Objectives: Orthotopic liver transplant remains technically challenging.
Materials and Methods: We performed whole graft orthotopic liver transplants with different anhepatic times (≤20 min, n = 19; vs 30 min, n = 9) and partial orthotopic liver transplants in rats including a male-to-male Sprague-Dawley group (n = 15), a male-to-male Lewis-to-Brown Norway group (n = 20), and a male-to-male Sprague-Dawley-to-Lewis group (n = 20); there was also a female-to-male Sprague-Dawley group (n = 19).
Results: For the groups with ≤20-minute or 30-minute anhepatic time, 14-day and 30-day survival rates were 94.7%, 89.5%, 88.9%, and 88.9%, respectively, and there was no difference in survival (P = .716). For 50% orthotopic liver transplants from the male-to-male Sprague-Dawley group, 14-day and 30-day survival rates were 93.3% and 86.7%, respectively, with no difference between whole and 50% graft orthotopic liver transplant. The 14-day and 30-day survival rates were, respectively, 30% and 10% for the Lewis-to-Brown Norway group and 30% and 6.6% for the Sprague-Dawley-to-Lewis group, with no differences between the 2 groups (P = .564). Most of the recipient rats died within 72 hours. Acute rejections and wound dehiscence were the causes of death. Recipients from the female-to-male Sprague-Dawley orthotopic liver transplant group died shortly after surgery.
Conclusions: Orthotopic liver transplants can be performed to achieve high success rates in the extended anhepatic time; however, orthotopic liver transplants from female Sprague-Dawley donor rats have a high risk of failure.
Key words : Anhepatic time, Partial orthotopic liver transplant, Sex mismatch
Orthotopic liver transplant (OLT) in rats is preferably used to study hemodynamic, ischemia-reperfusion injury, immunology, and regeneration. After its development by Lee and colleagues,1,2 it remains a difficult procedure despite many subsequent modifications.3-6 Herein we comprehensively summarized OLT in rats to simplify this procedure, especially for novices.
Materials and Methods
Orthotopic liver transplant in rats was performed to induce immunological tolerance through stem cells. Whole graft OLTs were conducted with different anhepatic times (≤20 minutes, n = 19; vs 30 minutes, n = 9), and partial OLTs were also performed for tolerance induction (Table 1) on the following groups: male donor rats to male recipient rats, including Sprague-Dawley (SD) (n = 15) to SD, Lewis to Brown Norway (BN) (n = 20), and SD to Lewis n = 20); as well as female SD donors to male SD recipients (sex-mismatch group, n = 19). However, the protocol of tolerance induction was not within the scope of this paper. Rats, including inbred SD, BN, and Lewis rats (200-400 g), used as donors and recipients were purchased from Beijing Vital River Laboratory Animal Technology Corporation. All animals were maintained in a temperature-controlled and light-controlled environment, with free access to standard food and bottled water; rats were fasted 12 hours before the operation. The experiments were conducted in compliance with the standards for animal use and care set by the Institutional Animal Care Committee of Henan Provincial People’s Hospital.
All surgical procedures were performed under nonsterile but clean conditions with a microscope. Inhalation of 5 volume percent isoflurane in 100% oxygen at a flow rate of 2 L/min was conducted with a modified mask for induction of anesthesia; 1 volume percent of isoflurane was inhaled through a modified mask connected to the anesthesia machine for maintenance anesthesia.
Donor surgical procedure and cuff preparations
All rats were placed supine with 4 limbs stabilized, disinfected, and draped. The entrance to the abdominal cavity was through a transverse subxiphoid incision; the abdominal aorta was clamped above the celiac trunk while both kidneys turned pale (Figure 1). Normal saline with heparin (5 mL, 50 μg/mL heparin) was injected into the aorta to heparinize and flush the liver; then, an additional 10 mL of lactated Ringer solution at 0 °C was injected into the portal vein (PV) to reflush the liver. The PV was dissected, the splenic vein was ligated, and the celiac trunk and common hepatic artery were carefully dissected. The splenic and left gastric arteries were separately ligated. A 3-mm long stent was inserted into the celiac trunk and secured.7 A 4-mm length of tubing (outer diameter 0.8 mm, inner diameter 0.6 mm) was cannulated into the bile duct and secured. Two adrenal veins were ligated, and the right renal vein was ligated, as well. The suprahepatic inferior vena cava (IVC) was cut close to the diaphragm, the infrahepatic IVC was cut at the level of the left renal veins, and the liver was explanted and immersed into 0 °C lactated Ringer solution for cuff preparation. The storage cold time was about 3 hours. The 2.4-mm-diameter cuff (2.1 mm inner diameter) was everted and secured on the PV, and the 2.8-mm-diameter cuff (2.4 mm inner diameter) was secured on the infrahepatic IVC2 (Figure 2). For the 50% grafts, the caudate lobes, the left lateral lobe, and the left portion of the median lobe were removed at the back table.
