protocol

Orthotopic mouse liver transplantation to study

liver biology and allograft tolerance
Shinichiro Yokota1,2,5, Shinya Ueki1,5, Yoshihiro Ono1, Naoya Kasahara2, Anglica Prez-Gutirrez1, Shoko Kimura1,
Osamu Yoshida1, Noriko Murase1, Yoshikazu Yasuda2, David A Geller1,3 & Angus W Thomson1,4
1Thomas E. Starzl Transplantation Institute, Department of

Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. 2Department of Surgery,
Jichi Medical University, Shimotsuke, Tochigi, Japan. 3Liver Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA. 4Department of
Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. 5These authors contributed equally to this work. Correspondence should
be addressed to A.W.T. (thomsonaw@upmc.edu).

2016 Macmillan Publihers Limited. All rights reserved.

Published online 2 June 2016; doi:10.1038/nprot.2016.073

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 ~23 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%.

INTRODUCTION
Development and applications of the protocol
Organ transplantation is the established treatment for patients
with end-stage organ disease. The importance of animal models
in transplantation is widely recognized among researchers in
the field to improve the understanding of transplant pathology
and to identify novel therapeutic strategies1. Orthotopic liver
transplantation in the mouse was first described by Qian
et al.2 at the University of Pittsburgh in 1991. This technique
was developed by applying the cuff technique principle previously used in orthotopic liver transplantation in the rat, which
simplified venous vascular anastomosis as compared with handsuture technique3. Since then, our laboratory and others have
used the model to elucidate mechanisms of liver lymphocyte
homeostasis4, transplant ischemiareperfusion injury510, liver
allograft tolerance1117, liver regeneration1820, and other liver
immunopathologies21,22.
The liver is a life-sustaining organ, and orthotopic liver transplantation in the mouse provides a more clinically relevant model
than heterotopic heart transplantation or single-lung transplantation, which are not life sustaining1. Moreover, with respect
to human/clinical relevance, the liver is a unique/tolerogenic
immune organ with hematopoietic and immune regulatory
properties that can promote operational tolerance in a significant proportion of stable human liver allograft recipients 2325.
Although the precise mechanisms of liver transplant tolerance
are yet to be elucidated, a better understanding of the unique
features of the liver as a tolerogenic organ has significant implications for an improved understanding of regulation of inflammatory and immune-mediated liver and other organ diseases and
their therapy. Thus, the mouse liver transplant model remains
an exceptionally useful and powerful tool with which to elucidate
the cellular and molecular mechanisms involved in inflammatory
liver disease and its resolution.
This procedure is of interest not only to microvascular
surgeons, gastroenterologists, hepatologists, and investigators

in the transplantation community, but also to those interested

in applying the model to study the regulation of liver immu
nopathology and liver biology in general. Because in adult mice
the liver is a hematopoietic organ, the liver transplant model is
also of considerable potential interest to investigators studying
hematopoiesis.
Overview of the protocol: advantages and limitations
The surgical procedure is divided into three main stages: (i) donor
surgery, (ii) back-table preparation of the liver graft, and (iii) the
recipient operation2,26. The final stage of the recipient operation is
the most difficult, as it requires efficient removal of the recipients
liver and implantation of the donor liver within 2030 min
to minimize the duration of portal vein (PV) clamping2. The
liver graft is placed orthotopically, thus occupying the normal
anatomical site in a manner similar to that seen in clinical transplantation in humans27, as opposed to heterotopic transplantation, in which the organ is placed in an alternative site. Thus,
the orthotopic transplantation model is more clinically relevant
than heterotopic transplantation, which is purely an experimental model. However, prolonged PV clamping in the orthotopic
liver transplantation model may lead to death of the recipient.
Thus, this model is considered surgically demanding compared
with other organ transplant models in mice such as heterotopic
heart transplantation and orthotopic kidney transplantation1,26.
We speculate that technical difficulty is the main reason that this
model has been used by only a few research groups in the world.
Here we describe techniques and materials in meticulous stepwise detail, including several modifications that have not been
described in the literature previouslye.g., surgical instruments,
preparation of vascular cuffs and bile duct stents, anesthetic
technique during donor and recipient operations, preparation
before the anhepatic phase, and the suprahepatic inferior vena
cava (SHIVC) suture during the anhepatic phase. We hope that
this will allow researchers who are not currently proficient with
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2016 Macmillan Publihers Limited. All rights reserved.

