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

high frequency ultrasound for the analysis of fetal and placental development in vivo

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

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

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

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

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

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

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

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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 ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

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

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

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

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

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

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

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

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

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

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

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

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

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

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

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

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

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

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

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

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

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

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

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

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

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

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

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

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

Transuterine Fetal Tracheal Occlusion Model in Mice

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

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

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