Recipient surgical procedure
The explant of the recipient liver was performed as for the donor. The adrenal veins were ligated, the infrahepatic IVC was exposed to the level of the right renal vein, and the proper hepatic artery was ligated. The back of the suprahepatic IVC was bluntly isolated to form a tunnel. The left subdiaphragmatic vein was ligated close to the diaphragm. The recipient PV and infrahepatic IVC were cross-clamped with microvascular clamps, and isoflurane was immediately decreased to 0.3 volume percent. A mosquito forceps was passed through the tunnel to cross-clamp the diaphragm ring to block the suprahepatic IVC (Figure 3). The suprahepatic IVC was then cut close to the liver, with 2 mm of the liver tissue in the back wall of the IVC retained; the PV and the infrahepatic IVC were transected, and the native liver was removed. The donor liver was orthotopically implanted. The suprahepatic IVC was anastomosed with continuous 8-0 nylon suture; once the anastomosis was complete, the mosquito forceps was removed, and an atraumatic vascular clamp was placed on the real suprahepatic IVC to occlude this again (Figure 4). The portal vein was reconnected by inserting the cuff into the recipient PV and secured, then the cross-clamps on the PV and the suprahepatic IVC were sequentially released to restore portal flow. The IVC was reanastomosed in the same manner as the PV. The anhepatic time generally ranged from 15 to 20 minutes for the ≤20-minute anhepatic group; for the group with 30-minute anhepatic time, the clamped PV was unclamped at 30 minutes. Then, 5 mL of lactated Ringer solution was injected through the penile vein while the liver graft was becoming bright red. The stent connecting the donor hepatic artery was inserted into the recipient common hepatic artery and secured8 (Figure 5). The stent was inserted into the recipient bile duct and fixed from both ends. The incision was closed in 2 layers. Ceftriaxone (100 mg/kg) was intraperitoneally injected daily until day 3 posttransplant. For the groups to induce tolerance, cyclosporine was subcutaneously injected daily at 4 mg/kg for 14 days, and recipients were placed into a cage with free access to 10% glucose, bottled water, and food. Oxygen was supplied to the recipients for about 4 hours under a warming lamp, and afterward the recipients were transferred to the regular cage.
For any recipient that lived for at least 8 hours postoperatively, the transplant surgery was considered successful.
Data for continuous variables are presented as means ± standard deviation. The cumulative survival rates were compared by the Kaplan-Meier method with SPSS software (version 22.0). Differences were considered significant at P < .05.
For our project, 28 whole liver and 74 half liver transplants (50% OLT) were performed. For the group with less than 20-minute anhepatic time, the 14-day and 30-day survival rates were 94.7%, and 89.5%, respectively; for the group with 30-minute anhepatic time, the 14-day and 30-day survival rates were 88.9% and 88.9%, respectively. There was no difference in survival for OLT with different anhepatic times (less than 20 minutes vs 30 minutes; P = .716) (Figure 6). Missed suturing was the cause of death in 2 cases, and the cause was unknown in 1 case. For the 50% OLT group from male SD to male SD, the 14-day and 30-day survival rates were 93.3% and 86.7%, respectively; the cause of death was blood loss after reperfusion in 2 cases, and there was no difference between whole graft OLT and 50% graft OLT. The 14-day survival rate for both the Lewis-to-BN group and the SD-to-Lewis group was 30%, and the 30-day survival rates were 10% and 6.6%, respectively, with no differences between the 2 rejection groups (P = .564; Figure 6).
