Name: a novel use of three-dimensional high-frequency ultrasonography for early pregnancy characterization in the mouse
Uploaded: 2017-10-24T13:00:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse at JoVE.com

We very much appreciate the assistance of Rong Zhao, Jie Li, and Yan Ying.

These studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and animal protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine under protocol number AN-4203.
1. Preparation of the Pregnant Mouse for Ultrasound
<ol>
	<li>Timed mating
	<ol>
		<li>Place the dam with a proven fertile male mouse overnight beginning after 1700. Separate dam and male by 0700 hours (h), regardless if vaginal plug is present, to ensure accurate dating of pregnancy. 
		NOTE: The morning after mating is considered 0.5 day post coitum (d.p.c.).</li>
	</ol>
	</li>
	<li>Preparation for ultrasound
	<ol>
		<li>Place the pregnant mouse in a sealed anesthesia induction container.</li>
		<li>Sedate with isoflurane (2%) inhaled anesthetic and oxygen (1&#160;L/min) until the animal loses the righting reflex (approximately 1-2 min depending on the size of the mouse) and spontaneous movements are not observed (besides respiration).</li>
		<li>Place sedated mouse in supine position in nose cone with isoflurane (2%) inhaled anesthetic and oxygen (1&#160;L/min). Apply ophthalmic ointment to the animal&#39;s eyes.</li>
		<li>With a cotton swab, apply depilatory cream to entire abdomen, sternum to vagina, and lateral to the flanks.</li>
		<li>Allow cream to remain for a maximum of 3 min, and then remove cream and hair with tissue. Ensure that all hair is removed, as any remaining hair will decrease image quality.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Ultrasound Stage
<ol>
	<li>Turn on the transducer.</li>
	<li>Attach 3-D motor stage motor stage to the transducer.
	<ol>
		<li>Plug into 3D motor by connecting the 3D motor cable to the 3D motor connector on the rear panel. Attach the 3D motor system to the mounting system using the quick release post on the top to connect to the imaging station, and the quick release mount on the bottom to affix the transducer clamp.</li>
	</ol>
	</li>
	<li>Attach the probe to the transducer clamp.</li>
</ol>
3. Begin Imaging the Pregnant Mouse
<ol>
	<li>Place mouse supine on the monitoring platform. The mouse continuously receives isoflurane anesthetic (between 1.5-2.5%) and oxygen (1&#160;L/min) via the nose cone. Gently tape all paws to heart rate monitoring pads on the platform.
	<ol>
		<li>Apply 1-2 mL of ultrasound transmission gel on abdomen.</li>
	</ol>
	</li>
	<li>Using the manual motor stage, position the ultrasound probe on the lower abdomen.
	<ol>
		<li>Locate the bladder, which should appear as a fluid-filled dark circle just cephalad to the vaginal opening.</li>
		<li>Once the bladder is located, move the probe very slowly cephalad to visualize the pregnant uterus, which should appear as a cylindrical shape with round areas at pregnancy sites. This can be described as looking like pearls on a chain.</li>
		<li>Once the pregnant uterus has been identified, begin 2-D imaging.</li>
	</ol>
	</li>
</ol>
4. The 2-D Ultrasound Imaging (Figure 1)
<ol>
	<li>Once the pregnant uterus has been identified, begin at the pregnancy site closest to the bladder and slowly and sequentially move cephalad to determine number and location of pregnancy sites.</li>
	<li>If the kidney, spleen, or liver are visualized, reposition the probe closer caudal (closer to the bladder) as the user has gone too far cephalad.</li>
	<li>Image the contralateral uterine horn in the same manner.</li>
