Successful Transplantation of Non-Flushed Liver Organ in Rats

doi:10.21203/rs.3.rs-3136117/v1

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Successful Transplantation of Non-Flushed Liver Organ in Rats

https://doi.org/10.21203/rs.3.rs-3136117/v1

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This study aims to investigate the feasibility of liver transplantation using non-flushed organs in a rat model. Healthy adult rats were divided into two groups: liver transplantation using non-flushed donor liver (group NF; n = 12); liver transplantation using donor liver flushed with UW solution (group controls; n = 12). Liver function and histological changes were assessed at 72 hours post-transplantation. Long-term survival after transplantation was also compared between two groups by the Kaplan-Meier analysis. No significant differences in liver biochemistry were detected between two groups at 72 hours after transplantation. A comparable liver histology was noted between the NF and the controls. There was no significant difference in the overall survival rate. Transplantation of non-flushed livers is no inferior to current donor organ preservation technology in rats.

Health sciences/Gastroenterology

Health sciences/Medical research

Orthotopic liver transplantation in rat (OLT), first described by Lee^1,2, had been a popular animal model to investigate various problems. Donor liver flushing and preservation represent the upstream preparation for liver transplantation in rat liver transplantation. It also provides needed time for the preparation of the recipient and revascularization of the donor liver^3,4. Cold in situ perfusion and subsequent static cold storage (SCS) of the donor liver remains the standard of preservation prior to rat liver transplantation surgery^5,6. Residual blood in the donor liver is usually flushed out to prime subsequent cold storage, and the organ temperature drops during in situ perfusion.

Previous studies have found that liver inflow occlusion for no more than 30 minutes is safe and feasible in liver surgery⁷. If a donor liver was implanted shortly after organ procurement, the donor liver would not have to be flushed and preserved in vitro. Based on this assumption, this study was designed to investigate the feasibility of transplantation of non-flushed donor liver in rat model.

Validation of the rat liver transplantation model using a flushing-free organ

All liver recipients in each group survived after operation. In an effort to validate the rat liver transplantation model using a flushing-free organ, necrosis lesions were assessed 72 hours after surgery. No necrotic lesions on the engrafted liver surface in each group were observed by the naked eye 72 hours after surgery (Fig. 1a and 1b). Microscopically, the liver graft in NF and controls had normal architectures, with minor (< 5% of hepatocytes) centro-lobular micro-vacuolar steatosis (Fig. 1c and 1d). Immunohistochemical staining of CD31 positive cells showed no significant difference in the capillaries of the hepatic lobule area between the two groups at 72 hours (Fig. 1e and 1f).

The serum AST, ALT and TBIL levels in NF was lower than that in group controls 72 hours after transplantation even though the differences were insignificant (AST: controls (445[143–745]U/L), and NF (259 [104–551] U/L), p = 0.16; ALT: control (201 [81–279] U/L), and NF (118 [45–264] U/L), p = 0.20; TBIL: controls (0.7[0.3–1.2]mmol/L), NF (0.6[0.3–0.9] mmol/L), p = 0.37. Figure 2).

Survival analysis

Animal survival curves in all groups are shown in Fig. 3. Overall, no significant differences in survival were detected between the two groups (p = 0.57). However, the survival rate in the NF group became higher than that of the controls at the 4th week after surgery.

Our study shows that a non-flushed liver can be directly implanted within 30 minutes after procurement in rat liver transplantation model. Using no-flushed donor liver may simplify the procedures of rat liver transplantation but not affect the postoperative outcome.

The current standard organ preservation method in liver transplantation relies on static cold storage (SCS) in which residual blood inside the donor liver is flushed out and filled with an appropriate hypothermic preservation solution that prevents the formation of microcirculation thrombus and makes the organ to be tolerant to hypothermia⁸. In fact, hepatic blood occlusion for no more than 30 minutes is safe in liver surgery. Thus, if ischemic duration of a donor liver could be greatly shortened by rapid revascularization, organ flushing and preservation may become optional. As shown in this study, the postoperative serum aminotransferase and bilirubin 72 hours post-transplantation in rats with non-flushed livers were comparable to those with flushed donor organs. In addition, there were no significant differences in liver histology between the two groups. In animal models of liver preservation, reperfusion injury after prolonged liver storage will lead to loss of viability of sinusoidal endothelial cells. However, no significant difference in the capillaries of the hepatic lobule area was observed in immunohistochemical staining of CD31 positive cells detection between the two groups 72 hours after transplantation.

Unexpectedly, serum AST, ALT, and TBIL levels in animals using non-flushed donor liver at 72 hours after liver transplantation were numerically, but not statistically, lower than that with flushed organs. A few possible explanations for this phenomenon are as follows. First, the rewarming mediated injury of the non-flushed donor liver after implantation is lighter than the donor liver flushed with cryogenic UW solution. However, no statistically significant differences in serum AST, ALT, and TBIL levels were detected between different groups, and the main reason for this finding is the long-term ischemia occurred in the donor livers likely derived from complicated vascular reconstruction. Second, the ischemia-reperfusion of the non-flushed donor livers resembles the process of ischemic preconditioning (IPC) in liver surgery. IPC, which can be either directly applied to a target organ⁹, or remotely to a distant vascular bed¹⁰, has been shown to reduce hepatic injury resulting from ischemia-reperfusion. Several studies have found that small IPC tissue injury releases adenosine and L-arginine, which interact with adenosine receptors and ham-oxygenase and endothelial nitric oxide synthase systems and reduces hepatic necrosis microcirculation post reperfusion^9,11,12. In addition, the long-term survival rate of rats with non-flushed livers was numerically, but not statistically, higher than rats with flushed donor livers. This finding is in line with previous clinical studies suggesting that transaminase levels at 72 hours after transplantation was associated with a long-term prognosis in liver transplantation patients¹³, suggesting that, the less liver injury, the better the outcome.

