- Email: [email protected]
156 SUBNORMOTHERMIC MACHINE PERFUSION REDUCES PRESERVATION DAMAGE OF LIVERS FROM NON-HEART BEATING DONORS: A NEW CHANGE FOR EXPANDING THE DONOR POOL?
S66
Poster Session − Thursday, April 23
However, its efficacy is still questioned. Phosphoinositide 3-kinase (PI3K) plays a key role in the development of IPC in rodent livers. In this study we evaluated PI3Kdependent signals in transplanted human livers submitted to IPC. Methods: The livers of 40 deceased donors were randomised to receive or not (Controls) IPC before recovery. PI3K activation was evaluated in biopsies obtained immediately before IPC and 2 hours after reperfusion by measuring the phosphorylation of the PI3K down-stream kinase PKB/Akt and the levels of phosphatase tensin-homologues deleted from chromosome 10 (PTEN), an antagonist of PI3K activity. Results: IPC significantly increased PKB/Akt phosphorylation (p = 0.01) and decreased PTEN levels (p = 0.03) in grafted livers 2 hr after reperfusion, but did not significantly ameliorate post-transplant reperfusion injury. By calculating T2hr/T0 PKB/Akt phosphorylation ratios we observed that 10 out 19 (53%) of the preconditioned grafts had PKB/Akt phosphorylation index above the control threshold (IPC-responsive), while in the remaining 9 PKB/Akt phosphorylation index was comparable to controls (IPC-non-responsive). T2hr/T0 PTEN ratios were also decreased (p0.03) only in IPC-responsive grafts. In patients receiving IPC-responsive organs, IPC significantly (p0.05) ameliorated AST and ALT 24−48 hr after transplant. Bilirubin levels were also improved in subjects transplanted with IPC-responsive livers, while prothrombin activity was unchanged. Conclusions: We propose that the capacity of IPC to fully activate PI3K signalling might be variably impaired in liver grafts obtained from deceased donors and this might account for the inconsistent responses to IPC observed following human liver transplantation. 154 THE CONTRIBUTIONS OF DE NOVO ARGININE GENERATION AND ARGINASE ACTIVITY TO WHOLE BODY ARGININE METABOLISM IN ACUTE LIVER FAILURE N. Davies1 , G. Ten Have2 , V. Sharma1 , L. Ytrebo3 , S. Sen1 , C. Rose4 , A. Revhaug3 , R.P. Mookerjee1 , R. Jalan1 , N. Deutz2 . 1 Institute of Hepatology, University College London, London, UK; 2 Center for Translational Research on Aging & Longevity, University of Arkansas for Medical Sciences, Little Rock, USA; 3 Department of Surgery, University of Northern Norway, Tromso, Norway; 4 Neuroscience Research Unit, University of Montreal, Montreal, Quebec, Canada E-mail: [email protected] Background: In acute liver failure (ALF) arginine concentrations are believed to be decreased, potentially limiting substrate for nitric oxide production. However, our prior observations in a porcine hepatic devascularisation model showed no significant change in systemic haemodynamics. To further characterize the metabolic fate of arginine in ALF, we systematically assessed de-novo arginine generation, inter-organ flux of arginine, and its metabolism by the arginase pathway. Methods: Female adult pigs (30−35Kg) were randomized into either Sham (N = 8) or ALF groups (N = 8). ALF was induced by hepatic devascularisation, and the animals sampled over 6 hours. Measurements: Plasma arginine, citrulline, ornithine levels (HPLC); arginase activity (colourimetric assay); nitric oxide (stable isotope labelled citrulline generation-HPLC). Whole body metabolic rates and inter organ flux were calculated after stable isotope-labelled amino-acid infusions and calculation of arteriovenous concentration differences across each organ. Results: Plasma arginine levels decreased by 50% of the basal level at T=6 hrs (p < 0.001), whereas plasma citrulline and ornithine levels progressively increased in the ALF group compared to Sham (p < 0.001 and p < 0.001, respectively). Calculation of the whole body rate of appearance of Arginine and nitric oxide showed no difference between the two groups. However, there was an increase in the rate of de novo arginine synthesis in ALF pigs. Inter-organ flux data showed citrulline is predominantly produced in the small intestine and consumed in the kidney. Arginine and Ornithine were both primarily produced in the kidney. Plasma arginase activity (liberating ornithine) significantly increased in the ALF group over 6 hrs, whereas no change was observed in Sham animals. Conclusions: In this model of ALF, we demonstrated that although de novo production of arginine is increased, whole body level of arginine and