the protocol to implement the procedure successfully. We have

also provided step-by-step videos (Supplementary Videos 18)
that depict the important stages of the donor surgery, cuffing, and
recipient surgery. Our objective is to enable research groups to
implement the mouse liver transplantation procedure with high
technical success and a high perioperative survival rate.
Although technically demanding, the mouse model provides
unique advantages over other, larger animal liver transplant
models, such as the rat and pig models, because the mouse
genome is well characterized and there is much greater
diversity and availability of both genetically modified animals
(knockout or transgenic) and research reagents at a relatively
low cost2,26.
Alternative methods
Although some institutions have developed an arterialized liver
graft, this adds a further level of difficulty to the procedure
described in this protocol. In addition, the benefit of arterialization in mouse liver transplantation remains controversial5,26,28.
One study from the University of Oxford28 found that graft arterialization did not have a major impact on liver injury, cytokine
mRNA expression, or graft survival compared with nonarterialized liver transplantation in a syngeneic donorrecipient combination (CBA/Ca CBA/Ca). However, studies from the University
Hospital of Zurich26 and UCLA5 showed that re-arterialization of
the graft significantly (P < 0.05) reduced liver injury and prolonged
survival with or without prolonged (24 h) cold preservation in a
syngeneic combination (BALB/c BALB/c). The difference in the
conclusions drawn regarding the impact of graft arterialization
may reflect differences in the mouse strains used or technical

differences between these groups. The impact of graft arterialization in allogeneic liver transplantation has not been examined.
Partial liver transplantation, or reduced-size liver transplantation, has also been developed in mice18,19. This model represents
clinical living-donor liver transplantation, which uses a reducedsize, partial liver graft, rather than a whole liver graft. However,
reduced-size liver transplantation is essentially the same operation as the intact liver transplantation procedure, except that it
requires hepatectomy, which can be achieved relatively easily by
ligating certain lobes (the left lateral, the caudate, and/or the
median lobe) with silk sutures18,19. Thus, we describe our protocol for nonarterialized, orthotopic mouse liver transplantation.
This protocol can be applied to establish both arterialized and
partial liver transplantation in mice.
Experimental design
Mouse weight. Ideally, donor mice should weigh between 25 and
30 g, because bile duct stent insertion is difficult for mice weighing less than 25 g. In addition, the PV and infrahepatic inferior
vena cava (IHIVC) may be too short for anastomosis. However,
mice weighing more than 35 g should be avoided, as recipients
with large amounts of intra-abdominal fat make the operation
more difficult.
Choosing appropriate mouse donor and recipient strains.
In contrast to allogeneic heart or skin transplantation, allogeneic liver transplantation in the mouse usually does not result in
rejection in most strain combinations11,13,29. In most published
work, the male C57BL/6 (H-2b) to C3H (H-2k) combination has
been used.

MATERIALS
REAGENTS
CRITICAL The reagents and microsurgical equipment listed represent the
authors preference. Similar setups and equipment can be used as alternatives
and are likely to be equally effective.
CRITICAL All of the reagents listed can be used and stored according to
the manufacturers instructions.
Appropriate pairs of mice. For example, male C57BL/6 (H-2b; donor)
and C3H (H-2k; recipient) mice, 812 weeks of age (preferred body weight
for donors is 2530 g, and preferred body weight for recipients is 3035 g)
(The Jackson Laboratory) ! CAUTION All experiments involving animals
must be performed in accordance with national and institutional regulations. This protocol has been approved by the University of Pittsburgh and
Jichi Medical University institutional animal care and use committees and
in accordance with criteria outlined in the Guide for the Care and Use of
Laboratory Animals, a publication of the US National Institutes of Health
(Supplementary Videos 18 were recorded at Jichi Medical University).
Isoflurane, United States Pharmacopeia (USP) liquid for inhalation, 250 ml
(Piramal Healthcare, cat. no. NDC 66794-017-25)
Heparin sodium injection, USP (1,000 USP units per ml; Fresenius Kabi,
cat. no. 504031)
0.9% (wt/vol) sodium chloride injection (saline), USP (Baxter Healthcare
Corporation, cat. no. 2B1324X)
Viaspan (Belzer University of Wisconsin solution) 1L (Teva Pharmaceuticals, cat. no. 004606)
Buprenex injectable (buprenorphine hydrochloride; Reckitt Benckiser
Pharmaceuticals, cat. no. NDC 12496-0757-1)
Cefazolin for injection, USP (APP Pharmaceuticals,
cat. no. NDC 63323-238-61)
PVP scrub solution (povidoneiodine 7.5%; Medline,
cat. no. NDC 12496-0757-1)
70% Ethanol (Decon Labs, cat. no. 2716)
1164 | VOL.11 NO.7 | 2016 | nature protocols

