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BACKGROUND: In this study, we investigated the influence of brain death on inflammatory response and the effects of brain death on liver function both directly after explantation and after reoxygenation. METHODS: The influence of brain death on liver function was studied in rats using a brain death model and the liver slice model to mimic reoxygenation. Liver function was assessed by measuring the ATP content and the ATP-driven urea synthesis. The activation of non-parenchymal cells was studied by measuring mRNA levels of IL-10, cytokine production (IL-10 and IL-1 beta) and inducible nitric oxide synthases (iNOS) upregulation (mRNA) and protein level. RESULTS: Brain death had no direct influence on the ATP content of the liver. However, it led to induction of several cytokines because of activation of non-parenchymal cells, which led to upregulation of iNOS and to nitric oxide metabolites production. It is known that cytokine production may influence the drug metabolism capacity; however, no influence of brain death on drug metabolism was observed. An explanation may be the relatively short experimental period. CONCLUSIONS: Kupffer cells seem to be activated during the onset of brain death induction; however, they become quiescent when liver slices of brain dead rats were reoxygenated during incubation. Other non-parenchymal cells, possibly the endothelial cells, remain activated during incubation and reoxygenation in slices from brain death donors but not in slices from control livers. Future experiments in our rat liver transplantation model need to elucidate the implications of these findings.

Precision-cut liver slices (PCLS) are an ex vivo model for metabolism and toxicity studies. However, data on the maintenance of the morphological integrity of the various cell types in the slices during prolonged incubation are lacking. Therefore, our aims were to characterize morphological and functional changes in rat PCLS during five days of incubation in a rich medium, RegeneMed®, and a standard medium, Williams' Medium E. Although cells of all types in the slices remain viable, profound changes in morphology were observed, which were more prominent in RegeneMed®. Slices underwent notable fibrosis, bile duct proliferation and fat deposition. Slice thickness increased, resulting in necrotic areas, while slice diameter decreased, possibly indicating cell migration. An increased proliferation of parenchymal and non-parenchymal cells (NPCs) was observed. Glycogen, albumin and Cyp3a1 were maintained albeit to a different level in two media. In conclusion, both hepatocytes and NPCs remain viable and functional, enabling five-day toxicity studies. Tissue remodeling and formation of a new capsule-like cell lining around the slices are evident after 3-4 days. The differences in effects between media emphasize the importance of media selection and of the recognition of morphological changes in PCLS, when interpreting results from toxicological or pharmacological studies. © 2015 Elsevier B.V.

In the present study we investigated the applicability of the liver slice model to study mechanisms of drug uptake. Four model compounds were investigated that enter hepatocytes via entirely different membrane transport mechanisms. Rhodamine B (RB), which enters hepatocytes by passive diffusion, was homogeneously distributed throughout the rat liver slice (250 mum thickness) within 5 min, indicating that the penetration rate into the slice and the diffusion rate into the cells are rapid. In contrast, lucigenin (LU), which is taken up by hepatocytes through adsorptive endocytosis, was detected in the inner cell layers after 15 min. Digoxin uptake into the slice showed a temperature-dependent component and was stereos electively inhibited by quinine, which is compatible with the involvement of a carrier-mediated uptake mechanism. The neo-glycoalbumin Lactose(27)-Human Serum Albumin (Lact(27)-HSA) and the negatively charged Succinylated-Human Serum Albumin (Suc-HSA) entered the slices and were taken up temperature-dependently into hepatocytes and endothelial cells, respectively. The liver slice preparation is a valuable tool to investigate the mechanisms of cellular uptake of drugs. Moreover, the precision-cut liver slices offer the unique possibility to study both hepatocyte and endothelial cell function in human and rat liver. (C) 2001 Elsevier Science Inc. All rights reserved.

Aims/Background: In liver transplantation, adequate function tests for donor livers and transplanted livers are of utmost importance to provide an objective basis for decision-making. Isolated hepatocyte and/or slice preparations from human donor liver tissue may be suitable to test the quality of the organ to be transplanted. Methods: Surgical waste material remaining after reduced size or split liver transplantation in children was used to prepare slices and isolated hepatocytes. The viability of these preparations as well as drug transport and metabolism functions were determined and related to graft function in 32 liver recipients. Results: The in vitro tests used in the present study apparently did not select non-viable livers. In vitro preparations of the primary non-function grafts which occurred in the investigated group showed normal viability, metabolic and uptake function. Conclusion: These results indicate that either the presently used viability tests are not sensitive enough to detect potential organ failure or that other factors besides the hepatocyte viability at the time of transplantation are of paramount importance to the graft function of the recipient, such as complications during and after transplantation or the viability of the non-parenchymal cells.

