Name: development of obliterative bronchiolitis in a murine model of orthotopic lung transplantation
Uploaded: 2012-07-10T12:00:00
Duration: 10 min 1 s
Description: Watch this Scientific Journal Video about Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation at JoVE.com

Funding sources: This work as supported by National Institutes of Health grants HL067177, HL096845, and P01AI084853 to D.S.W.

1. Donor Procedure
<ol>
 <li>All surgical procedures were performed utilizing sterile technique. No antibiotics are given to both donor and recipient mice.</li>
 <li>Induction of anesthesia of the donor mouse is initiated with 5% Isoflurane.</li>
 <li>The mouse is orotracheally intubated with a 20-gauge intravenous catheter and then placed on a rodent ventilator, using 100% oxygen at a rate of 125 breaths/minute and approximately 0.5 ml tidal volume (2% of its body weight).</li>
 <li>Anesthesia is maintained with inhaled 1-2% Isoflurane.</li>
 <li>The donor mouse is placed in a supine position, prepped with 70% alcohol.</li>
 <li>Laparosternotomy is performed as a combined midline and transverse incision.</li>
 <li>Heparin at 100 u/kg is injected into the Inferior vena cava (IVC), from just below the liver.</li>
 <li>The diaphragm is cut along the ventral costal attachment toward the spine, and the thoracic cavity is exposed by cutting both sides of the chest wall to the neck.</li>
 <li>After incising the IVC at the level of diaphragm, the right atrial appendage is cut and the lungs flushed with 2 ml of cooled (4 &deg;C) Lactated Ringer's injection and 0.1 ml of heparin via a transverse incision at the root of the pulmonary artery (PA) trunk.</li>
 <li>Arresting ventilation at two thirds of end-tidal inflation, the heart-lung block is excised and stored on ice (4 &deg;C).</li>
 <li>The donor left lung is prepared for the recipient by the attachment of cuffs. The pulmonary ligament is incised up to the Pulmonary Vein (PV). The hilum is brought out by removing esophagus and aorta.</li>
 <li>Identify the PA, located at the most cranial aspect of the hilum, as well as the attached main bronchus (Br). Carefully dissect the PA from the bronchus.</li>
 <li>PA cuff is made from a 24-gauge Intravenous (I.V.) catheter and cut to 0.5 mm in length with an extension of 0.7 mm.</li>
 <li>The entire surface of the cuff is then abraded to facilitate anastomosis.</li>
 <li>The cuff for the bronchus is derived from a 20-gauge IV catheter, and cut in length of 1.0 mm with an extension of 0.7 mm.</li>
 <li>Using the same materials, the PV cuff varies with the weight of the donor mouse. Specifically, for mice 24-27 grams the cuff size is 22-gauge, 0.7 mm in length with a 0.7 mm extension. For mice weighing 27-32 grams, the studies utilized a 20-gauge catheter that is 0.7 mm in length with a 0.7 mm extension.</li>
 <li>The cuffs are inserted into the distal ends of the PA, PV and Br and secured with a 9-0 suture.</li>
 <li>The donor lung is flushed and washed with sterile saline with sterile heparin before storage. The donor lung is then wrapped with sterile gauze soaked in sterile saline on ice (4 &deg;C), which keeps the lung very clean and sterile. </li>
 <li>A microvessel clamp is placed on the bronchus to prevent Lactated Ringer entry into the airway.</li>
</ol>
2. Recipient Procedure
<ol>
 <li>Induction of anesthesia and mechanical ventilation are the same as described for the donor above. </li>
 <li>The left chest wall is shaved and prepped with 70% alcohol and the surgical field is draped.</li>
 <li>A thoracotomy incision is made in the left third intercostal space, extending the incision dorsally close to the spine, and a microvessel clamp placed on the left pulmonary vessels and bronchus adjacent to the heart. You can see PA at the cranial aspect, PV at the caudal end of hilum and Br between them.</li>
 <li>Using gentle traction on the hemostat to cause mild tension on the PA, Br and PV, the left lung is pulled out from the thoracic cavity while leaving the central hilar structures clamped.</li>
 <li>The PA, PV and Br are isolated by blunt dissection followed by placing a 9-0 suture positioned loosely around the PA, PV and Br.</li>
 <li>After dissecting the PA completely from its adventitial sheath, a small transverse incision of approximately one fourth of the vessel's circumference is made into the anterior wall, leaving the continuation of the dorsal part of the artery intact.</li>
 <li>The donor lung, wrapped in cold, Lactated Ringer-soaked cotton gauze and prepared as described above, is then positioned into the thoracic cavity, and cuffs inserted into the recipient PA, PV and Br and secured with 9-0 suture.</li>
