Authors thank Northwestern University Comprehensive Transplant Center and the Feinberg School of Medicine Research Cores program for resource and funding support.&#160;Specifically, flow Cytometry and histology services were provided by the Northwestern University Flow Cytometry Core Facility and Mouse Histology and Phenotyping Laboratory, respectively, both of which are supported by NCI P30-CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. We thank Mr. Nate Esparza for proofreading this manuscript.

All procedures and animal use in this study were performed according to protocols approved by the Northwestern University Internal Animal Care and Use Committee (IACUC). In this study, 8 to 10 week old male CD45.2 and CD45.1 mice (both on BALB/c background, from Jackson laboratory) were used as spleen donors and recipients, respectively, to create syngeneic spleen transplantation models. All animals were housed in the sterile environment in the animal facilities of Northwestern University. The eye lubricant was applied to all mice post-anesthetization to prevent dryness.
1. Surgical Preparation, Anesthetization, and Analgesia Regimen
<ol>
	<li>Place a sterile disposable drape (45.7 cm x 66 cm) on the surgical platform. Gently grab the mouse, inject ketamine (50 mg/kg) and xylazine (10 mg/kg) intraperitoneally (i.p) for anesthesia, and inject 0.05 mg/kg buprenorphine subcutaneously for analgesia.</li>
	<li>Ensure the depth of anesthesia by toe-pinch, shave the hair in the whole abdomen area with a razor and place the mouse on the sterile surgical platform under the operating microscope at 6-10x magnification.</li>
</ol>
2. Donor Spleen Harvest
<ol>
	<li>Sterilize the abdomen with an alcohol prep pad, secure the limbs with surgical tape, and make a 3-4 cm midline vertical skin incision from the pubis to the xiphoid process with scissors.</li>
	<li>Retract the abdominal wall with sterile retractors made from paperclips. Move the intestines to the right flank of the abdomen (surgeon&#8217;s left) side with a sterile cotton swab to expose the spleen. Cauterize the short gastric vein attached to the spleen with a sterile low temperature cautery (Figure 1A). Place a piece of sterile gauze soaked with 37 &#176;C saline over the spleen to keep it moist (Figure 1B).</li>
	<li>Separate and mobilize the portal vein from the pancreatic tissue (Figure 1C) by ligating the portal vein branches (superior pancreaticoduodenal vein and right gastric vein); place a suture around the portal vein distal from the splenic vein (Figure 1D).</li>
	<li>Flip the spleen to the right side to expose the aorta and celiac trunk with the splenic artery (Figure 1E). Dissect and mobilize aortic-celiac-splenic artery by ligating the hepatic artery and gastric artery; place a suture around the aorta proximal to the celiac artery (Figure 1F-G).</li>
	<li>Inject 100 international units (IU) heparin into the inferior vena cava (IVC) to heparinize the whole body and wait 3 min to ensure the heparin take effects. Ligate the aorta proximal to celiac artery, transect the portal vein, and then perfuse the whole body using 10 mL of heparinized cold (4 &#176;C) saline (10 mL/20 s) from the abdominal aorta distal to celiac trunk (Figure 1H).</li>
	<li>Collect the spleen graft en bloc with the associated aortic-celiac-splenic segment and the portal vein along with a segment of splenic vein and a small portion of pancreatic tissue. Preserve the graft in 5 mL of 4 &#176;C saline before transplant. Euthanize the mouse by cervical dislocation.</li>
</ol>
3. Recipient Splenectomy and Spleen Graft Implantation
<ol>
