Martinus A. W. Maas

Amino Acid Release from Cerebral Cortex in Experimental Acute Liver Failure, Studied by In Vivo Cerebral Cortex Microdialysis

Abstract: Both increased γ‐aminobutyric acid (GABA)‐ergic and decreased glutamatergic neurotransmission have been suggested relative to the pathophysiology of hepatic encephalopathy. This proposed disturbance in neurotransmitter balance, however, is based mainly on brain tissue analysis. Because the approach of whole tissue analysis is of limited value with regard to in vivo neurotransmission, we have studied the extracellular concentrations in the cerebral cortex of several neuroactive amino acids by application of the in vivo microdialysis technique. During acute hepatic encephalopathy induced in rats by complete liver ischemia, increased extracellular concentrations of the neuroactive amino acids glutamate, taurine, and glycine were observed, whereas extracellular concentrations of aspartate and GABA were unaltered and glutamine decreased. It is therefore suggested that hepatic encephalopathy is associated with glycine potentiated glutamate neurotoxicity rather than with a shortage of the neurotransmitter glutamate. In addition, increased extracellular concentration of taurine might contribute to the disturbed neurotransmitter balance. The observation of decreasing glutamine concentrations, after an initial increase, points to a possible astrocytic dysfunction involved in the pathophysiology of hepatic encephalopathy.

Features of the Synovium of Individuals at Risk of Developing Rheumatoid Arthritis

Findings from previous studies have suggested that subclinical inflammation of the synovium does not coincide with the appearance of rheumatoid arthritis (RA)–specific autoantibodies. This study was undertaken to examine the relationship between the presence of autoantibodies, changes in the synovium, and development of arthritis over time in a markedly larger, prospective study.

Evaluation of a novel bioartificial liver in rats with complete liver ischemia: treatment efficacy and species-specific α-GST detection to monitor hepatocyte viability

BACKGROUND/AIMS There is an urgent need for an effective bioartificial liver system to bridge patients with fulminant hepatic failure to liver transplantation or to regeneration of their own liver. Recently, we proposed a bioreactor with a novel design for use as a bioartificial liver (BAL). The reactor comprises a spirally wound nonwoven polyester fabric in which hepatocytes are cultured (40 x 10(6) cells/ml) as small aggregates and homogeneously distributed oxygenation tubing for decentralized oxygen supply and CO2 removal. The aims of this study were to evaluate the treatment efficacy of our original porcine hepatocyte-based BAL in rats with fulminant hepatic failure due to liver ischemia (LIS) and to monitor the viability of the porcine hepatocytes in the bioreactor during treatment. The latter aim is novel and was accomplished by applying a new species-specific enzyme immunoassay (EIA) for the determination of porcine alpha-glutathione S-transferase (alpha-GST), a marker for hepatocellular damage. METHODS Three experimental groups were studied: the first control group (LIS Control, n = 13) received a glucose infusion only; a second control group (LIS No-Cell-BAL, n = 8) received BAL treatment without cells; and the treated group (LIS Cell-BAL, n = 8) was connected to our BAL which had been seeded with 4.4 x 10(8) viable primary porcine hepatocytes. RESULTS/CONCLUSIONS In contrast to previous comparable studies, BAL treatment significantly improved survival time in recipients with LIS. In addition, the onset of hepatic encephalopathy was significantly delayed and the mean arterial blood pressure significantly improved. Significantly lower levels of ammonia and lactate in the LIS Cell-BAL group indicated that the porcine hepatocytes in the bioreactor were metabolically activity. Low pig alpha-GST levels suggested that our bioreactor was capable of maintaining hepatocyte viability during treatment. These results provide a rationale for a comparable study in LIS-pigs as a next step towards potential clinical application.

The effects of benzodiazepine-receptor antagonists and partial inverse agonists on acute hepatic encephalopathy in the rat

Two benzodiazepine-receptor partial inverse agonists (Ro 15-4513, Ro 15-3505) and one benzodiazepine-receptor antagonist (flumazenil) were administered to rats with hepatic encephalopathy due to acute liver ischemia. Significant improvement (P less than 0.002) of both the clinical grade of hepatic encephalopathy and the electroencephalographic abnormalities was observed after administration of the benzodiazepine-receptor partial inverse agonists: comatose rats with no spontaneous righting reflex regained consciousness immediately after injection of the drug. Only slight improvement in clinical hepatic encephalopathy grade was seen after administration of 25 mg/kg of flumazenil. The present data strongly support a role of increased gamma-aminobutyric acid-ergic tone in the pathogenesis of acute hepatic encephalopathy and provide a rationale for trials of benzodiazepine-receptor partial inverse agonists to restore consciousness in hepatic encephalopathy in humans in the near future.

