Perfused Rat Liver Model Research Articles

Effect of cold ischemia–warm reperfusion on the cirrhotic rat liver

Cirrhosis is known to induce capillarization of the sinusoidal endothelial cells (SECs) and collagenization of the space of Disse, resulting in a reduced access of plasma and plasma-dissolved substances to hepatocytes due to their limited diffusion in the extravascular space. The aim of the present study was to use a well known effect of cold ischemia-warm reperfusion (CI-WR) on liver SECs, that is, their retraction and detachment, progressing to a denudation of the SEC lining. The disappearance of the capillarized SEC lining would improve the access of plasma and plasma-dissolved substances to the hepatocytes and consequently might improve the metabolic function of cirrhotic livers. This study was performed using the isolated perfused rat liver model subjected to 24-hour CI followed by a 60-minute WR in thioacetamide-induced cirrhosis. Liver microcirculation was evaluated using the multiple indicator dilution curve (MIDC) technique. Hepatocyte, SEC, and Kupffer cell functions were evaluated using specific elimination processes. As occurs in normal livers, CI-WR induced extensive SEC necrosis with a marked reduction of the hyaluronic acid elimination. By contrast, the hepatic microcirculation was not modified: vascular, extravascular, and the cellular spaces were similar before and following CI-WR. In addition, the hepatic metabolic functions, as evaluated by propranolol and taurocholate hepatic uptake, were neither improved nor decreased, as were Kupffer cell functions. The present data strongly suggest that capillarization of SECs plays a lesser role than collagenization of the space of Disse in the reduced exchange between sinusoids and hepatocytes in thioacetamide-induced cirrhotic rat livers, which appear to be quite resistant to CI-WR.

Effect of nutritional status on hepatocyte integrity and function in an ex vivo perfused rat liver model

Effect of nutritional status on hepatocyte integrity and function in an ex vivo perfused rat liver model

Preservation of steatotic livers: A comparison between cold storage and machine perfusion preservation

Liver grafts are frequently discarded due to steatosis. Steatotic livers can be classified as suboptimal and deteriorate rapidly during hypothermic static preservation, often resulting in graft nonfunction. Hypothermic machine perfusion (MP) has been introduced for preservation of donor livers instead of cold storage (CS), resulting in superior preservation outcomes. The aim of this study was to compare CS and MP for preservation of the steatotic donor rat liver. Liver steatosis was induced in male Wistar rats by a choline-methionine-deficient diet. After 24 hours hypothermic CS using the University of Wisconsin solution (UW) or MP using UW-Gluconate (UW-G), liver damage (liver enzymes, perfusate flow, and hyaluronic acid clearance) and liver function (bile production, ammonia clearance, urea production, oxygen consumption, adenosine triphosphate [ATP] levels) were assessed in an isolated perfused rat liver model. Furthermore, liver biopsies were visualized by hematoxylin and eosin staining. Animals developed 30 to 60% steatosis. Livers preserved by CS sustained significantly more damage as compared to MP. Bile production, ammonia clearance, urea production, oxygen consumption, and ATP levels were significantly higher after MP as compared to CS. These results were confirmed by histology. In conclusion, MP improves preservation results of the steatotic rat liver, as compared to CS.

The Possible Study of HuaXi-1 Solution for All Intra-abdominal Organ Preservation: A Study in Rats

