Bote G. Bruinsma

Subnormothermic Machine Perfusion for Ex Vivo Preservation and Recovery of the Human Liver for Transplantation

To reduce widespread shortages, attempts are made to use more marginal livers for transplantation. Many of these grafts are discarded for fear of inferior survival rates or biliary complications. Recent advances in organ preservation have shown that ex vivo subnormothermic machine perfusion has the potential to improve preservation and recover marginal livers pretransplantation. To determine the feasibility in human livers, we assessed the effect of 3 h of oxygenated subnormothermic machine perfusion (21°C) on seven livers discarded for transplantation. Biochemical and microscopic assessment revealed minimal injury sustained during perfusion. Improved oxygen uptake (1.30 [1.11–1.94] to 6.74 [4.15–8.16] mL O2/min kg liver), lactate levels (4.04 [3.70–5.99] to 2.29 [1.20–3.43] mmol/L) and adenosine triphosphate content (45.0 [70.6–87.5] pmol/mg preperfusion to 167.5 [151.5–237.2] pmol/mg after perfusion) were observed. Liver function, reflected by urea, albumin and bile production, was seen during perfusion. Bile production increased and the composition of bile (bile salts/phospholipid ratio, pH and bicarbonate concentration) became more favorable. In conclusion, ex vivo subnormothermic machine perfusion effectively maintains liver function with minimal injury and sustains or improves various hepatobiliary parameters postischemia.

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.

Tissue heterogeneity in structure and conductivity contribute to cell survival during irreversible electroporation ablation by “electric field sinks”

Irreversible electroporation (IRE) is an emerging, minimally invasive technique for solid tumors ablation, under clinical investigation for cancer therapy. IRE affects only the cell membrane, killing cells while preserving the extracellular matrix structure. Current reports indicate tumors recurrence rate after IRE averaging 31% of the cases, of which 10% are local recurrences. The mechanisms for these recurrences are not known and new explanations for incomplete cell death are needed. Using finite elements method for electric field distribution, we show that presence of vascular structures with blood leads to the redistribution of electric fields leading to the areas with more than 60% reduced electric field strength in proximity to large blood vessels and clustered vessel structures. In an in vivo rat model of liver IRE ablation, we show that cells located in the proximity of larger vessel structures and in proximity of clustered vessel structures appear less affected by IRE ablation than cells in the tissue parenchyma or in the proximity of small, more isolated vessels. These findings suggest a role for “electric field sinks” in local tumors recurrences after IRE and emphasize the importance of the precise mapping of the targeted organ structure and conductivity for planning of electroporation procedures.

A simplified subnormothermic machine perfusion system restores ischemically damaged liver grafts in a rat model of orthotopic liver transplantation.

BackgroundLiver donor shortages stimulate the development of strategies that incorporate damaged organs into the donor pool. Herein we present a simplified machine perfusion system without the need for oxygen carriers or temperature control, which we validated in a model of orthotopic liver transplantation.MethodsRat livers were procured and subnormothermically perfused with supplemented Williams E medium for 3 hours, then transplanted into healthy recipients (Fresh-SNMP group). Outcome was compared with static cold stored organs (UW-Control group). In addition, a rat liver model of donation after cardiac death was adapted using a 60-minute warm ischemic period, after which the grafts were either transplanted directly (WI group) or subnormothermically perfused and transplanted (WI-SNMP group).ResultsOne-month survival was 100% in the Fresh-SNMP and UW-Control groups, 83.3% in the WI-SNMP group and 0% in the WI group. Clinical parameters, postoperative blood work and histology did not differ significantly between survivors.ConclusionThis work demonstrates for the first time in an orthotopic transplantation model that ischemically damaged livers can be regenerated effectively using practical subnormothermic machine perfusion without oxygen carriers.

Supercooling enables long-term transplantation survival following 4 days of liver preservation

The realization of long-term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a new technique based on subzero nonfreezing preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to four days, thereby tripling the viable preservation duration.The realization of long–term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a novel technique based on sub–zero non–freezing tissue preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to 4 days, thereby tripling the viable preservation duration.

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

Determination and extension of the limits to static cold storage using subnormothermic machine perfusion

Background/Aims Static cold storage (SCS) of the liver for transplantation is limited by time. Continuation of metabolic activity leads to depletion of energy stores and loss of cellular function, which results in poor post-transplant function. Machine perfusion (MP) applied at the end of preservation may improve the viability of marginal liver grafts and provides information on the quality of the organ. We attempt to define the limits to SCS in terms of easily measurable perfusion parameters and investigate whether MP can improve liver viability. Methods Rat livers were cold-stored for 0, 24, 48, 72, and 120 h, after which they were treated with subnormothermic machine perfusion (SNMP). Livers cold-stored for 48 and 72 h were transplanted orthotopically with or without SNMP. During SNMP easily measurable parameters were monitored and adenosine triphosphate (ATP) content was measured following preservation and SNMP. Results ATP increased significantly during SNMP, but the recovered ATP content deteriorated with increased duration of SCS, with minimal improvement after 72 h of SCS. Vascular resistance during SNMP increased with extended preservation. After 48 h of SCS, orthotopic transplantation survival increased significantly from 50% to 100% with SNMP, but did not improve after 72 h.M Conclusions Vascular resistance and ATP recovery suggest a decrease in viability after 48 h of SCS. Survival data confirms the loss of post-transplant graft function and supports the use of ATP and vascular resistance as useful indicators. Further, we show that the recoverability of a liver using SNMP is limited to 48 h of SCS.

Surgical models of Roux-en-Y gastric bypass surgery and sleeve gastrectomy in rats and mice.

Bariatric surgery is the only definitive solution currently available for the present obesity pandemic. These operations typically involve reconfiguration of gastrointestinal tract anatomy and impose profound metabolic and physiological benefits, such as substantially reducing body weight and ameliorating type II diabetes. Therefore, animal models of these surgeries offer unique and exciting opportunities to delineate the underlying mechanisms that contribute to the resolution of obesity and diabetes. Here we describe a standardized procedure for mouse and rat models of Roux-en-Y gastric bypass (80–90 min operative time) and sleeve gastrectomy (30–45 min operative time), which, to a high degree, resembles operations in humans. We also provide detailed protocols for both pre- and postoperative techniques that ensure a high success rate in the operations. These protocols provide the opportunity to mechanistically investigate the systemic effects of the surgical interventions, such as regulation of body weight, glucose homeostasis and gut microbiome.

Metabolic profiling during ex vivo machine perfusion of the human liver

As donor organ shortages persist, functional machine perfusion is under investigation to improve preservation of the donor liver. The transplantation of donation after circulatory death (DCD) livers is limited by poor outcomes, but its application may be expanded by ex vivo repair and assessment of the organ before transplantation. Here we employed subnormothermic (21 °C) machine perfusion of discarded human livers combined with metabolomics to gain insight into metabolic recovery during machine perfusion. Improvements in energetic cofactors and redox shifts were observed, as well as reversal of ischemia-induced alterations in selected pathways, including lactate metabolism and increased TCA cycle intermediates. We next evaluated whether DCD livers with steatotic and severe ischemic injury could be discriminated from ‘transplantable’ DCD livers. Metabolomic profiling was able to cluster livers with similar metabolic patterns based on the degree of injury. Moreover, perfusion parameters combined with differences in metabolic factors suggest variable mechanisms that result in poor energy recovery in injured livers. We conclude that machine perfusion combined with metabolomics has significant potential as a clinical instrument for the assessment of preserved livers.

Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.