Orthotopic liver transplantation in the mouse is a powerful research tool that has led to important mechanistic insights into the regulation of hepatic injury, liver immunopathology, and transplant tolerance. However, it is a technically demanding surgical procedure. Setup of the orthotopic liver transplantation model comprises three main stages: surgery on the donor mouse; back-table preparation of the liver graft; and transplant of the liver into the recipient mouse. In this protocol, we describe our procedure in stepwise detail to allow efficient completion of both the donor and recipient operations. The protocol can result in consistently high technical success rates when performed by personnel experienced in the protocol. The technique can be completed in ~2–3 h when performed by an individual who is well practiced in performing mouse transplantation in accordance with this protocol. We have achieved a perioperative survival rate close to 100%.
At a glance
- Lessons and limits of mouse models. Cold Spring Harb. Perspect. Med. 3, a015495 (2013). , , &
- Orthotopic liver transplantation in the mouse. Transplantation 52, 562–564 (1991). , , , &
- Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 28, 47–50 (1979). &
- Complete differentiation of CD8+ T cells activated locally within the transplanted liver. J. Exp. Med. 203, 437–447 (2006). &
- Inflammatory responses in a new mouse model of prolonged hepatic cold ischemia followed by arterialized orthotopic liver transplantation. Liver Transpl. 11, 1273–1281 (2005). et al.
- Graft tumor necrosis factor receptor-1 protects after mouse liver transplantation whereas host tumor necrosis factor receptor-1 promotes injury. Transplantation 82, 1214–1220 (2006). et al.
- Critical role of interferon regulatory factor-1 in murine liver transplant ischemia reperfusion injury. Hepatology 51, 1692–1701 (2010). et al.
- Hepatic B7 homolog 1 expression is essential for controlling cold ischemia/reperfusion injury after mouse liver transplantation. Hepatology 54, 216–228 (2011). et al.
- CD39 expression by hepatic myeloid dendritic cells attenuates inflammation in liver transplant ischemia-reperfusion injury in mice. Hepatology 58, 2163–2175 (2013). et al.
- IRF-1 promotes liver transplant ischemia/reperfusion injury via hepatocyte IL-15/IL-15Ralpha production. J. Immunol. 194, 6045–6056 (2015). et al.
- Murine liver allograft transplantation: tolerance and donor cell chimerism. Hepatology 19, 916–924 (1994). et al.
- Split tolerance induced by orthotopic liver transplantation in mice. Transplantation 58, 1–8 (1994). et al.
- DAP12 deficiency in liver allografts results in enhanced donor DC migration, augmented effector T cell responses and abrogation of transplant tolerance. Am. J. Transplant. 14, 1791–1805 (2014). et al.
- Apoptosis within spontaneously accepted mouse liver allografts: evidence for deletion of cytotoxic T cells and implications for tolerance induction. J. Immunol. 158, 4654–4661 (1997). et al.
- Il-12 antagonism enhances apoptotic death of T cells within hepatic allografts from Flt3 ligand-treated donors and promotes graft acceptance. J. Immunol. 166, 5619–5628 (2001). et al.
- New insights into mechanisms of spontaneous liver transplant tolerance: the role of Foxp3-expressing CD25+CD4+ regulatory T cells. Am. J. Transplant. 8, 1639–1651 (2008). et al.
- PD-1/B7-H1 interaction contribute to the spontaneous acceptance of mouse liver allograft. Am. J. Transplant. 10, 40–46 (2010). et al.
- Reduced-size liver transplantation in the mouse. Transplantation 76, 496–501 (2003). , , , &
- Arterialized partial orthotopic liver transplantation in the mouse: a new model and evaluation of the critical liver mass. Liver Transpl. 9, 789–795 (2003). , , &
- Kupffer cell-dependent TNF-alpha signaling mediates injury in the arterialized small-for-size liver transplantation in the mouse. Proc. Natl. Acad. Sci. USA 103, 4598–4603 (2006). et al.
