Negin Karimian

Ex vivo Normothermic Machine Perfusion and Viability Testing of Discarded Human Donor Livers

In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. We have studied the feasibility of normothermic machine perfusion in four discarded human donor livers. Normothermic machine perfusion consisted of pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion for 6 h. Two hollow fiber membrane oxygenators provided oxygenation of the perfusion fluid. Biochemical markers in the perfusion fluid reflected minimal hepatic injury and improving function. Lactate levels decreased to normal values, reflecting active metabolism by the liver (mean lactate 10.0 ± 2.3 mmol/L at 30 min to 2.3 ± 1.2 mmol/L at 6 h). Bile production was observed throughout the 6 h perfusion period (mean rate 8.16 ± 0.65 g/h after the first hour). Histological examination before and after 6 h of perfusion showed well‐preserved liver morphology without signs of additional hepatocellular ischemia, biliary injury or sinusoidal damage. In conclusion, this study shows that normothermic machine perfusion of human donor livers is technically feasible. It allows assessment of graft viability before transplantation, which opens new avenues for organ selection, therapeutic interventions and preconditioning.

Criteria for Viability Assessment of Discarded Human Donor Livers during Ex Vivo Normothermic Machine Perfusion

Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37°C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial.

Hypothermic oxygenated machine perfusion prevents arteriolonecrosis of the peribiliary plexus in pig livers donated after circulatory death.

Background Livers derived from donation after circulatory death (DCD) are increasingly accepted for transplantation. However, DCD livers suffer additional donor warm ischemia, leading to biliary injury and more biliary complications after transplantation. It is unknown whether oxygenated machine perfusion results in better preservation of biliary epithelium and the peribiliary vasculature. We compared oxygenated hypothermic machine perfusion (HMP) with static cold storage (SCS) in a porcine DCD model. Methods After 30 min of cardiac arrest, livers were perfused in situ with HTK solution (4°C) and preserved for 4 h by either SCS (n = 9) or oxygenated HMP (10°C; n = 9), using pressure-controlled arterial and portal venous perfusion. To simulate transplantation, livers were reperfused ex vivo at 37°C with oxygenated autologous blood. Bile duct injury and function were determined by biochemical and molecular markers, and a systematic histological scoring system. Results After reperfusion, arterial flow was higher in the HMP group, compared to SCS (251±28 vs 166±28 mL/min, respectively, after 1 hour of reperfusion; p = 0.003). Release of hepatocellular enzymes was significantly higher in the SCS group. Markers of biliary epithelial injury (biliary LDH, gamma-GT) and function (biliary pH and bicarbonate, and biliary transporter expression) were similar in the two groups. However, histology of bile ducts revealed significantly less arteriolonecrosis of the peribiliary vascular plexus in HMP preserved livers (>50% arteriolonecrosis was observed in 7 bile ducts of the SCS preserved livers versus only 1 bile duct of the HMP preserved livers; p = 0.024). Conclusions Oxygenated HMP prevents arteriolonecrosis of the peribiliary vascular plexus of the bile ducts of DCD pig livers and results in higher arterial flow after reperfusion. Together this may contribute to better perfusion of the bile ducts, providing a potential advantage in the post-ischemic recovery of bile ducts.

Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures.

BACKGROUND & AIMS The peribiliary glands of large bile ducts have been identified as a niche of progenitor cells that contribute to regeneration of biliary epithelium after injury. We aimed to determine whether injury to the peribiliary glands of donor livers is a risk factor for development of non-anastomotic biliary strictures (NAS) after liver transplantation. METHODS In 128 liver transplant procedures, biopsies were taken from the donor bile duct and injury was assessed using an established histological grading system. Histological severity of injury was subsequently compared in liver grafts that later developed biliary structures vs. uncomplicated liver grafts. RESULTS Luminal biliary epithelial loss >50% was observed in 91.8% of the grafts before transplantation, yet NAS occurred in only 16.4%. Periluminal peribiliary glands were more severely injured than deep peribiliary glands located near the fibromuscular layer (>50% loss in 56.9% vs. 17.5%, respectively; p<0.001). Injury of deep peribiliary glands was more prevalent and more severe in livers that later developed NAS, compared to grafts without NAS (>50% loss in 50.0% vs. 9.8%, respectively; p=0.004). In parallel, injury of the peribiliary vascular plexus was more severe in livers that developed NAS, compared to grafts without NAS (>50% vascular changes in 57.1% vs. 20.3%; p=0.006). CONCLUSION Injury of peribiliary glands and vascular plexus before transplantation is strongly associated with the occurrence of biliary strictures after transplantation. This suggests that insufficient regeneration due to loss of peribiliary glands or impaired blood supply may explain the development of biliary strictures.