consequent nitric oxide production remains unchanged. This we show to be due to the significantly increased arginase activity manifest by the greater levels of ornithine in the ALF group. The effect of arginase in reducing plasma arginine and thereby, the substrate for NO synthesis, may result in the impaired organ perfusion typically observed in ALF, and contribute to multiple organ dysfunction. This work was partly supported by The Norwegian MRC. 155 INFLUENCE OF ESTROGEN ON THE EARLY RECOVERY OF HEPATOBILIARY SECRETORY FUNCTION AFTER ISCHEMIA/REPERFUSION INJURY IN RATS A.H. de Vries1 , F.A.M. Ponds1 , R.T.A. Padbury2 , R.J. Porte3 , V.B. Nieuwenhuijs3 , G.J. Barritt4 . 1 Departments of Medical Biochemistry and Surgery, 2 Department of Surgery, Flinders Medical Centre, Adelaide, Australia; 3 Department of Surgery, University Medical Center Groningen, Groningen, The Netherlands; 4 Department of Medical Biochemistry, Flinders Medical Centre, Adelaide, Australia E-mail: [email protected] Background: Ischemia/reperfusion injury (I/R injury) is a major problem in liver surgery, which causes liver dysfunction and liver failure after transplantation or hepatectomy. Recent studies have shown gender-related differences in I/R injury, in which estrogen is suggested as a ‘survival factor’ for hepatic cells through various pathways [1,2]. Aim: The aim of this study was to investigate the influence of estrogen during the early recovery of hepatobiliary secretory function after I/R injury of the liver. Methods: A rat model of segmental (60−70%) I/R was used, in which 60 minutes of ischemia was induced followed by 90 minutes of reperfusion. Male (M) and female (F) Sprague Dawley rats (n = 32) were randomly pre-treated 24 hours prior to I/R with a s.c. injection of ethanol-oil (M and F Control), 17b-estradiol (M-E2), estrogen receptor antagonist ICI 182,780 (F-ICI) or ICI 182,780 plus 17b-estradiol (F-ICI/E2). Bile flow, ALT, bilirubine and histology were analyzed, using One-Way ANOVA (Post Hoc Bonferroni). Results: Female rats appeared to have a significantly better recovery compared to male rats, reflected by the initial degree of bile flow recovery, area under the curve of bile flow and levels of ALT (P < 0.05). No significant differences were found between the other groups. Conclusions: Gender appeared to have a significant effect on the early recovery of hepatobiliary secretory function after liver I/R injury. However this effect cannot be explained by estrogen mediated pathways. References [1] Harada H, Pavlick KP, Hines IN et al. J Appl Physiol 91(6): 2816−22, 2001. [2] Eckhoff DE, Bilbao G, Frenette L et al. Surgery 132(2): 302−9, 2002.
156 SUBNORMOTHERMIC MACHINE PERFUSION REDUCES PRESERVATION DAMAGE OF LIVERS FROM NON-HEART BEATING DONORS: A NEW CHANGE FOR EXPANDING THE DONOR POOL? A. Ferrigno1 , V. Rizzo2 , E. Bonconpagni3 , P. Richelmi1 , D. Neri4 , E. Gringeri4 , I. Freitas3 , U. Cillo4 , M. Vairetti5 . 1 Internal Medicine and Therapeutics, 2 Biochemistry, 3 Animal Biology and IGM-CNR, University of Pavia, Pavia, 4 General Surgery, University of Padua, Padua, 5 Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy E-mail: [email protected] Background and Aim: Machine perfusion (MP) preservation may enhance donor pool by reclaiming marginal livers including organs from nonheart-beating donors (NHBDs). We recently reported that MP performed at 20ºC greatly enhanced the rat liver preservation of steatotic livers [1]. Here, we tested whether the organ preservation by MP at 20ºC can also enhance the functional integrity of rat livers obtained from NHBDs when compared with cold storage. To further support our hypothesis we also compared MP at 20ºC with hypothermic MP at 4ºC.