EQUIPMENT
JELCO IV catheter, 16-gauge 11/4 (Smiths Medical,
cat. no. 4042; Fig. 1a)
IV catheter, 20-gauge 11/4 (Excel International, cat. no. 26742; Fig. 1a)
Becton, Dickinson and Company (BD) Intramedic polyethylene tubing
(PE10; BD, cat. no. 427400; Fig. 1a)
Heavy-duty razor blade (Titan, cat. no. 11039)
Microscope-Leica Wild M650, 640 magnification (Leica Microsystems)
Tec 3 isoflurane funnel fill vaporizer (General Anesthetic Services)
Vetroson V-10 bi-polar electrosurgical unit (Summit Hill Laboratories)
Cautery, high-temperature fine tip (Bovie Medical Corporation,
cat. no. AA01)
Mosquito classic delicate hemostatic forceps (Codman,
cat. no. 30-4472; Fig. 1b)
Micro-retractor (Murdock; Roboz Surgical Instrument,
cat. no. RS-6550; Fig. 1b)
Nonwoven gauze sponges (Fisherband, cat. no. 870-PC-DBL; Fig. 1b)
Sterile cotton swab, double cone hard sharp point tip (Puritan Medical
Products Company, cat. no. 870-PC-DBL)
Microneedle holder (Aesculap Surgical Instruments, cat. no. FD231R; Fig. 1c)
McPherson tying forceps (Accurate Surgical & Scientific Instruments
Corporation, cat. no. ASSI-4388; Fig. 1c)
Weldon miniature bulldog clamps, curved jaws (V. Mueller, cat. no.
CH6077-002; Fig. 1c)
Johns Hopkins bulldog clamps (V. Mueller, cat. no. CH5151-1; Fig. 1c)
Vannas capsulotomy scissors (Accurate Surgical & Scientific Instruments
Corporation, cat. no. ASSI-21288; Fig. 1c)
Gill Iris forceps (V. Mueller, cat. no. OP3424; Fig. 1c)
Hoskin Mk II micro forceps with long curved jaws, 6-mm tying
platform (Accurate Surgical & Scientific Instruments Corporation,
cat. no. ASSI-2177; Fig. 1c)

protocol
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).

c
Needle holder

IHIVC cuff

McPherson forceps

PV cuff

Weldon miniture bulldog clamp

Johns Hopkins bulldog clamp

BD stent

2016 Macmillan Publihers Limited. All rights reserved.

Vannas capsulotomy scissors

Stevens tenotomy scissors (Accurate Surgical & Scientific Instruments

Corporation, cat. no. ASSI-9193)
Durmont #5 forceps (Fine Science Tools, cat. no. 11251-20)
Adson forceps (V. Mueller, cat. no. NL1400)
Jewelers forceps (Codman, cat. no. 30-6712)
Tube occluding forceps (Roboz Surgical Instrument, cat. no. 65-7480)
Vannas spring scissors, 3-mm cutting edge (Fine Science Tools,
cat. no. 15000-00)
Micro serrefine-straight/15 mm (Fine Science Tools, cat. no. 18055-03)
Micro serrefine clamp applicator with lock (Fine Science Tools,
cat. no. 18056-14)
2-0 Silk (Ethicon, cat. no. SA-85)
5-0 Silk (Braintree Scientific, cat. no. SUT-S106)
7-0 Silk (Braintree Scientific, cat. no. SUT-S103)
Micro AROSuture, sterile 10-0 nylon suture, 70 m, TAP points
(AROSurgical Instruments Corporation, cat. no. T4A10N07)
4-0 Vicryl (Ethicon, cat. no. J662H)
5-ml Syringe (BD, cat. no. 309646)
BD 1-ml insulin syringe U-100 slip tip (BD, cat. no. 329654)
BD 0.5-ml U-100 insulin syringe, 28-gauge 1/2 (BD, cat. no. 329461)
IV catheter 22-gauge 11/4 inches (Excel International, cat. no. 26746)
BD PrecisionGlide needle, 27-gauge 1/2 (BD, cat. no. 305109)
BD PrecisionGlide needle, 30-gauge 1/2 (BD, cat. no. 305106)

Gill iris forceps

Hoskin Mk II micro forceps with curved jaws

Blunt-tip L-shaped injector (custom-made)

CRITICAL All microsurgical equipment listed above should be sterilized
by autoclaving (>120 C) before use.
Insulated foam container without lid (for back-table preparation of the
liver graft), dimensions (length width height): inside, 18 15 4 cm;
outside, 21 18 5.5 cm; wall thickness, 1.5 cm (Marko Foam Products,
cat. no. IC-103)
Falcon tissue culture dish, 60 15 mm (Corning, cat. no. 353002)
EQUIPMENT SETUP
Injector setup To make a blunt-tip L-shaped injector, take the sharp tip off
a 30-gauge needle by holding the tip with mosquito forceps and bending it
back and forth. Once the sharp tip comes off, blunt and smooth the end of
the injector using a tool such as a flat metal file.