Injury to Peribiliary Glands and Vascular Plexus Before Liver Transplantation Predicts Formation of Non-Anastomotic Biliary Strictures

C1650 The Knockout of Rap1 Accelerates Liver Regeneration After Transplantation. C. Li, C. Lo, K. Ng, X. Qi, W. Geng, Y. Ma, X. Liu, H. Liu, K. Man. Surgery, The University of Hong Kong, Hong Kong, China. Introduction and aim: Impaired of liver regeneration severe affects the tissue repair following liver surgery. Cytokine, growth factor and metabolic networks play important roles in theprogression of liver regeneration. Rap1 is a part of the shelterin complex at mammalian telomeres, involved in protecting chromosome ends and promoting gene silencing. Recent research revealed its novel roles in the regulation of metabolism andinfl ammatory response through binding to extra-telomeric sites. We recently also showed that the knockout of Rap1 attenuated liver graft injury after transplantation. Here, we aimed to investigate the role of Rap1 in liver regeneration and to explore underlying mechanism. Methods:Clinically, the intragraft Rap1 expression and its correlation among liver function, infl ammatory cytokines/chemokines, and macrophage infi ltration were analyzed in human liver graft biopsies after transplantation. To investigate the direct role of Rap1 in hepatocyte proliferation and liver regeneration, Rap1 knockoutand wild type mice were subjected to major hepatectomy plus partial hepatic ischemia/ reperfusion injury (IRI). Mouse hepatocyte proliferation, histological damage, liver function and gene expressions were compared between Rap1 knockoutand wild type group. Results: Intrahepatic Rap1 expression was increased in small-for-size graft in comparison to normal graft (2.9 vs 1.9 folds of normal liver, p<0.05), and associated with higher expressions of infl ammatory cytokines/chemokines and more infi ltrations of macrophage and neutrophil at 2 hours after transplantation. Furthermore, overexpression of Rap1 was signifi cantly correlated with impaired liver function after transplantation (ALT: day 1, 3, 4 p<0.05; AST: day 0, 3, 4, 5 p<0.05). In mouse model, the knockout of Rap1 signifi cantly accelerated liver regeneration at day 2 after major hepatectomy and hepatic IRI compared to wild type group (Ki67: 56 vs 11/ HPF, p<0.05; PCNA: 46 vs 29/HPF, p<0.05). The knockout of Rap1 also attenuated histological damage and liver function. Furthermore, reduced expression of PPAR-α and increased expressions of G0S2 and IL1β were detected in Rap1 knockout mice. Conclusion: The knockout of Rap1 accelerates liver regeneration and associated with the regulation of cell cycle control, PPAR-α and cytokine signaling. Abstract# C1651 Injury of Peribiliary Glands and Vascular Plexus Before Liver Transplantation Predicts Formation of Non-Anastomotic Biliary Strictures. N. Karimian,1 S. op den Dries,1,3 A. Westerkamp,1 A. Gouw,2 J. Markmann,3 T. Lisman,1 H. Yeh,3 K. Uygun,3 P. Martins,3 R. Porte.1 1Department of Surgery, Section of Hepatopancreatobiliary Surgery and Liver Transplantation, University Medical Center Groningen (UMCG), Groningen, Netherlands; 2Department of Pathology, University Medical Center Groningen (UMCG), Groningen, Netherlands; 3Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston. C1651 Injury of Peribiliary Glands and Vascular Plexus Before Liver Transplantation Predicts Formation of Non-Anastomotic Biliary Strictures. N. Karimian,1 S. op den Dries,1,3 A. Westerkamp,1 A. Gouw,2 J. Markmann,3 T. Lisman,1 H. Yeh,3 K. Uygun,3 P. Martins,3 R. Porte.1 1Department of Surgery, Section of Hepatopancreatobiliary Surgery and Liver Transplantation, University Medical Center Groningen (UMCG), Groningen, Netherlands; 2Department of Pathology, University Medical Center Groningen (UMCG), Groningen, Netherlands; 3Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston. Peribiliary glands of large bile ducts have been identifi ed as a niche of progenitor cells that contribute to regeneration of biliary epithelium after injury. It is unknown whether injury of the peribiliary glands is a risk factor for the development of non-anastomotic biliary strictures (NAS) after liver transplantation. Moreover, it is unknown whether pretransplant biliary injury is different in livers donated after brain death (DBD) or cardiac death (DCD). In 128 liver transplant procedures, biopsies were taken from the extrahepatic bile duct and injury was assessed using a systematic histological grading system. Histological injury was correlated with the occurrence of posttransplant biliary strictures and a comparison wasmade between DBD (n=97) and DCD livers (n=29). Biliary epithelial loss >50% was observed in 91.8% of the grafts before transplantation, yet NAS occurred in 16.4%. Periluminal peribiliary glands were more severely injured than the deep peribiliary glands located near the fi bromuscular layer (>50% loss in 56.9% versus 17.5%, respectively; p<0.001). Injury of deep peribiliary glands was more prevalent and