01a: LIVER TRANSPLANTATION/SURGERY/ACUTE LIVER FAILURE − a. EXPERIMENTAL Materials and Methods: 11−12 week old male Wistar rats were used as liver donor. MP technique: livers were perfused for 6 hrs with UW-G modified pH 7.4 at 20ºC and 4ºC. Cold storage: livers were perfused in situ and preserved with UW solution at 4ºC for 6 hrs. Both MP and cold storage preserved livers were reperfused with Krebs-Heinselet buffer (2hrs at 37ºC). Aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) release and glutamate dehydrogenase (GDH) levels (index of mitochondria dysfunction) were evaluated. Parameters under evaluation were also bile production and oxygen consumption. Results: Livers preserved by MP at 20ºC show a significant lower hepatic damage at the end of reperfusion period as compared with MP at 4ºC and cold storage (LDH mU/min/g: 12.9±1.1 versus 23.7±4.6 and 22.1±1.9, p < 0.05). Release of GDH was significantly reduced and bile production was higher in livers preserved by MP at 20ºC compared with MP at 4ºC and cold storage (GDH mU/min/g: 4.1±0.6 versus 8.5±1.1 and 6.5±0.6, p < 0.05; Bile ml/g: 51±6 versus 21±7 and 37±6, p < 0.05). No significant difference in oxygen up-take was found. Conclusions: MP at 20ºC, improving cell survival, results in a betterquality preservation of livers obtained from NHBDs as compared with MP at 4ºC and conventional cold storage. MP under a moderate hypothermia might provide a new method for a successful utilization of marginal livers such as those obtained from NHBDs. (Supported by COFIN 2006 and FAR-UniPV). References [1] Vairetti et al. Subnormotherminc machine perfusion protects steatotic livers against preservation injury. Liver Transplantation 2008 (in press).
157 COMBINATION EFFECT OF MELATONIN AND DEXAMETHASONE ON LIVER ISCHEMIA/REPERFUSION INJURY IN RAT B. Hajipour1 , M.H. Somi2 , N. Ahmadi Asl3 , E. Estakhri4 , A.R. Nour Azar5 , M. Nasirizadeh5 , A. Ghorbani Haghjou6 , A.M. Vatankhah6 . 1 Scientific Association-Faculty of Medicine, Islamic Azad University, Tabriz Branch, 2 Liver and Gastroenterology Research Center, 3 Department of Physiology, 4 Department of Pathology, Tabriz University of Medical Sciences, 5 Department of Physiology, Islamic Azad University, Tabriz Branch, 6 Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran E-mail: [email protected] Background and Aims: Liver failure following ischemia-reperfusion (I/R) injury is a major concern in clinical conditions like liver surgery and transplantation. Different pharmacological preconditionings have used for attenuating hepatic I/R injury but regarding different passways involving in injury process, they were not so successful. The purpose of this study was to evaluate combination pretreatment of melatonin as an important endogenous antioxidant and dexamethasone a potent anti-inflammatory agent on liver injury of rats subjected to hepatic I/R. Materials and Methods: Male wistar rats (n = 60) were assigned to 5 groups of 12 animals each: Group 1: Sham; injected with (0.9% saline). Group 2: I/R; injected with (0.9% saline) and under went hepatic I/R. Group 3: I/R+MEL;hepatic I/R+ injected intraperitoneally (i.p.) melatonin (20 mg/kg). Group 4: I/R+DEX; hepatic I/R+ injected intravenously (i.v) dexamethasone (10 mg/kg). Group 5: I/R+MEL+DEX; hepatic I/R+ injected intraperitoneally (i.p, 20 mg/kg)) melatonin + injected intravenously (i.v) dexamethasone (10 mg/kg). Liver subjected to ischemia by clamping port triad for 30 minutes and was reperfused for 6 hours after ischemia by removing the clamps. Results: Level of glutathione peroxidase(GPx) and superoxide dismutase(SOD) were higher in I/R+MEL+DEX group comparing to I/R, I/R+MEL and I/R+DEX groups and they were higher in I/R+MEL group comparing to I/R and I/R+DEX groups significantly (P < 0.05, Table 1). Level of ALT, AST, hepatic tissue malundealdehyde (MDA) and liver injury index was lower in I/R+MEL+DEX group comparing to I/R, I/R+MEL and I/R+DEX groups, and in I/R+MEL was lower than I/R+DEX group significantly (P < 0.05). TNF-a level was lower in
