Name: orthotopic liver transplantation in rats
Uploaded: 2012-07-01T14:00:00
Duration: 18 min 25 s
Description: Watch this Scientific Journal Video about Orthotopic Liver Transplantation in Rats at JoVE.com

CT was supported by the Swiss National Science Foundation (SCORE grant 3232230-126233). The study was supported by the Art&egrave;res Foundation, the Astellas Foundation Europe and the Boninchi Foundation.

1. Donor Operation
<ol>
 <li>Anesthetize male rats weighing 200&plusmn;20 grams with isoflurane on cone mask (3% for the induction, 2% during the operation, 1 l/min air flow, FiO2 70%). </li>
 <li>Perform a large median and transverse laparotomy and cauterize the epigastric vessels on both sides. </li>
 <li>Cut the falciform ligament, and separate the left diaphragmatic vein from the suprehepatic vena cava. Finally, divide the diaphragmatic vein between two 7-0 silk ligatures. </li>
 <li>Cut the left triangular and the gastro-hepatic ligaments. </li>
 <li>Divide the hepato-esophageal ligament and artery between 7-0 ligatures (coagulation is a valid alternative). </li>
 <li>Isolate the infrahepatic vena cava down to the left renal vein. Separate the right renal vein from the surrounding tissues and divide it between a 10-0 and a 7-0 ligature, on its proximal and distal ends, respectively. </li>
 <li>Divide the right suprarenal vein between 7-0 ligatures and free the liver from its posterior ligaments by cutting under gentle traction. </li>
 <li>Isolate the gastrosplenic vein and divide it between a 10-0 and a 7-0 ligature, on its proximal and distal ends, respectively. </li>
 <li>Divide the proper hepatic artery between 7-0 ties. </li>
 <li>Isolate the duodeno-pancreatic vein and divided it between a 10-0 and a 7-0 ligature, on its proximal and distal end, respectively. </li>
 <li>Insert a 22G, 3.5 mm stent into the common bile duct by practicing a small incision at 1 cm distance from the hepatic hilum. Secure the stent in position with a 7-0 ligature. </li>
 <li>Inject 10 UI of heparin, diluted into 1 ml of normal saline, through the dorsal vein of the penis. </li>
 <li>Cannulate the vena porta (as far as possible from the hilum) with a 21G needle and gently flush the liver with 20 ml of cold ringer lactate (or other preservation solutions). At the same time, cut the vena cava below the renal veins to allow an adequate outflow. Liver cold perfusion should last between one and two minutes. </li>
 <li>Cut the vena porta below the duodeno-pancreatic vein. </li>
 <li>Cut the common bile duct distally to the ligature around the stent. </li>
 <li>Complete the incision of the infrahepatic vena cava. </li>
 <li>Cut the suprahepatic vena cava skim to the diaphragm and remove the liver. </li>
 <li>Place the liver into a basin filled with cold ringer lactate and lay the basin on an ice pad. </li>
</ol>
2. Graft Preparation
<ol>
 <li>Insert the vena porta into its cuff, then evert the vessel around the edge to expose the intima. Secure the everted segment on the cuff with a 7-0 ligature. </li>
 <li>Repeat the same operation on the infrahepatic vena cava. </li>
 <li>Position a micro-clamp (4-6 mm length) on the proximal part of the infrahepatic vena cava. This operation avoids blood loss after portal reperfusion. </li>
 <li>Overturn the liver and insert two prolene 8-0 sutures at the opposite lateral edges of the suprahepatic vena cava, from outside to inside. </li>
 <li>Store the graft (immersed into ringer lactate solution) in the refrigerator at 0-4 &deg;C. </li>
</ol>
3. Recipient Operation
<ol>
 <li>Anesthetize male rats weighing 200&plusmn;20 grams with isoflurane on cone mask (3% for the induction, 2% during the operation, 1 l/min air flow, FiO2 70%) and keep on a warm pad. Of note, recipient and donor weights should be matched (&plusmn;40 grams). </li>
 <li>Inject 10 ml of normal saline subcutaneously and 0.03 g of piperacillin/tazobactam intramuscular before laparotomy. </li>
 <li>Perform a midline xipho-pubic laparotomy. </li>
 <li>Reverse the xiphoid process with an auto-static forceps in order to better expose the liver. </li>
 <li>Cut the anterior ligaments and divide the left diaphragmatic vein between two 7-0 ties. </li>
 <li>Divide the hepato-esophageal ligament and artery between 7-0 ligatures (coagulation is a valid alternative). Not shown in the video (identical to donor operation). </li>
 <li>Isolate the infrahepatic vena cava down to the right renal vein. </li>
 <li>Divide the right suprarenal vein between 7-0 ligatures and free the liver from its posterior ligaments by cutting under gentle traction (not shown in the video (identical to donor operation)). </li>
 <li>Place a 7-0 ligature around the common bile duct, just below its division. Put another ligature one millimeter below and cut in between. </li>
 <li>Isolate the hepatic artery at the hilum and divide it between 7-0 ligatures. </li>
 <li>Gently separate left and right portal branches. Pay attention not to damage the common hepatic artery or the gastro-splenic vein. </li>
 <li>Position the caval ring around the vena cava, cranially to the right renal vein. Then secure the vessel wall to the ring by placing four cardinal 9-0 sutures. </li>
 <li>Repeat the same operation with the portal ring on the vena porta. </li>