Objective University of Wisconsin (UW) solution continues to be the most commonly used solution for intra-abdominal organs. However, it is expensive. Therefore, we have formulated HuaXi-1 (HX-1) solution. The aim of this research was to assess the effect of HX-1 solution compared to UW, Collins 2, and hypertonic citrate solutions on intra-abdominal organs. Methods The effects of HX-1 solution for all intra-abdominal organ preservation were studied in a noncirculated, isolated, perfused rat liver model, in rat pancreas isografts in diabetic rats, and in a kidney autotransplant model. The effects were investigated by measuring hepatic tissue water content, sinusoidal lining cell mortality, Krebs-Henseleits perfusate, aspartate aminotransferase, the number of livers secreting bile during isolated perfusion, blood glucose, glucose tolerance tests, serum insulin of the rat pancreas-transplant recipients, the maximum serum creatinine levels, kidney graft survival rates, and observing the morphological changes in liver, pancreas, and kidneys. Results HX-1 solution preserved rat liver well for 24 hours as effectively as UW, rat pancreas as well for 48 hours, and dog kidney as well for 72 hours. Conclusion The experimental findings indicated that HX-1 solution was effective to preserve all intra-abdominal organs; it may simplify cold storage of organs for transplantation.

Pharmacokinetic consequences of time-dependent inhibition using the isolated perfused rat liver model

Mathematical models exist to describe the pharmacokinetic changes caused by time-dependent inhibition (TDI) and these have been reported to predict accurately for several marketed drugs. However, their robustness using in-depth, carefully controlled pre-clinical studies has yet to be established. In the current study, the isolated perfused rat liver was employed to investigate the effects of TDI under carefully controlled conditions. A five-compartmental model was used to describe the observed data and bring context to in vitro TDI data (kinact and KI). Co-administration of midazolam with troleandomycin, mifepristone, erythromycin and the discovery compound, AZ-X, increased midazolam area under the curve (AUC) 3.2-, 2.5-, 1.6- and 1.0-fold, respectively, compared with AUC increases of 1.8-, 1.4-, 1.2- and 1.1-fold predicted by the model. These experimental findings, whilst modest in overall effect, support the use of this model in the rat and it is proposed that projections can be made for the likely clinical impact of novel time-dependent inhibitors in man based on predicted human pharmacokinetics and TDI potency determined in vitro.

SELECTIVE EFFECTS OF NITRIC OXIDE ON THE DISPOSITION OF CHLORZOXAZONE AND DEXTROMETHORPHAN IN ISOLATED PERFUSED RAT LIVERS

The rapid and direct effects of nitric oxide (NO) donors sodium nitroprusside (SNP) and isosorbide dinitrate (ISDN) on the hepatic and biliary disposition of chlorzoxazone (CZX), a marker of CYP2E1, and dextromethorphan (DEM), a marker of CYP2D1, were studied in a single-pass isolated perfused rat liver model. Livers (n = 30) were perfused with constant concentrations of NO donors (0-120 min) in addition to infusion of CZX or DEM (60-120 min), and periodical outlet and bile samples were collected. Both ISDN and SNP significantly reduced (30 and 60%, respectively) the hepatic extraction ratio of CZX and decreased (50 and 70%, respectively) the recovery of the CYP2E1-mediated metabolite, 6-hydroxychlorzoxazone, in the outlet perfusate and bile. As for DEM, both NO donors increased (up to 3.5-fold) the recovery of the CYP2D1-mediated metabolite dextrorphan (DOR) in the outlet perfusate. However, this was associated with a simultaneous decrease (50-75%) in the excretion of the metabolite into the bile, thus resulting in no change in the overall recovery of DOR as a result of NO donor treatment. The decrease in the biliary excretion of DOR was caused by NO-induced simultaneous reductions in both the conjugation of DOR and biliary clearance of DOR conjugate. Additionally, both SNP and ISDN significantly reduced the metabolism of DEM to 3-hydroxymorphinan, which is mostly regulated by CYP3A2. These studies in an intact liver model confirm the selectivity of the inhibitory effects of NO donors on cytochrome P450 enzymes, which was recently reported in microsomal studies, and expand these inhibitory effects to conjugation pathways.

