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Orthotopic mouse liver transplantation to study liver biology and allograft tolerance

Nature Protocols volume 11, pages 1163–1174 (2016)Cite this article

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Abstract

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%.

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Figure 1: Instruments used in the mouse liver transplantation model and diagram of mouse position during surgery.
Figure 2: Schematic drawing of mouse liver and demonstration of incisions for donor harvest.
Figure 3: Back-table preparation of the liver graft.
Figure 4: Schematic drawing of recipient hepatectomy illustrating where graft incisions occur.
Figure 5: Schematic drawings of venous anastomoses and bile duct reconstruction in the recipient operation.
Figure 6: Perfused liver graft immediately after completing all anastomoses in mouse liver transplantation.
Figure 7: Histology of normally functioning mouse liver grafts 30 d after transplantation.

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References

  1. Chong, A.S., Alegre, M.L., Miller, M.L. & Fairchild, R.L. Lessons and limits of mouse models. Cold Spring Harb. Perspect. Med. 3, a015495 (2013).

    Article  Google Scholar 

  2. Qian, S.G., Fung, J.J., Demetris, A.V., Ildstad, S.T. & Starzl, T.E. Orthotopic liver transplantation in the mouse. Transplantation 52, 562–564 (1991).

    Article  CAS  Google Scholar 

  3. Kamada, N. & Calne, R.Y. Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 28, 47–50 (1979).

    Article  CAS  Google Scholar 

  4. Klein, I. & Crispe, I.N. Complete differentiation of CD8+ T cells activated locally within the transplanted liver. J. Exp. Med. 203, 437–447 (2006).

    Article  Google Scholar 

  5. Shen, X.D. et al. Inflammatory responses in a new mouse model of prolonged hepatic cold ischemia followed by arterialized orthotopic liver transplantation. Liver Transpl. 11, 1273–1281 (2005).

    Article  Google Scholar 

  6. Conzelmann, L.O. 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).

    Article  CAS  Google Scholar 

  7. Ueki, S. et al. Critical role of interferon regulatory factor-1 in murine liver transplant ischemia reperfusion injury. Hepatology 51, 1692–1701 (2010).

    Article  CAS  Google Scholar 

  8. Ueki, S. et al. Hepatic B7 homolog 1 expression is essential for controlling cold ischemia/reperfusion injury after mouse liver transplantation. Hepatology 54, 216–228 (2011).

    Article  CAS  Google Scholar 

  9. Yoshida, O. et al. CD39 expression by hepatic myeloid dendritic cells attenuates inflammation in liver transplant ischemia-reperfusion injury in mice. Hepatology 58, 2163–2175 (2013).

    Article  CAS  Google Scholar 

  10. Yokota, S. et al. IRF-1 promotes liver transplant ischemia/reperfusion injury via hepatocyte IL-15/IL-15Ralpha production. J. Immunol. 194, 6045–6056 (2015).

    Article  CAS  Google Scholar 

  11. Qian, S. et al. Murine liver allograft transplantation: tolerance and donor cell chimerism. Hepatology 19, 916–924 (1994).

    Article  CAS  Google Scholar 

  12. Dahmen, U. et al. Split tolerance induced by orthotopic liver transplantation in mice. Transplantation 58, 1–8 (1994).

    Article  CAS  Google Scholar 

  13. Yoshida, O. 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).

    Article  CAS  Google Scholar 

  14. Qian, S. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Li, W. 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).

    Article  CAS  Google Scholar 

  16. Li, W. 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).

    Article  CAS  Google Scholar 

  17. Morita, M. et al. PD-1/B7-H1 interaction contribute to the spontaneous acceptance of mouse liver allograft. Am. J. Transplant. 10, 40–46 (2010).

    Article  CAS  Google Scholar 

  18. Conzelmann, L.O., Zhong, Z., Bunzendahl, H., Wheeler, M.D. & Lemasters, J.J. Reduced-size liver transplantation in the mouse. Transplantation 76, 496–501 (2003).

    Article  Google Scholar 

  19. Tian, Y., Graf, R., Jochum, W. & Clavien, P.A. Arterialized partial orthotopic liver transplantation in the mouse: a new model and evaluation of the critical liver mass. Liver Transpl. 9, 789–795 (2003).

    Article  Google Scholar 

  20. Tian, Y. et al. 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).

    Article  CAS  Google Scholar 

  21. Garuti, C. 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).

    Article  CAS  Google Scholar 

  22. Li, X.K. 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).

    Article  CAS  Google Scholar 

  23. Demetris, A.J. et al. Monitoring of human liver and kidney allograft tolerance: a tissue/histopathology perspective. Transpl. Int. 22, 120–141 (2009).

    Article  Google Scholar 

  24. Sanchez-Fueyo, A. & Strom, T.B. Immunologic basis of graft rejection and tolerance following transplantation of liver or other solid organs. Gastroenterology 140, 51–64 (2011).

