Abstract: 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 30min 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.
Abstract: In the present study, the effects of dexamethasone on cadmium-induced toxicity were evaluated in isolated rat hepatocytes. Hepatocytes were cultured for 24 h in William's E medium containing fetal calf serum (10%), insulin (0.1 IU/ml), and glucagon (0.01 microM) in the absence or presence of 0.1 microM dexamethasone. Cadmium chloride, 5 or 10 microM, was added to the medium and the toxicity was evaluated for up to 48 h after treatment. Lactate dehydrogenase (LDH) release, the reduced and oxidized glutathione ratio (GSH/GSSG), protein-SH groups, and lipid peroxidation levels were evaluated. Cadmium induced a dose- and time-dependent LDH release in control hepatocytes at 24 h (Cd 10 microM 42%) while hepatocytes pretreated with dexamethasone showed lower necrosis (Cd 10 microM 12% at 24 h). GSH/GSSH ratio and protein-SH groups were higher while lipid peroxidation was lower in dexamethasone-treated hepatocytes as compared with untreated cells. In conclusion, cadmium toxicity was associated with an increase in intracellular oxidative stress responsible for accelerated cell death. The use of dexamethasone prevented cadmium damage, suggesting that the cytoprotective action of this hormone is related to its effect in preventing changes in thiols such as glutathione and protein-SH groups.
Abstract: We tested whether rat liver preservation performed by machine perfusion (MP) at 20 degrees C can enhance the functional integrity of steatotic livers versus simple cold storage. We also compared MP at 20 degrees C with hypothermic MP at 8 degrees C, and 4 degrees 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 degrees C). MP at 4 degrees C and 8 degrees C reduced the fatty liver necrosis compared with cold storage but we further protected the organs using MP at 20 degrees 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 degrees 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 degrees C as compared with cold storage. In livers from lean rats, the adenosine triphosphate/adenosine diphosphate ratio appears better conserved by MP at 20 degrees 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 degrees 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 degrees C versus cold storage. MP at 20 degrees C improves cell survival and leads to a marked improvement in hepatic preservation of steatotic livers as compared with cold storage.
Abstract: We compared the susceptibility of liver grafts from lean and obese Zucker rats to preservation injury, using two organ-preservation techniques: conventional static preservation (SP) and machine perfusion (MP) preservation. SP: livers preserved by UW solution at 4, 8 or 20 degrees C for 6-h. MP: livers perfused for 6-h with an improved oxygenated Krebs-Henseleit solution (KH) at 4, 8 or 20 degrees C. Reperfusion with KH (2-h) was performed either with the SP or MP preserved livers. Fatty livers tolerate SP poorly at 4, 8 and 20 degrees C as compared with MP at the same temperatures. SP induced a decrease in the ATP/ADP ratio both at 8 and 20 degrees C in obese rats while an increase in energy status was found with MP at 8 and 20 degrees C. Nitrate/nitrite (NOx) concentration was higher and bile flow lower in livers preserved with SP than MP. In lean rats, no differences were observed between MP and SP as regards enzyme release, bile production and NOx levels except for SP at 20 degrees C in which high enzyme release and low bile flow were observed. In lean rats ATP/ADP was higher and NOx was lower with MP at 20 degrees C than with SP at 20 degrees C. To optimize steatotic liver preservation SP should be avoided because it is particularly detrimental as compared with MP.
Abstract: The effects of Celsior solution were compared with those of the University of Wisconsin solution (UW) after 18 or 48 hours of cold storage in a perfused rat liver model. Lactate dehydrogenase (LDH), hyaluronic acid (HA) uptake, thiobarbituric acid-reactive substances (TBARS), tissue reduced (GSH) and oxidized glutathione (GSSG) and ATP were evaluated. Histochemical in situ evaluation ofLDH and reactive oxygen species (ROS) were also performed. No significant difference in LDH release, HA uptake, TBARS, ATP levels and GSH/GSSG ratio were observed between UW and Celsior solution when the livers were preserved for 18 hours. By contrast, when preservation was performed for 48 hours, LDH release, TBARS and ROS formation were higher and the ATP levels, GSH/GSSG ratio and HA uptake were lower in the liver preserved by Celsior as compared with UW. Celsior solution was as effective as UW in liver preservation up to 18 hours but the superiority of UW over Celsior solution was obtained when liver was preserved for 48 hours.
