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>Turn on the ultrasound imaging system and the physiology monitoring unit.</li>
	<li>Connect the 30/45 MHz transducer.</li>
	<li>Place the corresponding scan head on its holder near the imaging platform.</li>
	<li>Select the Cardiac Measurement Program option.</li>
	<li>Place the ultrasound gel upside down in its pre-warming container set to 37 &#176;C.</li>
	<li>Confirm appropriate tubing system for anesthesia and verify the levels of oxygen and isoflurane.</li>
	<li>Disinfect the imaging platform and the working area.</li>
	<li>Set the heat level of the imaging platform to maintain constant body temperature and heart rate of the dams.</li>
</ol>
2. Pregnant Mouse Preparation
<ol>
	<li>Place the pregnant mouse (C57/BL6) dam in the anesthesia induction chamber.</li>
	<li>Induce anesthesia by using continuously delivered inhalational isoflurane (isoflurane 2%-3%) mixed with 100% oxygen (100% O2) at a flow rate of 200 mL/min in the induction chamber.</li>
	<li>Transfer the sedated animal onto the imaging platform in a supine position.</li>
	<li>Provide steady-state sedation by using a facemask connected to the anesthesia tubing system-delivering isoflurane (1.0% - 1.5%) mixed with 100% O2 at 200 mL/min. 
	CAUTION: Control the leakage of anesthetic gas by using a ventilation system equipped with a charcoal filter containing canister set.</li>
	<li>Tape the limbs gently to the embedded electrocardiographic electrodes after application of the electrode gel to achieve constant monitoring of maternal cardiac and respiratory rates.</li>
	<li>Adjust the level of isoflurane to maintain an average heart rate of (450 +/- 50 beats/min (bpm)).</li>
	<li>Maintain the body temperature within a range of 37.0 &#176;C +/- 0.5 &#176;C. Monitor the body temperature and heart rate being displayed on the physiology controller unit.</li>
	<li>Document the vital signs of the sedated mouse every 15 min throughout the imaging procedure.</li>
	<li>Assess the level of anesthesia by evaluating the mouse&#39;s posture, heart rate, and response to toe pinches.</li>
	<li>Apply ophthalmic balm (1 drop into each eye) to prevent eye dryness and corneal damage.</li>
	<li>Remove the fur from the mid-chest level to the lower limbs by using a depilatory cream to minimize ultrasound attenuation. Remove cream 1-1.5 min after application by alternating wet and dry gauze wipes to prevent damage to the skin.</li>
</ol>
3. Embryo Identification
<ol>
	<li>Palpate the abdominal wall gently to locate the fetuses and spread them out.</li>
	<li>Annotate each embryo on the dam&#39;s abdomen and define their anterior-posterior and dorsal-ventral orientations by using a marker.</li>
	<li>Use the cervix of the sedated dam as a landmark. Label the fetuses on the left and right uterine horns as L1, L2,L 3, etc. (left side) and R1, R2, R3, etc. (right side), respectively (Figure 1A). 
	CAUTION: Avoid spreading the fetuses forcefully. 1-2 fetuses in each litter may overlap with the others, making their positioning and imaging unreliable. Exclude these fetuses from the analysis.</li>
</ol>
4. Fetal Heart Visualization and Annotation
<ol>
	<li>Apply pre-warmed ultrasound gel on the abdomen and spread it carefully to avoid bubble formation. Add additional amount of gel on the area of scan imaging.</li>
	<li>Place the ultrasound probe on its mechanical holder and mobilize it gradually towards the skin to contact with the thick gel layer while looking for the beating heart using the scanning B-Mode (Figure 1).</li>
	<li>Click the scanning B mode button to obtain 2-D images. Use the bladder as a landmark to identify the first fetus positioned in the right or the left uterine horn and mark it as R1 or L1, respectively.</li>
	<li>Confirm the right and left orientation of the individual fetus in real time by moving the imaging platform in the horizontal plane. Scan from head to tail to annotate the snout, the limbs, and the spine as landmarks (Figure 1B, Video 1).</li>
	<li>Visualize the beating heart and annotate the left ventricle (LV) and the right ventricle (RV). Use color Doppler mode to optimize the heart visualization (Figure 1 C-G, Videos 1-2).</li>
	<li>Click the scanning B mode button to obtain a parasternal short axis-view, have the LV and RV displayed in their maximum diameter at the center of data acquisition frame. Start live imaging (Figure 1B-C).</li>
	<li>Change the orientation of the mouse with respect to scanning planes to obtain a longitudinal four-chamber view (Figure 1D). 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 diameter. Then start image acquisition.</li>
