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

Podsumowanie

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.

Streszczenie

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.  Despite relatively challenging techniques, this procedure can be performed with >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.

Wprowadzenie

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 splenectomy^4,5. 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 explored^7,8.

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 splenectomy^9,10. 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 rats^15,16. 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.

Protokół

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

1. 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.
2. 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.

2. Donor Spleen Harvest

1. 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.
2. Retract the abdominal wall with sterile retractors made from paperclips. Move the intestines to the right flank of the abdomen (surgeon’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 °C saline over the spleen to keep it moist (Figure 1B).
3. 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).
4. 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).
5. 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 °C) saline (10 mL/20 s) from the abdominal aorta distal to celiac trunk (Figure 1H).
6. 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 °C saline before transplant. Euthanize the mouse by cervical dislocation.

3. Recipient Splenectomy and Spleen Graft Implantation

1. Place a heating pad on the surgical platform and adjust the temperature to 37 °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.
2. Carefully move the intestine to right side of the mouse with a sterile cotton swab to expose the recipient’s spleen. Ligate the splenic vein and artery and remove the spleen.
3. Carefully move the intestine to left side of the mouse and cover the intestines with wet gauze (soaked with sterile 37 °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.
4. 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).
5. Clear the intraluminal blood or blood clot (in the aorta and IVC) with 500 μL of heparinized saline (10 units/mL).
6. Place the spleen graft in the right flank of the recipient mouse abdomen; carefully identify the donor’s aortic cuff and the donor’s portal vein. After making sure that the vessels are not twisted, cover the spleen graft with gauze soaked with cold (4 °C) saline.
7. Connect the donor’s aortic cuff to the proximal and distal apex of the recipient’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’s aortic cuff and the recipient’s aortaotomy (anterior wall) (Figure 2D). Turn the spleen graft over to the left side of the recipient; make the anastomosis between the donor’s aortic cuff and the recipient’s aortotomy (posterior wall) (Figure 2E).
8. Perform an anastomosis to connect the donor’s portal vein to the posterior wall of the recipient’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).
9. Release the vessel clamps and use sterile cotton swab to tamponade bleeding until the spleen color is recovered (Figure 2H).
10. 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’s portal vein and the recipient’s IVC first (step 4.8); then make an anastomosis between the donor’s aortic cuff and the posterior wall of the recipient’s aortotomy, using 2 to 3 continuous sutures in the inside of the aorta and close the suture on the outside of the aorta.

4. Animal Recovery

1. Inject 1 mL of warm saline subcutaneously via 4 separate locations (0.25 mL/location) after closing the abdomen.
2. Keep the mouse in a temperature-controlled incubator (30 °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 °C) underneath the cage. Keep the mouse post-surgery in a separate cage.

5. Post-surgical Pain Management

1. Inject 0.05 mg/kg buprenorphine subcutaneously 24 h and 48 h post-surgery to maintain analgesia regimen.

Wyniki

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...

Dyskusje

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 remodeling^21,22,23. These reports highlight the under-recognition role of the spleen in many chronic diseases, of which cardiovascular dise...

Materiały

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