Development of the Isolated Dual Perfused Rat Liver Model as an Improved Reperfusion Model for Transplantation Research

The Isolated Perfused Liver (IPL) model is a widely used and appreciated in vitro method to demonstrate liver viability and metabolism. Reperfusion is performed in a controlled setting, however, via the portal vein only. To study transplant related questions concerning bile and transport of bile, the in vitro Isolated dual Perfused Liver model is revisited. The IdPL is an in vitro reperfusion model, using both portal vein and hepatic artery. Livers from 12 Wistar rats were flushed with University of Wisconsin-organ preservation solution, procured and reperfused in either the conventional IPL-model (n = 6) or the new IdPL-model (n = 6). Liver injury, assessed by the release of aspartate amino transferase and lactate dehydrogenase, showed similar levels during both IPL and I dPL reperfusion, only alanine amino transferase showed an improvement. Cumulative bile production showed an improvement: 176.3 +/- 8.4 in the IdPL compared to 126.1 +/- 12.2 microg/g-liver in the IPL (p < 0.05). Clearance of phenol red (PR) and taurocholic acid (TC) remained similar. At 90 minutes reperfusion the PR clearance showed 0.11 +/- 0.01 and 0.11 +/- 0.02 mg/30min/g-liver and the TC clearance 1.01 +/- 0.10 and 1.01 +/- 0.07 micromol/ml/30min/g-liver in the IPL and IdPL, respectively. Increasing the reperfusion time beyond the normally used 90 minutes resulted in a significant increase in transaminases and LDH and a decrease in bile production, liver morphology remained intact and glycogen content was appropriate. In conclusion, the IdPL-model showed similar or better results than the IPL-model, but the liver could not endure an extended reperfusion time using the IdPL.

Low viscosity histidine-tryptophan-ketoglutarate graft flush improves subsequent extended cold storage in University of Wisconsin solution in an extracorporeal rat liver perfusion and rat liver transplantation model

Adequate flushing for liver donation requires large fluid volumes delivered at a high flow. This can be achieved more effectively with crystalloid solutions than with colloid-based solutions. This study examined the combination of initial histidine-tryptophan-ketoglutarate solution (HTK) graft flush and subsequent storage in University of Wisconsin solution (UW) to that of the single use of each solution. Livers from inbred Wistar rats were procured using aortic perfusion with UW or HTK for initial perfusion and reflushed after 30 minutes using either solution. In a third group, after perfusion with HTK, organs were reflushed with UW. A 60-minute in-vitro recirculating perfusion was performed after 24 hours of cold storage in the subsequent solution, as well as allotransplantation after 18 and 24 hours of cold storage. In extracorporeal perfusion, the HTK flush followed by UW storage was superior compared to the single use of either UW or HTK solution, as measured by portal venous pressure, bile flow, liver enzymes released into the effluent perfusate, glycerol leakage, and histological examinations. These data were consistent with the transplantation study. Histological damage and enzyme release after 5-day survival were lowest in the HTK flush and subsequent UW storage groups following 18 hours of cold storage; likewise, the 5-day survival was superior following 24 hours of cold storage. In conclusion, the combined use of HTK solution for initial graft rinse and subsequent storage in UW solution resulted in a cumulative protection. Choosing low-viscosity HTK solution for the initial organ flush may represent a feasible improvement in liver preservation, which also further reduces the required amount of UW solution.

Efficacy of Polyethylene Glycols in University of Wisconsin Preservation Solutions: A Study of Isolated Perfused Rat Liver

Recent reports argue that the performance of University of Wisconsin (UW) solution is limited by the presence of hydroxyethyl starch (HES) as an additive, since HES could be responsible for human red blood cell aggregation. We investigated the effect on rat liver preservation of replacing HES in UW solution by polyethylene glycols (PEG20 and PEG35) at two concentrations. An isolated perfused rat liver model was used. Six groups of preserved livers (n = 7 for each group) were compared to controls (nonpreserved livers, n = 7). The following preservation solutions were assayed: UW without oncotic supply, UW-HES (0.25 mmol/L), UW-PEG20 (0.03 and 0.25 mmol/L), and UW-PEG35 (0.03 and 0.25 mmol/L). After 24-hour cold storage, the livers were perfused for 120 minutes at 37°C with oxygenated Krebs-Henseleit solution. During perfusion, transaminase release, portal and bile flows, and bromosulfophthalein (BSP) clearance were assessed. Results showed that the omission of oncotic supply in UW statistically increased ALT and AST release in perfusate and decreased bile and portal flows. PEG addition in UW solution, especially PEG35 at 0.25 mmol/L, effectively protected the rat liver graft from the onset of hypothermic ischemia/reperfusion damage. In conclusion, data reported here reveal that oncotic supply is essential for liver preservation and that HES can be effectively replaced by PEG in UW solution.

