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 vaginal plug and treat the females immediately after plug confirmation. 
	NOTE: A plug is the sperm of the male in the vaginal orifice of the female.
	<ol>
		<li>Inject 250 &#181;L of PBS intraperitoneally in control Cpa3+/+ females.</li>
		<li>Inject 250 &#181;L of anti-CD122 (0.25 mg) intraperitoneally in MC-deficient Cpa3Cre/+ females. 
		Note: An injection of 0.25 mg of anti-CD122 depletes peripheral NKs and uNKs in MC-deficient Cpa3Cre/+ females as described previously22.</li>
	</ol>
	</li>
	<li>Wait until gd5. 
	NOTE: At gd5, there is the earliest possibility for implantation analysis.
	<ol>
		<li>Proceed with steps 2-5 for the ultrasound analysis.</li>
	</ol>
	</li>
	<li>Perform the ultrasound imaging at gd5, 8, 10, 12, and 14.</li>
</ol>
2. Preparation of the Ultrasound System
<ol>
	<li>Turn on the system (Figure 1A; main power on the back and computer standby at the left site), the heated platform (Figure 1B; at the control pad), and the gel warmer (Figure 1C). 
	NOTE: Ultrasound gel needs to warm up for approximately 0.5 h.</li>
	<li>Ensure that the isoflurane unit is filling sufficiently (Figure 1D).</li>
	<li>Open a New Study or New Series in an existing study in the browser. Fill in all the required information (owner, study name, series name, animal data) in the Study Info window. Click Ok.</li>
	<li>After clicking Ok, ensure that the B-mode imaging window appears and the imaging in B-mode begins automatically.</li>
</ol>
3. Mouse Handling
<ol>
	<li>Anesthetization of the mouse
	<ol>
		<li>Place the mouse in the knockdown box (Figure 1E), close the box, open the isoflurane tube to the knockdown box, and turn on the isoflurane (concentration 3.5%).</li>
		<li>When the mouse is anesthetized, lower (to concentration 1.5%) and redirect the isoflurane flow by opening the tube in the direction of the heating platform and close the flow to the knockdown box. 
		NOTE: To reach sufficient anesthesia, wait an additional 10 s after the mouse is no longer moving.</li>
		<li>Transfer the mouse quickly from the knockout box to the heating platform (Figure 1F) in a dorsal position, and gently position its nose in the anesthesia nose tube located on the top of the platform.</li>
	</ol>
	</li>
	<li>Fixation, depilation, and preparation of the mouse for measuring
	<ol>
		<li>Place eye protection cream in each eye of the mouse to prevent dry eyes.</li>
		<li>Place one drop of electrode gel on each of the four copper areas on the heated platform (Figure 1F).</li>
		<li>Tap the paws with surgical tape on the electrode gel-coated areas of the heating platform.</li>
		<li>Check ECG [optimal value = 450-550 beats/min (BPM)] and respiratory physiology at all times. 
		NOTE: By using a rectal probe, body temperature measurement is possible, but not necessary.</li>
		<li>Place depilatory cream at the abdomen of the mouse, rub the cream with a cotton swab and wait around 1 min. Remove the cream with a water-soaked compress. Repeat this step if not all hairs are gone.</li>
		<li>Apply the pre-warmed ultrasound gel on the depilated skin.</li>
	</ol>
	</li>
</ol>
4. Measurements and Acquisition of Images and Videos
<ol>
	<li>Hold the transducer (Figure 1G) in the hand or clamp it in the holding device (Figure 1H; holding device is recommended).</li>
	<li>Identify the bladder with the transducer and use it as reference point. Move the transducer to the left and right sites of the abdomen to trace implantations.</li>
	<li>B-mode for visualization of anatomical structures in 2D grayscale image
	<ol>
		<li>Move the transducer or heating platform table where the mouse is fixated until the first implantation is visible on the screen at its largest size.
		<ol>
			<li>Select Image Label and enter a name, or Frame Store (storage without name) to store single frames, or Cine Store to store a cineloop for whole implantation measurements.</li>
		</ol>
		</li>
		<li>Move the transducer or table to bring the placenta to a position where blood flow in the UmA is visible. Store a single frame or cineloop (see step 4.3.1.1) for placental measurements. 
		NOTE: Placental measurements are possible from gd10 onwards.</li>
		<li>Continue with all implantations using the same method.</li>
	</ol>
	</li>
	<li>Color doppler mode to visualize and determine the direction of blood flow
	<ol>
		<li>Press the Color button.</li>
		<li>Move the Color Box (in this area, the signal is visible) to the required position by using the trackball. If necessary, change the size of the box by pressing Update and move the trackball (to the right side/upward = bigger; to the left side/downward = smaller). When the box has the right size, press Select.</li>
		<li>Store single frames or cineloops as described in step 4.3.1.1.</li>
	</ol>
	</li>
	<li>Pulse-wave (PW) doppler mode to quantify blood flow through the vessels in the Arteria uterina (uterine artery, UA) and UmA
	<ol>
		<li>Locate the region of interest in the color doppler acquisition. 
		NOTE: The UA is located caudal to the bladder, and the UmA is located between the fetus and placenta.</li>
		<li>Press PW, and a dashed line will appear. Move this line to the blood vessel of interest and adjust the angle of the line using the &#8220;Doppler Angle&#8221; knob in line with the blood flow. Press Update. 
		NOTE: The angle between the direction of the blood flow and the transducer must be consistent in all animals, especially when using angles of greater than 60&#176; (here, 70&#176; for UAs and 45&#176; for UmAs were used).</li>
		<li>Store a cineloop of the appearing doppler lines in the PW doppler acquisition window.</li>
	</ol>
	</li>
</ol>
5. Reviewing and Finishing Data Acquisition and Saving a Series
<ol>
	<li>To review data, press Study Management. Scroll to the thumbnail image of interest and double-click Update.</li>
	<li>Press first Study Management then Close in the browser window to finish data acquisition and save a recorded series. 
	NOTE: After closing a series, it is not possible to store frames or cineloops in this series anymore.</li>
</ol>
6. Mouse Handling Following Acquisition of Data
<ol>
	<li>Remove the gel from the anaesthetized animal with the help of dry compresses.</li>
	<li>Remove the surgical tape carefully from the paws.</li>
	<li>Close the isoflurane tube (concentration 0%).</li>
	<li>Proceed with following ultrasound analysis at gd5, 8, 10, and 12.
	<ol>
		<li>Place the animal alone in a cage for a minimum of 5 min so it has time to wake up and orientate.</li>
		<li>Place the mouse back in the original cage. 
		NOTE: Do not turn off the isoflurane before removing the gel and surgical tape, as mice wake up very quickly (around 20 s) after turning off the isoflurane.</li>
	</ol>
	</li>
	<li>Proceed with the following ultrasound analysis at gd14.
	<ol>
		<li>Sacrifice the female before it wakes up by cervical dislocation. Open the animal, remove the uterus, separate the fetuses and placentas, and measure fetal and placental weights.</li>
	</ol>
	</li>
</ol>
7. Copying and Importing the Data
<ol>
	<li>Mark one or more series by clicking Export To and choose the storage space to copy data onto a hard disk.</li>
	<li>Open the software at a computer and click Copy From and select the study/series from the hard disk to import a study/series into the software.</li>
	<li>Analyze the data with the software.</li>
</ol>