PROCEDURE
Preparation of PV and inferior vena cava cuffs TIMING 510 min
1| Prepare the cuff for the PV from a 20-gauge angiocatheter using a razor blade. Although an 18-gauge angiocatheter
can be used as an alternative, we prefer a 20-gauge angiocatheter because of the ease of inserting the 20-gauge cuff
into the PV. The metal needle of the angiocatheter should be used as a cutting surface. The main body of the cuff should
be 2 mm in length. The extension handle can be 1 mm or longer, depending on the preference of the operator (Fig. 1a).
2| Make two shallow grooves on the main body of the cuff using a razor blade. One of these grooves will help secure the
attachment of the cuff to the donor PV with silk suture ligation during the back-table preparation. The second groove will
be helpful with the anastomosis of the PV during the recipient operation.
3| Repeat Steps 1 and 2 using a 16-gauge angiocathether to prepare the cuff for IHIVC.
Preparation of bile duct stent TIMING 5 min
4| Prepare the bile duct stent. Use a razor blade to cut the polyethylene tubing (PE10) into a trapezoid shape 2 mm in length
(Fig. 1a). Use a hard, blunt object (e.g., a ruler) to repeatedly push down the length of the tube to narrow its width to 0.30.5 mm.
CRITICAL STEP Be careful not to push down too hard, as this will cause narrowing of the lumen of the bile duct stent.
Narrowing or occlusion of the stent could cause obstruction of the bile duct and negatively influence graft recipients survival.
Donor surgery TIMING 4050 min
5| Anesthesia and laparotomy. Anesthetize the donor mouse by putting the animal in an induction chamber. Use the
appropriate amount (~12 ml per 2530 g of animal body weight) of isoflurane to anesthetize the animal. Observe the
respiration rate of the animal carefully. When a respiration rate of one breath per second is achieved, the mouse is ready to
be placed on the surgical table.
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6| Place the anesthetized mouse under the surgical microscope. Maintain the mouse under general anesthesia using
isoflurane inhalation. Adjust the vaporizer and use 34% for induction with continuous air or oxygen flow of 0.51 L/min.
Prep the abdominal wall with povidoneiodine first and then with 70% ethanol.
7| Make a midline abdominal incision from the xiphoid process to the pubis. Next, make a transverse incision and use
the bi-polar electrosurgical unit to stop bleeding from the abdominal wall. After making the incisions, decrease isoflurane to
0.51% for maintenance of anesthesia. Alternative concentrations of isoflurane may be used.
CRITICAL STEP It is critical to observe the respiration of the mouse during the operation to assess the depth of
anesthesia. Adjust the isoflurane vaporizer to maintain a respiration rate of approximately one breath per second.

2016 Macmillan Publihers Limited. All rights reserved.

8| Use mosquito forceps to hold the xiphoid process and retract toward the head of the mouse. Fix the mosquito forceps
in place using soft clay (Fig. 1b).
CRITICAL STEP See Supplementary Video 1 for additional guidance on preparation before liver graft harvest (Steps 819).
9| Wrap the small and large intestines using a wet nonwoven gauze sponge (6 6 cm) and place them on the left side of
the abdominal cavity.
10| Use a small (3.5 3.5 cm), wet nonwoven gauze sponge to gently retract the left and middle lobes of the liver toward
the xiphoid process to expose the caudate lobe (Fig. 1b).
11| Cut the ligament around the caudate lobe.
12| Expose the proper hepatic artery (PHA). Dissect the PHA using McPherson forceps (Fig. 1c) and ligate it with
10-0 nylon (Fig. 2a).
13| Dissect the extrahepatic bile duct using McPherson forceps and put 7-0 silk under the dissected portion of the bile
duct. Cut the anterior portion of the bile duct above the pancreas. Insert a bile duct stent into the bile duct and secure
it in place with 7-0 silk.
! CAUTION Make sure not to insert the bile duct stent too deep, to avoid obstructing the bile duct confluence (Fig. 2b).
? TROUBLESHOOTING
14| Gently retract the right lobe of the liver with a small, wet sponge toward the xiphoid process to expose the IHIVC.
15| Use a fine-tip cautery to dissect the retroperitoneum above the right renal artery and veins. Slightly displace the IHIVC
with a cotton swab to cauterize small veins merging into the IHIVC on the right side.
16| Remove the small sponge retracting the right lobe and use it to cover the PV to avoid injury to the PV. Dissect the left
side of the IHIVC above the left renal artery and veins. Slightly displace the IHIVC with a cotton swab to cauterize veins
merging into the IHIVC on the left side. Also cauterize the lumbar veins merging into the IHIVC.
17| Ligate the right adrenal vein with 7-0 silk. Ligation should be placed close to the IHIVC.
18| Skeletonize the right renal vein and separate it from the right renal artery. Put 10-0 nylon under the right renal vein.
19| Dissect the retroperitoneum and the fat on the IHIVC.
20| Inject 20 U (20 l) of heparin sodium using a 0.5-ml U-100 insulin syringe (28-gauge) via the penile vein.
CRITICAL STEP Avoid infusing any air bubbles. Even a small amount of air could cause air embolism and potentially be
fatal for the animal.
CRITICAL STEP See Supplementary Video 2 for additional guidance on donor liver perfusion and harvest (Steps 2034).
21| While waiting for 1 min for the heparin to distribute systemically, use a small, wet gauze to retract the duodenum to
expose the PV. Confirm the location of the pyloric vein and the splenic vein merging into the PV.
22| Slowly inject cold 4 C saline or UW solution to perfuse the donor liver from the IHIVC using a 5-ml syringe with a
27-gauge needle. The needle should be inserted slightly below where the left renal vein merges with the IHIVC. When blood
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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.