S67
I/R+MEL+DEX group comparing to other groups and it was lower in I/R+DEX group than I/R+MEL and I/R group significantly (P < 0.05). Table 1
SOD (U/mg protein) GPx (U/mg protein) MDA (nmol/ml) Liver tissue injury index
Sham
Hepatic I/R I/R+MEL
I/R+DEX
I/R+MEL+DEX
4.76±0.35 4.34±0.44 1.66±0.44 0.62±0.51
1.14±0.27 1.24±0.42 5.15±0.39 15.25±1.03
1.72±0.41 2.08±0.18 3.36±0.34 12.37±0.91
3.62±0.32 3.44±0.35 2.83±0.21 8.25±1.03
2.84±0.46 2.71±0.41 4.19±0.42 10.12±1.12
Conclusions: Combination therapy of melatonin and dexamethasone had better results in decreasing the liver injury comparing to administering each of them alone. Considering safety and effectiveness of this protocol it may be useful for protection of hepatic I/R injury. 158 DOWN REGULATION OF DIACYLGLYCEROL KINASE q ACTIVITY MEDIATES THE CYTOPROTECTIVE EFFECT OF HEPATIC PRECONDITIONING G. Baldanzi1 , E. Alchera2 , C. Imarisio2 , M. Gaggianesi1 , C. Dal Ponte2 , P. Nitti3 , C. Domenicotti3 , E. Albano2 , A. Graziani2 , R. Carini2 . 1 Department of Clinical and Experimental Medicine, 2 Department of Medical Sciences, University ‘A. Avogadro’ of Piemonte Orientale, Novara, 3 Department of Experimental Medicine, University of Genoa, Genoa, Italy E-mail: [email protected] Background and Aims: The understanding of the signal mediators of liver preconditioning is still incomplete. Diacylglycerol kinases (DGKs) participate to cell signalling by regulating diacylglycerol (DAG) level, but their biolological functions are mostly unknown. This study investigates the role of DGKq in the development of hepatocyte preconditioning. Methods: DGKs activity was evaluated in rat hepatocytes preconditioned by 10 min hypoxia followed by 10 min re-oxygenation or by 15 min treatment with the adenosine A2A receptors (A2aR) agonist CGS 21680. Results: Hypoxic preconditioning decreased respectively by 50% total DGKs activity and by 75% the DGK q activity. These effects were mimicked by hepatocyte treatment with adenosine or CGS21680. CGS21680 treatment steadily increased the level of intracellular DAG and induced the stimulation of the GTPase protein Rho. Rho blockage with Clostridrium boltulinum-derived C3 transferase, reverted the inhibition of DGK q and the resistance to hypoxic damage of preconditioned hepatocytes. The cytoprotective effect of hepatocyte preconditioning were additionally abolished by genetic inhibition of DGKq with siRNA. The DGKs inhibitor R59949 mimicked the cytoprotective action of preconditioning and its capacity to increase DAG levels and to activate the DAG-dependent PKC d and e and their downstream mediator p38 MAPK. Conclusions: A2A receptor stimulation, induces Rho A/C activation and inhibition of DGKq with decrease of DAG metabolism. This increases the resistance to hypoxia of preconditioned hepatocytes by maintaining the activation of the DAG-dependent survival signals. Our results indicate DGK as new pharmacological target to reproduce the protective effects of liver preconditioning. 159 EFFECTS OF ISCHEMIC PRE- AND POSTCONDITIONING ON HIF-1a, VEGF AND TGF-b EXPRESSION DURING WARM LIVER ISCHEMIA AND REPERFUSION A. Knudsen1 , A.-S. Kannerup1 , H. Grønbæk2 , P. Funch-Jensen1 , J. Frystyk3 , A. Flyvbjerg3 , F.V. Mortensen1 . 1 Department of Surgical Gastroenterology L, 2 Department of Gastroenterology V, 3 The Medical Research Laboratories, Clinical Institut, Aarhus University Hospital, Aarhus, Denmark E-mail: [email protected] Background and Aims: Ischemic pre- and postconditioning increase the ability of the liver, to tolerate ischemia/reperfusion (I/R) injuries. The
Poster Session − Thursday, April 23