Abstract: Autofluorescence spectroscopy is a promising and powerful approach for an in vivo, real time characterization of liver functional properties. In this work, preliminary results on the dependence of liver autofluorescence parameters on the nutritional status are reported, with particular attention to vitamin A and lipid accumulation in liver tissue. Normally fed and 24 h starving rats were used as animal models. Histochemical and autofluorescence analysis showed that lipids and vitamin A colocalize in the liver parenchyma. Fasting condition results in a parallel increase in both lipids and vitamin A. Autofluorescence imaging and microspectrofluorometric analysis carried out on unfixed, unstained tissue sections under 366 nm excitation, evidenced differences in both spectral shape and response to continuous irradiation between liver biopsies from fed and starving rats. As to photobleaching, in particular, fitting analysis evidenced a reduction of about 85% of the signal attributable solely to vitamin A during the first 10 s of irradiation. The tissue whole emission measured in fed and starving rat livers exhibited reductions of about 35% and 52%, respectively, that are closely related to vitamin A contents. The findings open interesting perspectives for the set up of an in situ, real time diagnostic procedure for the assessment of liver lipid accumulation, exploiting the photophysical properties of vitamin A.
Abstract: This study compares the effects of machine perfusion (MP) at different temperatures with simple cold storage. In addition, the role of Ca(2+) 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 CaCl(2)) at 4 degrees C, 10 degrees C, 20 degrees C, 25 degrees C, 30 degrees C, or 37 degrees C. For cold storage, livers were perfused in situ and preserved with Celsior solution at 4 degrees C for 6 hours. The reperfusion period (2 hours at 37 degrees 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 degrees 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 degrees C and 10 degrees C but not with MP at 20 degrees C. When a KH-1.25 mM CaCl(2) solution was used during MP at 20 degrees 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 CaCl(2). ATP levels were higher and morphology was better in liver preserved with KH-1.25 mM CaCl(2). MP at 20 degrees C with KH-1.25 mM CaCl(2) 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.
Abstract: Autofluorescence response to oxygen supply modulation has been investigated in livers of rats under the hypermetabolic state associated to a pathological condition-hyperthyroidism-that is known to enhance hepatocyte metabolic activities involving both NAD, i.e. oxidative pathways engaged in ATP synthesis, and NADP, i.e. reductive bio-synthesis and antioxidant functions. Experiments have been performed on rats in normal condition or submitted to long-term thyroxine (T(4)) administration. Histological inspection did not show any appreciable morphological alteration in liver parenchyma; biochemical analysis indicated an increase in both NADP(+) and NADPH contents. Autofluorescence properties have been monitored in vivo, via a fiber optic probe, on exposed livers both during induction of global ischemia and after restoration of blood circulation. Alteration of oxygen supply modulated liver autofluorescence properties, mainly as to NAD(P)H contribution, in dependence of changes in pyridine coenzymes redox state. With respect to euthyroid, hyperthyroid rat livers exhibited higher autofluorescence signals in all phases of the experiment, and a faster signal decay time upon reoxygenation. The results have been interpreted on the basis of a larger content of NADPH-the coenzyme not directly oxidized in respiratory processes and likely providing an almost constant autofluorescence background contribution-and of uncoupling effects facilitating the respiratory NADH oxidation, associated with the hyperthyroid condition. The results obtained in the liver hypermetabolic model provide interesting perspectives for a further improvement of the diagnostic implications of autofluorescence.
