1. Donor Operation
<ol>
	<li>The donor rat should be kept fasting for 24 hr (free access to water/glucose solution).</li>
	<li>To induce Isoflurane inhalation anesthesia, start with 2% on standard atomizer, and then reduce to 1% after performing laparotomy. Perform a toe pinch to check sedation.</li>
	<li>Shave the abdomen and clean with skin prep 3 times (Kodan). Then perform a median incision after subcutaneous administration of the analgesic.</li>
	<li>After the graft is wrapped in saline soaked gauze, separate the physiological adhesions between the pancreas and the ascending colon gently with a Q-tip (under the surgical microscope with 6x magnification).</li>
	<li>Ligate and divide the ileocecal &amp; right &amp; middle colic vessels using 7-0 silk. After the ascending colon is spread out to the right side of the superior mesenteric vein (SMV), the ileocecal, right and middle colic vessels can be identified for ligation and division using 7-0 ties.</li>
	<li>Retract the stomach upward, so that the entire SMV is straightened out and exposed. Use a mosquito clamp for retraction. The clamp is held by plasticine mass formed into shape as needed.</li>
	<li>Ligate and divide the pancreatico-duodenal veins coming from the SMV. All small pancreatico-duodenal veins originating from the SMV must be carefully identified, ligated with 7-0 silk and divided before the pancreatic tissue can be removed from the graft.</li>
	<li>Ligate and divide the lose connective tissue including all lymphatics between the SMV and the abdominal aorta. With the graft still on the right side of the abdomen, the lose connective tissue including all lymphatics between the SMV and the abdominal aorta is accessible. This connective tissue must be ligated using 7-0 silk and divided to avoid postoperative lymphorrhea from the intestinal graft.</li>
	<li>Ligate and divide the right renal artery. After the connective tissue is divided, the right renal artery becomes accessible and is ligated and divided using 7-0 silk.</li>
	<li>Systemically heparinise the rat using 200 units of heparin i.v. via the penile vein.</li>
	<li>Ligate the marginal arteries, and divide the small bowel at the duodeno-jejunal junction and at the terminal ileum.</li>
	<li>The aorta is ligated proximally to the origin of the SMA. The portal vein is transected at the confluence with the splenic vein. Then the graft is harvested with its vascular pedicle consisting of the SMA with an aortic segment.</li>
</ol>
2. Backtable Procedure
<ol>
	<li>Perfuse the graft using 3 ml of University of Wisconsin solution (UW) at 4 &deg;C via the aortic conduit and irrigate the intestinal lumen from the jejunal end with 30 ml of Uro-Nebacetin N solution at 4 &deg;C (lumen irrigation is mandatory).</li>
</ol>
Immediately after extracting the graft, the aortic conduit is used for perfusion with 3 ml chilled UW solution. For this, a 20 G i.v. catheter on a 10 ml syringe is used. The perfusate should be observed to flow out freely from the divided portal vein. For the intestinal irrigation with Nebacetin, a 50 ml syringe is used.
<ol start="2">
	<li>Store the graft in UW solution at 4 &deg;C during the preparation of the recipient.</li>
</ol>
3. Recipient Operation
<ol>
	<li>The recipient rat should be kept fasting for 24 hr (free access to water/glucose solution).</li>
	<li>To induce Isoflurane inhalation anesthesia, start with 2% on standard atomizer, and then reduce to 1% after performing laparotomy. Perform a toe pinch to check sedation.</li>
	<li>Shave the abdomen and clean with skin prep 3 times (Kodan). Then perform a median incision after subcutaneous administration of Carprofen 5 mg/kg s.c. for intraoperative analgesia.</li>
	<li>Wrap the recipient bowel in normal saline soaked gauze and place it on the recipient's chest.</li>
	<li>Open the retroperitoneum bluntly with Q-tips, and expose the abdominal aorta and inferior vena cava just below the renal vessels down to the level of the iliac vessels, if necessary also use microscissors. Ligate the small lumbar and spinal tribuaries from the aorta and vena cava using 7-0 silk to avoid blood loss. (To do this, change the surgical microscope zoom to 16x.)</li>
	<li>Cross-clamp the aorta and the IVC below the left renal vessels proximally and above the iliac bifurcation distally using microvessel clips. Only one clamp is used proximally as well as distally to clamp both vessels simultaneously. Incise both vessels anteriorly using a microknife and wash out remaining blood.</li>
	<li>Create an end-to-side anastomosis between the graft aortic segment and the recipient's infrarenal aorta using a continuous 10-0 Prolene suture.. Initially, the graft is placed on the the right side of the abdomen (the head of the rat positioned at 12 o'clock) to perform the back wall stitches of the arterial anastomosis and tying of the lower stay suture. Then, the graft is turned over to the left side of the abdomen (the head of the rat positioned at 9 o'clock) to expose and suture the front wall of the anastomosis.</li>
	<li>An end-to-side anastomosis between the graft portal vein and the recipient's IVC is performed likewise by running sutures using 10-0 Prolene. With the rat still lying sideways (head in 9 o'clock position), the graft is positioned on the left side of the abdomen and a lower stay suture is placed. The anastomosis is started with the back wall from inside the vessel. After the lower stay suture is tied, the front wall stitches can be performed from outside.</li>
	<li>Remove the distal clamps first, followed by the upper clamps. Any anastomotic bleeding is controlled by direct pressure using Q-tips. The graft should be checked for equal and quick reperfusion.</li>
	<li>Resect the entire recipient's small intestine after ligation of the mesenteric vessels. Recipients undergo subtotal enterectomy, preserving 2-3 cm of proximal jejunum and 1 cm of distal ileum.</li>
	<li>Restore enteric continuity by proximal (jejuno-jejunostomy) and distal (ileo-ileostomy) end-to end intestinal anastomoses using an interrupted one-layer suture with 6-0 Monocryl. Approximately 16 sutures are needed to complete the anastomoses.</li>
	<li>Irrigate the peritoneal cavity with normal saline until clean. Administer 2 ml of normal saline intraperitoneally for fluid replacement. Then close the abdomen using a continuous suture with 3-0 Vicryl for the muscle layer plus a continuous skin suture.</li>
	<li>In the postoperative period the rats should be kept fasting (with access to water and glucose solution) for another 24 hr then restarted on standard rat chow and water ad libitum. Analgesia with carprofen should be administered for 3 days (see dosage below).</li>
</ol>

