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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Research

JoVE Journal

Medicine

14.1K Views.  University of Bonn, Germany. Малый трансплантации кишечника стало общепринятым вариантом лечения для пациентов с необратимыми кишечной недостаточности. Наши экспериментальные модели ортотопической небольшой трансплантации кишечника у крыс служит надежным инструментом для решения о...

Video: Orthotopic Small Bowel Transplantation in Rats

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 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 ...

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. ...

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 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 ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

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. ...

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential model to investigate the mechanism of renal allograft rejection in pre-clinical studies and could aid in the development of novel approaches to improve the long-term survival of renal allografts. Donor kidney implantation in rat orthotopic renal transplantation is commonly performed by end-to-side anastomosis to recipients' aorta and inferior vena cava. In this model, the donor's kidney was implanted using end-to-end anastomosis to the recipients' renal artery and renal vein. The donor's ureter was anastomosed to the recipient's bladder in an end-to-side 'tunnel' method. This model contributes to better healing of ureter-bladder anastomosis and increases the recipients' survival by avoiding interference with blood supply and venous reflux of the lower body. This model can be used to investigate the mechanisms of acute and chronic immune and pathologic rejection of renal allografts. Here, the study describes the detailed protocols of this orthotopic renal transplantation between rats.

The rat orthotopic renal transplantation model contributes to investigating the mechanism of renal allograft rejection. The current model increases the recipients' survival without interference with blood supply and venous reflux of the lower body using an end-to-end anastomosis of kidney implantation and an end-to-side "tunnel" method of ureter-bladder anastomosis.

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential ...

Studying Murine Small Bowel Mechanosensing of Luminal Particulates

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion and absorption. For this reason, some of the motility patterns in the small bowel include segmentation for mixing of luminal contents and peristalsis for their propulsion. Physical properties of luminal contents modulate the patterns of small bowel motility. The mechanical stimulation of GI mechanosensory circuits by transiting luminal contents and underlying gut motility initiate and modulate complex GI motor patterns. Yet, the mechanosensory mechanisms that drive this process remain poorly understood. This is primarily due to a lack of tools to dissect how the small bowel handles materials of different physical properties. To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit. We gavage live mice with fluorescent liquid or tiny fluorescent beads. After 30 minutes, we dissect out the bowels to image the distribution of fluorescent contents across the entirety of the GI tract. In addition to high-resolution measurements of the geometric center, we use variable size binning and spectral analysis to determine how different materials affect small bowel transit. We have explored how a recently discovered "gut touch" mechanism affects small bowel motility using this approach.

To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion ...