Rat Liver Model Research Articles

Blockade of Fibronectin-α4β1 Adhesive Interactions Down-Regulates Cyclooxygenase-2 Inducible Nitric Oxide Synthase and Prolongs Recipient Survival in a 24-Hour Model of Cold Hepatic Ischemia-Reperfusion Injury

We investigated the effects of the connecting segment-1 (CS1) peptide, which blocks fibronectin (FN)-α4β1 integrin interactions, upon recipient survival and extent of tissue injury in a well-established rat liver model of ex vivo 24-hour cold ischemia followed by isotransplantation. In this model, CS1 peptides were administered through the portal vein of rat livers prior to and after cold ischemic storage. In addition, recipients of orthotopic liver transplants (OLT) received a dose of CS1 peptides 1 hour post-OLT. CS1 peptide therapy significantly inhibited the intragraft recruitment of T lymphocytes and neutrophil activation/infiltration, and repressed important mediators of inflammation, such as cyclooxygenase-2, and inducible nitric oxide synthase expression. Importantly, CS1 peptide therapy improved function/histological preservation of liver grafts and extended their 14-day survival from 50% in control to 100% in CS1-treated OLTs. Thus, CS1 peptide-mediated blockade of FN-α4β1 interaction protects against severe ischemia-reperfusion injury experienced otherwise by OLTs. These novel findings document the potential of targeting FN-α4β1 in vivo interaction for improving OLT outcomes.

Cyclic RGD Peptides With High Affinity for α5β1 Integrin Protect Genetically Fat Zucker Rat Livers From Cold Ischemia/Reperfusion Injury

We tested a hypothesis that interactions between fibronectin (FN), a key extracellular matrix component, and its integrin α5β1 receptor are important in the development of ischemia/reperfusion (I/R) injury of steatotic liver transplants. We examined the effect of a cyclic RGD peptide (cRGD), with high affinity for α5β1 integrin, in a well-established steatotic rat liver model of ex vivo cold ischemia followed by isotransplantation. In this model, cRGD peptides were administered through the portal vein of steatotic Zucker rat livers prior to and after cold ischemic storage. Lean Zucker recipients of fatty orthotopic liver transplants (OLTs) received an additional course of cRGD peptides 1 hour posttransplantation. cRGD peptide therapy significantly inhibited the recruitment of monocyte/macrophages, and repressed the expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase (iNOS), and MMP-9 expression. Importantly, cRGD peptide therapy improved the function and histologic preservation of steatotic liver grafts, extending their 14-day survival in lean recipients from 50% in untreated to 100% in cRGD-treated OLTs. Thus, cRGD peptide-mediated blockade of FN-α5β1 interaction protects against severe I/R injury otherwise experienced by steatotic OLTs.

Differential gene expression profiles in the steatosis/fibrosis model of rat liver by chronic administration of carbon tetrachloride

Global gene expression profile was analyzed by microarray analysis of rat liver RNA after chronic carbon tetrachloride (CCl 4) administration. Rats received 0.5 ml CCl 4/kg three times a week, and the liver samples were obtained after 0, 30, 60, and 90 days of injection. Histopathologic studies of liver tissues enabled the classification of the CCl 4 effect into mild and severe fatty liver/steatosis (30 and 60 days, respectively) and fibrosis/cirrhosis (90 days) stages. The expression levels of 4900 clones on a custom rat gene microarray were analyzed and the results were confirmed by semi-quantitative RT-PCR. Four hundred thirty-eight clones were differentially expressed with more than a 1.625-fold difference (which equals 0.7 in log2 scale) at one or more time points. Multiple genes involved in lipid metabolism and ribosome biogenesis showed differential transcript levels upon chronic CCl 4 administration, which was previously seen in acute rat model as well. In addition, a total of 149 clones were identified as fibrosis/cirrhosis-specific genes by either fold changes or Significance Analysis of Microarrays. In conclusion, we report microarray analysis results in rat liver upon chronic CCl 4 administration with a full chronological profile that not only covered fatty liver/steatosis but also later points of fibrosis/cirrhosis. These data will provide the insight of specific gene expression profiles that is implicated in the multistep process of fatty liver/steatosis and fibrosis/cirrhosis after chronic hepatotoxin exposure.

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.

Effects of emodin and double blood supplies on liver regeneration of reduced size graft liver in rat model

To study the influences of emodin and reconstruction of double blood supplies on liver regeneration of reduced size graft liver in rat model. A total of 45 SD-SD rat reduced size liver transplantation models were randomly divided into three groups (A-C). The conventional reduced size liver transplantation was performed on rats in group A, while the hepatic artery blood supply was restored in groups B and C. The emodin (1.5 mg/kg/d) was given by intraperitoneal route in group C only. The recipients were killed on the seventh day after the operation. The proliferative cell nuclear antigen (PCNA), TBil and ALT of serum were detected, and the pathological changes of liver cell were observed. The numbers of the rats that survived in A, B, and C group on the seventh day after operation were 14, 13, 13, respectively. The levels of TBil (31.5+/-5.2 micromol/L, 23.2+/-3.1 micromol/L vs 38.6+/-6.8 micromol/L), and ALT (5 351+/-1 050 nKat, 1300+/-900 nKat vs 5779+/-1202 nKat) in serum in groups B and C were lower than those in group A (P<0.05), while the expression of PCNA in groups B or C was higher than that in group A (22.0+/-3.5%, 28.2+/-4.2% vs 18.6+/-3.2%, P<0.05). The deeper staining nuclei, double nuclei, multi-nuclei and much glycogen were observed in liver cells of groups B and C, especially in group C, while fewer were found in liver cells of group A. The reconstruction of arterial blood supply is very important for rat liver regeneration after reduced size liver transplantation. Emodin has the effect of promoting liver regeneration and improving liver function in rats after reduced size transplantation. The possible mechanism is improving proliferation of liver cell and protecting liver cells from injury.

Machine perfusion preservation of the non–heart-beating donor rat livers using polysol, a new preservation solution

Aims The increasing shortage of donor organs has led to a focus on extended criteria donors, including the non–heart-beating donor (NHBD). An optimal preservation method is required to facilitate successful transplantation of these ischemically damaged organs. The recent literature has shown clear advantages of hypothermic machine perfusion (MP) over cold storage (CS). For MP, modified University of Wisconsin perfusion solution (UW-G) is often used, which, however, is known to cause microcirculatory obstruction, is difficult to obtain, and is expensive. Therefore, Polysol was developed as a MP preservation solution that contains specific nutrients for the liver, such as amino acids, energy substrates, and vitamins. The aim of this study was to compare Polysol with UW-G in a NHBD rat liver model. Methods After 24 hours hypothermic MP of NHBD rat livers using UW-G or Polysol, liver damage and function parameters were assessed during 60 minutes of reperfusion with Krebs-Henseleit buffer. Control livers were reperfused after 24 hours CS in UW. Results Liver enzyme release was significantly higher among the CS-UW group compared to MP using UW-G or Polysol. Flow during reperfusion was significantly higher when using Polysol compared to UW-G. Bile production and ammonia clearance were highest when using Polysol compared to UW-G. There was less cellular edema after preservation with Polysol compared to UW-G. Conclusions MP of NHBD rat livers for 24 hours using UW-G or Polysol resulted in less hepatocellular damage than CS in UW. MP of NHBD livers for 24 hours using Polysol is superior to MP using UW-G.

Fibronectin-α4β1 integrin interactions modulate p42/44 MAPK phosphorylation in steatotic liver cold ischemia-reperfusion injury

We investigated the effects of the connecting segment-1 (CS1) peptide, which blocks fibronectin (FN)-α4β1 integrin interactions upon cell signaling, leukocyte migration, and secretion of proinflammatory cytokines, in a well-established steatotic rat liver model using ex vivo cold ischemia followed by isotransplantation. In this model, CS1 peptides were administered through the portal vein of steatotic Zucker rat livers prior and after cold ischemic storage. Lean Zucker recipients of fatty orthotopic liver transplantation (OLT) received an additional 3-day course of CS1 peptides post-OLT. CS1 peptide-treated steatotic OLTs harvested at 1, 3, and 7 days showed moderated levels of p42/44 mitogen-activated protein kinase (MAPK) phosphorylation, comparable to those observed in steatotic naïve livers. In contrast, p42/44 MAPK phosphorylation was found up-regulated in 1- to 3-day damaged control OLTs. However, 7-day control OLTs were characterized by virtually lack of p42/44 MAPK phosphorylation. Lack of p42/44 MAPK phosphorylation in 7-day control OLTs was correlated with massive presence of leukocytes in the grafts and elevated levels of proinflammatory cytokines. CS1 peptide-treated OLTs at 7 days showed a profound decrease in T-cell (10 ± 3 vs 56 ± 20, P < .03) and monocyte/macrophage (+/++ vs +++) infiltration and significantly reduced levels of cytokine expression, such as IL-2 (approximately sixfold), and IFN-γ (approximately three- to fourfold), as compared with controls.

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.

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

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.

