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The Effects of Short-term Subnormothermic Perfusion After Cold Preservation on Liver Grafts From Donors After Circulatory Death

An Ex Vivo Rat Model

Kakizaki, Yuta, MD1; Miyagi, Shigehito, MD, PhD1; Shimizu, Kenji, MD, PhD1; Miyazawa, Koji, MD, PhD1; Nakanishi, Wataru, MD, PhD1; Tokodai, Kazuaki, MD, PhD1; Hara, Yasuyuki, MD, PhD1; Nakanishi, Chikashi, MD, PhD1; Unno, Michiaki, MD, PhD1; Kamei, Takashi, MD, PhD1; Goto, Masafumi, MD, PhD2; Satomi, Susumu, MD, PhD1

doi: 10.1097/TP.0000000000002080
Original Basic Science—Liver

Background We previously reported that short oxygenated warm perfusion before cold storage (CS) had improved the graft viability of rat livers from donors after circulatory death (DCD). In this study, we investigated the effectiveness of short-term oxygenated subnormothermic perfusion for different durations after CS in a rat DCD model.

Methods We used an isolated perfused rat liver system. In study 1: the grafts were retrieved from Wistar rats 30 minutes after cardiac arrest (thoracotomy), preserved in CS for 6 hours, and perfused with oxygenated subnormothermic (20-25°C) Krebs-Henseleit buffer for different durations (0, 15, 30, 60, and 90 minutes groups; n = 5 in each). In study 2: in addition to subnormothermic ex vivo liver perfusion (SELP), after 15-minute incubation at room temperature, the grafts were reperfused under normothermic condition for 60 minutes as a model of liver transplantation (0, 30, 60, and 90 minutes groups; n = 5 in each).

Results In study 1, portal flow, bile production and tissue adenosine triphosphate increased with perfusion duration. In study 2, SELP significantly improved portal flow volume (P <0.05), and bile production (P <0.05), decreased liver enzymes (P <0.05) and cytokines (P <0.0001), and increased tissue adenosine triphosphate (P <0.01). Histological examinations showed that additional SELP ameliorated tissue deterioration, preserved the parenchymal structure, and decreased apoptosis (P <0.01). Furthermore, scanning electron microscopy revealed that additional SELP alleviated sinusoidal endothelial cells and hepatic microvasculature.

Conclusions Even 30 minutes of SELP after CS rescued DCD livers from ischemia-reperfusion injury, which may help the viability of the grafts.

Does short-term oxygenated subnormothermic perfusion prior to cold storage improve the viability of DCD livers in a rat model? The data show that subnormothermic ex vivo perfusion prevented liver from ischemia-reperfusion injury that may improve organ viability.

1 Department of Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan.

2 Division of Transplantation and Regenerative Medicine, Tohoku University, Sendai, Japan.

Received 31 July 2017. Revision received 6 November 2017.

Accepted 9 November 2017.

This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and the Ministry of Welfare of Japan and a Grant from Tohoku University Graduate School of Medicine.

The authors declare no conflicts of interest.

K.Y., M.S., S.K., K.M., W.N., T.K., H.Y., N.C., U.M., and K.T. participated in study design. K.Y. participated in the acquisition of data. K.Y. and M.S. participated in the analysis and interpretation. K.Y. drafted the article. G.M., K.T., and S.S. participated in the revision.

Correspondence: Yuta Kakizaki, MD, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan. (

Liver transplantation (LTx) is a well-established treatment for end-stage liver diseases and acute liver failure. However, the shortage of donors and waitlist mortality are serious problems. To enlarge the donor pool, it is necessary to use livers from extended-criteria donors, such as steatotic donors, elderly donors, and donors after circulatory death (DCD). In fact, the liver donor pool in some European countries has increased by as much as 20% due to the rise in DCD transplants.1 However, the use of DCD livers is associated with a higher incidence of primary graft nonfunction (PNF)2,3 and biliary complications compared with donation after brain death.4,5 These limitations of using DCDs need to be resolved before successful incorporation in LTx programs.