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The authors have nothing to disclose.

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><tbody><tr><td>LEAF anti-Maus CD122 (IL-2Rb)</td><td>BioLegend</td><td>123204</td><td>Klon TM-&amp;beta;1; 500 &amp;micro;g</td></tr><tr><td>Vevo 2100 System</td><td>&amp;nbsp;FujiFilm VisualSonics Inc.</td><td></td><td>Transducer&amp;nbsp;MS550D-0421</td></tr><tr><td>Vevo LAB Software</td><td>&amp;nbsp;FujiFilm VisualSonics Inc.</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>PZN: 6497131</td><td></td></tr><tr><td>Electrode gel</td><td>Parker</td><td>12_8</td><td></td></tr><tr><td>Surgical tape</td><td>3M Transpore</td><td>1527-1</td><td></td></tr><tr><td>Eye cream</td><td>Bayer</td><td>PZN: 1578675</td><td></td></tr><tr><td>Cotton tipped applicators</td><td>Raucotupf</td><td>11969</td><td>100 pieces</td></tr><tr><td>Depilatory cream</td><td>Reckitt Benckiser</td><td>2077626</td><td></td></tr><tr><td>Compresses</td><td>Nobamed Paul Danz AG</td><td>856110</td><td>10 x 10 cm</td></tr><tr><td>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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

10.1K Views.  Otto-von-Guericke University. 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.

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

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 ...

Medicine

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 ...

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 ...

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 ...

State of the Art Cranial Ultrasound Imaging in Neonates

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a patient is unstable. In addition it is radiation-free and allows serial imaging. CUS possibilities have steadily expanded. However, in many neonatal intensive care units, these possibilities are not optimally used. We present a comprehensive approach for neonatal CUS, focusing on optimal settings, different probes, multiple acoustic windows and Doppler techniques. This approach is suited for both routine clinical practice and research purposes. In a live demonstration, we show how this technique is performed in the neonatal intensive care unit. Using optimal settings and probes allows for better imaging quality and improves the diagnostic value of CUS in experienced hands. Traditionally, images are obtained through the anterior fontanel. Use of supplemental acoustic windows (lambdoid, mastoid, and lateral fontanels) improves detection of brain injury. Adding Doppler studies allows screening of patency of large intracranial arteries and veins. Flow velocities and indices can be obtained. Doppler CUS offers the possibility of detecting cerebral sinovenous thrombosis at an early stage, creating a window for therapeutic intervention prior to thrombosis-induced tissue damage. Equipment, data storage and safety aspects are also addressed.

Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique.

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Bioengineering

High-frequency Ultrasound Imaging of the Abdominal Aorta

Source: Amelia R. Adelsperger, Evan H. Phillips, and <a href="https://www-jove-com.bdigitaluss.remotexs.co/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, 
...

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

Summary

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Chapters in this video

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

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

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

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

State of the Art Cranial Ultrasound Imaging in Neonates

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

High-frequency Ultrasound Imaging of the Abdominal Aorta