from the donor liver is flushed and the liver turns evenly
pale, stop the infusion. It usually requires 23 ml of
perfusion solution to achieve good perfusion of the graft.
Immediately cut the PV below the splenic vein to make an
outlet for the perfusion solution.
CRITICAL STEP Remove any air bubbles from the syringe
before infusion to avoid infusion of air into the graft
and subsequent air embolism. Air embolism of the donor
liver would be visible as multiple red patches, indicating
nonperfused areas. This would lead to poor graft function
after implantation.

e (Step 31)

GB

b (Steps13 and 23)

(Step 12)

PHA
LGA

Rt. adrenal vein

SpA
SpV

Rt. RV

d (Step 29)

Pyloric vein

Lumbar veins
(Step 25)

c
Lt. RV

23| Cut the bile duct below the area where the bile duct
stent is inserted (Fig. 2b). We prefer to cut the bile
duct after perfusion rather than before, to avoid bleeding before perfusion, which may compromise the cardiodynamic
stability of the donor and the condition of the graft.
24| Using a cotton swab and forceps, skeletonize the PV.
CRITICAL STEP Removal of fat from the PV will make the cuff attachment easier and also help avoid stenosis in the cuff.
? TROUBLESHOOTING
25| Ligate the pyloric vein and the splenic vein with 10-0 nylon. Leave one segment of 10-0 nylon on the splenic vein
for the cuff attachment. Cut the PV trunk below the splenic vein (Fig. 2c).
CRITICAL STEP Alternatively, this step can be performed before graft perfusion at Step 20 if preferred.
26| Ligate the right renal vein with 10-0 nylon and cut it.
27| Separate the right renal artery from the IHIVC.
28| Put a 10-0 nylon needle through the IHIVC above the left renal vein. Leave one segment of 10-0 nylon for the
cuff attachment.
29| Cut the IHIVC above the left renal vein (Fig. 2d).
30| Use a small, wet nonwoven gauze sponge to gently retract the liver to expose the gall bladder. Use 10-0 nylon with a
needle with a needle holder (Fig. 1c), and put the swaged end of the needle under the cystic duct and ligate it. Cut the gall
bladder with scissors or cauterize it with a cautery to drain bile.
31| Retract the liver downward to expose the SHIVC. After cutting the falciform ligament, cut the anterior wall of the SHIVC
first, and then cut the posterior wall of the SHIVC (Fig. 2e). If the confluence of the left phrenic vein and the SHIVC is close
to the liver, it is necessary to ligate and cut the left phrenic vein to avoid bleeding after SHIVC anastomosis during the
recipient operation. However, in our experience, it is not necessary to ligate the left phrenic vein in the majority of cases
when using C57BL/6 mice as donors.
CRITICAL STEP It is important to cut both the anterior and the posterior wall as close to the diaphragm as possible, to
preserve enough margin for anastomosis of the SHIVC in the recipient operation.
32| Cut the left triangle ligament and retract the left lateral lobe to the right side. Ligate the paraesophageal vessel located
at the cranial end of the caudate lobe with 7-0 silk.
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33| Cut the ligament and connective tissue between the liver and the retroperitoneum.
34| Harvest the whole liver and immediately place the organ in a container with cold (4 C) saline or UW solution. Discard
the carcass of the donor mouse according to institutional guidelines.
Back-table preparation of the liver graft TIMING 1530 min
35| Place crushed ice in an insulated foam container without a lid, and use a tissue culture dish on ice to prepare the liver
graft. The dish should be filled with cold (4 C) saline or UW solution. Carefully rotate the donor liver floating in the dish
so that the inferior surface faces upward. Clamp the handle of a Weldon miniature bulldog clamp (Fig. 1c) with a mosquito
clamp (Fig. 3a). Put soft clay on the handle of the mosquito clamp to fix it in place on the ice.
CRITICAL STEP See Supplementary Video 3 for additional guidance on cuff attachment (Steps 3540).