However, its efficacy is still questioned. Phosphoinositide 3-kinase (PI3K) plays a key role in the development of IPC in rodent livers. In this study we evaluated PI3Kdependent signals in transplanted human livers submitted to IPC. Methods: The livers of 40 deceased donors were randomised to receive or not (Controls) IPC before recovery. PI3K activation was evaluated in biopsies obtained immediately before IPC and 2 hours after reperfusion by measuring the phosphorylation of the PI3K down-stream kinase PKB/Akt and the levels of phosphatase tensin-homologues deleted from chromosome 10 (PTEN), an antagonist of PI3K activity. Results: IPC significantly increased PKB/Akt phosphorylation (p = 0.01) and decreased PTEN levels (p = 0.03) in grafted livers 2 hr after reperfusion, but did not significantly ameliorate post-transplant reperfusion injury. By calculating T2hr/T0 PKB/Akt phosphorylation ratios we observed that 10 out 19 (53%) of the preconditioned grafts had PKB/Akt phosphorylation index above the control threshold (IPC-responsive), while in the remaining 9 PKB/Akt phosphorylation index was comparable to controls (IPC-non-responsive). T2hr/T0 PTEN ratios were also decreased (p0.03) only in IPC-responsive grafts. In patients receiving IPC-responsive organs, IPC significantly (p0.05) ameliorated AST and ALT 24−48 hr after transplant. Bilirubin levels were also improved in subjects transplanted with IPC-responsive livers, while prothrombin activity was unchanged. Conclusions: We propose that the capacity of IPC to fully activate PI3K signalling might be variably impaired in liver grafts obtained from deceased donors and this might account for the inconsistent responses to IPC observed following human liver transplantation. 154 THE CONTRIBUTIONS OF DE NOVO ARGININE GENERATION AND ARGINASE ACTIVITY TO WHOLE BODY ARGININE METABOLISM IN ACUTE LIVER FAILURE N. Davies1 , G. Ten Have2 , V. Sharma1 , L. Ytrebo3 , S. Sen1 , C. Rose4 , A. Revhaug3 , R.P. Mookerjee1 , R. Jalan1 , N. Deutz2 . 1 Institute of Hepatology, University College London, London, UK; 2 Center for Translational Research on Aging & Longevity, University of Arkansas for Medical Sciences, Little Rock, USA; 3 Department of Surgery, University of Northern Norway, Tromso, Norway; 4 Neuroscience Research Unit, University of Montreal, Montreal, Quebec, Canada E-mail: [email protected] Background: In acute liver failure (ALF) arginine concentrations are believed to be decreased, potentially limiting substrate for nitric oxide production. However, our prior observations in a porcine hepatic devascularisation model showed no significant change in systemic haemodynamics. To further characterize the metabolic fate of arginine in ALF, we systematically assessed de-novo arginine generation, inter-organ flux of arginine, and its metabolism by the arginase pathway. Methods: Female adult pigs (30−35Kg) were randomized into either Sham (N = 8) or ALF groups (N = 8). ALF was induced by hepatic devascularisation, and the animals sampled over 6 hours. Measurements: Plasma arginine, citrulline, ornithine levels (HPLC); arginase activity (colourimetric assay); nitric oxide (stable isotope labelled citrulline generation-HPLC). Whole body metabolic rates and inter organ flux were calculated after stable isotope-labelled amino-acid infusions and calculation of arteriovenous concentration differences across each organ. Results: Plasma arginine levels decreased by 50% of the basal level at T=6 hrs (p < 0.001), whereas plasma citrulline and ornithine levels progressively increased in the ALF group compared to Sham (p < 0.001 and p < 0.001, respectively). Calculation of the whole body rate of appearance of Arginine and nitric oxide showed no difference between the two groups. However, there was an increase in the rate of de novo arginine synthesis in ALF pigs. Inter-organ flux data showed citrulline is predominantly produced in the small intestine and consumed in the kidney. Arginine and Ornithine were both primarily produced in the kidney. Plasma arginase activity (liberating ornithine) significantly increased in the ALF group over 6 hrs, whereas no change was observed in Sham animals. Conclusions: In this model of ALF, we demonstrated that although de novo production of arginine is increased, whole body level of arginine and