Does dietary ornithine α-ketoglutarate supplementation protect the liver against ischemia–reperfusion injury?

Nutritional supplementation with glutamine, arginine and their precursors has been proposed to contribute to the protection against ischemia-reperfusion-related injuries. The aim of this study was to evaluate in an isolated perfused rat liver model the preventive effect of a 4-day oral ornithine alpha-ketoglutarate (OKG) supplementation against warm ischemia-reperfusion (I-R) injury, and the involvement of nitric oxide synthesis. Rats were fed a controlled regimen supplemented with either OKG (5 g kg(-1); n=15) or an isonitrogenous mixture of non-essential amino acids (Control; n=6) for 4 days. Livers were subsequently prepared for isolated perfusion experiments, including a 45 min no-flow ischemic period. The OKG-treated group was divided into two groups according to the absence (OKG; n=8) or presence of a NO-synthase inhibitor, L-N(omega)-nitro-arginine methyl ester (OKG L-NAME; n=7) during liver perfusion. Liver cytolysis after ischemia was demonstrated by an elevated alanine aminotransferase release during the last 15 min of reperfusion that was significantly higher in the OKG-L-NAME group. Tumor necrosis factor alpha (TNF(alpha)) production was transiently increased only in the control group just after ischemia. At the end of the reperfusion period, liver superoxide dismutase activity was significantly lower in the OKG-L-NAME group compared to control animals. Dietary OKG administration had only a limited effect in this model of mild hepatic I-R, leading mainly to reduced TNF(alpha) production. As the content of lipid peroxidation products was not modified, it seems that OKG acts on the inflammatory response rather than on oxidative reactions. This action can tentatively be attributed to the role of OKG as a glutamine precursor rather than to the synthesis of arginine and nitric oxide.

Evaluation of CMU-1 preservation solutions using an isolated perfused rat liver model

CMU-1 is a new preservation solution with a low potassium concentration as well as low viscosity that is highly effective in reducing preservation injury. The purpose of this experiment is to compare the protective effect of CMU-1 solution with that of UW during cold preservation and normothermic reperfusion. Wistar rats were divided into two groups according to different preservation solution: CMU-1 group and UW group. After 6, 12 and 24 h cold storage of rat liver in different preservation solutions, the isolated perfused rat liver model was applied to reperfuse the liver for 120 min normothermically (37 degrees C) with Krebs-Henseleit solution, meanwhile the pH value of the preservation solution was measured. The perfusate was sampled for the evaluation of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH). At the end of the reperfusion, all of the bile product was collected, energy metabolic substrate and histological examination were performed. After preserving for 6 h, pH value of both groups did not change; after 12 h, both decreased but with no significant difference. After 24 h, pH value in UW solution group significantly decreased. The total adenine nucleotides level and AEC in liver tissue decreased with preservation time, but they were higher in CMU-1 group. And the amount of bile product after perfusion for 120 min in CMU-1 group was much more than that in UW group. However, there were no significant differences in ALT and LDH levels between two groups. Histology showed no difference. The preservation effect of CMU-1 solution is similar with that of UW solution. However, CMU-1 solution shows some advantages over UW solution in energy metabolism, preventing intracellular acidosis and bile product.