 <li>The hilar cross clamp is removed allowing for reperfusion and ventilation.</li>
 <li>After positioning the transplanted lung back into the recipient thorax, the thoracotomy incision is closed using a 5-0 suture.</li>
 <li>The mouse is allowed to recover from anesthesia. Buprenorphine (0.05-0.15 mg/kg) is administered immediately after surgery, and for every 8 hours for 2-3 days post-surgery.</li>
</ol>
3. Representative Results
Our experience has taught us that it requires several months of repeated practice to become proficient in the mouse lung transplant model. After proficiency is attained, we achieved a 96% (96/100 consecutive surgeries) perioperative survival rate with deaths occurring within seven days post operatively. Two deaths were due to bleeding that began intraoperatively, and pneumothraces were the cause of death in the other two mice. For all procedures, the warm ischemia time was 14.32 &plusmn; 3.14 minutes, and cold ischemia time was 58.51&plusmn; 18.06 minutes. Three orthotopic lung transplant groups were studied: isograft: C57BL/6&rarr;C57BL/6, allograft: C57BL/10&rarr;C57BL/6 and C57BL/6&rarr;C57BL/10. We used only male mice but our technique can be also applied to female mice, because there is no significant anatomical difference between the sexes.
Grading of rejection pathology was conducted in a blinded fashion utilizing standard criteria for clinical lung transplantation7(Table 1). Whereas we observed mild or no rejection in isograft (C57BL/6&rarr;C57BL/6), both allograft combinations developed comparable acute or chronic rejection (Figure 1). In contrast, OB was significantly more frequent in the C57BL/10&rarr;C57BL/6 than C57BL/6&rarr;C57BL/10 group by day28 (Table 1).
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>C57BL/6&rarr; 
 C57BL/6</td>
 <td>C57BL/10&rarr; 
 C57BL/6</td>
 <td>C57BL/6&rarr; 
 C57BL/10</td>
 <td>P-value</td>
 </tr>
 <tr>
 <td>Rejection Pathology 
 "A" Scores at day 28</td>
 <td>0.67&plusmn;0.89</td>
 <td>*3.33&plusmn;0.82</td>
 <td>*3.29&plusmn;0.76</td>
 <td>*P&gt;0.05</td>
 </tr>
 <tr>
 <td>OB/Total mice at day 21 and 28</td>
 <td>0/24 (0%)</td>
 <td>**14/34 (42.1%)</td>
 <td>**2/16 (12.5%)</td>
 <td>**P&lt;0.05</td>
 </tr>
 </tbody>
</table>
Table 1. Histological scores of acute rejection and prevalence of obliterative bronchiolitis post transplant. Scoring of acute rejection ("A" Scores) by standard criteria as described in representative results. Data represents the mean &plusmn; SD of "A" Scores at days 28 post transplantation. Data represents the quantity and percentage of mice in each group that developed OB at days 21 and 28 post transplantation.
<img alt="Figure 1" src="/files/ftp_upload/3947/3947fig1.jpg" /> 
Figure 1. Macroscopic findings and histopathology at 28 days post lung transplantation. Panel 1A represent macroscopic findings and H&amp;E stained Isograft lung and right na&iuml;ve lung. Panel 1B and 1C represents H&amp;E and Masson's Trichrome stained BL/10 lung allograft transplanted into BL/6 mouse recipient, which developed OB and non OB, respectively. The white arrow in 1B identifies lesions of OB. Panel 1D shows BL/6 lung allograft transplanted into BL/10 mouse recipient.
Main trouble shooting during the procedure were as follows.
<ol>
 <li>Blood flow disorder: Flush donor lung until the color becomes white in color. Use of excessive pressure could induce pulmonary edema. But do not push too much to prevent edema after transplant.</li>
 <li>Difficulty of cuff insertion into the donor: Ascertain that vasculature is of sufficient length and free of attached fat and connective tissues.</li>
 <li>Bleeding at anastomotic site: Use Q tips to apply pressure at affected site for approximately 5 minutes.</li>
 <li>Recipient PA blood flow obstruction: Mainly caused by torsion of the during cuff insertion. If air is observed within the PA then re-do of the anastomosis is necessary.</li>
 <li>Difficult PV anastomosis: Membrane of PV is very thin and easily tears. Select proper cuff size and insert straight to the recipient's PV very gently.</li>
 <li>Recipient PV blood flow obstruction: Make sure the position and direction of the cuff. Release the compression from the bronchial cuff.</li>
 <li>Pneumothorax: This may be caused by either bronchial perforation due to manipulation of the airway, or perforation of lung surface due to trauma associated with the transplant procedure. The former is treated with surgical closure of the hole within the airway wall; and the latter is treated by oversewing the leak on the lung surface using 10-0 nylon suture.</li>
</ol>