 <li>Inject 10 UI of heparin, diluted into 1 ml of normal saline, through the dorsal vein of the penis. </li>
 <li>Set the anesthesia on 0.25% and the FiO2 on 100%. </li>
 <li>Clamp the infrahepatic vena cava just above the right renal vein, then clamp the vena porta just above the gastro-splenic vein. Finally clamp the suprahepatic vena cava being sure to include at least 2 mm of diaphragm. </li>
 <li>Cut the suprahepatic vena cava close to the parenchyma. </li>
 <li>Cut the left and the right portal branches and divide the septum in between. </li>
 <li>Cut the infrahepatic vena cava close to the parenchyma, paying attention not to incise any of the sutures previously positioned. Remove the liver and place the graft into the abdominal cavity. </li>
 <li>Perform an end-to-end anastomosis between the suprahepatic vena cava of the graft and the recipient. Start with the running suture of the posterior wall from left to right, then proceed on the anterior wall from right to left. Keep the anterior suture loose until the end in order to allow a generous flushing of the interior lumen before tightening the ends. </li>
 <li>Insert the caudal branch of the approximator into the portal ring handle; then insert the vena porta of the graft into the slit of the cranial branch of the approximator, with the portal cuff finding its position into the slot. </li>
 <li>Close the approximator while flushing the recipient's vena porta with normal saline and allow the cuff to insert into the vessel. Secure the recipient's vena porta around its cuff with a 7-0 circumferential tie. </li>
 <li>Remove the approximator. Open the clamp on the suprahepatic vena cava and the vena porta in order: portal flow is now reestablished. </li>
 <li>Remove the portal ring by cutting the 9-0 ligatures with extra-fine scissors.</li>
 <li>Insert the caudal branch of the approximator into the caval ring handle; then insert the infrahepatic vena cava of the graft into the slit of the cranial branch of the approximator, with the caval cuff finding its position into the slot. </li>
 <li>Close the approximator while flushing the recipient's infrahepatic vena cava with normal saline and allow the cuff to insert into the vessel. Secure the recipient's vena cava around its cuff with a 7-0 circumferential tie. </li>
 <li>Remove the approximator. Open the microclamps on the infrahepatic vena cava of the graft and the recipient in order: venous flow is now reestablished. </li>
 <li>Perform a partial incision of the recipient's common bile duct, apply a gentle traction towards the head, and insert the free side of the stent previously inserted into the bile duct of the graft. Secure the stent with a circumferential 7-0 tie. </li>
 <li>Close the animal by layers with 5-0 sutures. Allow free water and food from wakening. </li>
 <li>Administer effective analgesia according to local institutional protocols. A minimum duration of 24 h is recommended. </li>
</ol>
Of note: end-to-end hepatic artery anastomosis can be performed, when required by the experimental design (the artery is not required for long-term survival after rat liver transplantation). 
4. Representative Results
The described technique could be successfully mastered by our team after approximately 10 training surgeries. It allowed 100% long-term (&gt;21 days) survival on 36 consecutive Lewis-to-Lewis and Dark Agouti-to-Lewis transplantations, after the short training period (a number of syngeneic liver graft recipients have been kept alive for over 12 months now). Post- syngeneic transplant day 1 liver function tests showed median aspartate transaminase (AST): 84 U/l (51.7 - 92.7) and median alanine transaminase (ALT): 172.5 U/l (127.5 - 240.2). The mean anhepatic phase (from vena porta clamping to graft re-perfusion) was 14 &plusmn;2 min, with the longest step being the suprahepatic vena cava anastomosis (running suture) time 9&plusmn;2 min.
The quick-linker system allowed the positioning of 1.55 mm-bore cuffs on vena porta and 2.40 mm-bore cuffs on vena cava (recipients weighing 200 &plusmn;20 g). By contrast, when we tried transplanting using Kamada's technique, they could not exceed 1.40 mm and 2.16 mm, respectively (rats matching for strain and weight). As shown in Figure 7, quick-linker rings are designed to keep recipient's vessels on optimal stretching, allowing minimum caliper and length wasting, which results in closer-to-physiological hemodynamic results. 
<img src="/files/ftp_upload/4143/4143fig1.jpg" alt="Figure 1" /> 
Figure 1. Rings measures.
<img src="/files/ftp_upload/4143/4143fig2.jpg" alt="Figure 2" /> 
Figure 2. Microclamps measures.
<img src="/files/ftp_upload/4143/4143fig3.jpg" alt="Figure 3" /> 
Figure 3. Quick-linker kit.
<img src="/files/ftp_upload/4143/4143fig4.jpg" alt="Figure 4" /> 
Figure 4. Quick-linker armed.
<img src="/files/ftp_upload/4143/4143fig5.jpg" alt="Figure 5" /> 
Figure 5. Quick-linker closed.
<img src="/files/ftp_upload/4143/4143fig6.jpg" alt="Figure 6" /> 
Figure 6. SHVC clamp.
<img src="/files/ftp_upload/4143/4143fig7.jpg" alt="Figure 7" /> 
Figure 7. Classic vs. quick-linker assisted cuff-anastomosis hemodynamics.
<img src="/files/ftp_upload/4143/4143fig8.jpg" alt="Figure 8" /> 
Figure 8. Donor and recipient's skin preparation and incision.