The effect of ethanol on the hepatic uptake of various cationic drugs was studied in isolated perfused rat livers, isolated rat hepatocytes and isolated rat liver mitochondria. In isolated rat hepatocytes and in isolated perfused rat livers, the uptake of the model organic cation tri-n-butylmethylammonium was found to be markedly stimulated by ethanol in a concentration-dependent fashion. The uptake of tri-n-butylmethylammonium at 1 mu M was increased to 120% and 137% at 0.5% (v/v, (=87 mM)) and 1% (v/v, (=174 mM)) ethanol, respectively. At 25 mu M, tri-n-butylmethylammonium uptake was increased to 124% and 152% at 0.5% (v/v) and 1% (v/v) of ethanol, respectively. The uptake of the organic cations azidoprocainamide methoiodide, vecuronium, ORG 9426 and ORG 6368, the anionic compound taurocholate and the uncharged compound ouabain was not markedly increased at these ethanol concentrations. The mechanism of action of ethanol on the uptake of tri-n-butylmethylammonium was further studied. Competitive inhibitors for the type I organic cation uptake system, procainamide ethobromide and verapamil, almost completely blocked uptake of tri-n-butylmethylammonium (1 mu M) in the presence of 1% (v/v) ethanol, indicating that carrier-mediated uptake is still involved and that additional passive diffusion is unlikely. Neither the plasma membrane potential nor the accumulation of the cation in mitochondria was altered after ethanol treatment, suggesting that potential driving forces for uptake and sequestration were not affected. Apart from ethanol, methanol (1%, v/v), propanol (1%, v/v) and dimethyl sulfoxide (1%, v/v) also stimulated the uptake of tri-n-butylmethylammonium (at 1 mu M) in isolated rat hepatocytes to 117%, 124% and 122% respectively. The results of our study indicate that ethanol selectively stimulates the uptake of the aliphatic organic cation tri-n-butylmethylammonium rather than through generally alterated hepatobiliary transport processes. (C) Journal of Hepatology.

Rat hepatocytes were preserved for 24hr with high recovery and good maintenance of viability and transport function both in University of Wisconsin (UW) solution and in various simplified UW solutions. Cell quality is somewhat affected after 48 hr of preservation in both the original UW solution and the simplified solutions. ATP content and uptake rate of taurocholic acid are more sensitive markers of cell viability than Trypan blue exclusion or the MTT test. A much less expensive solution than UW, containing only K+-lactobionate, KH2PO4, MgSO4 and raffinose, can be used successfully for preservation of rat hepatocytes for 24 hr for drug transport studies.

To investigate whether hepatobiliary transport of organic cations is under regulatory control, we studied transport of tri-n-butylmethylammonium in the isolated perfused rat liver and in isolated rat hepatocytes. Transport was investigated in the presence of modulators of the protein kinase C and the cyclic AMP second-messenger system. In the isolated perfused rat liver, it was observed that compounds modulating protein kinase C activity clearly affected the biliary excretion process of the cation tri-n-butylmethylammonium. Phorbol 12-myristate 13-acetate, a compound that directly stimulates protein kinase C, elevated the biliary excretion rate of tri-n-butylmethylammonium in a concentration-dependent manner, reaching a twofold increase at 60 nmol/L of the phorbol ester. The inactive derivative 4alpha-phorbol 12, 13-didecanoate (60 nmol/L) did not show any effect. Vasopressin (48 nmol/L), a receptor-mediated activator of protein kinase C, stimulated the excretion rate of the cation by about 50%. Staurosporin (1 mumol/L), an inhibitor of protein kinase C, clearly decreased the biliary excretion rate of the cation and also blocked its stimulation by phorbol 12-myristate 13-acetate. Neither phorbol 12-myristate 13-acetate nor vasopressin (at concentrations ranging from 10(-9) to 10(-6) mol/L) affected the initial uptake velocity of tri-n-butylmethylammonium in isolated hepatocytes and isolated perfused livers, whereas staurosporin (1 mumol/L) showed only a modest inhibition of the uptake of the cation. It is inferred that the effect of protein kinase C modulators on hepatobiliary transport of organic cations occurs at the level of carrier-mediated transport in the canalicular membrane. Because bile flow was only slightly affected by these agents, effects on biliary excretion rate of the cation are unlikely to be caused by changes in bile flow. With regard to the cyclic AMP second-messenger system, neither glucagon (concentration range of 10(-9) to 10(-6) mol/L), a receptor-mediated activator of adenylate cyclase, nor forskolin (100 mumol/L), a direct activator of adenylate cyclase and dibutyryl cyclic AMP (100 mumol/L), affected the biliary excretion rate and the hepatic uptake rate of the cation in these preparations. In conclusion, cell-to-bile transport of the organic cation tri-n-butylmethylammonium at the canalicular level is directly or indirectly regulated by protein kinase C. Neither the protein kinase C nor the cyclic AMP second-messenger systems seem to be involved in the hepatic uptake process of the cation.