This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. All authors have made substantial contributions to the conception and design of the study, acquisition, analysis, and interpretation of data, drafting the article, and revising it for important intellectual content and final approval of the version to be submitted. The authors thank Can Sabuncuo&#287;lu for his kind efforts on the production of the artwork of the surgical technique.

All experiments have complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80023, revised 1978). The procedure was approved with IACUC protocol #2016-0068 by the Cincinnati Children&#39;s Research Foundation Institutional Animal Care and Use Committee.
1. Preparation
<ol>
	<li>To mate age-matched wild-type (WT) C57BL/6 mice, place them in the same cage at 6:00 p.m. and separate them at 9:00 a.m. the next day.</li>
	<li>To determine embryonic day 0 (E0), look at the vaginal plug, which has a homogeneous outer zone attached to the vaginal wall and an inner zone that is fibrous and includes some spermatozoa that form entangled masses mixed with the fibers of the plug material.</li>
	<li>Record the weight of the mice at the time of mating.</li>
	<li>Re-weigh the mice on E10 to ensure ongoing pregnancy.</li>
	<li>Perform the surgery on E16.5 (early canalicular stage).</li>
	<li>Sterilize the instruments that are going to be used during surgery: scissors, needle holder, forceps, clamps, and surgical knives and handles.</li>
	<li>Pre-heat the surgery platform to 24 &#176;C and prepare warm saline (24 &#176;C) prior to surgery.</li>
	<li>Create a warm environment for recovery, and leave wet food inside the cage for the early feeding.</li>
	<li>Stay with the operated animals until they can feed themselves.</li>
	<li>Keep the operated mice alone in their individual cages after the surgery.</li>
</ol>
2. Anesthesia
<ol>
	<li>Apply subcutaneous 0.1 mg/kg of buprenorphine to the pregnant dams 1 h before the procedure.</li>
	<li>Use inhaled 5 mL/h of isoflurane for induction and 2 mL/h continuously during the procedure for anesthesia.</li>
	<li>Monitor the movements of the chins of the pregnant mice.</li>
</ol>
3. Laparotomy
<ol>
	<li>Clean the abdominal surface with alcohol and povidone-iodine. Maintain sterile conditions throughout the operation.</li>
	<li>Perform a vertical incision for the laparotomy of pregnant dams. Cut all layers separately.</li>
	<li>Identify uterine horns on each side.</li>
	<li>Determine the candidate fetuses for the surgery. 
	NOTE: Do not operate on the fetuses that are the nearest to the vagina.</li>
	<li>Operate on two fetuses in each uterine horn if there are an even number of fetuses on each side (4 most of the time), and on 1 fetus in each uterine horn if there are an odd number of the uterus(3 most of the time).</li>
</ol>
4. Tracheal occlusion
<ol>
	<li>Use 2.5x magnification glasses for visualization.</li>
	<li>Position the uterine horn in a transverse fashion.</li>
	<li>Take the pups, facing upward, between two fingers using the eyes of the pups and the tail as a guide to position the fetus.</li>
	<li>Apply gentle pressure to the pup&#39;s head to allow extension of the head and therefore, visualization of the neck.</li>
	<li>Use a 6.0 polypropylene suture with an atraumatic needle to perform TO (Figure 1). Keep the placenta on the side and far from the entrance and exit points of the needle.</li>
	<li>Insert the needle transversely through the side of the uterus away from the placenta through the 1/3rd anterior part of the neck.</li>
	<li>Move the needle gently until the midline of the neck and direct it to the anterior part, then exit the neck between the trachea and opposite the carotid sheath and uterus.</li>
	<li>Knot the suture, taking care to maintain the integrity of the membranes and uterine wall, and keep the umbilical cord safe during knotting.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61772/61772fig01.jpg" /> 
Figure 1:&#160;Tracheal occlusion.&#160;(A) The transuterine suture passing through the neck. (B) Schematic representation of the structures after the suture passes through and before the knot. Abbreviations: C = Carotid artery; J = Jugular vein; T =Trachea; E = Esophagus; V = Vertebra.&#160;<a href="https://www.jove.com/files/ftp_upload/61772/61772fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Abdominal wall closure
<ol>
	<li>Replace the uterine horn in the abdomen.</li>
	<li>Inject 2 mL of warm sterile saline into the peritoneal cavity before closure.</li>
	<li>Put a running 5/0 polyglactin suture to close the abdominal wall, and close the skin with a non-running silk suture.</li>
	<li>Apply 0.1 mg/kg of buprenorphine intraperitoneally for analgesia, and allow the recovery of the dam in a warm incubator.</li>
</ol>
6. Harvest
<ol>
	<li>Apply anesthesia to the pregnant dam, and harvest all fetuses at E18.5 by cesarean section.</li>
	<li>Check the viability of the fetuses by watching the movements of the fetuses.</li>
	<li>Use at least two different techniques for the euthanasia: carbon dioxide insufflation and cervical dislocation.</li>
	<li>Remove the bodies per the regulation of veterinary laboratory.</li>
	<li>Weigh all fetuses.</li>
	<li>Perform a vertical incision on the thorax for thoracotomy to remove the lungs.</li>
	<li>Dissect the lungs of embryos, and weigh them to calculate total lung to body weight ratio (LBWR = (left lung weight + right lung weight)/body weight x100).</li>
</ol>
7. Histology
<ol>
	<li>Snap-freeze the tissues in liquid nitrogen, optimal cutting temperature compound, and dry ice.</li>
	<li>Cut the samples in 10 &#181;m sections using a cryostat, and mount them on poly-lysine-coated slides.</li>
	<li>Bake the slides at 60 &#176;C overnight, and stain the baked slides with hematoxylin and eosin before mounting them for image acquisition at 10-20x magnification using a widefield microscope.</li>
</ol>
8. Tissue processing for protein and DNA analyses
<ol>
	<li>Snap-freeze the dissected fetal lungs, and homogenize them in 300 &#181;L of radioimmunoprecipitation assay buffer. Centrifuge at 4 &#176;C for 5 min at 18,000 &#215; g.</li>
	<li>Extract and quantify protein, DNA, and RNA10,12.</li>
</ol>