	<li>Exclude the non-optimal, oblique images from the final analysis. Click the Cini button to obtain continuous recording &#39;Cineloops&#39; for a minimum of 10 s, then save the recorded images.</li>
</ol>
5. Evaluating Fetal Heart Rate and Ventricular Function
<ol>
	<li>Click the scanning M-mode button to obtain cardiac images from four chamber planes (Video 3).</li>
	<li>View the list of the recordings for analysis once the images of all the embryos are complete.</li>
	<li>Exclude the non-optimal, oblique images from the final analysis.</li>
	<li>Click the analysis button to measure wall thickness and the left/right ventricular internal diameter at diastole (LVID, d; RVID, d) and systole (LVID, s; RVID, s), as shown in Figure 2.</li>
	<li>Determine the average fetal heart rate by playing each recorded M-mode tracing and calculating the measurement of one flow cycle to the following flow cycle (the spacing between adjacent peaks).</li>
	<li>Perform multiple measurements (at least 5 per tracing) to obtain the average heart rate (Figure 2).</li>
	<li>Measure the temporal changes between left ventricular internal diastolic diameter (LVID, d) and left ventricular internal diameter at end systole (LVID, s) throughout the cardiac cycle. Then calculate fractional shortening percentage (FS%) as follows: FS%=[(LVID,d-LVID,s)/LVID,d]x100.</li>
	<li>Perform multiple measurements (at least 5 per tracing) to obtain the average FS% values.</li>
</ol>
6. Evaluating Cardiopulmonary Flow Parameters
<ol>
	<li>Adjust the sector at an angle of acquisition less than 60o. Click the Doppler Button to perform pulsed wave Doppler measurements from the 2-D four chamber-imaging plane by using a 45-MHz transducer.
	<ol>
		<li>First, visualize the bifurcation of the pulmonary artery to identify the right outflow tract. Next, click pulsed wave Doppler button to obtain the flow pattern through the pulmonary and the aortic valves (Figure 3A, Video 4).</li>
	</ol>
	</li>
	<li>Obtain pulmonary flow measurements from pulsed wave Doppler tracing, including peak systolic velocity (PkV), acceleration time (AT), and ejection time (ET).</li>
	<li>Perform multiple measurements (at least 5 per tracing) to obtain average measurements as shown in Figure 3A (right).</li>
	<li>Calculate the AT/ET ratio for each outflow valve as an indicator of the outflow tracts patency and blood flow.</li>
	<li>Proceed to obtain the mitral and aortic flow patterns from 2-D apical four chamber views using the pulsed wave Doppler. First, identify the left atrial&#160;and left ventricular chambers. Next, place the pulsed wave Doppler sample volume for recording of mitral inflow Doppler patterns and measuring early diastolic velocity ( E) and atrial contraction velocity ( A) (Figure 3B)24,25.</li>
	<li>Adjust Doppler sample volume to obtain the aortic Doppler jet pattern. Use the aortic Doppler jet tracing to measure acceleration time (AT) and ejection time (ET) as shown in Figure 3B (right) (Video 5)</li>
</ol>
7. Evaluating the Feto-Placental Axis
<ol>
	<li>Use the color Doppler scan to visualize the uterine artery and feto-placental vascular tree by using the 45 MHz transducer (Figure 4A).</li>
	<li>Identify the umbilical vessels (two arteries and one vein) in the intra-amniotic segment of the umbilical cord, just after the cord exits from the fetal abdomen.</li>
	<li>Place pulsed wave Doppler sample volume to obtain the umbilical artery flow pattern (Figure 4A).</li>
	<li>Measure vascular peak flow parameters including acceleration time (AT), ejection time (ET), and peak flow velocity at end systole (PkV, s) using the pulsed wave Doppler scanning record (Figure 4B).</li>
	<li>Obtain 5 consecutive waveforms in each vessel, in the absence of fetal movements and&#160;maternal respiratory movements, to measure the average peak velocity for each vessel.</li>
	<li>Proceed to the next embryo.</li>
</ol>
8. Post-Imaging Animal Monitoring
<ol>
	<li>Turn off the isoflurane container following the completion of the imaging process.</li>
	<li>Continue monitoring body temperature, respiratory rate, and heart rate during the recovery phase.</li>
	<li>Remove the facemask and the connected tubing system once the dam starts spontaneous movements.</li>
	<li>Return the dam to the appropriate housing and continue observation according to standard institutional post-procedure protocols.</li>
	<li>Document the time to full resumption of normal activity.</li>
</ol>
9. Performance Requirements and Technical Considerations
<ol>
	<li>Limit the processing time for ~8 fetuses to approximately 1 h to avoid the adverse effects of prolonged anesthesia on vital signs and physiological parameters.</li>
	<li>Complete training with 8-10 pregnant mice to optimize techniques for image acquisition and flow patterns tracing in a short time frame.</li>
</ol>