Hypothermic machine perfusion of rat livers preserves endothelial cell function

This study was conducted to investigate the effect of starch in the preservation solution during hypothermic machine perfusion (HMP) on endothelial cell and hepatocyte functions in an isolated perfused rat liver model. Livers isolated from male Sprague-Dawley rats were perfused with the University of Wisconsin (UW) solution (HMP + starch group); modified UW solution (starch omitted) (HMP − starch group) at 0.4 mL/min per g liver; or simply stored in the UW solution (SCS group) at 4°C for 24 hours. Following preservation, livers from HMP + starch, HMP − starch, SCS, and control group (without preservation) were perfused with Krebs-Henseleit Buffer solution at 37°C for 30 minutes. Samples were taken every 10 minutes during 30-minute warm perfusion to assess hepatocyte and endothelial cell function and damage. After 24 hours of hypothermic preservation and 30 minutes rewarming, livers in the HMP + starch group displayed significantly lower lactate dehydrogenase levels and higher bile production. Endothelial cell function was also improved as indicated by hyaluronic acid uptake and shorter transient time for albumin observed in a multiple indicator dilution study. Liver wet and dry ratio and histological findings confirmed reduced edema formation in the tissue of the HMP + starch group livers compared with that of the HMP − starch and SCS group livers. These results suggest that HMP with the UW solution containing starch improve endothelial cell function and induce less hepatocellular damage following 24-hour preservation compared to SCS and HMP with the starch-free UW solution. These results also suggest that oncotic support may be an important component in preserving hepatic microcirculation in HMP.

Optimization of a new preservation solution for machine perfusion of the liver: Which is the preferred colloid?

Aims Machine perfusion (MP) has proven to be beneficial in experimental preservation of the liver. The modified University of Wisconsin solution (UW-Gluconate or UW-G) is used as the MP preservation solution of choice. We have developed Polysol, an enriched MP preservation solution based on a colloid. We sought to optimize Polysol by substituting the colloid hydroxyethylstarch (HES) with the colloids dextran and polyethylene glycol (PEG). Methods In an isolated perfused rat liver model, hepatocellular damage and liver function were assessed during reperfusion with Krebs-Henseleit buffer after 24 hours hypothermic MP using Polysol-HES, Polysol-dextran, or Polysol-PEG. Control livers were preserved by MP using UW-G. Results Compared to MP-UW-G, MP using Polysol resulted in significantly less damage and improved function during reperfusion. MP using Polysol-dextran or Polysol-PEG resulted in equal or less damage than Polysol-HES. Differences in ammonia clearance and bile production were not significant. Tissue edema was higher after MP using Polysol-HES as compared to Polysol-dextran and Polysol-PEG. Conclusions MP of rat livers for 24 hours using UW-G results in more extensive damage and reduced liver function compared to MP using Polysol. MP using Polysol-dextran or Polysol-PEG results in equal or even better preservation compared to Polysol-HES.

Effect of aging on liver functions—an experimental study in a perfused rat liver model

Background. Aging is associated with marked changes in the physiology of many organs. Aging of the liver has been little studied and findings are inconclusive. The purpose of this work was to determine the effect of aging on transport and metabolic functions of the liver as assessed by extraction ratio of indocyanine green (ICG) and urea flux respectively. Bile flow was also recorded. As ICG is removed exclusively by the liver without bioconversion, its clearance reflects hepatic functional mass. Methods. Livers from adult (3-month old) or old (24-month old) rats were perfused in a recirculating system for 90 min. At time 30 min, a bolus of 0.125 mg of indocyanine green was introduced in the perfusion buffer. At least every 10 minutes, the perfusion buffer was sampled for the measurements of ICG and urea. Bile flow was closely monitored throughout the experiment. Results. Extraction ratio of ICG was increased in livers from old rats (9.49±2.84 vs 3.70±1.56% of ICG extracted), whereas urea flux was diminished (0.33±0.06 vs 1.33±0.65 μmol/min/% of ICG extracted) and bile flow was unchanged (4.03±1.02 vs 3.57±1.34 μl/min). Conclusions. Aging does not affect the different functions of the liver in the same way. It increases hepatocellular uptake function, but decreases the metabolic function of hepatocytes and does not change excretion function. These discrepancies are likely to be of some importance in the study of drug metabolism and action, and so we suggest that results should be corrected for ICG extraction.

