Orthotopic mouse liver transplantation to study liver biology and allograft tolerance

Journal name:
Nature Protocols
Volume:
11,
Pages:
1163–1174
Year published:
DOI:
doi:10.1038/nprot.2016.073
Published online

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%.

At a glance

Figures

  1. Instruments used in the mouse liver transplantation model and diagram of mouse position during surgery.
    Figure 1: Instruments used in the mouse liver transplantation model and diagram of mouse position during surgery.

    (a) Cuffs for IHIVC and PV, and bile duct (BD) stent. (b) Schematic drawing of mouse position and setting for donor and recipient operations. Use mosquito forceps to hold the xiphoid process and retract toward the head of the mouse. Wrap the small and large intestines using a wet, nonwoven gauze sponge and gently retract the left and middle lobes of the liver toward the xiphoid process. (c) Instruments used throughout the protocol. Scale bars, 1 mm (a) and 1 cm (c).

  2. Schematic drawing of mouse liver and demonstration of incisions for donor harvest.
    Figure 2: Schematic drawing of mouse liver and demonstration of incisions for donor harvest.

    Labels indicate the following: (a) PHA incision line, (b) bile duct stent inserted in the common bile duct, (c) PV incision line, (d) IHIVC incision line, and (e) SHIVC incision line. Dashed lines represent the locations of incisions only, and solid lines represent the locations of tie and incision procedures. GB, gallbladder; LGA, left gastric artery; Lt. RV, left renal vein; Rt. RV, right renal vein; SpA, splenic artery; SpV, splenic vein.

  3. Back-table preparation of the liver graft.
    Figure 3: Back-table preparation of the liver graft.

    (a) Donor liver is placed with the inferior surface facing upward in a tissue culture dish placed on ice filled with cold (4 °C) saline or UW solution. The cuff attachment is set into position with a Weldon miniature bulldog clamp and a mosquito clamp fixed into place with soft clay. (b) To begin attachment of the cuff to the IHIVC, pull the 10-0 nylon tied to the IHIVC through the 16-gauge cuff. (c) Confirm the lumen of the IHIVC and fold the IHIVC over the 16-gauge cuff to expose the inner endothelial surface. (d) Appearance after cuff attachment of both the IHIVC and the PV. (e) Appearance after putting stay sutures on the SHIVC using 10-0 nylon on the bilateral edge. Experiments were performed under an institutional animal care and use committee–approved protocol.

  4. Schematic drawing of recipient hepatectomy illustrating where graft incisions occur.
    Figure 4: Schematic drawing of recipient hepatectomy illustrating where graft incisions occur.

    (a) PHA incision line. (b) Bile duct incision line; ligate the common bile duct with 7-0 silk as close to the confluence as possible and make the incision above the ligation. (c) PV incision line; ligate the right PV with 10-0 nylon and cut the bifurcation of the PV just above the right PV ligation after clamping the PV. (d) SHIVC incision line; cut the SHIVC as close to the liver as possible. (e) IHIVC incision line. Dashed lines represent a cut, solid lines represent a tie and cut procedure, and the solid double line represents a tie only. GB, gallbladder; LGA, left gastric artery; Lt. RV, left renal vein; Rt. RV, right renal vein; SpA, splenic artery; SpV, splenic vein.

  5. Schematic drawings of venous anastomoses and bile duct reconstruction in the recipient operation.
    Figure 5: Schematic drawings of venous anastomoses and bile duct reconstruction in the recipient operation.

    (a) SHIVC anastomosis. After placing the bilateral stay suture on the recipient SHIVC, start continuous suture on the posterior wall from the left side of the SHIVC and then continue the suture on the anterior wall from the right side. (b) PV anastomosis. Hold and pull the thread tied to the right PV and a stay suture on the left edge of posterior wall of the PV and slightly lift the anterior wall of the recipient's PV using an L-shaped injector; then insert the donor PV cuff. (c) Bile duct reconstruction; cut the anterior wall of the recipient bile duct and insert the bile duct stent into the recipient's bile duct.

  6. Perfused liver graft immediately after completing all anastomoses in mouse liver transplantation.
    Figure 6: Perfused liver graft immediately after completing all anastomoses in mouse liver transplantation.

    (a) Top, perfused liver graft immediately after completing all anastomoses. Bottom, a higher-power view (25× magnification) of the PV, the IHIVC, and the bile duct anastomoses in the field above (10× magnification). Scale bars, 5 mm. (b) Schematic drawing of transplanted liver after completing anastomoses. SMV, superior mesenteric vein. Experiments were performed under an institutional animal care and use committee–approved protocol.

  7. Histology of normally functioning mouse liver grafts 30 d after transplantation.
    Figure 7: Histology of normally functioning mouse liver grafts 30 d after transplantation.

    The tissue was fixed in 10% formalin for 24–48 h (at 4 °C). Hematoxylin and eosin staining was performed using paraffin-embedded tissue section. (a) Liver graft 30 d after syngeneic liver transplantation (C57BL/6 right arrow C57BL/6). (b) Liver graft 30 d after allogeneic liver transplantation (C57BL/6 right arrow C3H). In syngeneic transplantation, there are fewer infiltrating inflammatory cells in periportal and sinusoidal areas compared with allogeneic transplantation. Experiments were performed under an institutional animal care and use committee–approved protocol. Scale bars, 50 μm.

Videos

  1. Preparation before liver graft harvest (Steps 818)
    Video 1: 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.
  2. Donor liver perfusion and harvest (Steps 2033)
    Video 2: 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.
  3. Cuff attachment (Steps 3540)
    Video 3: 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.
  4. Stay suture attachment (Step 41)
    Video 4: Stay suture attachment (Step 41)
    The liver is rotated and the stay suture is put on the SHIVC, on the bilateral edge.
  5. Preparation before anhepatic phase (Steps 4355)
    Video 5: 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.
  6. The anhepatic phase (Steps 5671)
    Video 6: 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.
  7. IHIVC anastomosis (Steps 72 and 73)
    Video 7: 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.
  8. Bile duct anastomosis (Step 74)
    Video 8: 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.

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Author information

  1. These authors contributed equally to this work.

    • Shinichiro Yokota &
    • Shinya Ueki

Affiliations

  1. Thomas E. Starzl Transplantation Institute, Department of Surgery, 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

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Author details

Supplementary information

Video

  1. 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.
  2. 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.
  3. Video 3: Cuff attachment (Steps 35–40) (24.4 MB, Download)
    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.
  4. Video 4: Stay suture attachment (Step 41) (17.34 MB, Download)
    The liver is rotated and the stay suture is put on the SHIVC, on the bilateral edge.
  5. 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.
  6. 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.
  7. Video 7: IHIVC anastomosis (Steps 72 and 73) (26.86 MB, Download)
    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.
  8. Video 8: Bile duct anastomosis (Step 74) (27.96 MB, Download)
    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.

Additional data