consequent nitric oxide production remains unchanged. This we show to be due to the significantly increased arginase activity manifest by the greater levels of ornithine in the ALF group. The effect of arginase in reducing plasma arginine and thereby, the substrate for NO synthesis, may result in the impaired organ perfusion typically observed in ALF, and contribute to multiple organ dysfunction. This work was partly supported by The Norwegian MRC. 155 INFLUENCE OF ESTROGEN ON THE EARLY RECOVERY OF HEPATOBILIARY SECRETORY FUNCTION AFTER ISCHEMIA/REPERFUSION INJURY IN RATS A.H. de Vries1 , F.A.M. Ponds1 , R.T.A. Padbury2 , R.J. Porte3 , V.B. Nieuwenhuijs3 , G.J. Barritt4 . 1 Departments of Medical Biochemistry and Surgery, 2 Department of Surgery, Flinders Medical Centre, Adelaide, Australia; 3 Department of Surgery, University Medical Center Groningen, Groningen, The Netherlands; 4 Department of Medical Biochemistry, Flinders Medical Centre, Adelaide, Australia E-mail: [email protected] Background: Ischemia/reperfusion injury (I/R injury) is a major problem in liver surgery, which causes liver dysfunction and liver failure after transplantation or hepatectomy. Recent studies have shown gender-related differences in I/R injury, in which estrogen is suggested as a ‘survival factor’ for hepatic cells through various pathways [1,2]. Aim: The aim of this study was to investigate the influence of estrogen during the early recovery of hepatobiliary secretory function after I/R injury of the liver. Methods: A rat model of segmental (60−70%) I/R was used, in which 60 minutes of ischemia was induced followed by 90 minutes of reperfusion. Male (M) and female (F) Sprague Dawley rats (n = 32) were randomly pre-treated 24 hours prior to I/R with a s.c. injection of ethanol-oil (M and F Control), 17b-estradiol (M-E2), estrogen receptor antagonist ICI 182,780 (F-ICI) or ICI 182,780 plus 17b-estradiol (F-ICI/E2). Bile flow, ALT, bilirubine and histology were analyzed, using One-Way ANOVA (Post Hoc Bonferroni). Results: Female rats appeared to have a significantly better recovery compared to male rats, reflected by the initial degree of bile flow recovery, area under the curve of bile flow and levels of ALT (P < 0.05). No significant differences were found between the other groups. Conclusions: Gender appeared to have a significant effect on the early recovery of hepatobiliary secretory function after liver I/R injury. However this effect cannot be explained by estrogen mediated pathways. References [1] Harada H, Pavlick KP, Hines IN et al. J Appl Physiol 91(6): 2816−22, 2001. [2] Eckhoff DE, Bilbao G, Frenette L et al. Surgery 132(2): 302−9, 2002.
156 SUBNORMOTHERMIC MACHINE PERFUSION REDUCES PRESERVATION DAMAGE OF LIVERS FROM NON-HEART BEATING DONORS: A NEW CHANGE FOR EXPANDING THE DONOR POOL? A. Ferrigno1 , V. Rizzo2 , E. Bonconpagni3 , P. Richelmi1 , D. Neri4 , E. Gringeri4 , I. Freitas3 , U. Cillo4 , M. Vairetti5 . 1 Internal Medicine and Therapeutics, 2 Biochemistry, 3 Animal Biology and IGM-CNR, University of Pavia, Pavia, 4 General Surgery, University of Padua, Padua, 5 Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy E-mail: [email protected] Background and Aim: Machine perfusion (MP) preservation may enhance donor pool by reclaiming marginal livers including organs from nonheart-beating donors (NHBDs). We recently reported that MP performed at 20ºC greatly enhanced the rat liver preservation of steatotic livers [1]. Here, we tested whether the organ preservation by MP at 20ºC can also enhance the functional integrity of rat livers obtained from NHBDs when compared with cold storage. To further support our hypothesis we also compared MP at 20ºC with hypothermic MP at 4ºC.
