Name: high frequency ultrasound for the analysis of fetal and placental development in vivo
Uploaded: 2018-11-08T14:30:00
Description: Watch this Scientific Journal Video about High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo at JoVE.com

Many thanks to the Imaging Instrument company (especially to Magdalena Steiner, Katrin Suppelt, and Sandra Meyer) for their pleasant and fast support and for answering all our questions concerning the Imaging System and its usage promptly and completely. We are grateful to Prof. Hans-Reimer Rodewald and Dr. Thorsten Feyerabend (DKFZ Heidelberg, Germany) for providing the Cpa3 colony. Additionally, we thank Stefanie Langwisch, who was in charge of the mouse colonies and who generated the pictures in Figure 1.
The work and the Imaging System were funded by grants from the Deutsche Forschungsgemeinschaft (DFG) to A.C.Z. (ZE526/6-1 and AZ526/6-2) that were projects embedded in the DFG priority program 1394 &#34;Mast cells in health and disease.&#34;

All methods described here have been approved by the &#8220;Landesverwaltungsamt Sachsen Anhalt: 42502-2-1296UniMD.&#8221;
1. Experimental Procedure
<ol>
	<li>Mate 6 to 8-week-old female MC-deficient C57BL/6J-Cpa3Cre/+ (Cpa3Cre/+) mice and MC-sufficient C57BL/6J-Cpa3+/+ (colony controls; Cpa3+/+) with BALB/c males.</li>
	<li>Define the gestation day (gd) 0 after confirmation of the v...

<ol>
	<li>Abramowicz, J. S., Kremkau, F. W., Merz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraschall+in+der+Geburtshilfe:+Kann+der+F&#246;tus+die+Ultraschallwelle+h&#246;ren+und+die+Hitze+sp&#252;ren?.">Ultraschall in der Geburtshilfe: Kann der F&#246;tus die Ultraschallwelle h&#246;ren und die Hitze sp&#252;ren?.</a> Ultraschall in der Medizin. 33 (3), 215-217 (1980).</li><li>Jones, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echolocation.">Echolocation.</a> Current Biology. 15 (13), R484-R488 (2005).</li><li>Simmons, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sonar+receiver+of+the+bat.">The sonar receiver of the bat.</a> Annals of the New York Academy of Sciences. 188, 161-174 (1971).</li><li>Zala, S. M., Reitschmidt, D., Noll, A., Balazs, P., Penn, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-dependent+modulation+of+ultrasonic+vocalizations+in+house+mice+(Mus+musculus+musculus).">Sex-dependent modulation of ultrasonic vocalizations in house mice (Mus musculus musculus).</a> Public Library of Science ONE. 12 (12), e0188647 (2017).</li><li>W&#246;hr, M., Seffer, D., Schwarting, R. K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+Socio-Affective+Communication+in+Rats+through+Playback+of+Ultrasonic+Vocalizations.">Studying Socio-Affective Communication in Rats through Playback of Ultrasonic Vocalizations.</a> Current Protocols in Neuroscience. 75, 1-8 (2016).</li><li>Hasiniaina, A. F., 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=High+frequency/ultrasonic+communication+in+a+critically+endangered+nocturnal+primate,+Claire's+mouse+lemur+(Microcebus+mamiratra).">High frequency/ultrasonic communication in a critically endangered nocturnal primate, Claire's mouse lemur (Microcebus mamiratra).</a> American Journal of Primatology. , e22866 (2018).</li><li>Yeo, L., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+and+power+Doppler+combined+with+Fetal+Intelligent+Navigation+Echocardiography+(FINE)+to+evaluate+the+fetal+heart.">Color and power Doppler combined with Fetal Intelligent Navigation Echocardiography (FINE) to evaluate the fetal heart.</a> Ultrasound in Obstetrics &amp; Gynecology. 50 (4), 476-491 (2017).</li><li>Teichholz, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiography+in+valvular+heart+disease.">Echocardiography in valvular heart disease.</a> Progress in Cardiovascular Diseases. 17 (4), 283-302 (1975).</li><li>Zechner, P. 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=Lungensonographie+in+der+Akut-+und+Intensivmedizin.">Lungensonographie in der Akut- und Intensivmedizin.</a> Der Anaesthesist. 61 (7), 608-617 (2012).</li><li>Blank, W., Schuler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonografie+der+Schilddr&#252;se+-+Update+2017.">Sonografie der Schilddr&#252;se - Update 2017.</a> Praxis. 106 (12), 631-640 (2017).</li><li>Hansen, K. L., Nielsen, M. B., Ewertsen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonography+of+the+Kidney:+A+Pictorial+Review.">Ultrasonography of the Kidney: A Pictorial Review.</a> Diagnostics. 6 (1), (2015).</li><li>Older, R. A., Watson, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+anatomy+of+the+normal+male+reproductive+tract.">Ultrasound anatomy of the normal male reproductive tract.</a> Journal of Clinical Ultrasound. 24 (8), 389-404 (1996).</li><li>Reeves, J. J., Rantanen, N. W., Hauser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transrectal+real-time+ultrasound+scanning+of+the+cow+reproductive+tract.">Transrectal real-time ultrasound scanning of the cow reproductive tract.</a> Theriogenology. 21 (3), 485-494 (1984).</li><li>Sharma, M., Somani, P., Sunkara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+gall+bladder+by+endoscopic+ultrasound.">Imaging of gall bladder by endoscopic ultrasound.</a> World Journal of Gastrointestinal Endoscopy. 10 (1), 10-15 (2018).</li><li>Weskott, H. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraschall+in+der+Diagnostik+maligner+Lymphome.">Ultraschall in der Diagnostik maligner Lymphome.</a> Der Radiologe. 52 (4), 347-359 (2012).</li><li>Shirinifard, A., Thiagarajan, S., Johnson, M. D., Calabrese, C., Sablauer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Absolute+Blood+Perfusion+in+Mice+Using+Dynamic+Contrast-Enhanced+Ultrasound.">Measuring Absolute Blood Perfusion in Mice Using Dynamic Contrast-Enhanced Ultrasound.</a> Ultrasound in Medicine &amp; Biology. 43 (8), 1628-1638 (2017).</li><li>Quaia, E. <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+tissue+perfusion+by+contrast-enhanced+ultrasound.">Assessment of tissue perfusion by contrast-enhanced ultrasound.</a> European Radiology. 21 (3), 604-615 (2011).</li><li>Saw, S. N., Poh, Y. W., Chia, D., Biswas, A., Zaini Mattar, C. N., Yap, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+hemodynamic+wall+shear+stresses+in+human+umbilical+vessels+from+normal+and+intrauterine+growth+restricted+pregnancies.">Characterization of the hemodynamic wall shear stresses in human umbilical vessels from normal and intrauterine growth restricted pregnancies.</a> Biomechanics and Modeling in Mechanobiology. , (2018).</li><li>Kessler, J., Rasmussen, S., Godfrey, K., Hanson, M., Kiserud, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+growth+restriction+is+associated+with+prioritization+of+umbilical+blood+flow+to+the+left+hepatic+lobe+at+the+expense+of+the+right+lobe.">Fetal growth restriction is associated with prioritization of umbilical blood flow to the left hepatic lobe at the expense of the right lobe.</a> Pediatric Research. 66 (1), 113-117 (2009).</li><li>Laurin, J., Lingman, G., Mars&#225;l, K., Persson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+blood+flow+in+pregnancies+complicated+by+intrauterine+growth+retardation.">Fetal blood flow in pregnancies complicated by intrauterine growth retardation.</a> Obstetrics and Gynecology. 69 (6), 895-902 (1987).</li><li>Arduini, D., Rizzo, G., Romanini, C., Mancuso, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+blood+flow+velocity+waveforms+as+predictors+of+growth+retardation.">Fetal blood flow velocity waveforms as predictors of growth retardation.</a> Obstetrics and Gynecology. 70 (1), 7-10 (1987).</li><li>Meyer, 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=Chymase-producing+cells+of+the+innate+immune+system+are+required+for+decidual+vascular+remodeling+and+fetal+growth.">Chymase-producing cells of the innate immune system are required for decidual vascular remodeling and fetal growth.</a> Scientific Reports. 7, 45106 (2017).</li><li>Meyer, N., Sch&#252;ler, T., Zenclussen, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Ablation+of+Uterine+Natural+Killer+Cells+and+Uterine+Mast+Cells+in+Mice+Leads+to+Poor+Vascularization+and+Abnormal+Doppler+Measurements+That+Compromise+Fetal+Well-being.">Simultaneous Ablation of Uterine Natural Killer Cells and Uterine Mast Cells in Mice Leads to Poor Vascularization and Abnormal Doppler Measurements That Compromise Fetal Well-being.</a> Frontiers in Immunology. 8, 1913 (2017).</li><li>Evans, D. H., Jensen, J. A., Nielsen, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonic+color+Doppler+imaging.">Ultrasonic color Doppler imaging.</a> Interface Focus. 1 (4), 490-502 (2011).</li></ol>

Using our ultrasound system, we demonstrated fetal growth restriction in MC/NK-deficient mothers from gd10 on. Furthermore, at gd10 and 12, we observed reduced placental dimensions, and at gd14 the absence or reversion of end diastolic flow in the UmAs of some fetuses of uMC/uNK-deficient mice. This sign of poor vascularization was associated with a significant resistance index of the arteries indicating IUGR. Results confirm the important role of uMCs and uNKs in pregnancy and fetal well-being and in understanding the c...

Ultrasound is sound waves with frequencies above the audible range of the human ear, higher than about 20 kHz1. Animals like bats, wales, dolphins2,3, mice4, rats5, and mouse lemurs6 all use ultrasound for orientation or communication. Humans take advantage of ultrasound for several technical and medical applications. An ultrasound device is able to create the sound wave and distribute and represent the signal. If ultrasound encounters an obstacle, the sound is reflected, absorbed or can go through it. The application of ultrasound as an imaging method, called sonography, is used for the analysis of organic tissues in human or veterinary medicine like the heart (echocardiography)7,8, lung9, thyroid gland10, kidneys11, and urinary and reproductive tracts12,13; detecting gallstones14 and tumors15; and evaluating perfusion of blood vessels or organs16,17. Ultrasound is a standard method in prenatal care during pregnancy, and fetal developmental disabilities or impairments can be recognized early. Specifically, the growth of a fetus is closely monitored at regular intervals to recognize a possible IUGR. Finally, fetal blood flow can be monitored, as this can point out growth restrictions18,19,20,21.
A major advantage of ultrasound imaging compared to other methods like radiography is the sound&#39;s harmlessness of the tissues to be analyzed. This easy and fast method is non-invasive, painless, and can be used a number of times. The initial outlay of an ultrasound device is expensive; however, the consumable materials needed are cheap. The ultrasound system used in this manuscript is suitable for a range of animal models (i.e., mice and fish) While for humans an ultrasound device requires a frequency of 3-15 mHz, a frequency of 15-70 mHz is required for mice.
The present manuscript describes a protocol for the use of B-mode, color doppler mode, and pulse-wave doppler mode. The description includes preparation of the mice as well as performance, data acquisition, and storage. This method has been successfully applied to different mouse strains at all gestational days and can be used to investigate fetal and placental development as well as maternal and fetal blood parameters. Here, all applications are explained based on our studies employing pregnant MC/NK-deficient and control mice.

<table border="0" cellpadding="0" cellspacing="0" width="758">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LEAF anti-Maus CD122 (IL-2Rb)</td>
			<td style="width:204px;">BioLegend</td>
			<td style="width:151px;">123204</td>
			<td style="width:187px;">Klon TM-&#946;1; 500 &#181;g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vevo 2100 System</td>
			<td>&#160;FujiFilm VisualSonics Inc.</td>
			<td style="width:151px;"></td>
			<td>Transducer&#160;MS550D-0421</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vevo LAB Software</td>
			<td>&#160;FujiFilm VisualSonics Inc.</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Baxter</td>
			<td>PZN: 6497131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrode gel</td>
			<td>Parker</td>
			<td>12_8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical tape</td>
			<td>3M Transpore</td>
			<td>1527-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye cream</td>
			<td>Bayer</td>
			<td>PZN: 1578675</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton tipped applicators</td>
			<td>Raucotupf</td>
			<td>11969</td>
			<td>100 pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Depilatory cream</td>
			<td>Reckitt Benckiser</td>
			<td>2077626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Compresses</td>
			<td>Nobamed Paul Danz AG</td>
			<td>856110</td>
			<td>10 x 10 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasound gel</td>
			<td>Gello GmbH</td>
			<td>246000</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Individual components of the ultrasound system used in this manuscript are shown in Figure 1. Figure 2 shows representative ultrasound images acquired in B-mode at gd5, 8, 10, and 12 (B) and corresponding implantation area measurement results (A), demonstrating a significant reduced implantation area of anti-CD122-treated Cpa3Cre/+ mice from gd10 onwards.
<p class="jove_content" fo...

Watch this Scientific Journal Video about High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo at JoVE.com

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.

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 ...

This method is very important to answer key questions in the areas of Oncology, Neuro-Biology, Cardiology and Fetal Development, and of course also it's important in the area of reproductive research. The main advantages of this technique are it is non-invasive, painless, easy, fast and allows the follow-up of fetuses at relevant gestational time points throughout pregnancy. At least a half hour before the imaging procedure, turn on the ultrasound system, the heated platform, and the gel warmer.Open a new study or new series in an existing study in the browser. Enter all the required information in the study info window. Click OK, and confirm that the B-mode imaging window appears.When the ultrasound is ready, confirm a lack of response to toe pinch in an anesthetized mouse, and apply ointment to the animal's eyes. Place one drop of electro gel onto each of the 4 copper sections of the heated platform, and secure the paws to the electro gel coated sections of the platform. Remove the hair from the abdomen of the animal with depilatory cream and a cotton swab, washing the cream from the depilated skin after one minute with a water-soaked compress.Then apply pre-warmed ultrasound gel onto the exposed area. One reason for low signal intensity can be the low amount of gel placed between the mouse and the ultrasound beam. In our experience, a rather large amount of gel is necessary for a good signal quality.When the animal is ready, place the transducer over the bladder for use as a reference point, and move the heating platform table to the left and right sides of the abdomen to trace the fetal implantations in 2D B-mode, adjusting the transducer or platform as necessary until the first implantation is visible on the screen, at its largest size. Select Image Label, and enter a name for the image file. Adjust the position of the image, until the blood flow within the arteria umbilicalis of the placenta is in view, and store a single frame or cineloop for placental measurements.For color Doppler imaging to determine the direction of the blood flow, press the Color button, and use the trackball to move the color box to the appropriate position, clicking Update to adjust the size of the box as necessary. When the box is the correct size, click Select and store the image as experimentally appropriate. For pulsed wave Doppler quantification of the blood flow through the vessels, locate the region of interest in the color Doppler acquisition mode and click Pulse Wave.A dashed line will appear. Move the line to the blood vessel of interest and use the Doppler angle knob to adjust the angle of the line to align with the blood flow. For pulsed wave Doppler measurements, it is important to watch the angle between the direction of the blood flow and the ultrasound beam.Too high angles, or different angles between animals in the same experiment may lead to inaccurate velocity measurements. Then, click Update and store cineloop of the appearing Doppler lines in the pulsed wave Doppler acquisition window. To review the data, click Study Management, scroll to the thumbnail image of interest, and double click Update, then click Study Management again, and click Close to close the browser window, to finish the data acquisition and save a recorded series.To export the data, connect a hard disk, click Export To, to mark one or more series of data, and select the storage space for copying the data onto the hard disk. Here, representative ultrasound images acquired in the B-mode at gestational days 5, 8, 10 and 12, and their corresponding implantation area measurement results are shown, revealing a significant reduction in the implantation area in anti-CD122 treated mast cell-deficient mice from gestational day 10 onwards. B-mode imaging facilitates the imaging and measurement of individual parts of an implantation.For example, anti-CD122 treated mast cell-deficient animals demonstrate a significant reduction in the placental area, thickness and diameter compared to untreated mice at gestational days 10 and 12. In contrast, the placental area and diameter are similar between the groups at gestational day 14, and the placental thickness, a significant increase in the anti-CD122 treated transgenic animals compared to untreated mice at gestational day 14. Pulsed wave Doppler imaging of the arteria uterina for measurement of the peak systolic velocity and diastolic velocity, and calculated resistance index, reveal similar values between the treated and untreated animals.The resistance index of the arteria umbilicalis in anti-CD122 treated mast cell deficient animals, however, is significantly increased compared to untreated mice. While attempting this procedure, it is important to keep in mind, that you should do this every other day rather than every day, to avoid the stress, and the risk of repetitive narcotization of the animals. Following this procedure, other measurements, like 3D measurement, visualization and quantification of tissue movement over time, blood pressure measurements, and also ultrasound guided injections can be performed.After it's development, this method paved the way for physicians and researchers in the area of Obstetric and pre-natal care, because it's possible to follow up fetal development and to recognize possible abnormalities during pregnancy.

high-frequency-ultrasound-for-analysis-fetal-placental-development

Research

JoVE Journal

Developmental Biology

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 ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

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.

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.

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

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its ...

Transuterine Fetal Tracheal Occlusion Model in Mice

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Various animal models of congenital diaphragmatic hernia and fetal tracheal occlusion present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and availability of genetic tools. This model provides a new tool to study the impact of both tracheal occlusion and increased luminal pressure on lung development.

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). ...

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 ...