DCD livers inevitably suffer from warm ischemia-reperfusion (IR) injury. First, warm ischemia (WI) causes hypoxia and intracellular adenosine triphosphate (ATP) depletion. This causes hepatocyte swelling and microcirculatory disturbances. Hepatocytes release damage associated molecular patterns, such as high mobility group box 1, that activate Kupffer and dendritic cells.6 Second, reperfusion exacerbates IR injury when damage-associated molecular patterns are released. Furthermore, IR injury is more severe when reflow facilitates contact between these antigen presenting cells and primed T cells, neutrophils, and monocytes. These cells activation causes cytokine storms that damage hepatocytes and sinusoidal endothelial cells (SEC).7,8 Therefore, warm IR injury causes a feedback loop involving microcirculatory disturbances and cytokine storms, which cause PNF.9

Some reports have shown that machine perfusion (MP) improves the viability of DCD liver grafts.10,11 Theoretically, normothermic (around 37°C) ex vivo liver perfusion (NELP) reproduces the physiological environment, enabling the supply of oxygen and nutrients, removal of waste products, and assessment of graft viability. However, NELP requires a complex heated perfusion system, hindering its translation into the clinical institutions. The simplicity and low cost of cold storage (CS) have kept it the standard of care for transplantation centers. On the other hand, subnormothermic (20-25°C) ex vivo liver perfusion (SELP) can be used at room temperature, which reduces the need for strict temperature control in addition to lowering oxygen demand during perfusion. Therefore, SELP might be effective against warm IR injury and grafts perfused in this manner may be transplanted with good survival.12,13

The aim of this study was to evaluate the effectiveness of SELP after CS without oxygen carriers and to determine the optimal perfusion duration to maximize liver viability. In many reports of SELP and NELP, perfusion duration was set at more than 2 hours with no standard criteria.12-17 In the hypothermic perfusion study, 1 hour short-term perfusion was effective for rat DCD liver graft recovery after CS.18 On the other hand, we previously reported that NELP for 30 minutes before CS improved the viability of DCD livers.19-22 We hypothesized that even short-term perfusion has the potential to protect the liver graft against IR injury. In this study, we investigated the effects of SELP after CS on the rat DCD liver model according to the perfusion durations.

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Male Wistar rats (Japan SLC, Inc., Shizuoka, Japan), weighing 260 to 350 g, were used for the experiments. Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, which was prepared by the National Academy of Science. This study was approved by the Institutional Animal Care Committee of Tohoku University.

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Study Design

Study 1 assessed the effects of SELP. Grafts were retrieved 30 minutes after cardiac arrest induced by bilateral thoracotomy, preserved in cold University of Wisconsin (UW; DuPont Pharmaceuticals, Wilmington, DE) solution for 6 hours, and perfused with oxygenated subnormothermic Krebs-Henseleit buffer (KHB) without oxygen carriers. Samples were split into groups according to SELP durations of 0, 15, 30, 60, and 90 minutes (DCD, SELP-15, 30, 60, and 90; n = 5 in each group), and then collected. We also retrieved livers from heart beating (HB) donors as positive controls. In each group, 5 rats were investigated (Figure 1A).



Study 2 was added normothermic reperfusion for 60 minutes to study 1. Samples were split into groups according to SELP durations of 0, 30, 60, and 90 minutes (DCD, R-30, -60, and -90; n = 5, in each group). After SELP, the grafts were incubated for 15 minutes at 23°C to 26°C (mimicking the duration of anastomosis), reperfused with oxygenated normothermic KHB for 60 minutes at 37°C, and subsequently, samples were collected. We also reperfused HB livers as positive controls. In each group, 5 rats were investigated (Figure 1A).

In study 2, we did not perform the 15-minute SELP group according to the results of lactate level and ATP content in study 1 (see the Results of lactate level and ATP content). Additionally, in this study, we focused on evaluating the initial phase of warm IR injuries, for example, cytokine storms. Cytokine storms cause various phenomena with hepatic manifestations. Therefore, we considered the first 1 hour to be the most critical period.