more severe in livers that later developed NAS, compared to uncomplicated grafts (>50% loss in 50.0% versus 9.8%, respectively; p=0.004). In parallel, injury of the peribiliary vascular plexus was more severe in livers that developed NAS, compared to uncomplicated grafts (>50% vascular changes in 57.1% versus 20.3%; p=0.006). Comparison of DBD and DCD livers revealed signifi cantly more vascular injury in the latter (p=0.005). Conclusion: Injury of peribiliary glands and vascular plexus before transplantation is strongly associated with the occurrence of biliary strictures after transplantation. This suggests that insuffi cient regeneration due to loss of peribiliary glands and blood supply may explain the development of biliary strictures. Abstract# C1652 N-Acetylcysteine Treatment Attenuates ROS Mediated Endoplasmic Reticulum Stress and Apoptosis During Liver Ischemia Reperfusion Injury. H. Lu, F. Zhang, J. Rao, Y. Sun, X. Qian, L. Lu, X. Wang. Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University(NJMU); Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, Nanjing, China. Background: The protective effects of N-acetylcysteine (NAC) treatment have been demonstrated during ischemia reperfusion injury (IRI) in several organs, but its underlying mechanism has not been suffi ciently elucidated. Our previous data has shown that ER stress is critical for the development of liver IRI. This study investigated effects of NAC on ER stress and tissue injury during liver IRI. Methods: Mice were injected with NAC (300mg/kg, ip) 2 hours before ischemia. The level of reactive oxygen species (ROS) was analyzed by GSH and MDA. Hepatic injury was evaluated based on sALT and histopathology. Apoptosis was analyzed by TUNEL staining and Caspase-3 activities. ER stress molecules (GRP78, ATF4 and CHOP) were determined by real-time PCR and western blotting in vivo and in vitro. Antiapoptotic molecules related to ER stress (Bcl-2 and Bcl-xl) were assessed after reperfusion. To analyze the roles of NAC on ROS-mediated ER stress and apoptosis, LDH was examined in cultured hepatocytes treated by H2O2 or Thapsigargin (TG). Results: NAC treatment signifi cantly attenuated ROS-mediated liver injury after IRI. Importantly, ROS-mediated ER stress was signifi cantly inhibited in NAC treated mice after IRI. As demonstrated by experiments in vitro, NAC treatment signifi cantly reduced the upregulation of ER stress molecules after H2O2 treatment. Furthermore, NAC treatment signifi cantly reduced caspase-3 activity after reperfusion, which was in line with the protein expression of Bcl-2 and Bcl-xl. Similarly, NAC treatment signifi cantly inhibited LDH release from hepatocytes treated by H2O2 or TG. Conclusions: This work provides a new evidence for the protective effects of NAC treatment on hepatocytes from IRI. Through the inhibition of ROS-mediated ER stress, NAC may be critical for inhibiting the ER stress related apoptosis pathway. Abstract# C1653 Inhibition of 12/15-Lipoxygenase Prevents Cell Death After Hepatic Ischemia and Reperfusion. M. Drefs, M. Thomas, A. Khandoga, F. Haak, J. Andrassy, M. Guba, J. Werner, M. Rentsch. Department of General, Visceral, Transplantation, Vascular and Thoracic Surgery, Hospital of the University of Munich, Munich, Germany. Introduction Graft failure secondary to ischemia-reperfusion-injury (IRI) still represents a major complication in liver transplantion. 12/15-Lipoxygenase (LOX), a protein of the glutathione-peroxidase-4 (GPx-4) signaling cascade, has been proven to substantially mediate cerebral postischemic apoptosis. Aim of this study was to investigate the impact of 12/15-LOX inhibition on hepatic IRI and thus gain insights into GPx-4 dependent signaling in the liver. Methods Livers of C57BL/6 mice were exposed to 60 minutes of warm ischemia by clamping the common pedicle of the median and left lateral liver lobe and subsequent reperfusion for 90 minutes. Baicalein, an inhibitor of 12/15-LOX, was administered intraperitoneally 30 minutes before operation (group 1). Controls were treated with vehicle dimethylsulfoxide(DMSO) (group 2) or untreated (group 3).Tissue samples were analyzed by TUNEL assay and Western Blot for pro-apoptotic proteins p44/42 MAP kinase (ERK1/2), Jun-amino-terminal kinase (JNK), Poly-ADP-ribose polymerase (PARP) and Caspase-3. Results Analysis of hepatic cell death by fl uorescence TUNEL labeling showed a signifi cant reduction of apoptotic cells in liver samples pretreated with baicalein (group 1: -64.8%; p<0,001) and with DMSO (group 2: -23.2%) compared to untreated samples (group 1). Western Blot analysis revealed a considerable downregulation of ERK1/2 (-36,7%) and PARP (-73,8%), as well as a slight reduction of JNK and Caspase-3 after Baicalein administration. The pretreatment with DMSO also showed a slight down-regulation regarding the investigated pro-apoptotic protein cascade, however in a discrete fashion. Conclusion Inhibition of 12/15-LOX leads to a signifi cant decr