<ol>
	<li>Abu-Elmagd, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+Hundred+Intestinal+and+Multivisceral+Transplantations+at+a+Single+Center:+Major+Advances+With+New+Challenges.">Five Hundred Intestinal and Multivisceral Transplantations at a Single Center: Major Advances With New Challenges.</a> Ann. Surg. 250 (4), 567-56 (2009).</li><li>Berney, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+versus+systemic+drainage+of+small+bowel+allografts:+comparative+assessment+of+survival,+function,+rejection,+and+bacterial+translocation.">Portal versus systemic drainage of small bowel allografts: comparative assessment of survival, function, rejection, and bacterial translocation.</a> J. Am. Coll. Surg. 195 (6), 804 (2002).</li><li>Fishbein, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+transplantation.">Intestinal transplantation.</a> N. Engl. J. Med. 361 (10), 998 (2009).</li><li>Hernandez, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+portal+venous+outflow+better+than+systemic+venous+outflow+in+small+bowel+transplantation?+Experimental+study+in+syngeneic+rats.">Is portal venous outflow better than systemic venous outflow in small bowel transplantation? Experimental study in syngeneic rats.</a> J. Pediatr. Surg. 40 (2), 336 (2005).</li><li>Monchik, G. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+small+bowel+in+the+rat:+technical+and+immunological+considerations.">Transplantation of small bowel in the rat: technical and immunological considerations.</a> Surgery. 70 (5), 693 (1971).</li><li>Murase, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft-versus-host+disease+after+brown+Norway-to-Lewis+and+Lewis-to-Brown+Norway+rat+intestinal+transplantation+under+FK506.">Graft-versus-host disease after brown Norway-to-Lewis and Lewis-to-Brown Norway rat intestinal transplantation under FK506.</a> Transplantation. 55 (1), 1 (1993).</li><li>Murase, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulation+of+intestinal+transplant+with+allograft+irradiation+and+simultaneous+donor+bone+marrow+infusion.">Immunomodulation of intestinal transplant with allograft irradiation and simultaneous donor bone marrow infusion.</a> Transplant Proc. 31 (1-2), 565 (1999).</li><li>Pech, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perioperative+infliximab+application+ameliorates+acute+rejection+associated+inflammation+after+intestinal+transplantation.">Perioperative infliximab application ameliorates acute rejection associated inflammation after intestinal transplantation.</a> Am. J. Transplant. 10 (11), 2431 (2010).</li><li>Schaefer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+macrophages+are+involved+in+intestinal+transplantation-associated+inflammation+and+motoric+dysfunction+of+the+graft+muscularis.">Resident macrophages are involved in intestinal transplantation-associated inflammation and motoric dysfunction of the graft muscularis.</a> Am J Transplant. 7 (5), 1062 (2007).</li><li>Schaefer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+impact+of+organ+harvesting+and+ischemia-reperfusion+injury+within+the+graft+muscularis+in+rat+small+bowel+transplantation.+Transplant.">Mechanism and impact of organ harvesting and ischemia-reperfusion injury within the graft muscularis in rat small bowel transplantation. Transplant.</a> Proc. 38 (6), 1821 (2006).</li><li>Schaefer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+rejection+and+the+muscularis+propria+after+intestinal+transplantation:+the+alloresponse,+inflammation,+and+smooth+muscle+function.">Acute rejection and the muscularis propria after intestinal transplantation: the alloresponse, inflammation, and smooth muscle function.</a> Transplantation. 85 (10), 1465 (2008).</li><li>Xue, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+experience+of+refined+3-cuff+technique+for+orthotopic+small-bowel+transplantation+in+rat:+a+report+of+270+cases.">Surgical experience of refined 3-cuff technique for orthotopic small-bowel transplantation in rat: a report of 270 cases.</a> Am. J. Surg. 198 (1), 110 (2009).</li><li>Zhang, X. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+techniques+in+rat+heterotopic+small+bowel+transplantation.">Simplified techniques in rat heterotopic small bowel transplantation.</a> Transplant. Proc. 38 (6), 1840 (2006).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+establishment+of+a+new+en+bloc+combined+liver-small+bowel+transplantation+model+in+rats.+Transplant.">The establishment of a new en bloc combined liver-small bowel transplantation model in rats. Transplant.</a> Proc. 39 (1), 278 (2007).</li></ol>