<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>

No conflict of interest declared.

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><tbody><tr><td>Vevo 2100</td><td>VisualSonics, Toronto, Ontario, Canada</td><td>N/A</td><td>High Freequency Ultrasound Biomicroscopy. The set up is available in animal physiology core facility, division of molecular medicine, UCLA. USA</td></tr><tr><td>inbred mice (c57/BL6)</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 | Developmental Biology | Video

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 ...

fetal-mouse-cardiovascular-imaging-using-high-frequency-ultrasound

Research

Education

JoVE Journal

JoVE Core

Biology

Developmental Biology

Human Biology

Circulatory and Pulmonary Systems

SCIENTISTS IN ACTION

11.5K Views.  University of California, Los Angeles. 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.

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

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  ...

Medicine

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Murine Fetal Echocardiography

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

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

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

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

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 ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...

Ultrafast Doppler Ultrasound for Hepatic Microvasculature Analysis in Mice

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the microvasculature using Doppler ultrasound methods, imaging modalities employing the concept of ultrafast imaging are preferred. By increasing the frame rate of the ultrasound scanner to thousands of frames per second, it becomes possible to improve the sensitivity of the blood flow down to 2 mm/s and to obtain functional information about the microcirculation in comparison to a sensitivity of around 1 cm/s in conventional Doppler modes. While Ultrafast Doppler ultrasound (UFUS) imaging has become adopted in neuroscience, where it can capture brain activity through neurovascular coupling, it presents greater challenges when imaging the vasculature of abdominal organs due to larger motions linked to breathing. The liver, positioned anatomically under the diaphragm, is particularly susceptible to out-of-plane movement and oscillating respiratory motion. These artifacts not only adversely affect Doppler imaging but also complicate the anatomical analysis of vascular structures and the computation of vascular parameters. Here, we present a qualitative and quantitative imaging analysis of the hepatic vasculature in mice by UFUS. We identify major anatomical vascular structures and provide graphical illustrations of the hepatic macroscopical anatomy, comparing it to an in-depth anatomical assessment of the hepatic vasculature based on Doppler readouts. Additionally, we have developed a quantification protocol for robust measurements of hepatic blood volume of the microvasculature over time. To contemplate further research, qualitative vascular analysis provides a comprehensive overview and suggests a standardized terminology for researchers working with mouse models of liver disease. Furthermore, it offers the opportunity to apply ultrasound as a non-invasive complementary method to inspect hepatic vascular defects in vivo and measure functional microvascular alterations deep within the organ before unraveling blood vessel anomalies at the micron scale levels using ex vivo staining on tissue sections.

Ultrafast Doppler ultrasound (UFUS) with high spatial resolution (100 &#956;m) and sensitivity represents a suitable noninvasive imaging modality for obtaining a comprehensive qualitative overview of the hepatic vasculature. UFUS also facilitates quantitative measurements of the microvasculature, aiming to enhance our understanding of vascular disease mechanisms.

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the ...

High-frequency Ultrasound Imaging of the Abdominal Aorta

Source: Amelia R. Adelsperger, Evan H. Phillips, and <a href="https://www.jove.com/author/Craig+J._Goergen">Craig J. Goergen</a>, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
High-frequency ultrasound systems are used to acquire high resolution images. Here, the use of a state-of-the-art system will be demonstrated to image the morphology and hemodynamics of small pulsatile arteries and veins found in mice and rats. Ultrasound is a relatively inexpensive, portable, and versatile method for the noninvasive assessment of vessels in humans as well as large and small animals. These are several key advantages that ultraound offers compared to other techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), and near-infrared fluorescence tomography (NIRF). CT requires ionizing radiation and MRI can be prohibitively expensive and even impractical in some scenarios. NIRF, on the other hand, is limited by the penetration depth of light required to excite the fluorescent contrast agents.
Ultrasound has limitations in terms of imaging depth; however, this may be overcome by sacrificing resolution and using a lower frequency transducer. Abdominal gas and excess body weight can severely diminish image quality. In the first case, the propagation of sound waves is limited, while in the latter case, they are attenuated by overlying tissues, such as fat and connective tissue. As a result, no contrast or faint contrast may be observed. Finally, ultrasound is a highly user-dependent technique, requiring the sonographer to be familiar with anatomy and to be able to work around issues, such as the appearance of imaging artifacts or acoustic interference.

High-frequency Ultrasound Imaging and Image Acquisition

Source: Amelia R. Adelsperger, Evan H. Phillips, and Craig J. Goergen, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, 
...

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

Summary

Explore More Videos

Chapters in this video

Murine Echocardiography and Ultrasound Imaging

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Murine Fetal Echocardiography

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

High-frequency Ultrasound Imaging of the Abdominal Aorta