	<li>Place a heating pad on the surgical platform and adjust the temperature to 37 &#176;C. Place a sterile drape (45.7 cm x 66 cm) on top of the heating pad to create a sterile surgical platform. Repeat steps 2.1 and 2.2 for surgical preparation and anesthetization. Make a 3-4 cm midline incision and retract the abdominal wall as described in step 2.1 and step 2.2.</li>
	<li>Carefully move the intestine to right side of the mouse with a sterile cotton swab to expose the recipient&#8217;s spleen. Ligate the splenic vein and artery and remove the spleen.</li>
	<li>Carefully move the intestine to left side of the mouse and cover the intestines with wet gauze (soaked with sterile 37 &#176;C saline). Dissect and ligate the lumbar branches of the infrarenal aorta and IVC; cross-clamp the infrarenal aorta and IVC by using two 4 mm microvascular clamps.</li>
	<li>Place an 11-0 nylon suture through the infrarenal aorta (a full thickness) and retract to create an elliptical aortotomy by a single cut with microscissors (the length should match the diameter of the donor aorta, Figure 2A). Pierce the IVC using a 30 G needle to create an elliptical venotomy and extend the opening to donor portal vein-matched length using microscissors (Figure 2A).</li>
	<li>Clear the intraluminal blood or blood clot (in the aorta and IVC) with 500 &#956;L of heparinized saline (10 units/mL).</li>
	<li>Place the spleen graft in the right flank of the recipient mouse abdomen; carefully identify the donor&#8217;s aortic cuff and the donor&#8217;s portal vein. After making sure that the vessels are not twisted, cover the spleen graft with gauze soaked with cold (4 &#176;C) saline.</li>
	<li>Connect the donor&#8217;s aortic cuff to the proximal and distal apex of the recipient&#8217;s aortaotomy with two stay sutures (11-0 nylon suture, same as below) (Figure 2B,C). Make an anastomosis with 2-3 bites of continuous 11-0 nylon sutures between the donor&#8217;s aortic cuff and the recipient&#8217;s aortaotomy (anterior wall) (Figure 2D). Turn the spleen graft over to the left side of the recipient; make the anastomosis between the donor&#8217;s aortic cuff and the recipient&#8217;s aortotomy (posterior wall) (Figure 2E).</li>
	<li>Perform an anastomosis to connect the donor&#8217;s portal vein to the posterior wall of the recipient&#8217;s IVC, using 4 to 5 bites of continuous sutures on the inside of the IVC and then close the suture on the outside of the IVC (Figure 2F,G).</li>
	<li>Release the vessel clamps and use sterile cotton swab to tamponade bleeding until the spleen color is recovered (Figure 2H).</li>
	<li>Close the abdomen with a 5-0 synthetic absorbable vicryl suture in a continuous pattern. Close the skin layer with a 5-0 nylon suture in an interrupted pattern. 
	NOTE: For the steps 4.7-4.8, alternatively, make an anastomosis between the donor&#8217;s portal vein and the recipient&#8217;s IVC first (step 4.8); then make an anastomosis between the donor&#8217;s aortic cuff and the posterior wall of the recipient&#8217;s aortotomy, using 2 to 3 continuous sutures in the inside of the aorta and close the suture on the outside of the aorta.</li>
</ol>
4. Animal Recovery
<ol>
	<li>Inject 1 mL of warm saline subcutaneously via 4 separate locations (0.25 mL/location) after closing the abdomen.</li>
	<li>Keep the mouse in a temperature-controlled incubator (30 &#176;C) for the first few hours post-operation, monitor the mouse until it has regained sufficient consciousness, and then transfer the mouse to a new clean cage with regular food and water, with a heating pad (30 &#176;C) underneath the cage. Keep the mouse post-surgery in a separate cage.</li>
</ol>
5. Post-surgical Pain Management
<ol>
	<li>Inject 0.05 mg/kg buprenorphine subcutaneously 24 h and 48 h post-surgery to maintain analgesia regimen.</li>
</ol>

