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 t...

<ol>
	<li>Cesta, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+structure,+function,+and+histology+of+the+spleen.">Normal structure, function, and histology of the spleen.</a> Toxicologic Pathology. 34 (5), 455-465 (2006).</li><li>Mebius, R. E., Kraal, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+spleen.">Structure and function of the spleen.</a> Nature Reviews Immunology. 5 (8), 606-616 (2005).</li><li>Misiakos, E. 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>

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 dise...

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.

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			<td>Ketamine</td>
			<td>Wyeth</td>
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			<td></td>
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			<td>Xylazine</td>
			<td>Lloyd Laboratories</td>
			<td>139-236</td>
			<td></td>
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			<td>Heparin solution</td>
			<td>Abraxis Pharmaceutical Products</td>
			<td>504031</td>
			<td></td>
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			<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>
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			<td>ThermoCare Small Animal ICU System</td>
			<td>Thermocare, Inc.</td>
			<td></td>
			<td></td>
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			<td>Adson Forceps</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-5230</td>
			<td></td>
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			<td>Derf Needle Holder</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-7822</td>
			<td></td>
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			<td>Extra Fine Micro Dissecting Scissors</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-5881</td>
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			<td>Micro-clip</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-5420</td>
			<td></td>
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			<td>7-0 silk</td>
			<td>Braintree Scientific</td>
			<td>SUT-S 103</td>
			<td></td>
		</tr>
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			<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>
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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 f...

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 ...

The mouse model of spleen transplantation offers a great opportunity for uncovering the role of spleen cells in the regulation of local and systemic immune responses in inflammatory disease. The main advantage of this protocol is that by using congenic mice, spleen transplantation makes it possible to check the fate, longevity, and function of spleen cells. Before beginning the procedure, place a sterile disposable drape over the surgical platform and confirm the lack of response to toe pinch in the anesthetized donor mouse.Shave the hair from the entire abdominal area and place the mouse onto a sterile surgical platform under an operating microscope at a 6 to 10X magnification. Sterilize the exposed skin with an alcohol prep pad and secure the limbs with surgical tape. Enter the abdomen via a three to four centimeter midline vertical skin incision from the pubis to the xyphoid process.Cauterize the skin vessels and extend the incision to the lateral abdomen on both sides to achieve better exposure. Using a sterile cotton swab, move the intestines to the right flank of the abdomen to expose the spleen and use a sterile low temperature cautery to cauterize the short gastric vein attached to the spleen. Place a piece of sterile gauze soaked with 37 degree Celsius saline over the spleen to keep it moist and separate and mobilize the portal vein from the pancreatic tissue.Ligate the branches of the portal vein as demonstrated. Place a suture around the portal vein distal from the splenic vein and flip the spleen to the right side to expose the aorta and the splenic artery on the celiac trunk. Dissect and mobilize the aortic celiac splenic arteries and place a suture around the aorta proximal to the celiac artery.Inject 100 international units of heparin into the inferior vena cava or IVC to heparinize the whole body. After three minutes, ligate the hepatic and gastric arteries in the aorta proximal to the celiac artery. Transect the portal vein and perfuse the whole body with 10 milliliters of four degree Celsius heparinized saline from the abdominal aorta distal to celiac trunk.Collect the spleen graft and block with the associated aortic celiac splenic segment in the portal vein along with the segment of splenic vein and a small portion of pancreatic tissue. Then preserve the graft in five milliliters of four degree Celsius saline. Enter the recipient abdomen via midline incision after the surgical area is prepared as it is for the donor and remove recipient native spleen by ligating the splenic vein and artery.Cover the intestine tissues with a 37 degree Celsius soaked gauze and carefully move the intestine to the left side. Dissect and ligate the lumbar branches of the infrarenal aorta in the IVC and cross-clamp the infrarenal aorta in the IVC with two four milliliter microvascular clamps. Place an 11-0 nylon suture through the infrarenal aorta and retract the vessel to allow the creation of an elliptical aortotomy with microscissors.Use a 30 gauge needle to pierce the IVC to create an elliptical venotomy and extend the opening to the length of the donor portal vein. Flush the aorta and IVC with 500 microliters of heparinized saline to clear the intraluminal blood or blood clots from the vessels and place the donor spleen graft into the right flank of the recipient abdomen. Carefully identify the donor aortic cuff and portal vein.After confirming that the vessels are not twisted, cover the spleen graft with four degree Celsius saline soaked gauze. Using two 11-0 stay sutures, connect the donor aortic cuff to the proximal and distal apex of the recipient aortotomy and make an anastomosis with two to three bites of continuous 11-0 nylon sutures between the donor aortic cuff and the anterior wall of the recipient aortotomy. Turn the spleen graft to the left side of the recipient and make the anastomosis between the donor aortic cuff and the posterior wall of the recipient aortotomy.Perform the venous anastomosis between the donor portal vein and the recipient IVC with four to five bites of continuous sutures on each side. Suture the posterior wall within the vessel lumen first and then close the anterior wall using the same suture. Complete the anastomosis with a knot placed on the lower corner outside of the IVC.Now release the vessel clamps and use a sterile cotton swab to tamponade the bleeding until the spleen color is recovered. Close the abdomen with a 5-0 synthetic absorbable Vicryl suture in a continuous pattern and close the skin layer with a 5-0 nylon suture in an interrupted pattern. Then inject 250 microliters of warm saline subcutaneously into four separate locations and warm the mouse in a 30 degree Celsius temperature controlled incubator for the first few hours post operation with monitoring until the animal has regained sufficient consciousness for transfer to the home cage.Hematoxylin and eosin of spleen isografts days one and seven after transplantation reveals that the architecture of the spleen isografts remains intact during the first post operative week. Flow cytometric analysis to investigate donor spleen cell migration after transplantation demonstrates that approximately 50%of the spleen cells are donor derived at one day after transplantation and approximately 46%are recipient derived. Seven days after transplantation, the donor derived leukocytes account for approximately 32%of the total spleen cells while the recipient derived cells increased up to approximately 53%Donor splenic leukocytes also migrated into the lymph nodes, blood, and bone marrow as early as day one and are maintained to at least day seven generating a unique Chimera valuable for splenocyte trafficking research.Using genetically modified knock-in mice as spleen donors will allow the investigation of the roles of spleen cell derived mediators or key signaling pathways in disease processes. This model could be a powerful tool for exploring the mechanisms of spleen cell populations in response to pathogens, injury, inflammation, or transplant rejection.

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15.1K vistas.  Northwestern University. El modelo de ratón de trasplante de bazo ofrece una gran oportunidad para descubrir el papel de las células del bazo en la regulación de las respuestas inmunitarias locales y sistémicas en la enfermedad inflamatoria. La principal ventaja de este protocolo es que mediante el uso de ratones congénicos, el trasplante de bazo permite comprobar el destino, la longevidad y la función de las células del bazo. Antes de comenzar el procedimiento, coloque una cortina desechable estéril sobre la...