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Experimental Procedures

All rats were anesthetized with medetomidine hydrochloride (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) (0.15 mg/kg) + midazolam (Teva Pharma Japan Inc., Tokyo, Japan) (2 mg/kg) + butorphanol tartrate (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) (2.5 mg/kg) via the intraperitoneal route, and 1000 U/kg of heparin (AY Pharmaceuticals Co., Ltd., Tokyo, Japan) via the penile vein at laparotomy. The common bile duct was cannulated with a 22-gauge (G) polyethylene tube (Terumo, Tokyo, Japan) in all cases. All livers, except HB grafts, were subjected to 30 minutes of in situ WI after approximately 7 minutes in an agonal state induced by bilateral thoracotomy followed by cardiac arrest, as described previously.9,22 The portal vein was then cannulated with a 14-G polyethylene tube and the livers were flushed with 20-mL Ringer lactate solution (Lactec Injection; Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and 20-mL UW solution at 4°C. Grafts were retrieved, the infrahepatic inferior vena cava was ligated, and the suprahepatic inferior vena cava was cannulated with a 14-G short stent. All grafts were preserved in UW solution at 4°C for 6 hours. HB grafts were retrieved in the same manner as DCD grafts. Flushing with Ringer lactate solution before UW solution is an experimental technique to effectively wash out the blood.

After CS, grafts were perfused with oxygenated KHB (the oncotic pressure; 274-303 mOsm/kg) through the portal vein alone using a nonrecirculating perfusion machine at a pressure of 7 mm Hg (Perfusion System PS-1; Hugo Sachs Elektronik-Harvard Apparatus GmbH, March-Hugstetten, Germany). Carbogen (95% O2 and 5% CO2) was bubbled into KHB, creating a pO2 of 500 to 550 mm Hg in the perfusate according to the standardized condition in the isolated perfused rat liver (IPRL)23 (Figure 1B). There was almost no oxygen loss in this circuit, because the perfusion device used Tygon tubes (Saint-Gobain Co., Ltd., Tokyo, Japan) that have very low oxygen permeability. It cannot be ruled out that this bubbling may cause micro air embolism. However, all grafts were oxygenated by the bubbled perfusate in our perfusion system with 2 bubble traps. According to our previous studies, our perfusion method of oxygenation by bubbling before transplantation improved the survival rate.22,24 Therefore, we believe that bubbling has no distinct disadvantages.

The temperature for subnormothermic conditions was set at 23°C to 26°C and for normothermic conditions at 37°C. The outflow volume of perfusate and the bile production were measured, and samples were stored at −80°C until analysis.

Previously, although we used a recirculating circuit system, it was difficult to carry out the 1-hour perfusion because of the microcirculation obstacle (the experimental system did not fit with the recirculating perfusion). The IR injury in our DCD model was very severe with an agonal condition; therefore, the destroyed cells obstructed the microcirculation. We attempted to place a filter and challenged it again, but it became clogged and ultimately failed. For these reasons, we used the nonrecirculating system in this study. Also, the ideal reperfusion is using the blood (mimicking a transplant situation). However, using the perfusate containing the blood would necessitate controlling the blood density in all cases and may entail the sacrifice of an excessive number of rats in our nonrecirculating perfusion system. To bypass the technical challenges and to protect the animals, we decided to use noncellular perfusate in the present study according to IPRL guidelines.23

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Biochemical Examination

The aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β) and soluble intercellular adhesion molecule 1 (sICAM-1) levels in the perfusate of study 2 were measured using commercially available kits (AST and ALT from Wako Pure Chemical Industries, Ltd., Tokyo, Japan; LDH from Nipro Corporation, Osaka, Japan; TNF-α from Invitrogen Corporation, Carlsbad, CA; IL-1β from Thermo Scientific, Waltham, MA; sICAM-1 from R&D systems, Minneapolis, MN).

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Lactate Level and Hepatic Oxygen Consumption

The lactate levels in the perfusates in both study 1 and study 2 were measured using a blood gas analyzer (Rapid Point 405; Siemens, Tokyo, Japan). Also, we evaluated the oxygen supply in our IPRL model by measuring the partial oxygen pressure in the inflow and outflow of perfusates using a blood gas analyzer. We calculated the oxygen consumption using the following equation:

Where dSaO2 is the difference in the saturation of oxygen between inflow and outflow; dPaO2, the difference in the partial pressure of oxygen between inflow and outflow)

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Assay of ATP Content

ATP and adenosine diphosphate (ADP) levels in the liver tissue were measured by a high-performance liquid chromatography method as described previously.25 We also measured these levels in fresh livers from HB donors without CS, as positive controls in study 1.