<ol>
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P., Bagias, G., Liakakos, T., Machairas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+splenectomy:+Current+concepts.">Laparoscopic splenectomy: Current concepts.</a> World Journal of Gastrointestinal Endoscopy. 9 (9), 428-437 (2017).</li><li>Kristinsson, S. Y., Gridley, G., Hoover, R. N., Check, D., Landgren, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+risks+after+splenectomy+among+8,149+cancer-free+American+veterans:+a+cohort+study+with+up+to+27+years+follow-up.">Long-term risks after splenectomy among 8,149 cancer-free American veterans: a cohort study with up to 27 years follow-up.</a> Haematologica. 99 (2), 392-398 (2014).</li><li>Thai, L. H., 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=Long-term+complications+of+splenectomy+in+adult+immune+thrombocytopenia.">Long-term complications of splenectomy in adult immune thrombocytopenia.</a> Medicine (Baltimore). 95 (48), e5098 (2016).</li><li>Sinwar, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overwhelming+post+splenectomy+infection+syndrome+-+review+study.">Overwhelming post splenectomy infection syndrome - review study.</a> International Journal of Surgery. 12 (12), 1314-1316 (2014).</li><li>Rorholt, M., Ghanima, W., Farkas, D. K., Norgaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+cardiovascular+events+and+pulmonary+hypertension+following+splenectomy+-+a+Danish+population-based+cohort+study+from+1996-2012.">Risk of cardiovascular events and pulmonary hypertension following splenectomy - a Danish population-based cohort study from 1996-2012.</a> Haematologica. 102 (8), 1333-1341 (2017).</li><li>Crary, S. E., Buchanan, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+complications+after+splenectomy+for+hematologic+disorders.">Vascular complications after splenectomy for hematologic disorders.</a> Blood. 114 (14), 2861-2868 (2009).</li><li>Di Carlo, I., Pulvirenti, E., Toro, 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+new+technique+for+spleen+autotransplantation.">A new technique for spleen autotransplantation.</a> Surgical Innovation. 19 (2), 156-161 (2012).</li><li>Holdsworth, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+spleen+and+splenic+autotransplantation.">Regeneration of the spleen and splenic autotransplantation.</a> British Journal of Surgery. 78 (3), 270-278 (1991).</li><li>Tzoracoleftherakis, E., Alivizatos, V., Kalfarentzos, F., Androulakis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+of+splenic+tissue+reimplantation.">Complications of splenic tissue reimplantation.</a> Annals of the Royal College of Surgeons of England. 73 (2), 83-86 (1991).</li><li>Kato, 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=Transplantation+of+the+spleen:+effect+of+splenic+allograft+in+human+multivisceral+transplantation.">Transplantation of the spleen: effect of splenic allograft in human multivisceral transplantation.</a> Annals of Surgery. 246 (3), 436-444 (2007).</li><li>Aronovich, A., 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=Correction+of+hemophilia+as+a+proof+of+concept+for+treatment+of+monogenic+diseases+by+fetal+spleen+transplantation.">Correction of hemophilia as a proof of concept for treatment of monogenic diseases by fetal spleen transplantation.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (50), 19075-19080 (2006).</li><li>Cohen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splenosis;+review+and+report+of+subcutaneous+splenic+implant.">Splenosis; review and report of subcutaneous splenic implant.</a> Archives of surgery. 69 (6), 777-784 (1954).</li><li>Coburn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spleen+transplantation+in+the+rat.">Spleen transplantation in the rat.</a> Transplantation. 8 (1), 86-88 (1969).</li><li>Lee, S., Orloff, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+splenic+transplantation+in+the+rat.">A technique for splenic transplantation in the rat.</a> Surgery. 65 (3), 436-439 (1969).</li><li>Swirski, F. K., 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=Identification+of+splenic+reservoir+monocytes+and+their+deployment+to+inflammatory+sites.">Identification of splenic reservoir monocytes and their deployment to inflammatory sites.</a> Science. 325 (5940), 612-616 (2009).</li><li>Hsiao, H. 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=Spleen-derived+classical+monocytes+mediate+lung+ischemia-reperfusion+injury+through+IL-1beta.">Spleen-derived classical monocytes mediate lung ischemia-reperfusion injury through IL-1beta.</a> Journal of Clinical Investigation. 128 (7), 2833-2847 (2018).</li><li>Robbins, C. S., 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=Extramedullary+hematopoiesis+generates+Ly-6C(high)+monocytes+that+infiltrate+atherosclerotic+lesions.">Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions.</a> Circulation. 125 (2), 364-374 (2012).</li><li>Kim, E., Yang, J., Beltran, C. D., Cho, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+spleen-derived+monocytes/macrophages+in+acute+ischemic+brain+injury.">Role of spleen-derived monocytes/macrophages in acute ischemic brain injury.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 34 (8), 1411-1419 (2014).</li><li>Bronte, V., Pittet, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spleen+in+local+and+systemic+regulation+of+immunity.">The spleen in local and systemic regulation of immunity.</a> Immunity. 39 (5), 806-818 (2013).</li><li>Wang, N. P., 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=Recruitment+of+macrophages+from+the+spleen+contributes+to+myocardial+fibrosis+and+hypertension+induced+by+angiotensin+II.">Recruitment of macrophages from the spleen contributes to myocardial fibrosis and hypertension induced by angiotensin II.</a> Journal of the Renin-Angiotensin-Aldosterone System. 18 (2), 1470320317706653 (2017).</li><li>Tian, 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+spleen+contributes+importantly+to+myocardial+infarct+exacerbation+during+post-ischemic+reperfusion+in+mice+via+signaling+between+cardiac+HMGB1+and+splenic+RAGE.">The spleen contributes importantly to myocardial infarct exacerbation during post-ischemic reperfusion in mice via signaling between cardiac HMGB1 and splenic RAGE.</a> Basic Research in Cardiology. 111 (6), 62 (2016).</li><li>Jang, 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=Cutting+Edge:+Check+Your+Mice-A+Point+Mutation+in+the+Ncr1+Locus+Identified+in+CD45.1+Congenic+Mice+with+Consequences+in+Mouse+Susceptibility+to+Infection.">Cutting Edge: Check Your Mice-A Point Mutation in the Ncr1 Locus Identified in CD45.1 Congenic Mice with Consequences in Mouse Susceptibility to Infection.</a> Journal of Immunology. 200 (6), 1982-1987 (2018).</li></ol>

