Mariapia Vairetti

Subnormothermic machine perfusion protects steatotic livers against preservation injury: A potential for donor pool increase?†

We tested whether rat liver preservation performed by machine perfusion (MP) at 20°C can enhance the functional integrity of steatotic livers versus simple cold storage. We also compared MP at 20°C with hypothermic MP at 8°C, and 4°C. Obese and lean male Zucker rats were used as liver donors. MP was performed for 6 hours with a glucose and N‐acetylcysteine–supplemented Krebs‐Henseleit solution. Both MP and cold storage preserved livers were reperfused with Krebs‐Henseleit solution (2 hours at 37°C). MP at 4°C and 8°C reduced the fatty liver necrosis compared with cold storage but we further protected the organs using MP at 20°C. Necrosis did not differ in livers from lean animals submitted to the different procedures; the enzymes released in steatotic livers preserved by MP at 20°C were similar to those showed in nonsteatotic organs. The adenosine triphosphate/adenosine diphosphate ratio and bile production were higher and the oxidative stress and biliary enzymes were lower in steatotic livers preserved by MP at 20°C as compared with cold storage. In livers from lean rats, the adenosine triphosphate/adenosine diphosphate ratio appears better conserved by MP at 20°C as compared with cold storage. In steatotic livers preserved by cold storage, a 2‐fold increase in tumor necrosis factor‐alpha levels and caspase‐3 activity was observed as compared with organs preserved by MP at 20°C. These data are substantiated by better morphology, higher glycogen content, and lower reactive oxygen species production by sinusoidal cells in steatotic liver submitted to MP at 20°C versus cold storage. MP at 20°C improves cell survival and leads to a marked improvement in hepatic preservation of steatotic livers as compared with cold storage. Liver Transpl 15:20–29, 2009.

Calcium‐dependent DNA fragmentation in human synovial cells exposed to cold shock

Exposure of confluent human synovial McCoys cells to near‐freezing temperatures followed by rewarming at 37°C resulted in endonuclease activation and cell death characteristic of a suicide process known as apoptosis. Both DNA fragmentation and cell killing were dependent on a sustained increase in the cytosolic Ca2+ concentration. Sensitivity to cold shock‐induced endonuclease activation was critically dependent on the cell cycle (proliferative) status and limited to confluent cells, whereas cells in the logarithmic growth phase were completely resistant. However, DNA fragmentation was promoted in the proliferating McCoys cells pretreated with H‐7 or sphingosine, inhibitors of protein kinase C. In addition, phorbol ester, known to activate PKC, inhibited DNA fragmentation in the confluent cells. Our findings indicate that cold shock‐induced DNA fragmentation in McCoys cells is dependent on a sustained Ca2+ increase, and sensitivity to the process appears to be regulated by the status of protein kinase C.

Cold-induced apoptosis in isolated rat hepatocytes: protective role of glutathione.

Liver conservation for transplantation is usually made at 2-4 degrees C. We studied the effect of rewarming to 37 degrees C for up to 3 h of rat hepatocytes kept at 4 degrees C for 20 h, modulating intracellular glutathione (GSH) concentration either with a GSH precursor (N-acetyl-L-cysteine, NAC), or with GSH depleting agents (diethylmaleate and buthionine sulfoximine, DEM/BSO). Untreated hepatocytes showed time-dependent production of reactive oxygen species (ROS), lipid peroxidation, chromatin condensation and membrane blebbing, decrease in GSH concentration, and protein sulfhydryl groups. Fluorochromatization with Propidium Iodide (PI) and Annexin V (AnxV) of cells rewarmed for 1 h caused an increase of AnxV-positive cells without PI staining and any observed lactate dehydrogenase leakage. TUNEL and DNA-laddering tests were negative for all times and treatments, indicating that apoptosis may occur without DNA fragmentation. Cold preservation and rewarming in the presence of NAC induced a significant improvement in the morphology, less oxidative stress and apoptosis. Conversely, DEM/BSO caused a marked deterioration of morphology, increase of oxidative stress and apoptosis. These results suggested that marked changes in GSH status might play a critical role in triggering apoptosis during cold preservation of isolated rat hepatocytes. NAC, added before rewarming, might represent a therapeutic approach for preventing the early events of apoptosis during cold storage.