In conclusion, transplantation of non-flushed livers is no inferior to current donor organ preservation technology in rats.

Animals

Male Sprague Dawley rats weighing 250–280 g, were used as both organ donors and recipients. The animals were housed under standardized conditions with a 12/12 hour dark/light cycle, and they were free to access food (standard diet) and water. This study was carried out according to the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences and approved by the Committee on the Ethics of Animal Experiments of Xi'an Jiaotong University (Permit Number 2022 − 105). All experiments complied with the ARRIVE guidelines and were performed in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978).

Study Design

Donor rats were divided into two groups: non-flushed donor liver (NF; n = 12) and flushing donor organ with a University of Wisconsin (UW) solution (controls; n = 12). Twelve transplantations were performed in each group by two surgeons.

Surgical Procedure

Syngeneic orthotopic liver transplantations were performed using the Cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) and magnetic anastomosis technique for suprahepatic vena cava (SHVC) reconstruction¹⁴.

Anesthesia was induced with 5% isoflurane at a flow rate of 1.5 L/min in a Plexiglas box and maintained with 2 vol% isoflurane at a flow rate of 0.6–0.8 L/min on a cone mask in all rats.

Donor surgery: after dissecting the falciform ligament and the connective tissues around the liver infrahepatic vena cava (IHVC) and the left renal vein, 50 units of heparin diluted in 2 mL of normal saline solution were administered through a dorsal penile vein in all groups bbefore retrieval. The donor liver was flushed through the catheter in the aorta with 20 mL of UW solution containing 25 U of heparin (4°C) at a rate of 3 mL/min with an infusion pump, and the donor livers were not subjected to flushing in both the NF groups. Each liver graft was immersed in UW solution at 4℃, then implanted by attaching cuffs to the IHVC and the PV and a magnetic ring to the SHVC. The recipient was prepared in parallel by another surgeon, maintaining a constant graft ischemia time of 20 ± 5 minutes.

Recipient surgery: a rat abdomen was opened via a 4 cm midline incision, and the hepatic artery was ligated with an 8 − 0 prolene suture (Ethicon Inc., New Brunswick, NJ) after dissecting the connective tissues and ligaments around the liver. Then, all liver vessels were isolated after ligating the common bile duct. A microvessel clip was applied to the IHVC just above the right renal vein while another microvessel clip was applied to the PV at the level of the pyloric vein. SHVC and a part of the diaphragm were fastened with a glover bulldog clamp after flushing the residual blood out of the liver by slowly injecting 2 mL of saline through the PV. Once the liver was explanted, a magnetic ring was attached to the SHVC. MCA technique was used for the SHVC anastomosis, and the Cuff technique for the remaining anastomoses. Next, the abdominal cavity was rinsed with warm normal saline solution after the common bile duct was reconstructed with the stent technique. Finally, the abdominal incision was closed in two layers with a continuous 3 − 0 nonabsorbable surgical suture.

All the rats were placed in a clean cage after surgery and given cefuroxime (16 mg/kg) and buprenorphine (0.05 mg/kg) intraperitoneally every 12 hours for 3 days. Six rats in each group were sacrificed to obtain liver tissue and blood samples at 24 hours and 72 hours after surgery, respectively.

Liver Function Tests

Liver function tests, including serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBIL) levels were performed at 24 hours and 72 hours after transplant.

Histological Analyses

Liver tissues were fixed in 3% phosphate-buffered saline-buffered formaldehyde and embedded in paraffin, cut longitudinally into 4-mm sections, and transferred to polylysine-coated slides. Serial sections were stained with hematoxylin and eosin (H&E), and polyclonal anti-CD31 antibody.

Long-term Survival

Two more groups of rats were used to evaluate the effects of free donor liver flushing on long-term survival after liver transplantation. Donor livers in one group were flushed in situ with UW solution after donor systemic heparinization (controls; n = 20). The donor livers were not flushed after donor systemic heparinization (NF; n = 20).

Statistical Analysis

The numerical data was expressed as a median [min-max] and compared using the Wilcoxon test. The Log rank test was used to compute survival rates. A P-value less than 0.05 was considered statistically significant. All data was analyzed using Prism 6 (GraphPad Software Inc., CA, USA).

Acknowledgement

The authors thank Kang Liu and Hong-Fan Ding from the Medical school of Xi'an Jiaotong University, for their collaboration in the surgeries.

Author contribution statements

Lu Q., and Lv Y. designed the study; Yang L.F., Ren L. and Yu J.W. performed the research and acquired the data; Yang L.F., Ren L. analyzed the data; Lu Q. and Yu J.W. drafted the manuscript; Wan Y., Lu Q. and Lv Y. contributed significantly to the revision of the manuscript.

Data availability statement

The original data can be obtained from the corresponding author by email.

Disclosures

Nothing to report.

Role of the funding source

This work was supported by the Key R & D plan of Shaanxi Province (No. 2021GXLH-Z-047), and the Innovation Capacity Support Plan of Shaanxi Province (No. 2020TD-040).

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No competing interests reported.

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Version 1

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You are reading this latest preprint version

Successful Transplantation of Non-Flushed Liver Organ in Rats

Status:

Version 1

Abstract

Figures

Introduction

Result

Validation of the rat liver transplantation model using a flushing-free organ

Survival analysis

Discussion

Method

Animals

Study Design

Surgical Procedure

Liver Function Tests

Histological Analyses

Long-term Survival

Statistical Analysis

Declarations

References

Additional Declarations

Status:

Version 1