The authors have nothing to disclose.

Compelling evidence suggests that spleen-derived monocytes play an important role in sterile inflammatory processes such as atherosclerosis19, acute ischemic brain20 or lung injury18, as well as myocardial I/R injury and remodeling21,22,23. These reports highlight the under-recognition role of the spleen in many chronic diseases, of which cardiovascular disease is an important one (especially given it is the number one killer globally). The mouse model of spleen transplantation offers a great opportunity to discover the role of spleen-derived immune cells in various diseases as well as how they are primed in the spleen. For example, by using the mouse models of spleen transplantation, Swirski et al. found that in response to ischemic myocardial injury, spleen-derived monocytes increase their motility, migrate out of the spleen, adhere to injured tissue, and contribute to the wound healing17. Furthermore, this model is useful to address the longevities of mature immune cells in the spleen and underlying mechanisms and to explore therapeutic potentials of spleen transplantation.
Several aspects should be taken into consideration to improve the success of this protocol. First, it is critical to choose the proper experimental animals during the design process since the mouse weight, strain, and health condition could affect the difficulty of the surgical steps and experimental results. Our laboratory recommends using 8 to 12-week old mice with over 25 g weight to decrease the mortality that might be caused by bleeding. Second, during the spleen harvest procedure, too much manipulation of the spleen vessels could easily lead to breeding or vasospasm and potentially result in micro thrombosis in the spleen graft. Getting familiar with the anatomy of the mouse abdomen before surgery would be helpful to accelerate the learning process. Third, during the recipient surgery, the spleen graft should be always maintained moist and cool. Appropriate protection of spleen grafts could reduce the transplant ischemia/reperfusion (I/R) injury and prevent graft failure after transplantation. In addition, considering that the donor vessels for anastomosis are relatively long, positioning the spleen grafts properly before the anastomosis is critical to prevent twisting of the vessels. Moreover, the diameters of the recipient aortotomy and venotomy of IVC should be always comparable to those of the donor aorta and portal vein to ensure the proper blood flow and prevent thrombosis.
The average storage time of spleen grafts in 4 &#176;C saline is around 10 min. The total ischemic time should be limited to less than 50 min to ensure minimum transplant failure. We recommend using the University of Wisconsin (UW) cold storage solution to preserve the spleen graft, if over 1 h cold, ischemic time is needed in studies. It should be noted that a small portion of the pancreas intimately attach with the spleen, and attempt to remove the whole from the spleen grafts would easily lead to graft or vascular complications and markedly increase the operation time. We observed that the pancreas tissue attached to the spleen graft would undergo atrophy at POD 7 though some remained viable with the spleen grafts. Whether or not to remove the pancreas tissue attached with spleen grafts depends on the research purposes.
Availability of congenic mice has made it possible to track the origin of the spleen cells, donor versus recipient after transplantation. In this study, we used BALB/c CD45.2 and BALB/c CD45.1 congenic mice as spleen donors and recipients to create a syngeneic spleen transplantation model. Organ or tissue transplantation between these congenic mouse strains has been widely used to track the origin and the development of immune system. Despite the recent report regarding a point mutation associated with CD45.1 that influences NK cell response24, no transplant rejection was reported between these strain combinations. We observed a substantial influx of recipient cells into spleen grafts that occurred as soon as at POD 1. It is likely that recipient cells responded to the transplant I/R injury as the majority of the recipient cells were granulocytes. Our results showed that a relatively high percentage of splenic lymphocytes remained of donor origin. More interestingly, these lymphocytes migrated to (repopulated) in other lymphoid compartments (lymph node, bone marrow, and circulation). These findings prompt us to speculate that lymphocytes that originated from spleens are very important in the adaptive immunity. However, more investigations are required to delineate the distinct roles of splenic lymphocytes in adaptive versus innate immunity (Figure 4, Supplementary Figure 1 and Supplementary Figure 2).
The major limitation of this protocol is that it requires extensive microsurgical training for individuals with limited microsurgical experience to master this technique. Based on our learning experience in overall mouse solid organ transplant models (e.g., mouse heart, lung, or kidney transplant), it may take 6-10 months for a newly trained person (without any experimental microsurgical technique) to skillfully master this technique. Compared to mouse models of heart, or kidney transplantation, this model could be more challenging since it involves additional tissue dissection steps to isolate the spleen graft during the donor procedures. Moreover, the diameter of the donor portal vein (around 0.6 mm) is smaller than the IVC, which makes it more difficult for the anastomosis. Another limitation is that it is not clear if the grafted spleen would be massively invaded by recipient&#8217; cells at later transplant phase (e.g., POD 60). However, this model is also very attractive for its several inherent advantages. Firstly, mice and humans share a substantial range of similar genomes, thereby allowing for a relatively accurate representation of a realistic application. Moreover, comparing to the mouse model of non-vascularized spleen autotransplantation using the sections of splenic tissue, this vascularized spleen transplant model is less likely to develop complications such as aseptic necrosis of the splenic tissue and small bowel obstruction due to postoperative adhesions.
The mouse model of spleen transplantation has been previously reported by Swirski FK et al. However, the detailed information was not described.&#160; Our study provides a comprehensive step-by-step protocol of mouse spleen transplantation for interested researchers to follow and to mater this technique. Moreover, this protocol eliminates several unnecessary steps described in the report by Swirski FK et al. (e.g., the bile duct ligation) and introduces the 11-0 suture for anastomosis, which would help shorten the surgical time and prevent the bleeding.
In conclusion, this model could be a powerful tool to explore the mechanisms of the splenic cell population in responses to pathogens, injury, inflammation, or transplant rejection and is valuable for the test of the therapeutic potential of the spleen transplantation. With proper training and practice, this procedure can be performed with &#62;90% success.