Autofluorescence properties of isolated rat hepatocytes under different metabolic conditions

The contribution of endogenous fluorophores - such as proteins, bound and free NAD(P)H, flavins, vitamin A, arachidonic acid - to the liver autofluorescence was studied on tissue homogenate extracts and on isolated hepatocytes by means of spectrofluorometric analysis. Autofluorescence spectral analysis was then applied to investigate the response of single living hepatocytes to experimental conditions resembling the various phases of the organ transplantation. The following conditions were considered: 1 h after cells isolation (reference condition); cold hypoxia; rewarming-reoxygenation after cold preservation. The main alterations occurred for NAD(P)H and flavins, the coenzymes strictly involved in energetic metabolism. During cold hypoxia NAD(P)H, mainly the bound form, showed an increase followed by a slow decrease, in agreement with the inability of the respiratory chain to reoxidize the coenzyme, and a subsequent NADH reoxidation through alternative anaerobic metabolic pathways. Both bound/free NAD(P)H and total NAD(P)H/flavin ratio values were altered during cold hypoxia, but approached the reference condition values after rewarming-reoxygenation, indicating the cells capability to restore the basal redox equilibrium. A decrease of arachidonic acid and vitamin A contributions occurred after cold hypoxia: in the former case it may depend on the balance between deacylation and reacylation of fatty acids, in the latter it might be related to the vitamin A antioxidant role. An influence of physico-chemical status and microenvironment on the fluorescence efficiency of these fluorophores cannot be excluded. In general, all the changes observed for cell autofluorescence properties were consistent with the complex metabolic pathways providing for energy supply.

INTRANUCLEAR DISTRIBUTION, FUNCTION AND FATE OF GLUTATHIONE AND GLUTATHIONE-S-CONJUGATE IN LIVING RAT HEPATOCYTES STUDIED BY FLUORESCENCE MICROSCOPY

The availability of fluorescent probes to detect soluble and protein‐bound thiols has made it possible to investigate some aspects of reduced glutathione (GSH) metabolism and function in intact rat hepatocytes and in hepatocyte nuclei. Monochlorobimane (BmCl) has been employed to study the subcellular compartmentation of GSH and the formation and fate of the BmCl‐GSH conjugate. The occurrence of relatively high concentrations of GSH within the nuclear matrix has been inferred from fluorescence quantitation using image analysis. Concomitant biochemical studies have revealed the presence of a GSH‐stimulated ATP hydrolysis and of an ATP‐stimulated GSH accumulation in isolated nuclei, providing the molecular basis for nuclear glutathione compartmentation. The contemporary use of fluorescent probes to label nuclear free sulfhydryl groups, proteins and chromatin status led to the demonstration that intranuclear accumulation of glutathione may modulate the thiol/disulfide redox status of nuclear proteins and control chromatin compacting and decondensation. Microsc. Res. Tech. 36:243–252, 1997.

Machine perfusion at 20 °C reduces preservation damage to livers from non-heart beating donors

We previously reported that machine perfusion (MP) performed at 20°C enhanced the preservation of steatotic rat livers. Here, we tested whether rat livers retrieved 30 min after cardiac arrest (NHBDs) were better protected by MP at 20°C than with cold storage. We compared the recovery of livers from NHBDs with organs obtained from heart beating donors (HBDs) preserved by cold storage. MP technique: livers were perfused for 6h with UW-G modified at 20°C. Cold storage: livers were perfused in situ and preserved with UW solution at 4°C for 6h. Both MP and cold storage preserved livers were reperfused with Krebs-Heinselet buffer (2h at 37°C). AST and LDH release and mitochondrial glutamate dehydrogenase (GDH) levels were evaluated. Parameters assessed included: bile production and biliary enzymes; tissue ATP; reduced and oxidized glutathione (GSH/GSSG); protein-SH group concentration. Livers preserved by MP at 20°C showed significantly lower hepatic damage at the end of reperfusion compared with cold storage. GDH release was significantly reduced and bile production, ATP levels, GSH/GSSG and protein-SH groups were higher in livers preserved by MP at 20°C than with cold storage. The best preserved morphology and high glycogen content was obtained with livers submitted to MP at 20°C. Liver recovery using MP at 20°C was comparable to recovery with HBDs. MP at 20°C improves cell survival and gives a better-quality of preservation for livers obtained from NHBDs and may provide a new method for the successful utilization of marginal livers.

Exogenous melatonin enhances bile flow and ATP levels after cold storage and reperfusion in rat liver: Implications for liver transplantation

Abstract:   Although the use of melatonin in the transplantation field has been suggested, it has not been previously tested in a liver cold‐storage model. We used a rat liver model to study (a) the dose‐dependent effect of melatonin on bile production, and (b) the potential of melatonin to improve liver function after cold‐storage. Male Wistar rats were perfused with Krebs–Henseleit bicarbonate buffer (KHB) at 37°C without or with 25, 50, 100 and 200 μm melatonin. Each dose of melatonin stimulated bile production. For cold‐storage studies, livers were flushed with either University of Wisconsin (UW) or Celsior solution and stored for 20 hr at 4°C. Reperfusion (120 min) was performed with KHB at 37°C. In subsequent studies, 100 μm melatonin were added to the perfusate during the reperfusion period. ATP and melatonin levels in the tissue were measured. Bile analysis was performed by measuring melatonin, bilirubin and gamma‐glutamyl transpeptidase (γ‐GT) levels in the fluid. A dose‐dependent increase in bile secretion, associated with an enhanced melatonin and bilirubin levels in the bile were observed. Also, tissue levels of melatonin increased in a dose‐dependent manner. When melatonin was added during the reperfusion period, bile production and bile bilirubin levels increased both with UW and Celsior solutions. The analysis of γ‐GT in the bile showed an increase in the Celsior‐preserved liver and the addition of melatonin to the perfusate reduced this effect. Tissue ATP levels were higher when melatonin was added to the perfusion medium. Higher levels of melatonin in bile than in tissue were found. In conclusion, we demonstrate that melatonin improves significantly the restoration of liver function after cold‐storage and reperfusion.