Dual hypothermic oxygenated machine perfusion in liver transplants donated after circulatory death

Experimental studies have suggested that end‐ischaemic dual hypothermic oxygenated machine perfusion (DHOPE) may restore hepatocellular energy status and reduce reperfusion injury in donation after circulatory death (DCD) liver grafts. The aim of this prospective case–control study was to assess the safety and feasibility of DHOPE in DCD liver transplantation.

The origin of biliary strictures after liver transplantation: Is it the amount of epithelial injury or insufficient regeneration that counts?

Biliary complications continue to be a major problem after orthotopic liver transplantation (OLT). The incidence of biliary complications varies between 10% and 40% in different series and this type of complications is associated with frequent hospital admissions and high morbidity and mortality rates [1–3]. Among the variety of biliary complications that can occur after OLT, bile duct strictures are of the most concern. Bile duct strictures can be classified as anastomotic strictures (AS) or non-anastomotic strictures (NAS). Solitary strictures at the biliary anastomosis have been reported in 9%–12% of the patients [4–6], and NAS have been reported in 1%–20% of patients receiving a liver from donation after brain death and in up to 30% of patients receiving a liver from donation after cardiac death (DCD) [4,7,8]. NAS may occur in the extrahepatic donor bile duct as well as the intrahepatic bile ducts, but they are usually limited to the larger bile ducts. NAS may be accompanied by intraductal biliary sludge and cast formation. For many years researchers have been trying to understand the underlying mechanisms of AS and NAS. Current evidence suggests that AS are mainly related to the surgical technique and local ischemia of the distal bile duct stump, leading to fibrotic scarring of the anastomosis [1,6]. The etiology of NAS is thought to be multifactorial and three relevant types of biliary injury have been identified as a potential cause of NAS: ischemia/reperfusion related injury; immune-mediated injury; and cytotoxic injury caused by hydrophobic bile salts [1,9]. Current understanding of the occurrence of these types of biliary injury is that they originate mainly after transplantation of the liver. Depending on the severity of bile duct injury, the healing process may lead to scar formation and subsequent stricturing of the affected bile duct segments. Thus far, it remains unclear as to which extent bile ducts of donor livers are injured already before transplantation. In this issue of the Journal of Hepatology, Brunner et al. describe a clinical cohort study including 79 liver transplant procedures in

