Name: a mouse model of vascularized heterotopic spleen transplantation for studying spleen cell biology and transplant immunity
Uploaded: 2019-06-11T15:00:00.000+00:00
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Description: 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

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>

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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&amp;trade; 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&amp;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 ± 7% (mean ± SD, same as below) of the spleen cells were donor-derived and 46 ± 3% were recipient-derived. At POD 7, donor-derived leukocytes accounted for 32 ± 10 % of total spleen cells, and recipient-derived cells were up to 56 ± 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="Surgical procedure on portal vein, splenic artery; step-by-step dissection images, labeled veins/arteries." class="xfigimg" src="/files/ftp_upload/59616/59616fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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’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="Organ transplantation surgical procedure; step-by-step diagram; vascular connections; medical research." class="xfigimg" src="/files/ftp_upload/59616/59616fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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’s left side). Make the end-to-side anastomosis between donor aortic cuff and the anterior wall of the recipient’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’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’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="Histological comparison of lymph nodes: native vs. isograft POD1, POD7; microscope image." class="xfigimg" src="/files/ftp_upload/59616/59616fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
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&amp;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 μm. 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="Flow cytometry analysis chart showing donor cell distribution in spleen, LN, blood, BM, POD 1/7." class="xfigimg" src="/files/ftp_upload/59616/59616fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
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 — derived leukocytes in the indicated samples. (B) Percentages (mean ± 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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15.7K Views.  Northwestern University. Het muismodel van milttransplantatie biedt een geweldige kans om de rol van miltcellen in de regulatie van lokale en systemische immuunreacties bij ontstekingsziekten aan het licht te brengen. Het belangrijkste voordeel van dit protocol is dat door het gebruik van congene muizen, milttransplantatie maakt het mogelijk om het lot, levensduur, en functie van miltcellen te controleren. Voordat u met de procedure begint, plaatst u een steriel wegwerpgordijn over het chirurgische platform en bevest...

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

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Biology

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

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 ...

Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFR&#946;+MAdCAM-1- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.

This article describes a protocol for aggregating and encapsulating spleen cells within a semi-solid basement membrane matrix. Basement membrane matrix constructs can be used in three-dimensional culture for studying organoid development, or for in vivo transplantation and tissue regeneration studies.

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases ...

Developmental Biology

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Immunology and Infection

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

A Mouse Model of Vascularized Heterotopic Spleen Transplantation

Source: Wang, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59616">A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity.</a> J. Vis. Exp. (2019).
The video demonstrates a surgical method for generating a mouse model of vascularized heterotopic spleen transplantation. The abdominal cavity of an anesthetized donor mouse is surgically opened to remove the spleen. The inferior vena cava and infrarenal aorta are identified, their lumbar branches are ligated, and openings are made for the heterotopic transplantation of a spleen from a congenic donor mouse. Upon completing vascular anastomosis and restoring blood flow to the implanted spleen, the incision is closed, and the mouse is monitored for recovery.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

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