Name: Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease (Video) | JoVE Journal
Uploaded: 2010-01-20T18:15:00.000+00:00
Duration: 9 min 10 s
Description: Learn heterotopic and orthotopic tracheal transplantation in mice to study obliterative airway disease development. These surgical models enable researchers to investigate airway healing mechanisms and transplant complications.

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5 minutes

The authors thank Christiane Pahrmann (Lab Manager).

<ol>
 <li>Male Balb/C mice (8-12 weeks) are purchased from Charles River Laboratories (Sulzfeld, Germany). The mice are housed under conventional conditions, fed standard mice food and water ad libitum.</li>
 <li>2% Isofluran is used for anesthesia.</li>
</ol>
DONOR PREPARATION
<ol>
 <li>Shave the abdominal hair and disinfect the area using betaisodona.</li>
 <li>Under microscopic view, perform a midline cervical incision from the level of the larynx to the sternum.</li>
 <li>Remove the subcutaneous fat and the strap muscles to get a clear view of the trachea.</li>
 <li>Dissect the trachea from any surrounding tissues, like the esophagus, nerves, arteries, and connective tissue.</li>
 <li>Remove the whole trachea (from the larynx to the bifurcation).</li>
 <li>Flush the transplant with cold saline and store the graft at 4°C.</li>
 <li>The donor is euthanized by cervical dislocation following the harvest of the trachea.</li>
</ol>
RECIPIENT: HETEROTOPIC TRANSPLANTATION
<ol>
 <li>Shave the abdominal hair in a wide margin around the incision site and disinfect the area three times using betaisodona (betadine) followed by alcohol. Eyes should be lubricated with an ophthalmic ointment product to prevent the corneas from drying out.</li>
 <li>Perform a median laparotomy and place the intestine into a sterile, moistured glove.</li>
 <li>Spread the greater omentum carefully. Place the graft into the center and fixate it with a single suture using 8-0 (Prolene, Ethicon, Germany).</li>
 <li>Fully cover the transplant with the greater omentum and fix the graft with one single suture 8-0 (Prolene, Ethicon, Germany).</li>
 <li>Relocate the intestines back into the abdomen and flush with warm, sterile saline prior to closure.</li>
 <li>Close up in 2 layers - abdominal wall and skin layer with continuous pattern using 7-0 Prolene for the muscle and 7-0 Vicryl for the skin.</li>
</ol>
RECIPIENT: ORTHOTOPIC TRANSPLANTATION
<ol>
 <li>Shave the abdominal hair in a wide margin around the incision site and disinfect the area three times using betaisodona (betadine) followed by alcohol. Eyes should be lubricated with an ophthalmic ointment product to prevent the corneas from drying out.</li>
 <li>Divide the strap muscles to visualize the entire laryngotracheal complex.</li>
 <li>Carefully dissect the trachea from the surrounding tissues, take care to preserve the recurrent laryngeal nerves.</li>
 <li>Divide the trachea three rings caudal from the cricoid. The animal maintains physiologic respiration via the tracheostomy.</li>
 <li>Ensure clean tracheal edges in the recipient as well as in the graft.</li>
 <li>The graft is interposed between the recipient tracheal defects and orientated to maintain anatomic polarity.</li>
 <li>Using 8-0 (Prolene, Ethicon, Germany) anastomose the donor graft with the distal (mediastinal) trachea. The posterior aspect of the anastomosis is performed in continuous running fashion. The anterior aspect is then completed using interrupted stitches.</li>