<ol>
	<li>Asimacopoulos, P. J., Molokhia, F. A., Pegg, C. A., Norman, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+transplantation+in+the+rat.">Lung transplantation in the rat.</a> Transplant Proc. 3, 583-585 (1971).</li><li>Yoshida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+technique+of+experimental+lung+transplantation+in+rabbits.">Surgical technique of experimental lung transplantation in rabbits.</a> Ann. Thorac. Cardiovasc. Surg. 11, 7-11 (2005).</li><li>McDyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+murine+obliterative+bronchiolitis+in+transplant.">Human and murine obliterative bronchiolitis in transplant.</a> Proc. Am. Thorac. Soc. 4, 37-43 (2007).</li><li>Sato, M., Keshavjee, S., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+research:+animal+models+of+obliterative+bronchiolitis+after+lung+transplantation.">Translational research: animal models of obliterative bronchiolitis after lung transplantation.</a> Am. J. Transplant. 9, 1981-1987 (2009).</li><li>Okazaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+orthotopic+vascularized+aerated+lung+transplantation.">A mouse model of orthotopic vascularized aerated lung transplantation.</a> Am. J. Transplant. 7, 1672-1679 (2007).</li><li>Fan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+IL-17+Prevents+Obliterative+Bronchiolitis+in+Murine+Orthotopic+Lung+Transplantation.">Neutralizing IL-17 Prevents Obliterative Bronchiolitis in Murine Orthotopic Lung Transplantation.</a> Am. J. Transplant. 11, 911-922 (2011).</li><li>Stewart, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revision+of+the+1996+working+formulation+for+the+standardization+of+nomenclature+in+the+diagnosis+of+lung+rejection.">Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection.</a> J. Heart Lung Transplant. 26, 1229-1242 (2007).</li><li>Mizuta, T., Kawaguchi, A., Nakahara, K., Kawashima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+rat+lung+transplantation+using+a+cuff+technique.">Simplified rat lung transplantation using a cuff technique.</a> J. Thorac. Cardiovasc. Surg. 97, 578-581 (1989).</li><li>Jungraithmayr, W. M., Korom, S., Hillinger, S., Weder, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+orthotopic,+single-lung+transplantation.">A mouse model of orthotopic, single-lung transplantation.</a> J. Thorac. Cardiovasc. Surg. 137, 486-491 (2009).</li><li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+vascularized+right+lung+transplantation+in+the+mouse.">Orthotopic vascularized right lung transplantation in the mouse.</a> J. Thorac. Cardiovasc. Surg. , (2010).</li></ol>

No conflicts of interest declared.

