Name: transuterine fetal tracheal occlusion model in mice
Uploaded: 2021-02-05T13:00:00
Description: Watch this Scientific Journal Video about Transuterine Fetal Tracheal Occlusion Model in Mice at JoVE.com

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 deter...

<ol>
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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. 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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. 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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). ...

The most important advantages of this technique are reversibility, reduction in cost of the animals and maintenance, and a shorter pregnancy period. Moreover, animal mortality is reduced because there is no need for hysterectomy. And there are wider opportunities for genetic workup.This technique can be used for investigating the pathophysiology of the congenital diaphragmatic hernia. It can also be applied to understanding other diseases in the lung, such as cows and pulmonary hypoplasia. Begin by allowing the mice to mate in the same cage from 6:00 PM to 9:00 AM the next day.To determine E0, observe the vaginal plug which has a homogenous, outer zone attached to the vaginal wall and an inner zone that is fibrous and include some spermatozoa that form entangled masses mixed with the fibers of the plug material. Record the weight of the mice at the time of mating and on E10 to ensure ongoing pregnancy and perform the surgery at E16.5. Sterilize the instruments that are going to be used during the surgery.Preheat the surgery platform to 24 degrees Celsius and prepare warm saline prior to surgery. Create a warm environment for recovery and leave wet food inside the cage for the early feeding. Clean the abdominal surface with alcohol and povidone iodine and maintain sterile conditions throughout the operation.Perform a vertical incision for the laparotomy of pregnant dams and cut all layers separately. Identify the uterine horns on each side and determine the candidate fetuses for the surgery. Operate on two fetuses in each uterine horn if there is an even number of fetuses on each side.And on one fetus in each uterine horn if there is an odd number. Using 2X magnification glasses for visualization, position the uterine horn in a transverse fashion. Position the pups facing upward between two fingers using the eyes and the tail as a guide.Apply gentle pressure to the pup's head to allow extension of the head and visualization of the neck. Perform tracheal occlusion or TO using an atraumatic needle and a 6 by 0 polypropylene suture. Keep the placenta on the side and far from the entrance and exit points of the needle.Insert the needle transversely through the side of the uterus, away from the placenta through the one-third anterior part of the neck. Then move the needle gently to the midline of the neck. Direct it to the anterior part.Then exit the neck between the trachea and opposite the carotid sheath and uterus. Knot the suture, taking care to maintain the integrity of the membranes and uterine wall. Replace the uterine horn in the abdomen and inject two milliliters of warm sterile saline into the peritoneal cavity before closure.Place a running 5 by 0 polyglactin suture to close the abdominal wall. And close the skin with a non-running silk suture. Apply 0.1 microgram per kilogram of buprenorphine intraperitoneally for analgesia and allow the dam to recover in a warm incubator.Observe that the operated animal can feed itself and keep it alone in its individual cage. Harvest all the fetuses by E18.5 by ceasarean section and check their viability by watching their movements. Weigh all the fetuses.Perform a vertical incision on the thorax for a thoracotomy to dissect the lungs. And weigh them to calculate total lung to body weight ratio. Snap freeze the tissue in liquid nitrogen, optimal cutting temperature compound, and dry ice.Cut the samples in sections of 10 micrometers using a cryostat, and mount them on polylysine-coated slides. Bake the slides overnight at 60 degrees Celsius and stain the baked slides with hematoxylin and eosin. Image of the slides at 10 to 20X magnification, using a widefield microscope.For protein and DNA analyses, snap freeze the tissue and homogenized it in 300 microliters of radioimmunoprecipitation assay buffer. Centrifuge at 18, 000 x g and 4 degrees Celsius for five minutes. Then extract and quantify protein, DNA, and RNA.The mean body weight, lung weight, and LBWR were higher in the TO group than in the control group. Lung DNA amounts and the DNA to protein ratio were higher in the TO group. No difference was observed in lung RNA, and protein amounts were lower in the TO group than in the control group.Histological analyses of the E18.5 control lungs showed the late or early saccular stage of lung development with developing airspaces and thickened interstitium between epithelial surfaces. The lungs in the TO group had dilated central and distal airspaces with subjectively higher numbers of nuclei. When attempting this protocol, We fix the sutures in a way that the eyes of the fetus are visible and the neck is extended so that the needle does not disrupt the adjusted structures.The removal of the structures to the rebirth of the fetus in the nitrile fan and knockout models of CDH will be the future applications of this technique.

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