The spleen is the largest secondary lymphoid organ in the body and is critical in the immune and hematopoietic systems. Its functions are primarily carried out by two morphologically distinct compartments, the red pulp and the white pulp1. The red pulp is a three-dimensional meshwork of venous sinuses and splenic cords that consist of reticular fibers, reticular cells, and associated macrophages. This unique structure allows the red pulp to act as an effective blood filter that removes foreign materials and old or damaged erythrocytes. The white pulp includes follicles, marginal zone, and the periarteriolar lymphoid sheaths (PALS) and is an important site for antigen trapping and processing, lymphocyte homing, transformation, proliferation, and maturation2. Nevertheless, the spleen has commonly been considered as a dispensable organ because other lymphatic organs, such as lymph nodes, can also carry out some of its functions and the loss of spleen does not usually lead to death. Splenectomy has therefore been widely performed as a therapeutic method for patients with splenic injury or benign hematologic diseases3. However, patients with splenectomy face a number of long-term complications. Bacterial infections are the best-recognized complications of splenectomy4,5. Recently, the overwhelming post-splenectomy sepsis has been recognized as an intensive complication of splenectomy associated with a high mortality6. Moreover, recent epidemiological studies indicate that splenectomy may be associated with the occurrence of cardiovascular diseases, suggesting that further physiological functions of the spleen remain to be explored7,8.
Both spleen autotransplantation and spleen allotransplantation have been utilized in the clinic. Currently, spleen autotransplantation by implanting sections of splenic tissue into pouches created in the greater omentum is considered as the only possibility for preserving splenic function after traumatic splenectomy9,10. However, the efficacy of this surgery is debatable as post-surgery complications like aseptic necrosis of the splenic tissue and small bowel obstruction due to postoperative adhesions could occur11. Spleen allotransplantation is involved in multivisceral transplantation12. Clinical evidence from multivisceral transplantation suggests that spleen allotransplantation may play a protective role in small bowel allograft rejection without causing graft-versus-host disease (GVHD)12. Yet literature regarding the beneficial effect of spleen allotransplantation as a component of multivisceral transplantation is still limited and the underlying mechanisms remain to be defined. In 2006, Yair Reisner et al. reported that transplanting pig embryonic spleen tissue that has no T cells to mice could cure hemophilia A, a genetic disease without causing GVHD13, supporting that spleen transplantation holds therapeutic promise in certain diseases. Therefore, there is a need for further investigations on the therapeutic potential of spleen transplantation.
Animal models of spleen transplantation are valuable to explore the unappreciated function of the spleen-derived immune cells in disease progression as well as to test the potential therapeutic effect of spleen transplantation. Experimental whole spleen transplant models have been documented since early 1900s, as reviewed by Cohen14. In 1969, Coburn Richard J. and Lee et al. detailed the technique of spleen transplantation in rats15,16. More recently, Swirski FK et al. described a mouse model of spleen transplantation17. Compared to rat models, mouse models of spleen transplantation are more attractive due to its several inherent advantages. For example, by utilizing a mouse model, we can access an expansive variety of reagents unavailable to that of rat models. Moreover, by using congenic mice (e.g., mice with CD45.1/CD45.2 background), a syngeneic spleen transplantation makes it possible to track the fate, longevity, and function of splenocytes18. Based on the work by Swirski FK et al.17, we further established this simplified and enhanced protocol of spleen transplantation in mice. The protocol described below combines both reliability and feasibility in a standardized manner and can be utilized as a tool to study spleen biology and transplant immunity.

