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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	<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.
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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 ...

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.

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