THE OPTIMIZATION OF A NEW PRESERVATION SOLUTION FOR MACHINE PERFUSION OF THE LIVER: WHICH COLLOID IS PREFERRED?

P38 Aims: Machine perfusion (MP) proves to be beneficial in the preservation of the liver. Currently the modified University of Wisconsin solution (UW-Gluconate) is used as the MP preservation solution of choice. However, in our view this solution does not contain a sufficient amount of substrates for the decreased liver metabolism at 4 degrees. Therefore we have developed Polysol, a MP preservation solution based on a colloid, containing the necessary nutrients for the liver. In a previous study we have shown that Polysol results in equal or even better quality liver preservation when compared to UW-Gluconate. We sought to optimize Polysol by substituting the colloid Hydroxyethylstarch (HES), which is expensive, difficult to obtain and causes microvasculatory obstructions. We therefore compared HES with the colloids Dextran and Polyethyleneglycol (PEG). Methods: In an isolated perfused rat liver model, hepatocellular damage was assessed during reperfusion after 24 hours hypothermic MP with P-HES, P-Dextran or P-PEG. To determine hepatocellular damage ALT and LDH levels were measured during 60 minutes of normothermic reperfusion with oxygenated KHB. Liver function was assessed using bile production, ammonia clearance and oxygen consumption. Control livers were preserved for 24 hours by MP with UW-G. Results: Compared to the control (UW-G) MP with either P-HES, P-Dextran or P-PEG resulted in significantly less hepatocellular damage, higher flow, bile production and oxygen consumption during reperfusion. MP with P-Dextran resulted in less hepatocellular damage (U/L) as compared to MP with P-HES: ALT (t=40) 2.17±0.98 vs 3.50±3.51, respectively. Livers perfused with P-PEG also sustained less hepatocellular damage as compared to P-HES: ALT (t=40) 2.20±1.30 vs 3.50±3.51 respectively. Oxygen consumption (mmHg) was increased after MP with P-Dextran as compared to P-HES: (t=10) 447.80±53.10 vs 399.28±30.64. Neither these differences, nor differences in ammonia clearance or bile production were significant. Conclusions: 24 hours MP of livers with UW-G results in more extensive hepatocellular damage and reduced liver function when compared to MP with either Polysol-HES, Polysol-Dextran or Polysol-PEG. MP with Polysol-Dextran or Polysol-PEG results in equal or even less hepatocellular damage when compared to MP with Polysol-HES. Therefore substituting HES in Polysol with either Dextran or PEG can be considered feasible.

