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><tbody><tr><td>Buprenorphine&amp;nbsp;</td><td>Par Pharmaceutical</td><td>NDC 42023-179-05</td><td>For regional anesthesia</td></tr><tr><td>Isoflurane&amp;nbsp;</td><td>&amp;nbsp;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&amp;nbsp;</td><td>Macherey-Nagel, D&amp;uuml;ren, Germany</td><td>740952.5</td><td>DNA isolation</td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</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&amp;nbsp;</td><td>VWR&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</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. De belangrijkste voordelen van deze techniek zijn omkeerbaarheid, verlaging van de kosten van de dieren en onderhoud en een kortere zwangerschapsperiode. Bovendien wordt de diersterfte verminderd omdat hysterectomie niet nodig is. En er zijn bredere mogelijkheden voor genetisch onderzoek.Deze techniek kan worden gebruikt voor het onderzoeken van de pathofysiologie van de aangeboren diafragmatische hernia. Het kan ook worden toegepast om andere ziekten in de long te begrijpen, zoals koe...

Video: Transuterine Fetal Tracheal Occlusion Model in Mice

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Medicine

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Biology

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Bioengineering

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Neuroscience

Repeated Orotracheal Intubation in Mice

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator&#8217;s ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ...

Exploring Obliterative Airway Disease in Lung Transplants

Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially reported by our team, this model has gained use in the study of OAD due to its high technical reproducibility and suitability for investigating immunological behaviors and therapeutic interventions.
In the IPTT model, a rodent tracheal graft is directly inserted into the recipient's lung through the pleura. This model is distinct from the heterotopic tracheal transplantation (HTT) model, wherein grafts are transplanted into subcutaneous or omental sites, and from the orthotopic tracheal transplantation (OTT) model in which the donor trachea replaces the recipient's trachea. 
Successful implementation of the IPTT model requires advanced anesthetic and surgical skills. Anesthetic skills include endotracheal intubation of the recipient, setting appropriate ventilatory parameters, and appropriately timed extubation after recovery from anesthesia. Surgical skills are essential for precise graft placement within the lung and for ensuring effective sealing of the visceral pleura to prevent air leakage and bleeding. In general, the learning process takes approximately 2 months.
In contrast to the HTT and OTT models, in the IPTT model, the allograft airway develops airway obliteration in the relevant lung microenvironment. This allows investigators to study lung-specific immunological and angiogenic processes involved in airway obliteration after lung transplantation. Furthermore, this model is also unique in that it exhibits tertiary lymphoid organs (TLOs), which are also seen in human lung allografts. TLOs are comprised of T and B cell populations and characterized by the presence of high endothelial venules that direct immune cell recruitment; therefore, they are likely to play a crucial role in graft acceptance and rejection. We conclude that the IPTT model is a useful tool for studying intrapulmonary immune and profibrotic pathways involved in the development of airway obliteration in the lung transplant allograft.

Author Spotlight: Investigating the Key Factors of Obliterative Bronchiolitis After Lung Transplantation

The murine intrapulmonary tracheal transplantation (IPTT) model is valuable for studying obliterative airway disease (OAD) after lung transplantation. It offers insights into lung-specific immunological and angiogenic behavior in airway obliteration after allotransplantation with high reproducibility. Here, we describe the IPTT procedure and its expected results.

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially ...

Transuterine Fetal Tracheal Occlusion Model in Mice

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