	<li>Freeze the image when the ultrasound frame is in the center of the implantation/pregnancy site to save for later analysis and measurements. 
	NOTE: It takes less than one second for the image to be frozen and saved for later analysis.</li>
	<li>Measure implantation distance using the hyperechoic decidualization reaction as a marker by manually clicking on the &#34;measure&#34; tool first, and then clicking on the location of one implantation site. Next, drag the cursor to next implantation site and click to draw a line that the computer will automatically report its distance. The software will then report what the manual measurement is. This step is not computerized, but relies on the user to mark the distance between decidualized sites by drawing a line between the decidualized sites, which the program will provide a measurement for.</li>
	<li>Measure implantation size, gestational sac size, and fetal pole size.</li>
</ol>
5. Fetal Heart Rate
NOTE: At 9.5 d.p.c., the fetal heart beat should be clearly visualized.
<ol>
	<li>While keeping the probe very still, turn on the pulse wave Doppler and position this over the visible heartbeat.</li>
	<li>Record the cardiac heart beat pulsations. 
	NOTE: Apart from the heart, this procedure can also record pulsations in the umbilical cord.</li>
</ol>
6. 3-D Ultrasound Acquisition
<ol>
	<li>After the pregnant uterus has been visualized on 2-D imaging, position the probe in an area that is in the approximate mid-point of the desired 3-D image. For example, if one were imaging an apple in 3-D, the probe should initially be placed where the apple core is predicted to be located (i.e. middle of object).</li>
	<li>With the probe in a position in the middle of the desired image, obtain a 3-D acquisition. The 3-D motor stage will travel this distance across the uterus in a series of steps or frames, with the intention of capturing in entirety what the investigator desires.</li>
	<li>Confirm that the intended structures are completely captured in the 3-D scanning before completing the real-time ultrasound portion. Save this 3-D information for post-processing at a later time. The total time for 2-D and 3-D ultrasound acquisition is approximately 10-20 minutes when performed by an experienced user.</li>
</ol>
7. 3-D Reconstitution Post-processing (Figure 2)
<ol>
	<li>Load the desired data for 3-D image processing.</li>
	<li>Choose &#34;parallel and rotational methods&#34;, which will load all the 3D image frames into one 3-D &#34;box&#34; that the user will then trace the desired object in, frame-by-frame. Choose step size 0.08 mL. Begin at one end of the image block and scroll through to familiarize with the images that have been captured within space.
	<ol>
		<li>Start at one end of the image and manually trace the outline of the object.</li>
		<li>Scroll to next 2-D image slice or &#34;frame&#34; and manually trace the outline of the object.</li>
		<li>Continue this process until all frames have been manually annotated/traced by the user.</li>
		<li>Click &#34;finish&#34; to obtain the 3-D image and total volume calculations.</li>
	</ol>
	</li>
	<li>Choose between mesh and solid overlay, which is an arrow in the top left corner.</li>
	<li>Choose to keep or remove the surrounding ultrasound image information for clarity. Each 3-D reconstruction may take between 10 and 20 min.</li>
</ol>
8. Post-procedural Care
<ol>
	<li>When the ultrasound is complete, turn off the anesthesia gas, remove the mouse from the platform and gently wipe and wash away any ultrasound gel from the animal.</li>
	<li>Place the mouse back in the cage in a prone position in a padded area. Monitor until the animal is awake and moving spontaneously.</li>
</ol>