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Assay of Oxidative Stress

Malondialdehyde (MDA) levels in the liver were measured to evaluate the intensity of oxidative stress. Livers retrieved after normothermic perfusion in study 2 were immediately frozen and homogenized with 5-mM butylhydroxytoluene in a 20-mM Tris-hydrochloric acid buffer. MDA content per 1-g protein was measured by spectrometry with a commercially available kit (BIOXYTECH MDA-586; Oxis International Inc., Foster City, CA).

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Morphological Analysis

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Light Microscopy

After normothermic reperfusion, liver tissue samples were immediately obtained from the left lobe, preserved in 4% paraformaldehyde, paraffinized, and cut into 3-μm sections. Examination with optical microscopy was performed using 10 sections stained with hematoxylin and eosin from each group.

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Detection of Apoptosis

Apoptotic cells were identified using an Apop Tag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon International Inc., Billerica, MA) using the terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL) method. Positive cells were counted in 10 random high-power fields (×200) for each liver specimen, and the number of cells per mm2 was calculated.

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Scanning Electron Microscopy

After normothermic reperfusion, additional livers from each group were perfused with 1% glutaraldehyde/4% paraformaldehyde via the portal vein. After fixation, the samples were cut into 1-mm3 cubes. The samples were postfixed with 1% OsO4, dehydrated, substituted, and freeze-dried with acetate iso-amyl, ion-sputter-coated, then examined with an S-3200 microscope (Hitachi High-Technologies, Tokyo, Japan).

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Statistical Analysis

All calculations were made with the JMP Pro ver. 12.0 software package (SAS Institute, Cary, NC). The results are presented as mean ± standard deviation (SD). Statistical analysis was performed using one-factor analysis of variance and Tukey honest significant different test. A P value less than 0.05 was considered statistically significant.

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Inhibition of Ischemia-Reperfusion Injury by SELP

In study 1, portal flow volume and bile production increased according to perfusion duration (Table 1A). The rate of change in portal flow volume and bile production increased well only in the initial 30 minutes, after which the rate was almost constant. In study 2, portal flow volume and bile production were significantly increased in the additional SELP groups compared to those in the DCD group (Table 1B). The levels of AST and LDH in the collected perfusate of the additional SELP groups were significantly lower than those in the DCD group (Table 1B). The ALT levels in the R-30 group were also significantly lower than that in the DCD group (Table 1B). TNF-α IL-1β and sICAM-1 levels in the perfusate were significantly decreased in the additional SELP groups compared with those in the DCD group (Table 1B, Figure 2A). MDA was significantly decreased in the R-90 group than that in the DCD group (Figure 2B). In study 1, the lactate levels were significantly lowered in the SELP-30, SELP-60 and SELP-90 groups compared with SELP-15 group (Figure 2C). Additionally, the lactate levels were significantly lowered in the R-30, R-60, and R-90 compared with DCD group (Figure 2D).







In study 1, the ATP content increased with the duration in the additional SELP groups, and the ATP content in the SELP-30, SELP-60, and SELP-90 groups were significantly higher than that in the DCD groups (Figure 3A). Similarly, the ADP/ATP ratios in the SELP-30, SELP-60, and SELP-90 groups were significantly lower than that in the DCD groups (Figure 3B). Additionally, the ATP content in the SELP-30 group exceeded that of the fresh liver. The rate of change in the ATP content increased well only in the initial 30 minutes, after which, the rate was almost constant. In study 2, the ATP content in the R-30, R-60, and R-90 groups was significantly higher than that in the DCD group (Figure 3C). In addition, the ADP/ATP ratio in the R-30, R-60, and R-90 groups was significantly lower than that in the DCD group (Figure 3D).



According to the results of lactate levels and ATP content in study 1, we decided not to investigate the SELP-15 group in study 2.

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Morphological Examination

During histological examination, vacuolization of hepatocytes and narrow sinusoidal spaces were distinctly observed in the DCD group. The additional SELP groups showed less tissue deterioration and preservation of the structure of the parenchyma, similar to that seen in the HB group (Figure 4A). TUNEL-positive cells in the DCD group were significantly more than in the HB group, however, those in the additional SELP groups were significantly fewer than in the DCD group (Figure 4B, C).