 <li>Remove any secretions from the airway.</li>
 <li>The proximal anastomosis is then completed in the same way as the distal one.</li>
 <li>Ensure integrity of the airway and adequate, spontaneous breathing.</li>
 <li>Relocate the strap muscles and close the subcutaneous tissue and skin layer using 6-0 sutures (Vicryl, Ethicon, Germany) with continuous pattern.</li>
<li>Use injection anesthesia for the recipient, therefore the animal will retain physiological respiration through the trachea. A combination of 75/1/0.2 mg/kg of propofol, medetomidine and fentanyl, respectively, is used for i.p. anaesthesia in mice.</li> 
</ol>
<img src="/files/ftp_upload/1437/1437Fig1.jpg" alt="Tracheal reconstruction experiment in biological tissue; sequence showing airway implant process." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1: Donor trachea. 1A: Donor trachea in situ after preparation.
 1B: Excised donor trachea. 
1C: Donor trachea after explantation.
<img src="/files/ftp_upload/1437/1437Fig2.jpg" alt="Surgical procedure images showing internal structures; identifies tissues and sutures." data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2: Heterotopic Model. 2A: The graft is positioned in the center of the greater omentum. 
2B: The graft is fixed on both ends with a single suture. 
2C: The graft is wrapped into the greater omentum and fixed with a single suture.
<img src="/files/ftp_upload/1437/1437Fig3.jpg" alt="Laryngeal dissection process; surgical method; experimental setup for vocal fold examination." data-altupdatedat="1787630408000" data-alt="Figure 3"> Figure 3: Orthotopic Model. 3A: The graft is interposed between the recipient tracheal defects and the posterior wall is anastomosed in continuous running fashion. 
3B: The anterior wall is completed using a single suture.
<img src="/files/ftp_upload/1437/1437Fig4.jpg" alt="Histological sections, stained tissue; microscopy image; comparative analysis A and B." data-altupdatedat="1787630408000" data-alt="Figure 4"> Figure 4: Histology. 4A: Recovered heterotopic transplanted trachea after 28 days in the H+E staining (15x). Note the 100% luminal obliteration. 
4B: Recovered orthotopic transplanted trachea after 60 days in the H+E staining (15x). Maximal achieved luminal obliteration is approximately 45%.
<table border="1">
	<tbody>
		<tr>
 <td></td>
 <td>Heterotopic Tracheal Transplantation Model</td>
 <td>Orthotopic Tracheal Transplantation Model</td>
		</tr>
 <tr>
			<td>Advantages</td>
			<td>+ Easy to perform 
				+ Luminal obliteration with complete airway occlusion after 28 days 
				+ No physical affection of animals by OAD 
		 </td>
 <td>
 + Physical ventilation of the graft 
 + Inhaled drug administration possible 
 + Strong immunological reactions such 
 as alloreactive IgM antibody production 
 + Physiological thoracic milieu 
 + Tracheal-tracheal anastomosis imitates the 
 clinical setting
 </td>
		</tr>
		<tr>
 <td>Disadvantages</td>
 <td>- No ventilation of transplanted trachea 
 - No evaluation of inhaled pathogens possible 
 - Inhibition of mucociliary clearance and retained secretions 
 - Peritoneal microenvironment instead of thoracic milieu 
 </td>
 <td>
 - Surgical training necessary 
 - Luminal obliteration with luminal occlusion app. 45% after 60 days 
 - Animals may develop symptoms of OAD 
 </td>
		</tr>
	</tbody>
</table>
Table 1: Advantages and Disadvantages of Heterotopic and Orthotopic Tracheal Transplantation.

