System.">High-Frequency Ultrasound for the Study of Early Mouse Embryonic Cardiovascular System.</a> Reprod Sci. 22 (12), 1649-1655 (2015).</li><li>Deneke, T., Lawo, T., von Dryander, S., Grewe, P. H., Germing, A., Gorr, E., Hubben, P., Mugge, A., Shin, D. I., Lemke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+determination+of+the+optimized+atrioventricular+delay+in+patients+with+implanted+biventricular+pacing+devices.">Non-invasive determination of the optimized atrioventricular delay in patients with implanted biventricular pacing devices.</a> Indian Pacing Electrophysiol J. 10 (2), 73-85 (2010).</li><li>Kono, M., Kisanuki, A., Ueya, N., Kubota, K., Kuwahara, E., Takasaki, K., Yuasa, T., Mizukami, N., Miyata, M., Tei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+global+systolic+dysfunction+has+a+significant+role+in+the+development+of+diastolic+heart+failure+in+patients+with+systemic+hypertension.">Left ventricular global systolic dysfunction has a significant role in the development of diastolic heart failure in patients with systemic hypertension.</a> Hypertens Res. 33 (11), 1167-1173 (2010).</li></ol>

Cardiovascular malformations and diseases are substantially influenced by genetic factors and environmental elements19. We have previously demonstrated a significant impact of maternal caloric restriction, initiated during the second trimester, on feto-placental circulatory flow and fetal cardiac function9.
Prenatal hypoxia is another common stress factor during fetal development that may tremendously affect the feto-placental physiology and&#160...

Congenital malformations of the heart are heterogeneous structural defects that occur during early cardiac development. Current technical advances of operational procedures have led to significant improvements in the survival rates of&#160;infants with CHDs1,2. However, quality of life is often compromised secondary to&#160;prolonged hospitalization and needs for staged surgical repair procedures1,2,3,4,5. Prenatal detection of the underlying molecular mechanisms of CHDs is crucial in order to plan early interventions, to carry out new prevention strategies, and to improve the lifelong outcomes6,7.
Although multiple genetic and environmental factors have been implicated in CHDs pathogenesis, establishing the causality remains an unmet need to improve diagnostic, therapeutic, and preventive strategies1,8,9,10,11,12. Furthermore, examining the roles of in utero&#160;stress factors and epigenetic modifiers opens new venues for future investigations11,12. The last decade has indeed witnessed rapid advances in next generation sequencing technology including single nucleotide polymorphism (SNP) microarray, whole exome sequencing, and genome-wide methylation studies,&#160;their utilization in studying the genetic causes of complex human diseases, including CHDs1,8,9,10,11&#160;paving the way to identify&#160;novel mutations and genetic variants that have not yet been tested for their pathogenicity in suitable animal models.
Among the different disease model systems, mouse&#160;is the animal model of choice, not only for investigating mechanisms of CHDs during early cardiogenesis13,14,15,16, but also to elucidate their impact on cardiac chamber maturation and function at late gestation in prenatal and perinatal stress factors. Hence, performing in vivo phenotypic characterization of a mutant fetal mouse heart, during both early and late stages of development, is crucial to understand the role of these genetic variations&#160;and environmental factors on&#160;cardiac development, and the potential future impact on chamber specific maturation processes in mice.
Early detection and accurate diagnosis of cardiac defects during development is critical for interventional planning17,18. Being safe, simple, portable and repeatable, fetal sonography has indeed become the standard imaging technique for cardiac evaluation in the clinic. Fetal circulation assessment using Doppler ultrasound has been widely used in clinical practice&#160;not only for the detection of&#160;cardiac defects, but also to detect vascular abnormalities, placenta insufficiency and&#160;intrauterine growth restriction, and to assess the fetal well-being in response to in utero insults including hypoxemia, maternal illness, and drug toxicity17,18. In parallel to its&#160;value in evaluating human defects and diseases, ultrasound assessment of fetal mice has gained increasing utility in experimental settings19,20,21,22,23. In particular, fetal heart ultrasound (echocardiography) allows sequential in vivo visualization of the developing heart. Many experimental studies have used ultrasound-imaging technology to observe fetal cardiovascular development in transgenic fetal mice. Doppler ultrasound has been particularly useful to elucidate the pathophysiological parameters, such as the flow patterns in fetal circulation under physiological challenges or disease conditions10,19. In both humans and animals, abnormal blood flow or oxygen supply to the fetus can result from various conditions that can disrupt fetal environment&#160;in utero&#160;and affect the fetoplacental axis, including placental&#160;abnormalities, maternal hypoxia, gestational diabetes, and pharmaceutically induced vascular constriction15,22. Therefore, establishing standardized methods for performing Doppler ultrasounds on fetal mice will tremendously empower future studies of CHDs by facilitating&#160;monitoring&#160;flow patterns and key hemodynamic indices of the cardiovascular circuits during different stages of cardiac development in genetic mouse models.
High frequency ultrasound has emerged as a powerful tool to measure the developmental&#160;and physiological parameters of the cardiovascular system in mouse models&#160;and human diseases18. This technology has been further refined in recent years. We and other researchers have demonstrated the feasibility of this system for conducting ultra-high frequency ultrasound studies on the fetal mouse heart15,19,20,21,22,23. The system is equipped with Doppler color flow mapping and linear array transducers that generate two-dimensional, dynamic images at high frequency (30 to 50 MHz) frame rates. These advantages, compared to low frequency ultrasound systems and the prior generation of high frequency ultrasound21,22, provide the necessary sensitivity and resolution for in-depth assessment of the fetal circulatory system, including comprehensive characterization of heart structures, chamber function, and flow indices of fetal mice in experimental settings. Herein, we outline methods to perform rapid assessment of cardiopulmonary circulation and feto-placental circulation at embryonic day E18.5 in vivo by using a high frequency&#160;system. We chose a 30/45 MHz transducer that provides an axial resolution of approximately 60 &#181;m and a lateral resolution of 150 &#181;m. However, a higher frequency transducer (40/50 MHz) can be chosen to analyze earlier developmental stages by following a similar methodological approach. The selected M-mode allows the visualization of tissues in motion at high temporal resolution levels (1,000 frames/s). Finally, we&#160;demonstrate the feasibility of high ultrasound for detailed comprehensive phenotypic characterization of fetal cardiovascular hemodynamic status and function in mice at baseline and in response to prenatal hypoxia stress.