2016 Macmillan Publihers Limited. All rights reserved.

36| Clamp the extension handle of a 16-gauge cuff with a bulldog clamp and hold it in place (Fig. 3a). Pull the 10-0 nylon
tied to the IHIVC at Step 25 through the 16-gauge cuff, and also pull the IHIVC through the cuff (Fig. 3b).
37| Confirm the lumen of the IHIVC and fold the IHIVC over the 16-gauge cuff to expose the inner endothelial surface.
CRITICAL STEP The extension handle of the cuff should be on the ventral side of the IHIVC to avoid twisting.
? TROUBLESHOOTING
38| Secure the IHIVC to the cuff by ligating it with 7-0 silk (Fig. 3c). Two ties should be enough to secure the IHIVC to
the cuff. Ligation should be at one of the grooves made on the cuff body to avoid detachment of the ligation from the cuff.
Cut excess 7-0 silk close to the tie.
39| Put 5-0 silk around the IHIVC between the attached cuff and the right inferior lobe, and make one tie.
CRITICAL STEP This tie should be tight enough to prevent bleeding from the IHIVC immediately after PV anastomosis,
but it should be loose enough to release after the IHIVC anastomosis.
40| Attach a 20-gauge cuff to the PV in a similar manner as the IHIVC cuff attachment at Step 39 (Fig. 3d).
CRITICAL STEP The extension handle of the cuff should be on the ventral side of the PV. Avoid twisting of the PV by
having the pyloric vein and the splenic vein at 3 oclock
and 6 oclock.

41| Rotate the liver and expose its superior side. Put a stay
suture on the SHIVC using 10-0 nylon on the bilateral edge
(Fig. 3e). Use a Johns Hopkins bulldog clamp (Fig. 1c) to
hold the stay suture on both sides.
CRITICAL STEP See Supplementary Video 4 for additional
guidance on stay suture attachment (Step 41).
Recipient operation TIMING 7090 min
42| Anesthesia and laparotomy. Anesthetize the recipient
mouse, make a midline incision, wrap the intestine with wet
gauze, and expose the liver in the same way as in the donor
surgery (Steps 510). Use a micro-retractor to retract the
abdominal wall (Fig. 1b).
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.
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43| Cut the ligament around the caudate lobe. Cut the falciform ligament. Use a small, wet gauze to retract down the liver
to expose left triangle ligaments for cutting.
CRITICAL STEP See Supplementary Video 5 for additional guidance on preparation before anhepatic phase (Steps 4355).
44| Ligate the left phrenic vein with 10-0 nylon.
CRITICAL STEP Put the needle through the diaphragm with suture placed as close to the left phrenic vein as possible.
45| Expose the paraesophageal vessels and ligate with 7-0 silk.
46| Retract the liver with a small, wet gauze to expose the retroperitoneal space below the SHIVC. First, cut the ligament
between the liver and the retroperitoneum using Vannas capsulotomy scissors (Fig. 1c). Then, use Gill iris forceps (Fig. 1c)
to dissect the space between the liver and the retroperitoneum.

2016 Macmillan Publihers Limited. All rights reserved.

47| Put the liver back in its normal position. Carefully put Gill iris forceps behind the liver just below the SHIVC. Put 2-0 silk
through behind the SHIVC.
48| Expose the IHIVC, cauterize the small veins behind the IHIVC, and cauterize the lumbar veins in the same manner
as in Steps 1416.
49| Cauterize the right adrenal veins.
50| Use Gill iris forceps to dissect behind the IHIVC. Make sure that the IHIVC is free from any retroperitoneal structure.
51| Expose the hilum with a small, wet gauze. Ligate the PHA with 10-0 nylon (Fig. 4a).
52| Dissect the common bile duct and isolate from the PV. Ligate with 7-0 silk as close to the confluence as possible,
and cut above the ligation (Fig. 4b). Use a cotton tip to gently separate the bile duct from the PV.
53| Ligate the right PV with 10-0 nylon (Fig. 4c, solid line). Leave one end long enough to use as the stay suture during
the anhepatic phase.
54| Prepare for the anhepatic phase by adjusting the anesthesia so that the respiration rate is about one breath per second.
CRITICAL STEP Anesthesia adjustment needs to be done before clamping of the IHIVC and the PV in Step 56. If the
anesthesia is too deep and the respiration is slower than one breath per second, the respiration may stop and the animal
may die during the anhepatic phase.
55| Using a 5-ml syringe filled with cold (4 C) saline and
with a 22-gauge catheter attached, slowly flush the liver graft
from the PV to remove UW solution. Use 23 ml to perfuse.
CRITICAL STEP Avoid allowing any air into the graft.

d (Step 58)

56| Place microclamps first on the IHIVC and then on the PV.
CRITICAL STEP Place clamps as low as possible to leave
the margin for anastomoses as long as possible. Expose the
SHIVC and gently pull 2-0 silk to create space to put the jaw

c (Steps 53 and 59)

GB

b (Step 52)
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.

a (Step 51)
PHA

(Step 59)
Rt. adrenal vein

LGA
Pyloric vein
SpA
SpV

Rt. RV

Lt. RV

Lumbar veins

nature protocols | VOL.11 NO.7 | 2016 | 1169

protocol
of a Weldon miniature bulldog clamp behind the SHIVC. Place the bulldog clamp on the diaphragm slightly above the
border between the diaphragm and the SHIVC. Firmly press on the jaws of the bulldog clamp with forceps to make sure
that the bulldog clamp is closed tightly. A Satinsky clamp can be used instead of a bulldog clamp, if one with appropriate
size for clamping mouse SHIVC is available.
CRITICAL STEP See Supplementary Video 6 for additional guidance on the anhepatic phase (Steps 5671).
57| Cut the anterior wall of the SHIVC from the right edge as close to the recipient liver as possible so as to keep a margin
for anastomosis as long as possible.
58| Cut the posterior wall of the SHIVC, also from the right edge.
CRITICAL STEP Leave some liver parenchyma on the posterior wall, as it helps avoid tearing of the SHIVC during the
posterior wall suture (Fig. 4d).