Orthotopic lung transplantation in mice is challenging due to microsurgical demands and extreme fragility of tissues. Introduction of the cuff technique has allowed for the widespread use of the orthotopic lung transplantation in rats8. This became the basis for the development of the orthotopic lung transplant model in mice in our lab. In mice and rats, unlike humans, the left lung contains only one lobe and makes up only 25% of the total lung mass. This makes left-single lung transplantation feasible in the murine model without the need for a circulatory support system.
Our preliminary surgeries revealed that ventilation and perfusion of the transplanted lung was highly dependent on the size of PV anastomosis. Specific PV anastomotic cuff size were utilized to match donor and recipients as reported9. Inappropriate cuff sizing resulted in either atelectasis of the transplanted lung or dehiscence of the PV anastomosis. Okazaki et al reported the use of combined vessel ligation and clipping the bronchus5. In the current study we use an aneurysm clip for all hilar structures, which we suggest may shorten warm ischemic time. One limitation of the orthotopic left lung transplant in the murine model is that recipient animals can survive after alloimmune-mediated necrosis of their allografts5. Therefore, survival studies are not feasible and graft assessment depends on histological examination of the transplanted lung10.
Using this technique, we have developed a preclinical obliterative bronchiolitis model in mice. Specifically, the donor mouse is C57BL/10 and the recipient is C57BL/6. This suggests the role of minor, and not major, histocompatibility antigens in obliterative bronchiolitis pathogenesis6. Furthermore, we have reported neutralizing IL-17 prevents obliterative bronchiolitis in the murine model. This model represents a novel research tool for the examination of lung transplantation and the advancement of clinical lung transplants.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Zeiss Opmi 6SFC-1880</td>
 <td>Prescott's Inc</td>
 <td>PMZ014</td>
 </tr>
 <tr>
 <td>Harvard Rodent Ventilator</td>
 <td>Harvard Apparatus</td>
 <td>55-7058</td>
 </tr>
 <tr>
 <td>20-gauge I.V. catheter</td>
 <td>Terumo Medical Corporation</td>
 <td>100510D</td>
 </tr>
 <tr>
 <td>22-gauge I.V. catheter</td>
 <td>Terumo Medical Corporation</td>
 <td>081015S</td>
 </tr>
 <tr>
 <td>24-gauge I.V. catheter</td>
 <td>Terumo Medical Corporation</td>
 <td>100522A</td>
 </tr>
 <tr>
 <td>9/0 Meth Blue Virgin Silk</td>
 <td>Ashaway Line &amp; Twine Mfg. CO</td>
 <td>MBVS-90</td>
 </tr>
 <tr>
 <td>10-0 Alcon Surgical Suture</td>
 <td>Alcon Laboratories, Inc</td>
 <td>8065-192101</td>
 </tr>
 <tr>
 <td>Black Braided Silk 5-0/18inches</td>
 <td>Henry Schein</td>
 <td>M652630</td>
 </tr>
 <tr>
 <td>Heparin Sodium 1000 units/ml</td>
 <td>APP Pharmaceuticals</td>
 <td>407156</td>
 </tr>
 <tr>
 <td>Betadine Solution</td>
 <td>Purdue Product</td>
 <td>67618-150</td>
 </tr>
 <tr>
 <td>Lactated Ringer's injection</td>
 <td>Hospira</td>
 <td>NDC 0409-7953-03</td>
 </tr>
 </tbody>
</table>

<table border="1">
 <tbody>
 <tr>
 <td>Strain of the mice</td>
 <td>Vendor</td>
 <td>Age, Weight</td>
 <td>Gender, Comments</td>
 </tr>
 <tr>
 <td>C57BL/10</td>
 <td>Harlan Sprague-Dawley</td>
 <td>8-12 weeks, 25-30g</td>
 <td>Male, Donor</td>
 </tr>
 <tr>
 <td>C57BL/6</td>
 <td>Harlan Sprague-Dawley</td>
 <td>8-12 weeks, 25-30g</td>
 <td>Male, Donor or recipient</td>
 </tr>
 </tbody>
</table>
Table 2. Table of mice, specific reagents and equipment.

development of obliterative bronchiolitis in a murine model of orthotopic lung transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Watch this Scientific Journal Video about Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation at JoVE.com

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

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.

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

development-obliterative-bronchiolitis-murine-model-orthotopic-lung

Research

JoVE Journal

Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...