Abstract: Free radicals play a central role in the development of liver ischemia/reperfusion (I/R) injury. Reduced glutathione (GSH) is the main hepatic free radical scavenger. Brain-dead patients exhibit abnormalities of endocrine status. Many clinicians administer thyroid hormones to improve the transplantation outcomes. We previously reported that thyroxine (T(4)) pretreatment decreased rat liver tissue GSH, which was associated with increased liver I/R-induced damage. In this study, we investigated whether the reduction in GSH by T(4) pretreatment affected cell viability during anoxia or oxidative stress in suspensions of isolated hepatocytes. Furthermore, we evaluated the levels of GSH in isolated livers from hypothyroid rats preserved at 0-1 degrees C and reperfused. Thyroid hormone modulation was obtained by T(4) or 6-propylthiouracil (PTU) treatment. Isolated hepatocytes from T(4)-pretreated rats that underwent anoxia and oxidative stress, which was induced by tert-butylhydroperoxide, displayed progressive, time-dependent loss of cell viability, which was greater than that in hepatocytes in non-T(4)-pretreated rats. A significant decrease in GSH levels was observed in isolated hepatocytes obtained from hyperthyroid rats compared with those from euthyroid rats. In contrast, administration of the antithyroid drug PTU increased liver concentrations of GSH at the end of reperfusion thereby improving liver function after cold storage. These results may yield new protective strategies in the management of brain-dead organ donors.
Abstract: Hypothermic machine perfusion (MP) of the liver has been reported to improve graft function reclaiming marginal livers, such as those from non-heart-beating donors. Livers from obese donors often have fatty infiltrates and are more susceptible to hypothermic conditions. No data exist about MP at temperatures >4 degrees C. This study evaluated liver function after organ preservation by comparing MP at 20 degrees C with conventional cold storage. METHODS: For MP, rat livers were perfused for 6 hours using an oxygenated Krebs-Henseleit (KH) solution at 20 degrees C (pH 7.4). For cold storage, livers were perfused in situ and preserved with Celsior solution at 4 degrees C for 6 hours. The reperfusion period with KH (2 hours at 37 degrees C) was performed under the same conditions both among livers preserved by MP or cold storage. Hepatic enzyme release (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], and gamma-glutamyl transferase [GGT]), bile production, and ATP levels were measured during MP and reperfusion. RESULTS: At the end of reperfusion, livers preserved by MP showed significantly decreased liver damage compared with cold storage: AST, 18 +/- 4 vs. 45 +/- 6 mU/mL (P < .01); ALT, 1.5 +/- .07 vs. 6 +/- 0.5 mU/mL (P < .01); and LDH, 82 +/- 2 vs. 135 +/- 29 mU/mL (P < .05). No difference was observed between bile production between MP and cold storage. High levels of biliary GGT and LDH were found in cold preserved livers. ATP levels were higher in livers preserved with MP compared with those preserved by cold storage. CONCLUSIONS: MP at 20 degrees C resulted in a better quality of liver preservation, improving hepatocyte survival, compared with conventional cold storage. This may provide a new method for successful utilization of marginal livers, in particular fatty livers.
Abstract: In a previous investigation, reperfusion with a melatonin-containing medium was demonstrated to enhance bile production and tissue ATP levels in rat livers, cold-preserved with University of Wisconsin (UW) or Celsior solutions, with respect to melatonin-free reperfusion; lipid peroxidation products in the perfusate were not influenced by the indole. This was ascribed to an increased efficiency of the hepatocyte mitochondria induced by melatonin. Reactive oxygen species (ROS) normally leak from the electron transfer chain in mitochondria and excessive ROS production is presumed to mediate ischemia-reperfusion (I/R) damage. A histochemical reaction was used to demonstrate ROS on the same model. Compared to the lobular zonation of ROS in control livers, the stained area of cold-preserved livers reperfused without melatonin was restricted to a narrow portal region, in keeping with the much lower ATP content. When reperfusion was performed with melatonin, the liver morphology was improved and the ROS reaction in hepatocytes more intense, though not reaching the control liver pattern. Sinusoidal cells were poorly-stained in both cases. In conclusion, with this different approach, melatonin was confirmed to improve mitochondrial performance and to discriminate parenchymal from sinusoidal cell behavior. Our observations confirm that melatonin mitigates I/R injury and support its potential in 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 degrees C without or with 25, 50, 100 and 200 microM 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 degrees C. Reperfusion (120 min) was performed with KHB at 37 degrees C. In subsequent studies, 100 microM 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 (gamma-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 gamma-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.