The cause of death was missed suturing in 2 cases, and the cause of death was unknown in 1 case. For the group with 50% OLT from male SD to male SD, the 14-day and 30-day survival rates were 93.3% and 86.7%, the cause of death was blood loss after reperfusion, and there was no difference between whole graft and 50% graft OLT. The 14-day survival rate for both the Lewis-to-BN group and the SD-to-Lewis group was 30%, and the 30-day survival rates for the 2 groups were 10% and 6.6%, respectively (Figure 6). The cause of death for the group with rejections was bile leakage and wound dehiscence. Most of the recipient male SD rats with OLT from female SD donors died of graft failure in 3 days (Figure 7). Liver histological examinations of long-term survival rats are not shown for whole graft, 50% graft, the female graft, and the rejection groups.
Lee and colleagues1 developed the methods for OLT in the rat with the cuff method, which has been extensively used in basic science studies of liver transplant; however, microsurgically, this method remains challenging, although valuable improvements have been made.5-8 In this study, we modified some techniques; so far, modifications have been infrequently described in the literature. Our methods described here showed longer-term survival in rat recipients. For the incision, a transverse approach is better than a midline incision to expose the surgical field without a magnetic fixator retraction system, and it is less difficult, especially for 1 surgeon.
Donor quality is of paramount importance to liver transplant, and better flushing is a prerequisite.9 It is common to flush the liver through the PV as described in previous studies; however, this sort of flushing technique led to uneven perfusion, especially in the periphery of the liver. We propose that it is better and more efficient to flush the liver first through the aorta, followed then by flush through the PV.
Cuff-facilitated OLT techniques as described by Kamada and colleagues are well accepted.2,10-12 A 14-gauge catheter (1.54 mm in diameter) has been generally used as the cuff for the portal vein or IVC.13,14 However, this catheter is too small for most rats such that it will bring about the portal pressure increase with time after OLT, and this may have a negative effect on long-term survival, especially for rats over 300 g. We propose that the diameter of cuff for PV should be bigger for the heavier rats, and the cuff with 2.8 mm or greater diameter should be used for IVC. Dark Agouti rats have smaller vessels, and the cuff could be smaller (Figure 2). The cuff typically consists of a body and an extension, each with a length of 2 mm; however, we made the cuff with a longer body (3 mm) and a shorter extension (1 mm or less) to better facilitate insertion of the cuff into the recipient PV.
For liver transplant in rats, the reconstruction of the suprahepatic IVC is the most complicated step; suturing is the predominant procedure to date and should be completed as soon as possible because respiration is usually affected.15,16 Some modifications were made, including cuff anastomosis to simplify this critical step; magnetic anastomosis is in nature the cuff technique and efficiently shortens anhepatic time to benefit rat survival; however, there is difficultly to evert the suprahepatic IVC over the cuff and secure it, as the suprahepatic IVC is so short.17 Shortened anhepatic time is important for graft viability in animal and human OLT. In the literature, 26 minutes is reported to be the ceiling of anhepatic time in rat OLT.18-21
We revisited the anhepatic time documented in the literature, in which longer clamping of the diaphragm affected respiration and caused large movements in rat recipients; anesthesia had to be added to complete this reconnection rapidly, and higher mortality was shown with prolonged anhepatic duration.22 In our study, we changed clamping the diaphragmatic ring as applied to the suprahepatic IVC reconnection, thus safely extending anhepatic time to 30 minutes. As a result, this could simplify procedures and achieve higher success rates; generally, no more than 15 minutes is needed to complete this suturing anastomosis, with the diaphragm unclamped and real suprahepatic IVC clamped using another clamp, reducing the time of clamping of the diaphragm ring to benefit breathing in rats. This procedure can extend anhepatic time to 30 minutes, allowing surgeons a longer time to complete the anastomosis.
Our study showed no difference in survival rates between the groups with 30-minute and 20-minute anhepatic time. We consider that different anhepatic times have little impact on long-term survival. Magnetic anastomosis of the suprahepatic IVC brought about the shortest anhepatic time (about 10 minutes), although a foreign metal was introduced, which is not suitable for later imaging examinations. Ether mask anesthesia is a rather crude procedure in which anesthesia is uncontrolled and may account for higher intraoperative mortality rates; therefore, the ether mask technique should be abandoned.2,14,23
Portal vein and IVC reconnection with the cuff is less difficult than previously described. Because the hepatic artery supplies blood to the biliary system, its reconstruction is especially important to study chronic rejection or graft dysfunction in which immunological and nonimmunological factors may be involved; hence, OLT without arterialization precludes these similar studies. Without arterialization, OLT may lead to derangement of liver structure and therefore would not mimic clinical practice.24-27 Bile duct reconstruction is considered the final and least difficult procedure; it is recommended that a shorter donor bile duct can better avert biliary complications.