No conflicts of interest declared.

While intestinal transplant models in rats have been described as early as in the 1970ies 5 most of the recently employed models involve heterotopic intestinal transplantation using different techniques 13. While the heterotopic procedures in general have the advantage of easier microsurgical techniques and easier accessibility of the graft for assessment, heterotopic intestinal transplantation has the big disadvantage of not taking into account the multiple interactions of the transplanted s...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>University of Wisconsin (UW) solution (ViaSpan)</td>
 <td>Bristol-Myers Squibb </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Uro-Nebacetin N solution</td>
 <td>Nycomed</td>
 <td>6967855</td>
 <td></td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Ratiopharm</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Carprofen (Rimadyl)</td>
 <td>Pfizer</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Prolene 10-0 unresorbable suture</td>
 <td>Ethicon</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Monocryl 6-0 resorbable suture</td>
 <td>Ethicon</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vicryl 3-0 resorbable suture</td>
 <td>Ethicon</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>i.v. Catheter G 20 1.1x33 mm</td>
 <td>Braun</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>i.v. Catheter G 22 0.9x25 mm</td>
 <td>Braun</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Kodan Skin Prep</td>
 <td>Sch&uuml;lke</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl 0.9% Infusion solution</td>
 <td>Braun</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Curved forceps small </td>
 <td>FineScienceTools</td>
 <td>11009-13 </td>
 <td></td>
 </tr>
 <tr>
 <td>Micro forceps curved </td>
 <td>AESCULAP </td>
 <td>BD 333 </td>
 <td></td>
 </tr>
 <tr>
 <td>Micro forceps curved </td>
 <td>AESCULAP </td>
 <td>FD281R </td>
 <td></td>
 </tr>
 <tr>
 <td>Micro forceps straight 1 </td>
 <td>WPI</td>
 <td>5</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro forceps straight 2 </td>
 <td>WPI</td>
 <td>2</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro needle holder </td>
 <td>WPI </td>
 <td>14081 </td>
 <td></td>
 </tr>
 <tr>
 <td>Micro scissors</td>
 <td>FineScienceTools</td>
 <td>15006-09 </td>
 <td></td>
 </tr>
 <tr>
 <td>Micro scalpel </td>
 <td>MANI</td>
 <td>Ophthalmic knife</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro clamps</td>
 <td>AESCULAP </td>
 <td>FB329R </td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Normal postoperative course
The transplanted animals should recover quickly from the procedure under a heat lamp for approximately 1 hr. Hypothermia is a major cause of postoperative mortality and should be carefully avoided intra- and postoperatively. Intraoperative fluid losses must be replaced by s.c. injection of 2.5 ml normal saline plus 2.5 ml Glucose 5% every 8 hr for the first 24 hr. The rats should also have free access to glucose solution (or gel diet) and water p.o. for the first 24 hr...

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

Orthotopic Small Bowel Transplantation in Rats

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 ...

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14.1K Views.  University of Bonn, Germany. 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.