01a: LIVER TRANSPLANTATION/SURGERY/ACUTE LIVER FAILURE − a. EXPERIMENTAL Materials and Methods: 11−12 week old male Wistar rats were used as liver donor. MP technique: livers were perfused for 6 hrs with UW-G modified pH 7.4 at 20ºC and 4ºC. Cold storage: livers were perfused in situ and preserved with UW solution at 4ºC for 6 hrs. Both MP and cold storage preserved livers were reperfused with Krebs-Heinselet buffer (2hrs at 37ºC). Aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) release and glutamate dehydrogenase (GDH) levels (index of mitochondria dysfunction) were evaluated. Parameters under evaluation were also bile production and oxygen consumption. Results: Livers preserved by MP at 20ºC show a significant lower hepatic damage at the end of reperfusion period as compared with MP at 4ºC and cold storage (LDH mU/min/g: 12.9±1.1 versus 23.7±4.6 and 22.1±1.9, p < 0.05). Release of GDH was significantly reduced and bile production was higher in livers preserved by MP at 20ºC compared with MP at 4ºC and cold storage (GDH mU/min/g: 4.1±0.6 versus 8.5±1.1 and 6.5±0.6, p < 0.05; Bile ml/g: 51±6 versus 21±7 and 37±6, p < 0.05). No significant difference in oxygen up-take was found. Conclusions: MP at 20ºC, improving cell survival, results in a betterquality preservation of livers obtained from NHBDs as compared with MP at 4ºC and conventional cold storage. MP under a moderate hypothermia might provide a new method for a successful utilization of marginal livers such as those obtained from NHBDs. (Supported by COFIN 2006 and FAR-UniPV). References [1] Vairetti et al. Subnormotherminc machine perfusion protects steatotic livers against preservation injury. Liver Transplantation 2008 (in press).
157 COMBINATION EFFECT OF MELATONIN AND DEXAMETHASONE ON LIVER ISCHEMIA/REPERFUSION INJURY IN RAT B. Hajipour1 , M.H. Somi2 , N. Ahmadi Asl3 , E. Estakhri4 , A.R. Nour Azar5 , M. Nasirizadeh5 , A. Ghorbani Haghjou6 , A.M. Vatankhah6 . 1 Scientific Association-Faculty of Medicine, Islamic Azad University, Tabriz Branch, 2 Liver and Gastroenterology Research Center, 3 Department of Physiology, 4 Department of Pathology, Tabriz University of Medical Sciences, 5 Department of Physiology, Islamic Azad University, Tabriz Branch, 6 Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran E-mail: [email protected] Background and Aims: Liver failure following ischemia-reperfusion (I/R) injury is a major concern in clinical conditions like liver surgery and transplantation. Different pharmacological preconditionings have used for attenuating hepatic I/R injury but regarding different passways involving in injury process, they were not so successful. The purpose of this study was to evaluate combination pretreatment of melatonin as an important endogenous antioxidant and dexamethasone a potent anti-inflammatory agent on liver injury of rats subjected to hepatic I/R. Materials and Methods: Male wistar rats (n = 60) were assigned to 5 groups of 12 animals each: Group 1: Sham; injected with (0.9% saline). Group 2: I/R; injected with (0.9% saline) and under went hepatic I/R. Group 3: I/R+MEL;hepatic I/R+ injected intraperitoneally (i.p.) melatonin (20 mg/kg). Group 4: I/R+DEX; hepatic I/R+ injected intravenously (i.v) dexamethasone (10 mg/kg). Group 5: I/R+MEL+DEX; hepatic I/R+ injected intraperitoneally (i.p, 20 mg/kg)) melatonin + injected intravenously (i.v) dexamethasone (10 mg/kg). Liver subjected to ischemia by clamping port triad for 30 minutes and was reperfused for 6 hours after ischemia by removing the clamps. Results: Level of glutathione peroxidase(GPx) and superoxide dismutase(SOD) were higher in I/R+MEL+DEX group comparing to I/R, I/R+MEL and I/R+DEX groups and they were higher in I/R+MEL group comparing to I/R and I/R+DEX groups significantly (P < 0.05, Table 1). Level of ALT, AST, hepatic tissue malundealdehyde (MDA) and liver injury index was lower in I/R+MEL+DEX group comparing to I/R, I/R+MEL and I/R+DEX groups, and in I/R+MEL was lower than I/R+DEX group significantly (P < 0.05). TNF-a level was lower in