To evaluate sinusoidal ultrastructure, we performed scanning electron microscopy. In the HB liver, most SECs were well preserved, with their Disse spaces intact. In the DCD livers, the SECs were rough and disrupted, and obstruction of the microvasculature and enlarged Disse spaces were observed. In contrast, smooth SECs and well-preserved Disse spaces were observed in the additional SELP groups. In the R-30 group, vacuolization of hepatocytes was distinctly observed compared with the R-60 and R-90 groups (Figure 5).



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In this study, we investigated the effects of short-term SELP after CS according to perfusion duration. Additional SELP significantly improved graft function with increased bile production, increased liver tissue ATP content, recovered ADP/ATP ratios, and protected SECs and hepatic microcirculation. Even 30-minute periods of SELP were effective in protecting against IR injury. This study is the first report that reveals the effects of short-term SELP after CS according to perfusion durations, without the use of oxygen carriers, in a rat DCD model with an agonal phase.

Although MP preservation has recently become the focus of intensive investigation, its optimal parameters have not been sufficiently determined (for example, the critical temperature and the perfusion duration).26 In contrast to NELP and hypothermic MP, SELP does not require strict temperature control and may thus be more translatable to clinical practice. We had previously reported that NELP before CS was beneficial in rat DCD liver grafts.22 However, this technique’s clinical translation would be difficult because it might be challenging to perfuse the grafts at the donor institution. Additionally, in clinical scenarios, CS might be necessary during graft transportation. Therefore, the most feasible approach may be to perfuse the grafts in a subnormothermic manner after CS.

We chose the subnormothermic condition for 3 reasons: first, SELP is simpler and more affordable than other approaches. Second, the higher the temperature is raised, the lower the dissolved oxygen content is in the perfusates. NELP requires specific perfusates that contain erythrocytes or oxygen carriers to maintain the liver’s aerobic metabolism. Third, oxygen carriers in the perfusates lead to an increase in the viscosity. MP with high viscosity perfusates increases portal vein pressure at the same level of portal flow, which results in shear-related damage to the SECs.27 In this study, we did not need to use oxygen carriers in the perfusates.23 Although low temperature raises the perfusates’ viscosity in general, the viscosity of KHB is almost constant without oxygen carriers. Furthermore, Fujita et al28 reported that liver oxygen consumption dropped to approximately 30% of baseline at 21°C. In fact, SELP might be effective for protection against warm IR injury, allowing transplantation of liver grafts with good survival.12,13 We selected the subnormothermic condition based on these studies. This condition imposes lower metabolic demands on the graft while maintaining the minimal level of metabolism needed to allow for function and viability.

Before starting this experiment, we evaluated the oxygen supply in our IPRL model and in vivo models. We can consider oxygen consumption as the difference between the amounts of oxygen in the inflow and outflow because there was almost no oxygen loss in this circuit using Tygon tubes that have very low oxygen permeability. We measured PaO2 of inflow and outflow and in our IPRL model at 37°C. The PaO2 of inflow was 500 to 550 mm Hg and PaO2 of outflow was approximately 150 mm Hg. It is speculated that the amount of dissolved oxygen to the perfusate was sufficient compared with another study.23 Furthermore, we calculated the average flow rate in our experiments, which was 3.76 ± 0.97 mL/min per g-liver at 37°C and 3.25 ± 0.71 mL/min per g-liver at 23°C to 26°C, which were within the limits of the IPRL guidelines23 and exceeded that of the other study (3.0 mL/min per g-liver at 37°C and 1.0 mL/min per g-liver at 20-24°C).29 On the other hand, according to a previous study of MP using DCD liver, the highest oxygen consumption rate was in the fresh liver, followed by DCD liver with MP, and DCD only.12 Therefore, we measured the oxygen consumption rate of fresh liver (in vivo model) by sampling portal and hepatic vein oxygen content. The physiological blood flow in the rat portal vein at 37°C is 1.25 mL/g-liver per minute.23 The oxygen consumption rate was calculated using the above equation, and was 46.6 μL/min per g-liver in our IPRL model (Hb = 0) and 51.8 μL/min per g-liver in the in vivo model (Hb = 10, dSaO2 = 0.3, d PaO2 = 40), respectively. We also infer that the oxygen consumption rate of DCD livers may theoretically decrease compared with that of the fresh liver. Therefore, we believe that the oxygen supply in our experiments was sufficiently enough not only at 37°C but also at 23°C to 26°C, because the increasing the amount of dissolved oxygen should be higher and the oxygen consumption rate should be lower in the latter condition compared with the former condition.