<ol><li>Adams, B., Berry, G., Huang, X., Shorthouse, R., Brazelton, T., Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunosuppressive+therapies+for+the+prevention+and+treatment+of+obliterative+airway+disease+in+heterotopic+rat+trachea+allografts%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Immunosuppressive therapies for the prevention and treatment of obliterative airway disease in heterotopic rat trachea allografts.</a> Transplantation. 69, 2260-2266 Forthcoming.</li>
<li>Adams, B., Brazelton, T., Berry, G., Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+respiratory+epithelium+in+a+rat+model+of+obliterative+airway+disease%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">The role of respiratory epithelium in a rat model of obliterative airway disease.</a> Transplantation. 69, 661-665 Forthcoming.</li>
<li>Deuse, T., Schrepfer, S., Reichenspurner, H., Hoyt, G., Fischbein, M., Robbins, R., Pelletier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Techniques+for+experimental+heterotopic+and+orthotopic+tracheal+transplantations+%26%23x2013%3B+when+to+use+which+model%3B+Transplant+Immunology+17%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Techniques for experimental heterotopic and orthotopic tracheal transplantations &#x2013; when to use which model; Transplant Immunology 17.</a> , 255-261 (2007).</li>
<li>Hele, D., Yacoub, M., Belvisi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+heterotopic+tracheal+allograft+as+an+animal+model+of+obliterative+bronchiolitis%5BTitle%5D)+AND+%22Respiratory+Research%22%5BJournal%5D)">The heterotopic tracheal allograft as an animal model of obliterative bronchiolitis.</a> Respiratory Research. 2, 169-183 Forthcoming.</li>
<li>Hertz, M., Jessurun, J., King, M., Savic, S., Murray, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproduction+of+the+obliterative+bronchiolitis+lesion+after+heterotopic+transplantation+of+mouse+airways%5BTitle%5D)+AND+%22American+Journal+of+Pathology%22%5BJournal%5D)">Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways.</a> American Journal of Pathology. 142, (1993).</li>
<li>McDyer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+and+murine+obliterative+bronchiolitis+in+transplant%5BTitle%5D)+AND+%22Proceedings+of+the+American+Thoracic+Society%22%5BJournal%5D)">Human and murine obliterative bronchiolitis in transplant.</a> Proceedings of the American Thoracic Society. 4, 37-43 Forthcoming.</li>
<li>Schrepfer, S., Deuse, T., Hoyt, G., Sheikh, A., Hoffmann, J., Reichenspurner, H., Robbins, R., Pelletier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+orthotopic+tracheal+transplantation%3A+the+stanford+technique%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">Experimental orthotopic tracheal transplantation: the stanford technique.</a> Microsurgery. 27, 187-189 (2007).</li>
</ol>