<table>
	<tbody>
		<tr>
			<td>Ketamine</td>
			<td>Wyeth</td>
			<td>206205-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd Laboratories</td>
			<td>139-236</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin solution</td>
			<td>Abraxis Pharmaceutical Products</td>
			<td>504031</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection grade normal saline</td>
			<td>Hospira Inc.</td>
			<td>NDC 0409-4888-20</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Pharmco Products Inc.</td>
			<td>111000140</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoCare Small Animal ICU System</td>
			<td>Thermocare, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Forceps</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-5230</td>
			<td></td>
		</tr>
		<tr>
			<td>Derf Needle Holder</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-7822</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Micro Dissecting Scissors</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-clip</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-5420</td>
			<td></td>
		</tr>
		<tr>
			<td>7-0 silk</td>
			<td>Braintree Scientific</td>
			<td>SUT-S 103</td>
			<td></td>
		</tr>
		<tr>
			<td>11-0 nylon on 4-mm (3/8) needle</td>
			<td>Sharpoint DR4</td>
			<td>AK-2119</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD45.2 antibody</td>
			<td>BD Bioscience</td>
			<td>553772</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD45.1 antibody</td>
			<td>BD Bioscience</td>
			<td>553776</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD11b antibody</td>
			<td>BD Bioscience</td>
			<td>557657</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms B220 antibody</td>
			<td>BD Bioscience</td>
			<td>553089</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms Ly6C antibody</td>
			<td>eBioscience</td>
			<td>48-5932-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms Ly6G antibody</td>
			<td>BD Bioscience</td>
			<td>561236</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms F4/80 antibody</td>
			<td>BD Bioscience</td>
			<td>565614</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD11c antibody</td>
			<td>BD Bioscience</td>
			<td>558079</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD3 antibody</td>
			<td>eBioscience</td>
			<td>48-0032-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD4 antibody</td>
			<td>BD Bioscience</td>
			<td>552051</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD8 antibody</td>
			<td>BD Bioscience</td>
			<td>563786</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD&#8482; Fixable Violet Dead Cell Stain Kit</td>
			<td>Thermo Fisher</td>
			<td>L34955</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse model of vascularized heterotopic spleen transplantation for studying spleen cell biology and transplant immunity