S67
I/R+MEL+DEX group comparing to other groups and it was lower in I/R+DEX group than I/R+MEL and I/R group significantly (P < 0.05). Table 1
SOD (U/mg protein) GPx (U/mg protein) MDA (nmol/ml) Liver tissue injury index
Sham
Hepatic I/R I/R+MEL
I/R+DEX
I/R+MEL+DEX
4.76±0.35 4.34±0.44 1.66±0.44 0.62±0.51
1.14±0.27 1.24±0.42 5.15±0.39 15.25±1.03
1.72±0.41 2.08±0.18 3.36±0.34 12.37±0.91
3.62±0.32 3.44±0.35 2.83±0.21 8.25±1.03
2.84±0.46 2.71±0.41 4.19±0.42 10.12±1.12
Conclusions: Combination therapy of melatonin and dexamethasone had better results in decreasing the liver injury comparing to administering each of them alone. Considering safety and effectiveness of this protocol it may be useful for protection of hepatic I/R injury. 158 DOWN REGULATION OF DIACYLGLYCEROL KINASE q ACTIVITY MEDIATES THE CYTOPROTECTIVE EFFECT OF HEPATIC PRECONDITIONING G. Baldanzi1 , E. Alchera2 , C. Imarisio2 , M. Gaggianesi1 , C. Dal Ponte2 , P. Nitti3 , C. Domenicotti3 , E. Albano2 , A. Graziani2 , R. Carini2 . 1 Department of Clinical and Experimental Medicine, 2 Department of Medical Sciences, University ‘A. Avogadro’ of Piemonte Orientale, Novara, 3 Department of Experimental Medicine, University of Genoa, Genoa, Italy E-mail: [email protected] Background and Aims: The understanding of the signal mediators of liver preconditioning is still incomplete. Diacylglycerol kinases (DGKs) participate to cell signalling by regulating diacylglycerol (DAG) level, but their biolological functions are mostly unknown. This study investigates the role of DGKq in the development of hepatocyte preconditioning. Methods: DGKs activity was evaluated in rat hepatocytes preconditioned by 10 min hypoxia followed by 10 min re-oxygenation or by 15 min treatment with the adenosine A2A receptors (A2aR) agonist CGS 21680. Results: Hypoxic preconditioning decreased respectively by 50% total DGKs activity and by 75% the DGK q activity. These effects were mimicked by hepatocyte treatment with adenosine or CGS21680. CGS21680 treatment steadily increased the level of intracellular DAG and induced the stimulation of the GTPase protein Rho. Rho blockage with Clostridrium boltulinum-derived C3 transferase, reverted the inhibition of DGK q and the resistance to hypoxic damage of preconditioned hepatocytes. The cytoprotective effect of hepatocyte preconditioning were additionally abolished by genetic inhibition of DGKq with siRNA. The DGKs inhibitor R59949 mimicked the cytoprotective action of preconditioning and its capacity to increase DAG levels and to activate the DAG-dependent PKC d and e and their downstream mediator p38 MAPK. Conclusions: A2A receptor stimulation, induces Rho A/C activation and inhibition of DGKq with decrease of DAG metabolism. This increases the resistance to hypoxia of preconditioned hepatocytes by maintaining the activation of the DAG-dependent survival signals. Our results indicate DGK as new pharmacological target to reproduce the protective effects of liver preconditioning. 159 EFFECTS OF ISCHEMIC PRE- AND POSTCONDITIONING ON HIF-1a, VEGF AND TGF-b EXPRESSION DURING WARM LIVER ISCHEMIA AND REPERFUSION A. Knudsen1 , A.-S. Kannerup1 , H. Grønbæk2 , P. Funch-Jensen1 , J. Frystyk3 , A. Flyvbjerg3 , F.V. Mortensen1 . 1 Department of Surgical Gastroenterology L, 2 Department of Gastroenterology V, 3 The Medical Research Laboratories, Clinical Institut, Aarhus University Hospital, Aarhus, Denmark E-mail: [email protected] Background and Aims: Ischemic pre- and postconditioning increase the ability of the liver, to tolerate ischemia/reperfusion (I/R) injuries. The