2016 Macmillan Publihers Limited. All rights reserved.

59| Cut the bifurcation of the PV just above the right PV ligation, and then cut the IHIVC as close to the liver as possible
so as to leave the margin as long as possible for anastomosis (Fig. 4c,e).
60| Hold the liver by hand and gently lift it. Cut the ligament and connective tissue between the liver and the
retroperitoneum. Discard the harvested liver according to institutional guidelines.
61| Rotate the operating table 90 so that the recipients head is on the left side in the operators view. Use cotton
tips to wipe any blood from the retroperitoneal surface, and make sure that there is no active bleeding.
62| Place the graft in the recipients abdominal cavity by holding the bulldog clamp on the stay sutures attached to the
bilateral edge of the SHIVC.
63| Put a suture on the bilateral edge of the recipients SHIVC.
CRITICAL STEP Ligate the stay suture on the left edge of SHIVC, and clamp the loose end of the suture with tube-occluding
forceps. Place the forceps on soft clay, and gently pull toward the left side of the recipient so that the posterior wall of the
SHIVC becomes straight.
? TROUBLESHOOTING
64| Start continuous suture on the posterior wall from the left side of the SHIVC (Fig. 5a). It takes ~78 stitches.
The stitches should be placed closer together at both edges of the SHIVC to avoid bleeding.
CRITICAL STEP The posterior wall of SHIVC is extremely easy to tear. Avoid any abrupt movement when tightening
the suture.
65| Continue the suture on the anterior wall from the right side of the SHIVC. Before closing the anterior wall of the SHIVC,
flush any air from the SHIVC cavity to avoid air thrombosis, which can be fatal for the recipient. Close the anterior wall.
Ligate stay sutures on the bilateral edge of the SHIVC.
CRITICAL STEP Ligation of stay sutures should not be too
tight, as this may cause narrowing of the SHIVC and outflow
a
b
block of the graft.
66| Turn the operating table 90 so that the recipients
head is now in a midline position.

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 recipients 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 recipients bile duct.
1170 | VOL.11 NO.7 | 2016 | nature protocols

protocol
67| Retract the liver with a wet gauze and expose the liver hilum. Move the intestine upward so that the recipients PV is
close to the donor PV.
68| Clamp the thread tied to the right PV with a Johns Hopkins bulldog clamp. Place the bulldog clamp on the right side of
the recipient so that the recipient PV is in an upright position.

2016 Macmillan Publihers Limited. All rights reserved.

69| Place a stay suture on the left edge of the posterior wall of the PV. Use a Johns Hopkins bulldog clamp to hold the
suture, and place the bulldog on the left side of the recipient.
CRITICAL STEP The anterior wall of the PV should be arching downward and should not overlap with the posterior wall.
Nonoverlapping anterior and posterior walls make the insertion of the PV cuff easier.
70| Put 7-0 silk around the recipient PV, and make one knot. Using an L-shaped injector, slightly lift the anterior wall of
the recipients PV, hold the extension handle by Hoskin Mk II micro forceps (Fig. 1c), and insert the donor PV cuff (Fig. 5b).
Fix the donor PV cuff in the recipient PV with 7-0 silk placed around the recipient PV.
CRITICAL STEP Insert the cuff while flushing the recipients PV with saline using an L-shaped injector. This helps remove
air bubbles and avoids any air embolism. Avoid any abrupt movement, and make sure that PV anastomosis is completed
without any torsion.
? TROUBLESHOOTING
71| Release the clamp on the PV first, and then the one on the SHIVC. The graft should be perfused and systemic circulation
should be restored.
72| Put a stay suture on the bilateral edge of the recipient IHIVC so that the anterior wall is slightly hanging downward
and not overlapping the posterior wall. Put a 7-0 silk around the recipient IHIVC and make one knot. Use an L-shaped
injector to slightly lift the anterior wall of the recipients IHIVC, and insert the donor IHIVC cuff. Fix the donor IHIVC cuff
in the recipients IHIVC with 7-0 silk.
CRITICAL STEP Insert the cuff while flushing the recipients IHIVC with saline using an L-shaped injector to remove air.
In contrast to rat liver transplantation, we have found that it is unnecessary to evacuate some blood at the end of PV or
IHIVC anastomosis in the mouse liver transplantation model.
CRITICAL STEP The IHIVC anastomosis is performed in a similar manner as the PV anastomosis. See Supplementary
Video 7 for additional guidance on IHIVC anastomosis (Steps 72 and 73).
73| Release the 5-0 silk tied around the donor IHIVC first and then release the clamp on the recipient IHIVC.
74| Expose the hilum and use tubeoccluding forceps to pull down the
thread fixing the donor bile duct stent.
Put 7-0 silk around the recipient bile
duct and make one knot. Hold the
recipient bile duct so that it becomes
straight and parallel to the donor bile
duct. Cut the anterior wall of the

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
committeeapproved protocol.