    Article  Google Scholar 

  25. Feng, S. et al. Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants. JAMA 307, 283–293 (2012).

    Article  CAS  Google Scholar 

  26. Tian, Y., Rudiger, H.A., Jochum, W. & Clavien, P.A. Comparison of arterialized and nonarterialized orthotopic liver transplantation in mice: prowess or relevant model? Transplantation 74, 1242–1246 (2002).

    Article  Google Scholar 

  27. Kamada, N. The immunology of experimental liver transplantation in the rat. Immunology 55, 369–389 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Steger, U., Sawitzki, B., Gassel, A.M., Gassel, H.J. & Wood, K.J. Impact of hepatic rearterialization on reperfusion injury and outcome after mouse liver transplantation. Transplantation 76, 327–332 (2003).

    Article  Google Scholar 

  29. Starzl, T.E. & Lakkis, F.G. The unfinished legacy of liver transplantation: emphasis on immunology. Hepatology 43, S151–S63 (2006).

    Article  CAS  Google Scholar 

  30. Qian, S., Fung, J.J., Demetris, A.J. & Starzl, T.E. Allogeneic orthotopic liver transplantation in mice: a preliminary study of rejection across well-defined MHC barriers. Transplant. Proc. 23, 705–706 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Benseler, V. et al. The liver: a special case in transplantation tolerance. Semin. Liver Dis. 27, 194–213 (2007).

    Article  CAS  Google Scholar 

  32. Morita, M. et al. Rejection triggers liver transplant tolerance: involvements of mesenchyme-mediated Immune Control Mechanisms. Hepatology 62, 915–931 (2015).

    Article  CAS  Google Scholar 

  33. Yokota, S., Yoshida, O., Ono, Y., Geller, D.A. & Thomson, A.W. Liver transplantation in the mouse: insights into liver immunobiology, tissue injury and allograft tolerance. Liver Transpl. 22, 536–546 (2016).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the US National Institutes of Health (NIH) (grants P01 AI81678 and R56 AI118777 to A.W.T. and grant T32 AI74490 to O.Y.) and by the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research C-26461926 to N.K.). S.Y. was supported by funds from Jichi Medical University (Shimotsuke, Tochigi, Japan). O.Y. was supported by an American Society of Transplantation Basic Science Fellowship. We thank S. Qian and M. Morita (Department of Immunology, Lerner Research Institute, Cleveland Clinic) for valuable advice. We thank T. Teratani (Jichi Medical University) for valuable input.

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Author notes

  1. Shinichiro Yokota and Shinya Ueki: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Shinichiro Yokota, Shinya Ueki, Yoshihiro Ono, Angélica Pérez-Gutiérrez, Shoko Kimura, Osamu Yoshida, Noriko Murase, David A Geller & Angus W Thomson

  2. Department of Surgery, Jichi Medical University, Shimotsuke, Tochigi, Japan

    Shinichiro Yokota, Naoya Kasahara & Yoshikazu Yasuda

  3. Liver Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA

    David A Geller

  4. Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Angus W Thomson

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Contributions

S.Y. and A.W.T. conceived and designed the outline of the manuscript. S.Y., S.U., Y.O., N.K. and A.P.-G. wrote the manuscript and prepared the pictures and images. S.K., O.Y., N.M., Y.Y., D.A.G. and A.W.T. revised and provided input during final editing of the manuscript.

Corresponding author

Correspondence to Angus W Thomson.

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The authors declare no competing financial interests.

Supplementary information

Preparation before liver graft harvest (Steps 8–18)

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. (MP4 29000 kb)

Donor liver perfusion and harvest (Steps 20–33)

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. (MP4 26891 kb)

Cuff attachment (Steps 35–40)

IHIVC is folded over the cuff and is secured with 7-0 silk; 5-0 silk is placed around the IHIVC; the 10-0 nylon tied to the PV is pulled through the cuff; PV is folded over the cuff. (MP4 24988 kb)

Stay suture attachment (Step 41)

The liver is rotated and the stay suture is put on the SHIVC, on the bilateral edge. (MP4 17761 kb)

Preparation before anhepatic phase (Steps 43–55)

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. (MP4 27445 kb)

The anhepatic phase (Steps 56–71)

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. (MP4 24988 kb)

IHIVC anastomosis (Steps 72 and 73)

Stay suture is placed in the bilateral edge of IHIVC; 7-0 silk is placed around recipient IHIVC; anterior wall of IHIVC is lifted and cuff is inserted; the silk tied around donor IHIVC is released; the clamp is released. (MP4 27514 kb)

Bile duct anastomosis (Step 74)

7-0 silk is placed around the recipient bile duct; the anterior wall of the bile duct is cut; the stent is inserted and secured with the 7-0 silk. (MP4 28640 kb)

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Yokota, S., Ueki, S., Ono, Y. et al. Orthotopic mouse liver transplantation to study liver biology and allograft tolerance. Nat Protoc 11, 1163–1174 (2016). https://doi.org/10.1038/nprot.2016.073

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