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone splenectomy regardless of precipitating causes are prone to develop an overwhelming post-splenectomy infection and experience increased risks of deep venous thrombosis and malignancies. Recently, epidemiological studies indicated that splenectomy might be associated with the occurrence of cardiovascular diseases, suggesting that physiological functions of the spleen have not yet been fully recognized. Here, we introduce a mouse model of vascularized heterotopic spleen transplantation, which not only can be utilized to study the function and behavioral activity of splenic immune cell subsets in different biologic processes, but also can be a powerful tool to test the therapeutic potential of spleen transplantation in certain diseases. The main surgical steps of this model include donor spleen harvest, the removal of recipient native spleen, and spleen graft revascularization. Using congenic mouse strains (e.g., mice with CD45.1/CD45.2 backgrounds), we observed that after syngeneic transplantation, both donor-derived splenic lymphocytes and myeloid cells migrated out of the graft as early as post-operative day 1, concomitant with the influx of multiple types of recipient cells, thus generating a unique chimera. &#160;Despite relatively challenging techniques, this procedure can be performed with &#62;90% success rate. This model allows tracking the fate, longevity, and function of splenocytes during steady state and in a disease setting following a spleen transplantation, thereby offering a great opportunity to discover the distinct role for spleen-derived immune cells in different disease processes.