<table border="0" cellpadding="0" cellspacing="0" width="1606">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:189px;">Vevo 2100&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:291px;">VisualSonics, Toronto, Ontario, Canada</td>
			<td style="width:301px;">N/A</td>
			<td style="width:827px;">High Freequency Ultrasound Biomicroscopy. The set up is available in animal physiology core facility, division of molecular medicine, UCLA. USA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">inbred mice (c57/BL6)&#160;&#160;</td>
			<td>Charles River Laboratories</td>
			<td>N/A</td>
			<td>Inbread wild type mouse strain</td>
		</tr>
	</tbody>
</table>

fetal mouse cardiovascular imaging using a high-frequency ultrasound (30/45mhz) system

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Statistical analyses of cardiac and hemodynamic indices were performed offline. The means of 5 consecutive measurements in 3 optimal images were calculated. The data were expressed as Mean &#177; SEM. Student&#39;s t-test was used to infer intergroup comparisons. A P value of &#8804;0.05 was considered statistically significant.
Following the above protocol, we characterized the impact of chronic exposure to pr...

Watch this Scientific Journal Video about Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System at JoVE.com

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

The overall goal of the following experiment is to study the structure and hemodynamic parameters of the fetal circulation, using a high-frequency ultrasound system. This method provides comprehensive phenotyping of cardiac morphology and flow indices during development in real time, and allows studying of congenital heart defects in experimental settings. Compared to low frequency ultrasound systems, this technique provides higher sensitivity and resolution by generating two-dimensional dynamic images at high-frequency frame rates.To begin, turn on the ultrasound imaging system and the physiology monitoring unit. Then connect the 30-45 megahertz transducer, and place the corresponding scan head on its holder near the imaging platform. Next select the cardiac measurement program option.Place the ultrasound gel upside down in its pre-warming container, set to 37 degrees Celsius. Then confirm the appropriate tubing system for anesthesia, and verify the levels of oxygen and isoflurane. Disinfect the imaging platform and the working area.Finally, set the heat level of the imaging platform to maintain a constant body temperature of 37 degrees Celsius for the dam. Transfer the sedated dam from anesthesia induction chamber to the imaging platform, and place the animal in a supine position. Provide steady state sedation with isoflurane, using a face mask connected to the anesthesia tubing system.Apply electrode gel to the embedded electrocardiographic electrodes. Then gently tape the limbs to the electrodes for constant monitoring of maternal cardiac and respiratory rates. Monitor the dam's temperature and heart rate, displayed on the physiology controller unit.Adjust the level of isoflurane to maintain an average heart rate close to 450 beats per minute. Confirm the appropriate level of anesthesia by evaluating the mouse's posture, heart rate, and response to toe pinches. Then use a depilatory cream to remove the fur from the mid-chest level to the lower limbs, to minimize ultrasound attenuation.Palpate the abdominal wall gently to locate the fetuses and spread them out. Avoid spreading the fetuses forcefully. Using the cervix as a landmark, annotate each embryo on the dam's abdomen, and define their orientations.Exclude those fetuses where the orientation is unreliable. Label the fetuses on the left uterine horn with the prefix L, and those on the right uterine horns with an R prefix, as shown here. Apply pre-warmed ultrasound gel on the abdomen, and spread it carefully, to avoid bubble formation.Add additional amount of gel to the area of scanning. Place the ultrasound probe on its mechanical holder, and move it gradually towards the abdomen to contact the thick gel layer, while looking for the beating heart, using B mode scanning. Confirm the right and left orientation of the individual fetus in real time, by moving the imaging platform in the horizontal plane.Scan the fetus from head to tail to annotate the snout, the limbs, and the spine, as landmarks. Visualize the beating heart and annotate the left ventricle and the right ventricle. Switch to color Doppler mode to optimize the heart visualization.Click the B-Mode button to obtain a peristernal short access view. Ensure that the left and right ventricles are displayed in their maximum diameter at the center of the data acquisition frame, and start live-imaging. Change the scanning planes to obtain a longitudinal four chamber view.First identify the remaining structures of the heart, such as the atria, interventricular septum, and left and right outflow tracts. Next have the ventricular and atrial chambers displayed in their maximum length. Start image acquisition by clicking the Cine button, to obtain continuous recording cine-loops for a minimum of 10 seconds.Next click the scanning M-Mode button to obtain cardiac images from four chamber planes to evaluate fetal heart rate and ventricular function. Exclude any non-optimal images. Then click the Analysis button to measure the ventricular wall thickness and internal diameter at diastoli and systoli.First adjust the sector to an angle of acquisition less than 60 degrees, to visualize the bifurcation of the pulmonary artery and identify the right outflow tract. Next click Pulsed Wave Doppler button to obtain the flow pattern through the pulmonary and the aortic valves. Next place the pulse wave Doppler sample volume to record aortic mitral flow Doppler patterns, and measure early diastolic velocity and atrial contraction velocity.Use the color Doppler scan to visualize the uterine artery and fetal placental vascular tree, using the 45 megahertz transducer. Place the pulsed wave Doppler sampler volume to obtain the umbilical artery flow pattern. Use the pulsed wave Doppler scanning record to measure vascular peak flow parameters, including acceleration time, ejection time, and peak flow velocity at end systoli.Turn off the isoflurane container, following the completion of the imaging process. Continue monitoring body temperature, respiratory rate, and heart rate during the recovery phase. Document the time to full resumption of normal activity.The data in this table demonstrates the feasibility of using high-frequency ultrasound, as outlined in this protocol, to perform a comprehensive phenotyping of fetal heart morphology and function, and fetal placenta circulatory parameters, and their alterations in fetal mice exposed to chronic hypoxia in utero. Following this procedure, other methods like speckle tracing and stain analysis can be employed in order to answer additional questions, like measuring regional myocardial function of the developing myocardium in future studies. After watching this video, you should have a good understanding of how to operate high-frequency ultrasound effectively and to evaluate fetal cardiac morphology and circulatory parameters systematically in real time.

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11.4K 조회수.  University of California, Los Angeles. The overall goal of the following experiment is to study the structure and hemodynamic parameters of the fetal circulation, using a high-frequency ultrasound system. This method provides comprehensive phenotyping of cardiac morphology and flow indices during development in real time, and allows studying of congenital heart defects in experimental settings. Compared to low frequency ultrasound systems, this technique provides higher sensitivity and resolution by generating two-dimensional dyna...