We consider that the present perfusion experiments are more objective and suitable after CS on liver grafts from DCD as the initial concept. Although 1-hour reperfusion as a transplantation model is short, we focused on evaluating the initial phase of warm IR injury, which occurs via complex pathological network. We believe that the suppression of the beginning of inflammatory cascades on IR injury by SELP contributes not only to reduce the risk of PNF but also to improve critically ill conditions during the perioperative period.

We hypothesized that even short-term perfusion has the potential to protect the liver graft against IR injury based on our previous studies19-22 and on the Zurich group study.18 Excessive perfusion may cause damage to the liver grafts, such as the shear-related damage. There are reports which have investigated biomarkers of the graft’s recovery, such as tissue ATP,13 bile production,30,31 and lactate/pH in the perfusates.32 Increased ATP content indicates the recovery of hepatocytes’ mitochondrial function. Bile production requires the recovery of a functioning network among SECs, hepatocytes, and cholangiocytes. In this study, additional SELP for 30 minutes significantly increased bile production and ATP content compared with that in the DCD group of 60-minute reperfusion. Moreover, we demonstrated that SELP could maintain the morphological architecture of DCD liver grafts both microscopically and ultrastructurally. As a result of the microcirculation’s recovery, the lactate level in the perfusates significantly decreased compared with the DCD group. We have taken these results to indicate the recovery of the liver grafts’ viability. On the other hand, we did not find any disadvantages for excessive SELP for up to 90 minutes.

We have been concerned about the possibility of deleterious effects from oxidation, including the production of reactive oxygen species (ROS) in the mitochondria. However, MDA decreased with increasing SELP duration. The lack of ATP severely impacts mitochondrial permeability transition functionality and can ultimately trigger cell death through apoptotic pathways.33 NELP before CS improves mitochondrial function in DCD liver grafts, as reported previously.20,21 In this study, SELP was performed after CS and similarly decreased the rate of apoptosis. Moreover, ICAM-1 is reported to overexpress on the SECs and hepatocytes as a result of IR injury.34 sICAM-1 was significantly decreased in the additional SELP groups, which may indicate that SELP possibly prevented the progression of IR injury.

Our SELP technique is expected to remove vasoactive substances due to the use of a nonrecirculating perfusion system. It might be possible that the release of liver enzymes and cytokines decreases after reperfusion because of washing out during SELP. Clinically, the release of vasoactive proinflammatory factors from the DCD grafts into the systemic circulation is associated with severe hemodynamic instability. This release is also harmful to other organs, such as the lungs and kidneys.35 Furthermore, these substances recirculate and are injurious to the implanted grafts themselves. We consider our SELP technique to have the potential for alleviation of postreperfusion syndrome (PRS) in DCD liver grafts.36

There were several limitations to this study. First, an ex vivo reperfusion model enables us to avoid interference from immunological alloreactions and PRS. Second, we used the noncellular perfusate as reperfusion due to the technical limitations and the protection of animals. In vivo rat and large animal studies are necessary. Third, we did not perfuse the hepatic artery and evaluate biliary complications related to DCD liver grafts. We think that single perfusion without the hepatic artery may cause biliary complications after implantation, but we did not measure the level of LDH in the bile and could not evaluate the extent of the damage. Fourth, we could not investigate the damage resulting from excessive perfusion in this study. In fact, we did not find any disadvantages to 90 minutes of SELP.

In conclusion, short-term SELP after CS might rescue DCD livers in an agonal state from IR injury. Although the viability of the grafts remains unclear in the absence of a transplant model, further investigations using transplant models should be performed in the next step with the aim to expand the donor liver pool.

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The authors thank Chikako Sato and Yasuko Furukawa for their technical assistance. The authors also thank Biomedical Research Unit of Tohoku University Hospital for technical support.

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