Bile duct stent

Duodenum
PV cuff
Pancreas head

SMV
IHIVC cuff

nature protocols | VOL.11 NO.7 | 2016 | 1171

protocol
bile duct with scissors and insert the bile duct stent into the recipients bile duct (Fig. 5c). Ligate the bile duct stent
in place with 7-0 silk to complete the bile duct anastomosis. A representative photograph and a schematic drawing of
a transplanted liver graft after completion of all anastomoses are shown in Figure 6.
CRITICAL STEP See Supplementary Video 8 for additional guidance on bile duct anastomosis (Step 74).
75| Abdominal closure. Put 23 ml of warm saline in the abdominal cavity. Make sure that there is no intra-abdominal bleeding.
76| Close the abdominal incision in two layers, using a continuous running 4-0 vicryl.
Animal recovery TIMING 46 h
77| Place the recipient mouse in a warm cage to recover. Use buprenex and cefazolin as indicated in institutional guidelines.

2016 Macmillan Publihers Limited. All rights reserved.

78| Observe the recipient mouse every 12 h for the first 46 h to make sure that it is fully awake and does not show any
signs of pain before returning the cage to the housing location.
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
Table 1 | Troubleshooting table.
Step

Problem

Possible reason

Possible solution

13

Difficulty inserting the stent

into the bile duct

Not able to visualize the inner lumen

of the common bile duct

Hold the anterior wall of the bile duct with forceps

and confirm the inner lumen before insertion

24

Difficulty removing fat from

the PV

Cotton tip is wet and cannot apply

enough friction to remove fat

Use a dry cotton tip when removing fat from vessels

37

Difficulty folding the vessel

over the cuff

Poor visualization of the inner lumen

of the IHIVC

Conduct cuff attachment underwater to keep the

lumen open for visualization

63

Difficulty visualizing the

posterior wall because
the anterior wall is
overlapping

Stay sutures on the bilateral edge

of recipient SHIVC were placed
inappropriately

Place stay sutures on the posterior wall of the

recipient SHIVC

70

The donor PV inserted into

the recipient PV slips out

The recipient PV is not close enough

to the donor PV

Move the recipients small intestine upward to

the hilum of the donor liver

TIMING
Steps 13, preparation of PV and inferior vena cava cuffs: 510 min
Step 4, preparation of bile duct stent: 5 min
Steps 534, donor surgery: 4050 min
Steps 3541, back-table preparation of the liver graft: 1530 min
Steps 4276, recipient operation: 7090 min
Steps 77 and 78, animal recovery: 46 h
ANTICIPATED RESULTS
We have trained four surgeons in the past 5 years to perform mouse liver transplantation proficiently. All of these
surgeons had experience and were proficient in liver transplantation in the rat before they started training in mice.
It took ~50 operations for each of them to achieve consistent success (i.e., survival of the recipient beyond 7 postoperative
days in a healthy condition) using the mouse model. Hematoxylin and eosinstained sections of normally functioning
liver grafts obtained 30 d after syngeneic (C57BL/6 C57BL/6) or allogeneic (C57BL/6 C3H) mouse liver transplantation
are shown in Figure 7a,b. In allogeneic transplantation, there is mild inflammatory infiltration in the periportal and
sinusoidal areas as compared with syngeneic transplantation, as described in a previous paper30. Mouse liver allograft
transplantation has been studied extensively at our institution, and we have shown that liver allografts between most
mouse strain combinations survive over 100 d and exhibit a state of tolerance without the use of immunosuppressive
1172 | VOL.11 NO.7 | 2016 | nature protocols

protocol
Figure 7 | Histology of normally functioning mouse liver grafts 30 d
after transplantation. The tissue was fixed in 10% formalin for 2448 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 C57BL/6). (b) Liver graft 30 d
after allogeneic liver transplantation (C57BL/6 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 committeeapproved protocol. Scale bars, 50 m.

2016 Macmillan Publihers Limited. All rights reserved.

agents11,13,29. Cellular and molecular mechanisms underlying

liver transplant tolerance have not been fully elucidated,
but those that have been described in the mouse and other
species are discussed in recent articles and reviews24,3133.

Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments 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.
AUTHOR 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.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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