For partial OLT in rats, some liver lobes are removed; for the caudate, ligation was made with suture.28 For resection of the left lateral lobe and the right portion of the median lobe, suture ligation was made by 2 halves and an additional suture tie was made to prevent blood loss.
In clinical practice, the rate of female donation is increasing worldwide.29,30 Although female rat donors had favorable outcomes in our studies and other studies, the underlying mechanism is unclear; changes in the cytosolic content of both the estrogen and androgen receptors of the liver have been implicated, showing greater susceptibility to reperfusion injury in the immediate postoperative period.15
For the Lewis-to-BN group and the SD-to-Lewis group, 2 combinations were used to study immunology, and tolerance was induced through stem cells (not within the scope of this paper). The unfavorable outcomes for these 2 groups in our report were possibly because of early withdrawal of immunosuppression; however, further studies are needed to explain this. In addition, wound dehiscence was common in BN rats and uncommon in SD or Lewis recipients, and this is possibly species-specific. Also, we postulated that the sutures for closure of the abdominal wall could lead to dermatitis and cause itch and edema; this may also cause self-inflicted bite wounds in rats.
It was debatable whether postoperative administration of antibiotics in rat OLT is essential, and the use of an antibiotic agent excreted to bile should be considered during the early postoperative period.28 We chose to use antibiotics for 4 consecutive days because of the complexity of our procedure and because it is beneficial to survival, especially in partial OLT.
When systemic circulation was restored, 6 to 10 mL of additional normal saline were injected through the penile vein of male rats or the IVC of female rats, which could improve circulation; this led to faster breathing in rats, with the liver graft turning bright red, representing success of liver implantation.
Warming is necessary to mitigate trauma in rat recipients, especially during the first 24-hour period. Additional oxygen supply is also beneficial because, in partial OLT, liver regeneration will initiate, and this is an oxygen-consuming process; this also facilitates awakening from anesthesia and helps to mitigate operational trauma.31
The rat OLT and partial OLT described here achieved favorable outcomes, even with extended anhepatic time. However, OLT from female SD donor rats had a high risk of mortality for the male recipient rats.
Volume : 19
Issue : 9
Pages : 956 - 962
DOI : 10.6002/ect.2020.0364
From the Section 6 of Hepatopancreaticobiliary Surgical Department, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Zhengzhou, Henan, People’s Republic of China
Acknowledgements: We are grateful for support from Dr. Fangzhou, Liu, Dr. Xiaoying Guo, Hui Zhang from Henan Academy of Chinese Traditional Medicine. This study was funded by project 23456 of Henan Provincial People’s Hospital. Other than described, the authors have not received any funding or grants in support of the presented research or
for the preparation of this work and have no additional declarations of potential conflicts of interest.
Author contributions: HZ wrote the draft and performed orthotopic liver transplants. HL revised the draft. SW performed statistical analysis. GT cared for the rats. YC collected all references. GC funded and coordinated the study. SZ conceived, designed, and finalized the study and performed orthotopic liver transplants.
*Huibo Zhao and Hao Li contributed equally to this study.
Corresponding author: Shaotang Zhou or Guoyong Chen, 7 Weiwu Road, Jinshui district, Zhengzhou 450003, People’s Republic of China
Phone: +86 371 65580013
E-mail: email@example.com or firstname.lastname@example.org
Table 1. Donor and Recipient Weights and Survival Rates
Figure 1. Flushing the Liver First Through the Aorta, Which Was Blocked Above the Celiac Trunk
Figure 2. Cuffs for Portal Vein and Inferior Vena Cava
Figure 3. Clamping the Diaphragmatic Ring of the Rat
Figure 4. Clamping the Real Suprahepatic Inferior Vena Cava of the Rat With a Separate Vessel Clip
Figure 5. Hepatic Artery Reconstruction (arrow)
Figure 6. Survival Rates for 5 Different Groups
Figure 7. Poor Survival for Rat Liver Transplant with Female Sprague-Dawley Donors