The entire procedure of mouse spleen transplant can be completed within 90 min by experienced microsurgeons. Our laboratory has performed over 100 spleen transplants in mice. The success rate is over 90%, as defined by the survival of both recipient mouse and the spleen graft to post-operative day (POD) 1 or POD 7 (our study endpoint). The survival of the spleen graft was confirmed by the macroscopic appearance and flow cytometry analysis of the splenocytes. Based on our experience, the flow cytometry analysis (LIVE/DEAD Cell Viability Assays) is very sensitive to determine whether a spleen graft is survived, as the majority of the spleen cells would be dead if the spleen grafts were necrotic. The technical challenges of this procedure, common complications, and their troubleshooting are summarized in Table 1.
To test the graft morphology post-transplantation, Haemotoxylin and Eosin (H&#38;E) staining was performed in spleen isografts of BALB/c mice at POD 1 and POD 7. The representative pictures are shown in Figure 3. The architecture of the spleen isografts remained intact during the first postoperative week. The red pulp, white pulp, and the marginal zone were still clear and distinguishable. To investigate the cell migration after spleen transplantation, flow cytometry was performed at POD 1 and POD 7 to examine the phenotypes of leukocytes in the spleen isografts, lymph nodes, blood, and bone marrow. As shown in Figure 4A, at POD 1, 51 &#177; 7% (mean &#177; SD, same as below) of the spleen cells were donor-derived and 46 &#177; 3% were recipient-derived. At POD 7, donor-derived leukocytes accounted for 32 &#177; 10 % of total spleen cells, and recipient-derived cells were up to 56 &#177; 13%. We also observed that spleen leukocytes migrated into the lymph nodes, blood, and bone marrow as early at day 1 and maintained at day 7 (Figure 4B), generating a unique chimera valuable for splenocyte trafficking research.
Table 1: Troubleshooting methods.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59616/59616fig1.jpg" /> 
Figure 1: Donor spleen harvest. (A) Cauterize the short gastric vein attached to the spleen. (B) Place a small piece of sterile warm wet gauze over the spleen to keep it moist. (C) Dissect and isolate the portal vein behind the pancreas. (D) Ligate the side branches of the portal vein and place a suture round the portal vein distal to the splenic vein. The dashed lines represent the location transecting later for the anastomosis with the recipient IVC. (E) Flip the spleen over to the right side of the abdomen (surgeon&#8217;s left) to expose the aorta and celiac trunk with its branches including splenic artery. (F) Dissect and mobilize aortic -celiac -splenic artery by ligating the two other branches. (G) Place a suture around the aorta proximal to the celiac artery. The dashed lines represent the location transecting later used for the anastomosis with recipient abdominal aorta. (H) After ligating the aorta and transecting the portal vein, perfuse the spleen graft with 10 mL of heparinized saline through the aorta. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59616/59616fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59616/59616fig2.jpg" /> 
Figure 2: Spleen graft implantation. (A) After isolation and cross clamping the aorta and IVC, make a longitudinal aortotomy and venotomy in the aorta and IVC, respectively. (B-D) Place the spleen graft on the right side of the abdomen (surgeon&#8217;s left side). Make the end-to-side anastomosis between donor aortic cuff and the anterior wall of the recipient&#8217;s aortotomy, using 2-3 bites of continuing suture. (E) Turn the spleen graft over to the left flank of the recipient; repeat the previous procedure between the donor aortic cuff and the posterior wall of the recipient&#8217;s aortotomy and close the suture on the outside of aorta. (F-G) Make an end-to-side anastomosis between the donor portal vein and the posterior wall of the recipient&#8217;s IVC, using 4 to 5 bites of continuous 11-0 nylon suture in the inside of the IVC and then close the suture on the outside of the IVC. (H) After completing the anastomosis, release the vessel clamps and place some cotton buds to help stop the bleeding. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59616/59616fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59616/59616fig3.jpg" /> 
Figure 3: Representative histology of spleen isografts on day 1 and day 7 post-operation. Syngeneic spleen transplantations were performed using BALB/c mice. Spleen isografts were harvested on day 1 and day 7 post-transplantation and fixed in 10% formalin for 48 h. H&#38;E staining was performed using paraffin-embedded tissue section. The representative histology shows that the spleen isografts remain intact at 1 week post-transplantation. Scale bar = 250 &#956;m. POD, post-operative day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59616/59616fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59616/59616fig4.jpg" /> 
Figure 4: The chimera created by syngeneic spleen transplantation. Three syngeneic spleen transplantations were performed using BALB/c CD45.2 mice as donors and BALB/c CD45.1 mice as recipients. The spleen grafts, blood, lymph node (LN), and bone marrow (BM) in recipient mice were harvested on day 1 and day 7 post-transplantation. Single cell isolation and flow cytometry analysis were performed to analyze the phenotype of the cells. (A) Representative dot plots (gating from live singlets) showing the percentages of donor or recipient &#8212;&#160;derived leukocytes in the indicated samples. (B) Percentages (mean &#177; SEM) of the indicated populations in donor- derived cells in the indicated samples at day 1 and day 7. POD = post-operative day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59616/59616fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Representative plots (n = 3) showing the percentages of donor-derived versus recipient-derived lymphocytes in the transplanted spleen, lymph node, blood, and bone marrow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59616/supplemental_figures.pdf">Please click here to download this file.</a>
Supplementary Figure 2: Representative plots (n = 3) showing the percentages of donor-derived versus recipient-derived CD11b+Ly6C+monocytes in the transplanted spleen, lymph node, blood, and bone marrow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59616/supplemental_figures.pdf">Please click here to download this file.</a>

Watch this Scientific Journal Video about A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity at JoVE.com

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone ...

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15.6K Views.  Northwestern University. This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

Video: A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

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

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

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

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

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

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

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

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 ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

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

A Mouse Model of in Utero Transplantation

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

A Modified Method for Heterotopic Mouse Heart Transplantion

Murine Heterotopic Heart Transplant Technique

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Orthotopic Hind Limb Transplantation in the Mouse

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique