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>
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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.

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		<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>
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			<td height="21" style="height:21px;">Electrode gel</td>
			<td>Parker</td>
			<td>12_8</td>
			<td></td>
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		<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>
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		<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>
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			<td height="21" style="height:21px;">Compresses</td>
			<td>Nobamed Paul Danz AG</td>
			<td>856110</td>
			<td>10 x 10 cm</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ultrasound gel</td>
			<td>Gello GmbH</td>
			<td>246000</td>
			<td></td>
		</tr>
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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.
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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 ...

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