<ol>
	<li>Starzl, T. E., Marchioro, T. L., Porter, K. 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+and+clinical+observation+after+homotransplantation+of+the+whole+liver.">Experimental and clinical observation after homotransplantation of the whole liver.</a> Revue internationale d'hepatologie. 15, 1447 (1965).</li><li>Starzl, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+liver+transplantation.">Evolution of liver transplantation.</a> Hepatology. 2, 614S-636S (1982).</li><li>Kamada, N., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+rat.+Technique+using+cuff+for+portal+vein+anastomosis+and+biliary+drainage.">Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage.</a> Transplantation. 28, 47 (1979).</li><li>Kashfi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+various+techniques+of+orthotopic+liver+transplantation+in+the+rat.">A review of various techniques of orthotopic liver transplantation in the rat.</a> Transplantation proceedings. 37, 185-188 (2005).</li><li>Hori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+and+innovative+techniques+for+liver+transplantation+in+rats:+A+surgical+guide.">Comprehensive and innovative techniques for liver transplantation in rats: A surgical guide.</a> World Journal of Gastroenterology: WJG. 16, 3120 (2010).</li><li>Oldani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+rat+liver+transplantation+using+Quick+Linker+system:+a+preliminary+result.">A novel technique for rat liver transplantation using Quick Linker system: a preliminary result.</a> Journal of Surgical Research. 149, 303-309 (2008).</li><li>Nosaka, T., Bowers, J. L., Cay, O., Clouse, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+complications+after+orthotopic+liver+transplantation+in+rats.">Biliary complications after orthotopic liver transplantation in rats.</a> Surg. Today. 29, 963-965 (1999).</li><li>Li, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+incidence+of+biliary+complications+in+rat+liver+transplantation:+can+we+avoid+it.">High incidence of biliary complications in rat liver transplantation: can we avoid it.</a> World J. Gastroenterol. 17, 3140-3144 (2011).</li><li>Peng, Y., Gong, J. P., Yan, L. N., Li, S. B., Li, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+two-cuff+technique+for+orthotopic+liver+transplantation+in+rat.">Improved two-cuff technique for orthotopic liver transplantation in rat.</a> Hepatobiliary Pancreat. Dis. Int. 3, 33-37 (2004).</li><li>Urata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+survival+in+rat+liver+transplantation+with+extended+cold+preservation:+role+of+portal+vein+clamping+time.">Decreased survival in rat liver transplantation with extended cold preservation: role of portal vein clamping time.</a> Hepatology. 28, 366-373 (1998).</li></ol>

No conflicts of interest declared.