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The authors have nothing to disclose.

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. However it should be noted that histological methods would still be considered more optimal when characterization at a more magnified and more cellular level is desired, or when gene and protein analysis is required. With a growing number of new mouse models displaying implantation defects and early pregnancy loss19,20,21,22, the ability of this advanced ultrasound technique to detect early pregnancies from 5.5 d.p.c onwards provides clear advantages over earlier ultrasound methods that were limited to detection at mid-pregnancy12,13. Moreover, this method detects cardiac motion as soon as the heart starts to beat, between 8.5-9.5 d.p.c23,24 allowing for confirmation of viable fetuses. The ability to follow early pregnancy events in the same animal means fewer animals are required per experiment and more accurate analysis can be performed with such longitudinal experimental designs25,26,27.
It should be noted that the three most critical steps in the protocol are: (1) knowing the exact gestational day; (2) the ability of the operator to locate the uterus and position the probe over the correct anatomical site; and (3) accurate techniques to produce a 3-D reconstruction. The following trouble shooting guidelines have proven helpful: When it is difficult to locate the uterus on ultrasound, the user can start by ensuring that the bladder is identified first. The bladder is usually seen as a black circle, and is immediately cephalad to the vaginal opening. Even in mice that have undergone micturition immediately prior to the ultrasound, the bladder is never completely empty and thus (in our experience) can be a reliable marker. Once the bladder is identified, the probe should be slowly moved cephalad until the uterus is seen. If the bladder is small or unable to be visualized, the user can also attempt to visualize the uterus by starting at the ovary, which can be found at the flanks, inferior to the kidney. Using this systematical approach, each implantation site and fetal development can be observed, as in Figure 1. On occasion, it may be difficult to obtain the desired region of the uterus during ultrasonography due to the manner in which the uterus is positioned in the abdomen, as shown in Figure 2 by different shapes and positions of the uterus in vivo. The user can tilt the mouse platform plane to shift or realign the uterus internally to a more favorable position in which the desired uterine region can be captured by ultrasound. Finally, the user can confirm that the desired uterine images were obtained during ultrasonography by quickly scrolling through the acquired ultrasound images soon after the ultrasound session is completed. A minor limitation is that the 3-D mesh/solid overlay for visual reconstruction cannot be performed during the ultrasound session. To ensure that the ultrasound images being obtained are accurate, we recommend that as the researchers are gaining proficiency, the animals are euthanized after ultrasound in order to correlate the accuracy of the ultrasound findings to gross tissue specimen measurements and anatomy.
While we describe here the modeling of normal murine pregnancy, future applications of this technique will be applied to quantify abnormal early pregnancy events. HFUS and 3-D reconstruction will permit investigators to detect and monitor the number, size and locations of pregnancies which are abnormal in growth and/or development. For example, non-invasive phenotypic characterization of abnormalities&#8212;i.e. defective endometrial decidualization, aberrant embryo implantation distribution, and inadequate trophoblastic invasion and growth&#8212;displayed by an increasing number of genetically engineered mice can now be longitudinally analyzed without pregnancy interruption using this imaging methodology.

Recurrent early pregnancy loss is one of the most common complications after conception and affects approximately 1% of couples trying to conceive1,2. The underlying mechanisms of early pregnancy loss are varied: from intrinsic embryonic abnormalities and maternal comorbidities to defects in endometrial receptivity1,3,4. Because of their genetic tractability, mouse models have been widely utilized for investigations of early embryo implantation and pregnancy. Furthermore, the short gestational time of the mouse and the ability to perform large-scale studies have ensured the growing utility of the mouse in addressing key clinical questions in human reproductive medicine5. That said, the vast majority of murine experimental designs still require numerous dams to be euthanized on sequential gestational days to quantify and analyze implantation site location, number, size, and spacing patterns during pregnancy6,7,8, thereby precluding longitudinal studies on the same animal.
In the clinic, ultrasound is a reliable and invaluable tool to monitor human fetal viability and development in a non-invasive manner9,10,11. More recently, high-frequency ultrasound (HFUS) has begun to find limited applications in the mouse as a method for monitoring fetal viability and growth during pregnancy12,13,14. The recent technological advances in ultrasound imaging have permitted the application of three-dimensional (3-D) data for visual reconstruction of animal organs and subsequent monitoring of pathologies15,16,17. Use of this advanced imaging technology has markedly improved the power to detect smaller volume fluctuations, to reduce inter-animal variability, and to monitor the progression of a pathology or the efficacy of a therapeutic intervention17. While the primary utility of this technology has been to monitor malignancy progression in oncomouse models15,16, 3-D HFUS imaging has only recently been used to quantitate and monitor the active growth of embryo implantation and fetal development in the mouse uterus18.
Here, we demonstrate how to perform HFUS imaging to produce 2-D and 3-D data to generate reconstructions of the early pregnant mouse uterus. We demonstrate the utility of this novel method to detect these early embryonic implantation events without the need for pregnancy termination, allowing researchers to collect data in a non-invasive manner.