The Mitochondrial Apoptosis-induced Channel (MAC) Corresponds to a Late Apoptotic Event

We have investigated the mechanism responsible for mitochondria permeabilization occurring during cell apoptosis. We have developed an in vivo model of apoptotic rat liver. Mitochondria appeared as an homogenous population in control liver. On the contrary, mitochondria varied in size, morphology, and the matrical density in apoptotic liver. Mitochondria were purified from control and apoptotic livers. In control conditions, a single mitochondrial population was identified; whereas three populations of mitochondria were purified from apoptotic liver. Our data show that these apoptotic populations correspond to early, intermediate, and late apoptotic mitochondria, which are characterized by an increasing extent of permeabilization of their outer membrane and a gradual enrichment in oligomerized Bax protein. Remarkably, a new ionic channel was observed in apoptotic but not in control mitochondria. The biophysical and pharmacological properties of this channel are in good agreement with those reported for a previously described mitochondrial apoptosis-induced channel (MAC) (Pavlov, E. V., Priault, M., Pietkiewicz, D., Cheng, E. H., Antonsson, B., Manon, S., Korsmeyer, S. J., Mannella, C. A., and Kinnally, K. W. (2001) J. Cell Biol. 155, 725-731). However, MAC activity was only observed in the late apoptotic mitochondrial population. Thus, our study establishes that MAC activity is related to the overall apoptotic process but corresponds to a late event.

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.

Intra-arterial administration of sodium borocaptate (BSH)/lipiodol emulsion delivers B-10 to liver tumors highly selectively for boron neutron capture therapy: experimental studies in the rat liver model

Purpose Boron neutron capture therapy (BNCT) is particle radiotherapy with alpha ( 4He) particle and recoiled lithium nucleus ( 7Li) derived from a reaction of boron ( 10B) and thermal neutron. We investigated applying BNCT to malignant liver tumors. The purpose of the present study was to reveal the efficacy for administration of emulsion of a boron compound (sodium borocaptate; BSH) and lipiodol via a hepatic artery using a rat liver tumor model. Methods and materials Rat liver tumors were developed by direct injection of Walker 256 cells into the liver parenchyma. BSH (75 mg/kg)/lipiodol (0.3 mL/kg) emulsion was administered via the hepatic artery. Boron concentrations in the tumors, liver, and blood were measured at 1, 6, and 12 h after administration. Neutron capture radiography (NCR) was taken to confirm the selective accumulation of 10B in the liver tumors. Results Boron concentrations in the liver tumors and the tumor/liver (T/L) boron concentration ratio at 1, 6, and 12 h after administration of BSH/lipiodol emulsion (concentration: T/L ratio) were 479.2 ppm: 4.0, 197.3 ppm: 14.9, and 96.5 ppm: 6.6, respectively. Highly selective irradiation was clearly demonstrated by the NCR images. Conclusions Intra-arterial administration of BSH/lipiodol emulsion is effective method for delivering high concentration of 10B selectively to the liver tumors.

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.

Biliverdin Therapy Protects Rat Livers From Ischemia and Reperfusion Injury

Heme oxygenase (HO-1) provides a cellular defense mechanism during oxidative stress and catalyzes the rate-limiting step in heme metabolism that produces biliverdin (BV). The role of BV and its potential use in preventing ischemia/reperfusion injury (IRI) had never been studied. This study was designed to explore putative cytoprotective functions of BV during hepatic IRI in rat liver models of ex vivo perfusion and orthotopic liver transplantation (OLT) after prolonged periods of cold ischemia. In an ex vivo hepatic IRI model, adjunctive BV improved portal venous blood flow, increased bile production, and decreased hepatocellular damage. These findings were correlated with amelioration of histological features of IRI, as assessed by Suzuki's criteria. Following cold ischemia and syngeneic OLT, BV therapy extended animal survival from 50% in untreated controls to 90% to 100%. This effect correlated with improved liver function and preserved hepatic architecture. Additionally, BV adjuvant after OLT decreased endothelial expression of cellular adhesion molecules (P-selectin and intracellular adhesion molecule 1), and decreased the extent of infiltration by neutrophils and inflammatory macrophages. BV also inhibited expression of inducible nitric oxide synthase and proinflammatory cytokines (interleukin 1beta, tumor necrosis factor alpha, and interleukin 6) in OLTs. Finally, BV therapy promoted an increased expression of antiapoptotic molecules independently of HO-1 expression, consistent with BV being an important mediator through which HO-1 prevents cell death. In conclusion, this study documents and dissects potent cytoprotective effects of BV in well-established rat models of hepatic IRI. Our results provide the rationale for a novel therapeutic approach using BV to maximize the function and thus the availability of donor organs.