Biliary complications after orthotopic liver transplantation

Purpose of reviewThe incidence, pathogenesis and management of the most common biliary complications are summarized, with an emphasis on nonanastomotic biliary strictures (NAS) and potential strategies to prevent NAS after liver transplantation. Recent findingsNAS have variable presentations in time and localization, suggesting various underlying pathogeneses. Early-onset NAS (presentation within 1 year) have shown to be largely related to ischemia-induced bile duct injury, whereas late-onset NAS [>1 year after orthotopic liver transplantation (OLT)] have more immune-mediated causes. Cytotoxic hydrophobic bile salts and impaired biliary HCO3− secretion may also play a role in the occurrence of NAS. Recently, insufficient biliary epithelial regeneration capacity after transplantation has also been suggested to play a major role in the pathogenesis of NAS. A potential strategy to prevent NAS has been proposed to be preservation by machine perfusion instead of classical static cold storage. Although machine perfusion has been shown to be a better preservation method for the liver parenchyma, efficacy in preventing ischemic injury of the biliary epithelium is largely unknown. SummaryThe potential advantages of machine perfusion are very promising as it may provide better protection of the vulnerable bile ducts against ischemia–reperfusion injury. Clinical trials will be needed to demonstrate the impact of machine perfusion in reducing the incidence of biliary complications, especially NAS, after OLT.

Oxygenated Hypothermic Machine Perfusion After Static Cold Storage Improves Hepatobiliary Function of Extended Criteria Donor Livers

Background The mechanism through which oxygenated hypothermic machine perfusion (HMP) improves viability of human extended criteria donor (ECD) livers is not well known. Aim of this study was to examine the benefits of oxygenated HMP after static cold storage (SCS). Methods Eighteen ECD livers that were declined for transplantation underwent ex situ viability testing using normothermic (37°C) machine perfusion (NMP) after traditional SCS (0°C-4°C) for 7 to 9 hours. In the intervention group (n = 6), livers underwent 2 hours of oxygenated HMP (at 12°C) after SCS and before NMP. Twelve control livers underwent NMP without oxygenated HMP after SCS. Results During HMP, hepatic ATP content increased greater than 15-fold, and levels remained significantly higher during the first 4 hours of NMP in the HMP group, compared with controls. Cumulative bile production and biliary secretion of bilirubin and bicarbonate were significantly higher after HMP, compared with controls. In addition, the levels of lactate and glucose were less elevated after HMP compared with SCS preservation alone. In contrast, there were no differences in levels of hepatobiliary injury markers AST, ALT, LDH, and gamma-GT after 6 hours of NMP. Hepatic histology at baseline and after 6 hours of NMP revealed no differences in the amount of ischemic necrosis between both groups. Conclusions Two hours of oxygenated HMP after traditional SCS restores hepatic ATP levels and improves hepatobiliary function but does not reduce (preexisting) hepatobiliary injury in ECD livers.

Normothermic Machine Perfusion of Discarded Liver Grafts

To the Editor:We want to thank Knaak et al. (1) for their interest in ourresearchworkandfortheircomplimentsonourstudy(2).Inresponse to their minor comments we would like to makethe following remarks:(1) We disagree that the rise in ALT within the first hour ofperfusion can be interpreted as ongoing ischemiaduring that time period. We used a closed perfusionsystem and concentrations of ALT are a result ofcumulative washout of transaminases from the graftafter cold preservation, as has been shown in severalanimal studies (3). Values obtained in our ex vivosystem cannot be interpreted the same way as in vivovalues in blood of patients, as the perfusion circuitcontains only 2 L compared to an average of 5 L ofblood in vivo. Moreover, we strongly disagree thatthere was ongoing ischemic damage due to hypoper-fusion during the first hour. First, as shown in Figure 4there were no histological signs of zone III injury after6 h of perfusion. Second, it is a mistake to extrapolateflow data obtained from in vivo studies in cirrhoticpatientswith portal hypertensionsuchas by Kelly etal.one-to-one to the ex vivo settings used in our system.O