<ol>
	<li>Wright, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+PaedSurg+Research+Collaboration.+Management+and+outcomes+of+gastrointestinal+congenital+anomalies+in+low,+middle+and+high+income+countries:+protocol+for+a+multicentre,+international,+prospective+cohort+study.">Global PaedSurg Research Collaboration. Management and outcomes of gastrointestinal congenital anomalies in low, middle and high income countries: protocol for a multicentre, international, prospective cohort study.</a> BMJ Open. 9, 030452 (2019).</li><li>Aydin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approach+for+prenatally+diagnosed+congenital+anomalies+that+requires+surgery.">Current approach for prenatally diagnosed congenital anomalies that requires surgery.</a> Turkish Clinics Journal of Gynecology and Obstetrics. 27, 193-199 (2016).</li><li>Nolan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemorrhage+after+on-ECMO+repair+of+CDH+is+equivalent+for+muscle+flap+and+prosthetic+patch.">Hemorrhage after on-ECMO repair of CDH is equivalent for muscle flap and prosthetic patch.</a> Journal of Pediatric Surgery. 54 (10), 2044-2047 (2019).</li><li>Aydin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+diaphragmatic+hernia:+the+good,+the+bad,+and+the+tough.">Congenital diaphragmatic hernia: the good, the bad, and the tough.</a> Pediatric Surgery International. 35 (3), 303-313 (2019).</li><li>Ayd&#305;n, E., &#214;zler, O., Burns, P., Lim, F. Y., Peir&#243;, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+congenital+diaphragmatic+hernia-associated+musculoskeletal+deformities.">Left congenital diaphragmatic hernia-associated musculoskeletal deformities.</a> Pediatric Surgery International. 35 (11), 1265-1270 (2019).</li><li>Ayd&#305;n, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+primary+repair+is+not+enough:+a+comparison+of+synthetic+patch+and+muscle+flap+closure+in+congenital+diaphragmatic+hernia.">When primary repair is not enough: a comparison of synthetic patch and muscle flap closure in congenital diaphragmatic hernia.</a> Pediatric Surgery International. 36 (4), 485-491 (2020).</li><li>Wilson, M., Difiore, J. W., Peters, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+fetal+tracheal+ligation+prevents+the+pulmonary+hypoplasia+associated+with+fetal+nephrectomy:+Possible+application+for+congenital+diaphragmatic+hernia.">Experimental fetal tracheal ligation prevents the pulmonary hypoplasia associated with fetal nephrectomy: Possible application for congenital diaphragmatic hernia.</a> Journal of Pediatric Surgery. 28 (11), 1433-1440 (1993).</li><li>Mudri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+tracheal+occlusion+on+Wnt+signaling+in+a+rabbit+model+of+congenital+diaphragmatic+hernia.">The effects of tracheal occlusion on Wnt signaling in a rabbit model of congenital diaphragmatic hernia.</a> Journal of Pediatric Surgery. 54 (5), 937-944 (2019).</li><li>Khan, P. A., Cloutier, M., Piedboeuf, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracheal+occlusion:+a+review+of+obstructing+fetal+lungs+to+make+them+grow+and+mature.">Tracheal occlusion: a review of obstructing fetal lungs to make them grow and mature.</a> American Journal of Medical Genetics. Part C, Seminars in Medical Genetics. 145 (2), 125-138 (2007).</li><li>Chomczynski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reagent+for+the+single-step+simultaneous+isolation+of+RNA,+DNA+and+proteins+from+cell+and+tissue+samples.">A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples.</a> Biotechniques. 15 (3), 532-537 (1993).</li><li>Beurskens, N., Klaassens, M., Rottier, R., De Klein, A., Tibboel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+animal+models+to+human+congenital+diaphragmatic+hernia.">Linking animal models to human congenital diaphragmatic hernia.</a> Birth Defects Research Part A: Clinical and Molecular Teratology. 79 (8), 565-572 (2007).</li><li>Varisco, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+reversal+of+epidermal+growth+factor+receptor+and+ephrin+signaling+following+tracheal+occlusion+in+rabbit+model+of+congenital+diaphragmatic+hernia.">Excessive reversal of epidermal growth factor receptor and ephrin signaling following tracheal occlusion in rabbit model of congenital diaphragmatic hernia.</a> Molecular Medicine. 22, 398-411 (2016).</li></ol>

The authors have nothing to disclose.

This method describes a surgical procedure of fetal tracheal occlusion in mice and its impact on lung development. There are some critical steps in the protocol that should be carefully performed for successful TO. The warmth of the platform on which the surgery takes place and the saline introduced into the peritoneal cavity is crucial for the progression of the pregnancy. In addition, a slight pressure has to be applied to the head of the pups to ensure exposure of the neck.
A 6.0 polypropyl...

Congenital diaphragmatic hernia (CDH) occurs in 1:2500 pregnancies and results in pulmonary hypoplasia and neonatal pulmonary hypertension1,2,3,4,5,6. Fetal tracheal occlusion (TO) is an established prenatal therapy in severe CDH patients involving fetoscopy in the 26-30th gestational week in which a balloon is placed just above the carina and then removed in the 32nd gestational week. This temporary TO induces fetal lung growth and improves survival. Congenital High Airway Obstruction Syndrome is a lethal condition associated with lung hyperplasia, which inspired surgeons to perform artificial occlusion of the trachea to promote retention of the secreted epithelial fluid. This occlusion increased luminal pressure and induced lung growth7. However, the occlusion should be reversed to enable epithelial cell maturation.
Various animal models of CDH and TO - ovine, rabbit, rat, and mouse - have been developed 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. Although the surgical technique used for the ovine model is very similar to that used in humans and could be reversed, the major drawbacks of this model are the expense of the animal, the long gestational period, and the limited number of surgeries possible. The rabbit model has a shorter gestational period and is less expensive than the sheep model. However, the rabbit model is irreversible8,9. The murine model has the lowest cost, the highest number of fetuses per pregnancy, the best-characterized genome, and widely available tools for cellular and molecular analyses. However, a key drawback is the lack of reversibility of the TO, preventing full understanding of the impact of TO. Herein, a method is presented that combines all the advantages of the previously mentioned models and creates an easy, potentially reversible, and minimally invasive rodent TO model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buprenorphine&#160;</td>
			<td>Par Pharmaceutical</td>
			<td>NDC 42023-179-05</td>
			<td>For regional anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>&#160;Halocarbon Life Sciences</td>
			<td>NDC 66794-017-25</td>
			<td>For general anesthesia</td>
		</tr>
		<tr>
			<td>Magnification glasses</td>
			<td>USA Medical-Surgical</td>
			<td>SLR-250LBLK</td>
			<td>At least 2.5x</td>
		</tr>
		<tr>
			<td>Nikon 90i microscope</td>
			<td>Nikon</td>
			<td>3417</td>
			<td>Motorized Fluorescence</td>
		</tr>
		<tr>
			<td>Nucleospin Tissue Kit&#160;</td>
			<td>Macherey-Nagel, D&#252;ren, Germany</td>
			<td>740952.5</td>
			<td>DNA isolation</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>Thermo Fisher, IL, USA</td>
			<td>23225</td>
			<td>Protein quantification</td>
		</tr>
		<tr>
			<td>Polyglactin suture</td>
			<td>Ethicon</td>
			<td>VCP451H</td>
			<td>4-0, 24 mm, cutting</td>
		</tr>
		<tr>
			<td>Polylysine slides&#160;</td>
			<td>VWR&#160;</td>
			<td>48382-117</td>
			<td>Microscope adhesion slides</td>
		</tr>
		<tr>
			<td>Polypropylene suture</td>
			<td>Ethicon</td>
			<td>Y432H</td>
			<td>6-0, 13 mm 1/2c Taperpoint</td>
		</tr>
		<tr>
			<td>RIPA buffer&#160;</td>
			<td>Sigma-Aldrich, Missouri, USA</td>
			<td>R0278-50ml</td>
			<td>Protein isolation</td>
		</tr>
		<tr>
			<td>Silk suture</td>
			<td>Ethicon</td>
			<td>VCP682G</td>
			<td>4-0, 24 mm, cutting</td>
		</tr>
		<tr>
			<td>Trizol&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>15596026</td>
			<td>RNA isolation</td>
		</tr>
	</tbody>
</table>

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.

This study examined 37 fetuses: 20 (54.1%) as TO vs. 17 (45.9%) as control. As the trachea could not be occluded in 4 fetuses in the TO group, they were excluded from the study. There was no significant difference in mortality in both groups: 4 fetuses (25%) in the TO group and 2 fetuses (12%) in the control group (p=0.334, odds ratio (OR) 2.5, 95% confidence interval (CI) 0.39-16.05). The mean body weight, lung weight, and lung to body weight ratio (LBWR) were higher in the TO group than...

Watch this Scientific Journal Video about Transuterine Fetal Tracheal Occlusion Model in Mice at JoVE.com

Transuterine Fetal Tracheal Occlusion Model in Mice

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). ...

transuterine-fetal-tracheal-occlusion-model-in-mice

Research

JoVE Journal

Developmental Biology

3.0K Views.  Cincinnati Children’s Hospital Medical Center. 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.

Video: Transuterine Fetal Tracheal Occlusion Model in Mice

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as endotoxins, bacteria, umbilical cord occlusions, hypoxia and various pharmacological treatments. The life-saving clinical practices of glucocorticoid treatment in fetuses at risk of premature birth and the therapeutic hypothermia have been developed in this model. This is due to the unique amenability of the non-anesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation and in vivo organ imaging. Here we describe the surgical instrumentation procedure required to achieve a stable chronically instrumented non-anesthetized fetal sheep model including characterization of the post-operative recovery from blood gas, metabolic and inflammation standpoints.

The chronically instrumented non-anesthetized fetal sheep model is used to study human fetal development in health and disease, because it permits surgical placement and maintenance of catheters and electrodes, repetitive blood sampling, substance injection, recording of bioelectrical activity, and in vivo imaging. We describe the procedures required to establish this model.

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

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 ...

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 ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

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 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 ...

Culturing and Measuring Fetal and Newborn Murine Long Bones

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy human bones makes particularly valuable the use of mammalian models, such as mouse and rat, to look into different aspects of bone growth and homeostasis. Additionally, the development of sophisticated genetic tools in mice allows more complex studies of long bone growth and asks for an expansion of techniques used to study bone growth. Here, we present a detailed protocol for ex vivo murine bone culture, which allows the study of bone and cartilage in a tightly controlled manner while recapitulating most of the in vivo process. The method described allows the culture of a range of bones, including tibia, femur, and metatarsal bones, but we have focused mainly on tibial culture here. Moreover, it can be used in combination with other techniques, such as time-lapse live imaging or drug treatment.

Here, we present a method for ex vivo culture of long murine bones at both fetal and newborn stages, suitable for analyzing bone and cartilage development and homeostasis in controlled conditions while recapitulating the in vivo process.

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...