HYPOTHERMIC MACHINE PERFUSION OF RAT LIVERS PRESERVES ENDOTHELIAL CELL FUNCTION

P26 Aims: The present study was conducted to investigate the effects of starch content of the preservation solution during hypothermic machine perfusion (HMP) on hepatic microcirculation and heptocellular function in an isolated perfused rat liver model. Methods: Livers isolated from male Sprague-Dawley rats (200-250g) were perfused with the University of Wisconsin (UW)-lactobinate solution (HMP group, n=6), modified UW solution (starch omitted) (HMP w/o starch group, n=6) at 0.4 ml/min/gliver or statically stored in the UW solution (SCS group, n=5) at 4°C for 24 hr. Following preservation, livers from HMP, HMP w/o starch, SCS and control group (without preservation, n=7) were perfused with Krebs-Henseleit Buffer solution at 37°C for 30 min. Samples were taken every 10 min during 30 min warm perfusion to assess hepatocellular damage and function (LDH and bile production). Endothelial cell function and permeability were determined by hyaluronic acid (HA) uptake and multiple indicator dilution (MID) method, respectively. Results: After 24hrs hypothermic preservation, HMP livers showed significant lower initial LDH level (110.1 ± 9.8 U/L) than HMP w/o starch (271.9± 46.7 U/L) and SCS groups (725.1 ± 138.3 U/L) (P < 0.05). After 30min rewarming, HMP displayed significantly lower LDH level (220.1 ± 55.1 U/L in HMP vs. 1371.3±215.9 U/L in HMP w/o starch, 884 ± 71.9 U/L in SCS, P < 0.05), higher bile production (0.82 ± 0.14 in HMP vs. 0.56 ± 0.05 in HMP w/o starch, 0.32 ± 0.08 in SCS unit: μl/min/g liver dry wt, P < 0.05). Endothelial cell function was also improved as indicated by HA uptake (18.6±2.3% in HMP vs. 8.9±2.4% in HMP w/o starch, 13.9±3.6% in SCS) and shorter transient time for albumin were observed in MID experiments (23 s in HMP vs. 49 s in HMP w/o starch, 43 s in SCS, P < 0.05) after 30 min rewarming. Liver wet and dry ratio and histological findings also confirmed ameliorated edema in the tissue of the HMP liver compared with the HMP w/o starch and SCS livers. Conclusions: These results suggest that the HMP with the UW solution improved endothelial cell function compared with SCS and HMP w/o starch following 24hr preservation resulting in less hepatocellular damage. These results also suggest that oncotic support appears to be an important component in preserving hepatic microcirculation.

Effects of antihyperlipidemic agents on hepatic insulin sensitivity in perfused Goto-Kakizaki rat liver.

We previously reported that the Goto-Kakizaki (GK) rat, an animal model of type 2 diabetes, has hepatic insulin resistance using a perfused rat liver model. Pioglitazone, eicosapentaenoic acid (EPA), and fenofibrate are antihyperlipidemic agents and improve glucose tolerance. There have been few studies showing that these agents directly improve hepatic insulin sensitivity in type 2 diabetes mellitus. The aim of this study was to explore the effects of these agents on hepatic insulin sensitivity directly using a perfused GK rat liver model. GK rats were treated with oral pioglitazone (6 or 10 mg/kg body weight), EPA (1 or 2 g/kg body weight), or fenofibrate (30 mg/kg body weight) for 2 weeks. Livers were perfused in situ with glucagon or with glucagon and insulin, and hepatic glucose outputs were measured. In the pioglitazone-treated GK rats, blood glucose levels were significantly decreased. In the pioglitazone- and EPA-treated GK rats, insulin infusion significantly attenuated hepatic glucose output stimulated by glucagon. In the fenofibrate-treated GK rats, fat deposits in the hepatocytes were decreased, and glucose output elicited by glucagon was significantly decreased compared with that in the untreated GK rats, whereas insulin infusion did not affect glucose output by glucagon. These findings suggest that pioglitazone and EPA may improve glucose tolerance by directly increasing hepatic insulin sensitivity, while fenofibrate may improve glucose tolerance by improving hepatic glycogen metabolism in the GK rats. We previously reported that the Goto-Kakizaki (GK) rat, an animal model of type 2 diabetes, has hepatic insulin resistance using a perfused rat liver model. Pioglitazone, eicosapentaenoic acid (EPA), and fenofibrate are antihyperlipidemic agents and improve glucose tolerance. There have been few studies showing that these agents directly improve hepatic insulin sensitivity in type 2 diabetes mellitus. The aim of this study was to explore the effects of these agents on hepatic insulin sensitivity directly using a perfused GK rat liver model.

Sinusoidal endothelial cell and hepatocyte death following cold ischemia-warm reperfusion of the rat liver.