Correlation between the liver temperature employed during machine perfusion and reperfusion damage: Role of Ca2+

This study compares the effects of machine perfusion (MP) at different temperatures with simple cold storage. In addition, the role of Ca2+ levels in the MP medium was evaluated. For MP, rat livers were perfused for 6 hours with Krebs‐Henseleit (KH) solution (with 1.25 or 2.5 mM CaCl2) at 4°C, 10°C, 20°C, 25°C, 30°C, or 37°C. For cold storage, livers were perfused in situ and preserved with Celsior solution at 4°C for 6 hours. The reperfusion period (2 hours at 37°C) was performed under the same conditions used for MP‐preserved and cold storage–preserved livers. Hepatic enzyme release, bile production, adenosine triphosphate (ATP) levels, and morphology were evaluated during MP and reperfusion. MP at 37°C caused marked enzyme release; the same findings were obtained during reperfusion. By contrast, MP temperature lowering induced a significant decrease in liver damage. High levels of biliary gamma‐glutamyltransferase and lactate dehydrogenase were found with MP at 4°C and 10°C but not with MP at 20°C. When a KH–1.25 mM CaCl2 solution was used during MP at 20°C, very low enzyme release was observed and significantly lower hepatic damage was present at the end of the reperfusion period in comparison with cold storage. The same results were obtained when ruthenium red, a calcium uniporter blocker, was added to KH–2.5 mM CaCl2. ATP levels were higher and morphology was better in liver preserved with KH–1.25 mM CaCl2. MP at 20°C with KH–1.25 mM CaCl2 resulted in better quality liver preservation, improving hepatocyte and endothelial biliary cell survival, in comparison with cold storage. This raises the need to reconsider the temperature and calcium levels to be used during liver MP. Liver Transpl 14:494–503, 2008.

Selective blockade of mGlu5 metabotropic glutamate receptors is protective against acetaminophen hepatotoxicity in mice

BACKGROUND/AIMS mGlu5 metabotropic glutamate receptor antagonists protect rat hepatocytes against hypoxic death. Here, we have examined whether mGlu5 receptor antagonists are protective against liver damage induced by oxidative stress. METHODS Toxicity of isolated hepatocytes was induced by tert-butylhydroperoxide (t-BuOOH) after pretreatment with the mGlu5 receptor antagonists, MPEP, SIB-1757 and SIB-1893. The effect of these drugs was also examined in mice challenged with toxic doses of acetaminophen. RESULTS Addition of tBuOOH (0.5 mM) to isolated hepatocytes induced cell death (70+/-5% at 3 h). Addition of MPEP or SIB-1893 to hepatocytes reduced both the production of reactive oxygen species (ROS) and cell toxicity induced by t-BuOOH (tBuOOH=70+/-5%; tBuOOH+MPEP=57+/-6%; tBuOOH+SIB-1893=40+/-4%). In mice, a single injection of acetaminophen (300 mg/kg, i.p.) induced centrilobular liver necrosis, which was detectable after 24 h. MPEP (20 mg/kg, i.p.) substantially reduced liver necrosis and the production of ROS, although it did not affect the conversion of acetaminophen into the toxic metabolite, N-acetylbenzoquinoneimine. MPEP, SIB-1893 and SIB-1757 (all at 20 mg/kg, i.p.) also reduced the increased expression and activity of liver iNOS induced by acetaminophen. CONCLUSIONS We conclude that pharmacological blockade of mGlu5 receptors might represent a novel target for the treatment of drug-induced liver damage.

Haloperidol-induced changes in glutathione and energy metabolism: effect of nicergoline.

The aim of this study was to evaluate the possible effects of nicergoline, a semisynthetic ergot derivative, on the biochemical changes observed during chronic treatment with haloperidol in male Sprague-Dawley rats. Chronic treatment with haloperidol induced a significant decrease in the cellular glutathione (GSH) content in selected areas of the brain (cerebellum, striatum and cortex) and in the liver. Prolonged nicergoline administration was able to antagonize the haloperidol-induced GSH decrease, maintaining the GSH concentration at levels comparable to those observed in the control group. Analysis of the energy charge revealed changes similar to those observed for GSH: haloperidol induced a significant decrease in ATP and energy charge that was completely reversed by repeated nicergoline administration. In conclusion, chronic treatment with the classical antipsychotic haloperidol induces profound biochemical changes in the brain and in the liver. Nicergoline treatment is able to counteract the haloperidol-induced decrease in GSH levels and energy charge, suggesting a potential role of the drug in the treatment of neuroleptic-induced side effects.