The present rat liver transplantation model can be easily established. In our experience, a few key points help minimize the risk of death (&#x2264;20%). Early deaths, occurring within the first three hours from reperfusion, are most often due to a bowel infarction, as a consequence of a long portal clamping. It is therefore recommended to keep the anhepatic phase within the ideal limit of 15 minutes (20 minutes still acceptable). Central air embolism can also be a cause of sudden death and it is compulsory to flush all veins prior to anastomosis. 
Deaths occurring between postoperative day one and three are often due to liver failure or thrombosis of the vena cava. One should therefore avoid any aggressive graft flushing before the explantation in the donor (we flush with 20 ml over approximately 60 seconds) and ensure an appropriate cold preservation (full immersion into 0-4 &deg;C ringer lactate solution). The risk of thrombosis can be decreased by minimizing the manipulation of the intima and preserving its integrity. We do not recommend the use of anticoagulation after surgery.
Deaths between postoperative day four and nine are often related to bacterial cholangitis7. Based on our experience, a single dose of piperacilline/tazobactam (0.03 g) given before laparotomy is enough to minimize the risk of common bile duct infection.
Death is a rare event after day 10. However, due to absence of arterial flow, some bile duct problems can be observed with cholastasis8. Depending on the needs of the investigator an arterialization may be considered9. Of note, a cold ischemia time up to 24 hours can be considered as acceptable10.
The quick-linker system was originally designed to perform liver transplantation with three cuff anastomoses (very short anhepatic phase). However, the three-cuff technique requires the use of very small calipers for the suprahepatic vena cava (SHVC) cuff and it has not been widely accepted due to the risk of liver outflow problems11-14. We therefore favor a quick-linker assisted three-cuff technique only when warm ischemia times shorter than ten minutes are required. In most studies however, implantation phases up to 15-18 minutes are acceptable, allowing the use of the two-cuff technique, and a more physiological SHVC drainage. As shown in this video paper, we routinely use the quick-linker system in the second part of the implantation only. While some extra surgical time is required to position the quick-linker handles, this part of the procedure is associated with minimal risks and allows for a shortening of the critical anhepatic phase thanks to easier cuffs insertions. Other advantages of the quick-linker system are a better graft-recipient vessel alignment and the use of wider cuffs with better hemodynamic results compared to the previously described techniques (including the one described by Kamada et al.)6(Fig 7).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
 <tbody>
 <tr>
 <td> Tool 				/ Reagent</td>
 <td> Company</td>
 <td> Catalogue 				n.</td>
 <td> Comment</td>
 </tr>
 <tr>
 <td>Portal 				cuff</td>
 <td> <a href="http://altecweb.com" target="_blank">altecweb.com</a> 				or
 <a href="http://smiths-medical.com" target="_blank">smiths-medical.com</a></td>
 <td> Coil 				01-96-1729
 800/100/420</td>
 <td> Bore 				: 1.55mm 
 
 Wall 				: 0.30mm 
 
 Length: 				3.5 mm</td>
 </tr>
 <tr>
 <td>Infrahepatic 				v. cava cuff</td>
 <td> <a href="http://altecweb.com" target="_blank">altecweb.com</a> 				or
 <a href="http://smiths-medical.com" target="_blank">smiths-medical.com</a></td>
 <td> Coil 				01-96-1733
 800/100/540</td>
 <td> Bore 				: 2.40mm 
 
 Wall 				: 0.30mm
 
 Length: 				3.5 mm</td>
 </tr>
 <tr>
 <td>Common 				bile duct stent</td>
 <td>common 				22G venflon</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>7-0 				silk ties</td>
 <td><a href="http://georgetiemann.com" target="_blank">georgetiemann.com</a></td>
 <td>160-1226-7/0</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10-0 				prolene</td>
 <td><a href="http://ethicon.com" target="_blank">ethicon.com</a></td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>9-0 				prolene</td>
 <td><a href="http://ethicon.com" target="_blank">ethicon.com</a></td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>8-0 				prolene</td>
 <td><a href="http://ethicon.com" target="_blank">ethicon.com</a></td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Straight 				micro forceps</td>
 <td>s-and-t.net</td>
 <td>FRC-15RM-8 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Curved 				micro forceps</td>
 <td>s-and-t.net</td>
 <td>FRS-15-RM-8 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dissection 				needle-holder</td>
 <td>s-and-t.net</td>
 <td>B-15-8.2 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anastomosis 				needle-holder</td>
 <td>rumex.net</td>
 <td>8-021T</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro 				clamps</td>
 <td><a href="http://finescience.com" target="_blank">finescience.com</a></td>
 <td>18055-03</td>
 <td>6 				x 1mm</td>
 </tr>
 <tr>
 <td>Micro 				clamps applicator</td>
 <td><a href="http://finescience.com" target="_blank">finescience.com</a></td>
 <td>18057-14</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Suprahepatic 				v. cava clamp</td>
 <td><a href="http://pharmap.ch" target="_blank">pharmap.ch</a></td>
 <td>13.349.14</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Quick-Linker 				rings</td>
 <td> <a href="http://dltchiropody.co.uk" target="_blank">dltchiropody.co.uk</a></td>
 <td> PAR 				10</td>
 <td> modified 				scalpel blades</td>
 </tr>
 <tr>
 <td> Quick-Linker 				approximator</td>
 <td>pharmap.ch</td>
 <td> UD-04-275</td>
 <td> modified 				Kocher's forcep</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Orthotopic Liver Transplantation in Rats at JoVE.com

Orthotopic Liver Transplantation in Rats

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 ...

Research

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Medicine

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.

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 ...

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 Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

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 ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

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 ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the&#160;in vivo&#160;validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. ...