- Hepcidin expression does not rescue the iron-poor phenotype of Kupffer cells in Hfe-null mice after liver transplantation. Gastroenterology 139, 315–322 (2010). et al.
- Fulminant hepatitis by Fas-ligand expression in MRL-lpr/lpr mice grafted with Fas-positive livers and wild-type mice with Fas-mutant livers. Transplantation 71, 503–508 (2001). et al.
- Monitoring of human liver and kidney allograft tolerance: a tissue/histopathology perspective. Transpl. Int. 22, 120–141 (2009). et al.
- Immunologic basis of graft rejection and tolerance following transplantation of liver or other solid organs. Gastroenterology 140, 51–64 (2011). &
- Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants. JAMA 307, 283–293 (2012). et al.
- Comparison of arterialized and nonarterialized orthotopic liver transplantation in mice: prowess or relevant model? Transplantation 74, 1242–1246 (2002). , , &
- The immunology of experimental liver transplantation in the rat. Immunology 55, 369–389 (1985).
- Impact of hepatic rearterialization on reperfusion injury and outcome after mouse liver transplantation. Transplantation 76, 327–332 (2003). , , , &
- The unfinished legacy of liver transplantation: emphasis on immunology. Hepatology 43, S151–S63 (2006). &
- Allogeneic orthotopic liver transplantation in mice: a preliminary study of rejection across well-defined MHC barriers. Transplant. Proc. 23, 705–706 (1991). , , &
- The liver: a special case in transplantation tolerance. Semin. Liver Dis. 27, 194–213 (2007). et al.
- Rejection triggers liver transplant tolerance: involvements of mesenchyme-mediated Immune Control Mechanisms. Hepatology 62, 915–931 (2015). et al.
- Liver transplantation in the mouse: insights into liver immunobiology, tissue injury and allograft tolerance. Liver Transpl. 22, 536–546 (2016). , , , &
- Video 1: Preparation before liver graft harvest (Steps 8–18) (28.32 MB, Download)
- Proper hepatic artery is ligated; ligament around the caudate lobe is cut; stent is placed in extrahepatic bile duct; retroperitoneum is dissected and lumbar veins are cauterized; right adrenal vein is ligated; 10-0 nylon is placed around the right renal vein.
- Video 2: Donor liver perfusion and harvest (Steps 20–33) (26.26 MB, Download)
- Heparin is injected via the penile vein; donor liver is perfused from IHIVC; PV and bile duct are cut; pyloric vein is ligated; PV is skeletonized; splenic vein is ligated; PV is cut below the splenic vein. Right renal vein is ligated; stitch is placed in IHIVC; IHIVC is cut above the left renal vein; cystic duct is ligated and gall bladder is cut. Falciform ligament is cut; anterior and posterior wall of SHIVC are cut; paraesophageal vessels are ligated; ligament and connective tissue are cut.
- Video 5: Preparation before anhepatic phase (Steps 43–55) (26.8 MB, Download)
- Ligament around caudate lobe is cut; left phrenic vein is ligated; paraesophageal vessels are ligated; the space between the liver and the retroperitoneum is dissected; 2-0 silk is placed behind the SHIVC; adrenal artery is cauterized behind the IHIVC; common bile duct is dissected and ligated with 7-0 silk; right PV is ligated.
- Video 6: The anhepatic phase (Steps 56–71) (24.4 MB, Download)
- IHIVC is clamped; PV is clamped; bulldog clamp is placed on the diaphragm; anterior and posterior wall of SHIVC are cut; bifurcation of PV is cut; IHIVC is cut; ligament and connective tissue are cut. Suture is placed in the bilateral edge of SHIVC; stay suture on the left edge is tied; continuous suture on the posterior wall is started; air is flushed from SHIVC; anterior wall is closed; and suture is tied with the stay suture in the left lateral edge. For the portal vein anastomosis, a stay suture is placed on the posterior wall of PV; 7-0 silk is placed around recipient PV; the anterior wall of the recipient's PV is lifted; and the cuff is inserted. The clamp on the PV is released and the SHIVC is declamped.