Sonja Schrepfer has received a research grant from the Deutsche Forschungsgemeinschaft (DFG) (SCHR992/3&#x2013;1).]
All animals received human care in compliance with the Principles of Laboratory Animal Care, experiments on animals were performed in accordance with the guidelines and regulations set forth by the National Society for Medical research and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health publication 85-23, revised 1985).
All animals were obtained from Charles River Laboratories (Sulzfeld, Germany) and were maintained in the animal care facilities of the University Hospital Hamburg Eppendorf. The animals received standard chow and water ad libitum.

Mice are available in different transgeneic and knockout model, and therefore suitable to study mechanistic questions related to OAD (4). 
 Both models of tracheal transplantation shown in this video can be used as reliable models for studying OAD development. 
 However, each model demonstrates advantages and limitations.
The heterotopic tracheal transplantation is easy to perform and does not require special surgical training (3, 5). After het...

heterotopic and orthotopic tracheal transplantation in mice used as models to study the development of  obliterative airway disease

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 ...

Both the heterotopic and Orthotopic tracheal transplantations begin with the removal of the donor trachea for the heterotopic transplantation. An abdominal incision is made in the recipient and the intestines are placed on a glove. The donor trachea is placed into the center of the greater omentum and fixed by two stitches for the orthotopic transplantation.After a cervical incision is made in the recipient, the trachea is freed of surrounding tissue and cut. Then the donor trachea is inserted between the two ends of the recipient trachea and the trachea are stitched together. Hi, I'm Sonya Refa.I'm professor in Immunology and cardiac surgery in the Department of Cardiovascular Surgery at the University Heart Center in Hamburg. My laboratory is focusing on transplant immunology and stem cell immunobiology. Today we will introduce you to Moon Auto topic and topic transplantation model of the twig here.Hello, I'm from the TSI lab. We are using the also topic and the heterotopic trachea transplantation mouse models to study the mechanisms behind the development of observative airway diseases. And I'm Carrie Wan, a student at Stanford University and a summer fellow in Drer's TSI Laboratory.So let's get started. Before beginning this procedure shaved the cervical hair and disinfect the area using beta isod. Confirm that the donor mouse is completely anesthetized by a lack of the toe pinch reflex.Under microscopic view, perform a midline cervical incision from the level of the larynx to the sternum. Remove the subcutaneous fat as well as the strap muscles to get a clear view of the trachea. Dissect the trachea from any surrounding tissues such as the esophagus, nerves, arteries, and connective tissue.Remove the whole trachea from the larynx to the bifurcation. Flush the transplant trachea with cold saline and store the graft at four degrees Celsius. After confirming a lack of the toe pinch reflex in the anesthetized recipient mouse, shave the abdominal hair in a wide margin around the incision site and disinfect the area.Using beta is Sedona and alcohol. Perform a median laparotomy and place the intestine into a moisture glove. Spread the greater omentum.Carefully place the graft into the center and fixate it with two eight oh proline sutures. Fully cover the transplant with the greater omentum and fix the graft with a single eight o prolene suture. Relocate the intestines back into the abdomen.Close up the incision in two layers, the abdominal wall and skin layer with continuous pattern seven O proline sutures. After confirming that the recipient mouse is fully anesthetized using IP applicated injection, anesthesia, shave and disinfect the cervical area as previously shown, perform a midline cervical incision from the level of the larynx to the sternum. Divide the thymus and strap muscles to visualize the entire laryn tracheal complex.Carefully dissect the trachea from the surrounding tissues. Taking care to preserve the recurrent laryngeal nerves. Spread the corvus and divide the trachea.Three rings coddle from the cricoid. The animal will maintain physiologic respiration via the tracheostomy. Ensure clean tracheal edges in the recipient as well as in the graft by dissecting the tracheas under microscopic view.The graft is interposed between the recipient tracheal defects and oriented to maintain anatomic polarity using eight o proline sutures. Anastomosis the donor graft with the distal or mediastinal trachea. The posterior aspect of the anastomosis is performed in continuous running fashion.The anterior aspect is then completed using interrupted sutures. Then remove any secretions from the airway. The proximal anastomosis is then completed in the same way as the distal one.Perform the posterior aspect in continuous running fashion and the anterior aspect with interrupted sutures. Ensure integrity of the airway and adequate spontaneous breathing. Relocate the strap muscles and close the skin layer using six oh continuous pattern Vicryl sutures when the heterotopic transplanted trachea is recovered after 28 days and analyzed histologically by h and d staining 100%luminal obliteration is observed when the orthotopic transplanted trachea is recovered after 60 days and analyzed histologically by h and d staining, a maximal luminal obliteration of approximately 45%is achieved.We have just shown you two different models to investigate the development of literative airway disease. The appropriate model should be chosen by the study hypothesis. Our lab favors the also topic one since the physiological airflow imitates the clinical setting.So that's it. Thanks for watching and good luck with your experiments.

heterotopic-orthotopic-tracheal-transplantation-mice-used-as-models

Research

JoVE Journal

Biology

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

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.

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Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

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This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

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

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Transuterine Fetal Tracheal Occlusion Model in Mice

The aim of this manuscript is to describe the microsurgical steps required to perform a heterotopic laryngeal transplant in mice. The advantages of this mouse model compared to other animal models of laryngeal transplantation are its cost-effectiveness and the availability of immunologic assays and data.

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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: A Model for Investigating Obliterative Airway Disease After Lung Transplantation

Learn heterotopic and orthotopic tracheal transplantation in mice to study obliterative airway disease development. These surgical models enable researchers to investigate airway healing mechanisms and transplant complications.

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease (Video) | JoVE Journal

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

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