<table><tbody><tr><td>VisualSonics Vevo 2100 Ultrasound Imaging Platform/Machine</td><td>VisualSonics, inc.</td><td>VS-11945</td><td></td></tr><tr><td>Vevo Imaging Station</td><td>VisualSonics, inc.</td><td>SA-11982</td><td></td></tr><tr><td>Aquasonic 100 Ultrasound Transmission Gel</td><td>Parker</td><td>#SKU PLI 01-08</td><td></td></tr><tr><td>Isoflurane (IsoThesia) 100mL bottle</td><td>Henry Shein</td><td>#29404</td><td></td></tr><tr><td>PuraLubenAnimal Ophthalmic Ointment</td><td>Dechra</td><td>#12920060</td><td></td></tr></tbody></table>

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.

As demonstrated in Figure 1, the high frequency ultrasound can detect implantation site development beginning as early as the 5.5 d.p.c time point. Using the lighter hyperechoic decidualized endometrium as a marker of implantation sites at 6.5 d.p.c allows the number of implantation sites and spacing of these sites to be quantified. As the pregnancy progresses to 7.5 d.p.c., a darker hypoechoic gestational sac and fetal pole are also easily identifiable.
As shown in Figure 2, the completed post-processing composition of the three-dimensional reconstruction of the mouse uterus is able to be visualized. At 6.5 d.p.c, the 3-D high frequency ultrasound composition in both mesh and solid overlay formats can be used to provide a visual depiction. Another example, this time at 7.5 d.p.c, demonstrates the final composite image of another uterus with both mesh and solid overlay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56207/56207fig1.jpg" /> 
Figure 1: High Frequency Ultrasound detection of implantation site development and monitoring of fetal growth throughout gestation. As shown in Figure 1A, on 5.5 d.p.c. the lighter hyperechoic decidualized endometrium (D) allows the number of implantation sites and spacing to be quantified. As shown in Figure 1B, the decidualization sites (D) in another uterine horn are joined by the uterine horn (U). By 7.5 d.p.c., the darker hypoechoic sac (GS) and fetal pole (F) are easily identifiable Figure 1C. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56207/56207fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56207/56207fig2.jpg" /> 
Figure 2: The three-dimensional reconstruction of the mouse uterus during early pregnancy. At 6.5 d.p.c, as shown in Figure 2A, 3-D high frequency ultrasound in both mesh and solid overlay formats are shown. At 7.5 d.p.c, as shown in Figure 2B, 3-D high frequency ultrasound images with both mesh and solid overlay formats are shown. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56207/56207fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.1K Views.  Baylor College of Medicine. Muizen worden veel gebruikt om te studeren zwangerschapsduur biologie. Beëindiging van de zwangerschap is echter vereist voor dergelijke studies die zich verzet tegen longitudinale onderzoek en vereist het gebruik van grote aantallen dieren. Daarom beschrijven we een niet-invasieve techniek van hoge-frequentie Ultrasonografie voor de vroegtijdige opsporing en het toezicht na implantatie gebeurtenissen in de zwangere muis.

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

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 ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

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

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

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 ...

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

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

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

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

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

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

Cancer Research

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 ...

High-frequency Ultrasonography Based Early Pregnancy Characterization: A Technique to Study Embryo Implantation and Pregnancy Progression in Pregnant Murine Model

Source: Peavey, M. C. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56207" target="_blank">A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse</a>. J. Vis. Exp. (2017)
In this video, we demonstrate high-frequency ultrasonography to image embryo implantation sites in a pregnant murine model. This imaging technique successfully detects viable pregnancy and characterizes embryo-implantation sites during early murine pregnancy.

Source: Peavey, M. C. et al. A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse. J. Vis. ...

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

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