Cold ischemia-warm reperfusion (CI-WR) injury of the liver is characterized by marked alterations of sinusoidal endothelial cells (SECs), whereas hepatocytes appear to be relatively unscathed. However, the time course and mechanism of cell death remain controversial: early versus late phenomenon, necrosis versus apoptosis? We describe the occurrence and nature of cell death after different periods of CI with University of Wisconsin (UW) solution and after different periods of WR in the isolated perfused rat liver model. After 24- and 42-hour CI (viable and nonviable livers, respectively), similar patterns of liver cell death were seen: SEC necrosis appeared early after WR (10 minutes) and remained stable for up to 120 minutes. After 30 minutes of WR, apoptosis increased progressively with WR length. Based on morphological criteria, apoptotic cells were mainly hepatocytes within liver plates or extruded in the sinusoidal lumen. In addition, only after 42-hour CI were large clusters of necrotic hepatocytes found in areas of congested sinusoids. In these same livers, the hepatic microcirculation, evaluated by means of the multiple-indicator dilution technique, revealed extracellular matrix disappearance with no-flow areas. In conclusion, different time courses and mechanisms of cell death occur in rat livers after CI-WR, with early SEC necrosis followed by delayed hepatocyte apoptosis. These processes do not appear to be of major importance in the mechanism of graft failure because they are similar under both nonlethal and lethal conditions; this is not the case for the loss of the extracellular matrix found only under lethal conditions and associated with hepatocyte necrosis.

Oxidation Products of Vitamin E in the Peroxidation of Liposomal and Biological Systems

The oXidation products of vitamin E, α-tocopherol, during the peroxidation of unsaturated lipids in the peroxidation of liposomal systems, human plasma, and some biological tissues have been reViewed. Free radical reactions of α-tocopherol take place via the α-tocopheroxyl radical as an intermediate. If suitable free radical is present, a non-radical product can be formed from the coupling of the free radical with the or-tocopheroxyl radical. The reaction products of α-tocopherol with lipid-peroxyl radicals, such as phosphatidylcholine-peroxyl radicals and cholesteryl ester-peroxyl radicals, are 8a-(lipid-dioxy)-α-tocopherones, which are hydrolyzed to or-tocopherylquinone. The other product-forming pathway yields 2, 3-and 5, 6-epoxy-α-tocopherylquinones and their precursors, epoxyhydroperoxy-α-tocopherylquinones. Other vitamin E compound, γ-tocopherol, is superior to α-tocopherol in the detoXification of reactive nitrogen oxide species and forms 5-nitro-γ-tocopherol as the reaction product. The oxidation products of vitamin E have been detectedin liposomal systems, plasma samples, a perfused rat liver model, and human atherosclerotic lesions. Thus, the formation of these products provides us with much information onthe antioxidant function of vitamin E in biological systems.

Efficacy of preconditioning with N-acetylcysteine against reperfusion injury after prolonged cold ischaemia in rats liver in which glutathione had been reduced by buthionine sulphoximine.

To investigate the ability of N-acetylcysteine (NAC) to prevent cold ischaemic-reperfusion injury and improve hepatic integrity in a glutathione-depleted condition. Open laboratory study. University hospitals, Japan and France. 40 male Wistar rats. To produce a glutathione-depleted liver, buthionine sulphoximine (BSO) was injected intraperitoneally 2 hours before either NAC or 5% dextrose was infused 15 minutes before the liver was harvested. We used an isolated perfused rat liver model that had undergone prolonged hypothermic ischaemia, cold-storage for 48 hours and reperfusion for 120 minutes. Concentrations of hepatic enzymes released into samples of perfusate, concentration of adenosine triphosphate in liver tissue, concentrations of reduced and oxidized glutathione in perfusate, and bile production. The concentrations of the hepatocellular enzymes and oxidised glutathione in the perfusate samples were significantly reduced in the NAC group compared with the 5% dextrose group. Bile production improved significantly in the NAC group compared with the 5% dextrose group. The concentration of reduced glutathione in liver tissue was not increased by NAC. In a glutathione-depleted liver NAC prevented hepatic injury and improved liver integrity after a cold ischaemic-reperfusion injury, by acting not as a substrate for glutathione synthesis but as a direct free radical scavenger.