Abstract: Wilson's disease (WD) is an inherited disorder, characterized by selective copper deposition in liver and brain, chronic hepatitis and extra-pyramidal signs. In this study, we investigated changes of biochemical markers of oxidative stress and apoptosis in liver, striatum and cerebral cortex homogenates from Long-Evans Cinnamon (LEC) rats, a mutant strain isolated from Long Evans (LE) rats, in whom spontaneous hepatitis develops shortly after birth. LEC and control (LE) rats at 11 and 14 weeks of age were used. We determined tissue levels of glutathione (GSH/GSSG ratio), lipid peroxides, protein-thiols (P-SH), nitric oxide metabolites, activities of caspase-3 and total superoxide-dismutase (SOD), striatal levels of monoamines and serum levels of hepatic amino-transferases. We observed a decrease of protein-thiols, GSH/GSSG ratio and nitrogen species associated to increased lipid peroxidation in the liver and striatum - but not in the cerebral cortex - of LEC rats, accompanied by dramatic increase in serum amino-transferases and decrease of striatal catecholamines. Conversely, SOD and caspase-3 activity increased consistently only in the cortex of LEC rats. Hence, we assume that enhanced oxidative stress may play a central role in the cell degeneration in WD, at the main sites of copper deposition, with discrete pro-apoptotic conditions developing in distal areas.
Abstract: Ischemia-reperfusion injury, a major cause of organ metabolic alterations and consequent dysfunction in liver transplantation, could be overcome by optimizing organ preservation procedures. The potential of autofluorescence analysis was investigated with the aim to define parameters suitable for in vivo monitoring tissue functionality. Spectrofluorometric analysis was performed on explanted rat livers during cold storage, under standard (4 degrees C University of Wisconsin medium for 20 h) and purposely damaging (4 degrees C Eurocollins medium for 20, 43 and 72 h) preservation conditions, and reperfusion (rewarming-reoxygenation). For both preservation conditions, cold hypoxia caused a signal amplitude increase, mainly attributable to NAD(P)H, and a spectral shape modification, ascribable to changes in the relative contributions of NAD(P)H and flavins, as a result of the tissue reduced state enhancement. Upon rewarming-reoxygenation the autofluorescence signal decreased with a rate depending on the preservation conditions. The time constant changed according to the extent of the liver functionality impairment, as assessed by conventional biochemical and histochemical analyses, thus providing a parameter exploitable for an in situ, real time monitoring of the efficacy of experimental preservation procedures.
Abstract: Hypothermia induces injury in its own right, but the mechanisms involved in the cell damage are still unclear. The aim of this study was to test the effects that glutathione (GSH) depletion induces on cell death in isolated rat hepatocytes, kept at 4 degrees C for 20 h, by modulating intracellular GSH concentration with diethylmaleate and buthionine sulfoximine (DEM and BSO). Untreated hepatocytes showed Annexin V stained cells (AnxV(+)), scarce propidium iodide stained cells (PI(+)) and presented a low level of lactate dehydrogenase (LDH) leakage after 20 h at 4 degrees C and rewarming at 37 degrees C. When DEM and BSO were added before cold storage, we observed a few AnXV(+) cells and an increase in PI(+) cells associated with LDH release in the incubation medium. Conversely, the addition of DEM and BSO only during rewarming caused a marked increase in cell death by apoptosis. Production of reactive oxygen species (ROS) and thiobarbituric acid species (TBARS), associated with a decrease in GSH concentrations, was higher when DEM and BSO were added before cold storage. Cells treated with DEM and BSO before cold storage showed lower ATP energy stores than hepatocytes treated with DEM and BSO only during rewarming. Pretreatment of hepatocytes with deferoxamine protected against apoptotic and necrotic morphology in conditions of GSH depletion. These results suggest that pretreatment of hepatocytes with DEM and BSO before cold storage induces necrosis, while the treatment of hepatocytes only during rewarming increases apoptosis. In both conditions, iron represents a crucial mediator of cell death.
Abstract: We evaluated the effects of nicergoline on antioxidant defense enzymes (detoxifying enzymes), during chronic treatment with haloperidol in rats. Chronic use of haloperidol (10 weeks, 1.5 mg/kg/day) induces a significant decrease in glutathione reductase, glutathione peroxidase and superoxide dismutase activity, in selected areas of the brain. Co-administration of nicergoline (20 days, 10 mg/kg/day) significantly restored the activity of these enzymes to levels comparable to those observed in control rats. These observations suggest beneficial effects of nicergoline in the prevention and in the treatment of haloperidol-induced side effects.
Abstract: 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.