Distribution and activity of glutamine synthase and carbamoylphosphate synthase upon enlargement of the liver lobule by repeated partial hepatectomies

Glutamine synthase and carbamoylphosphate synthase show a strikingly heterogeneous and fully complementary distribution in the rat liver. In the human liver, however, there is a midlobular zone where both enzymes are absent. The diameter of the human liver lobule is approximately twice the size of the rat lobule. To investigate whether lobule size is a major determinant for the expression patterns of glutamine synthase and carbamoylphosphate synthase, Wistar strain rats were partially hepatectomized 3 times, at weekly or monthly intervals. Due to hepatic regeneration the cross-sectional area of the liver lobules increased twofold. However, a midlobular zone which lacked expression of both glutamine synthase and carbamoylphosphate synthase did not develop in these livers, thus showing that lobular size is not a major determinant for the distribution patterns of glutamine and carbamoylphosphate synthase. The twofold increase in the cross-sectional area of the liver lobule was associated with a similar reduction in the relative number of glutamine synthase-positive cells and in the enzyme content of the liver, indicating that the regeneration process does not affect the pericentral pattern of glutamine synthase expression. After regeneration was complete, the glutamine synthase content in the liver was restored to its original value, demonstrating a twofold increase in the cellular concentration of glutamine synthase-positive hepatocytes. An increase in the diameter of the liver lobule was only seen after the first partial hepatectomy. Liver growth following subsequent partial hepatectomies can be explained by an increase in the length of the liver lobule and/or by splitting of liver lobules. The zonal distribution of DNA replication, which is characteristic of the first partial hepatectomy, is lost after repeated partial hepatectomies. Furthermore, evidence was obtained that the signal for inducing DNA synthesis may originate at the level of single liver units.

Neocortical dialysate monoamines of rats after acute, subacute, and chronic liver shunt

Abstract: Intracerebral microdialysis was applied to monitor the neocortical extracellular levels of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, the neurotransmitters dopamine (DA), noradrenaline (NA), and serotonin (5‐HT), and the metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) in rats with various forms of experimental hepatic encephalopathy (HE). The extracellular aromatic amino acid levels were clearly increased in acute, subacute, and chronic HE. No changes compared with controls in the neocortical DA release could be detected in the three experimental HE rat models investigated. The NA release showed a significant increase only in the subacute HE group. These data suggest that HE may not be associated with any major reduction of neocortical DA or NA release as previously suggested. In acute and subacute HE, decreased extracellular DOPAC but elevated 5‐HIAA concentrations were seen. In chronic HE, elevations of both DOPAC and 5‐HIAA were observed. Neocortical 5‐HT release did not change in subacute and chronic HE, whereas it decreased in acute HE compared with control values. Significant increase in extracellular concentrations of 5‐HIAA and of the 5‐HIAA/5‐HT ratio in the present study are in agreement with previously reported increases in 5‐HT turnover in experimental HE. However, a substantially increased 5‐HT turnover in experimental HE does not appear to be related to an increase in neuronal neocortical 5‐HT release.

The effects of ammonia and portal-systemic shunting on brain metabolism, neurotransmission and intracranial hypertension in hyperammonaemia-induced encephalopathy

BACKGROUND/AIMS The pathogenetic factors contributing to encephalopathy in portacaval shunted rats with hyperammonaemia were studied. METHODS Hyperammonaemia was induced by ammonium-acetate infusions in portacaval shunted rats (2.8 mmol.kg bw-1.h-1; AI-portacaval shunted rats) and in sham-portacaval shunted rats (6.5 mmol.kg bw-1.h-1; AI-NORM rats). Severity of encephalopathy was quantified by clinical grading and EEG spectral analysis. Changes in brain metabolites were assessed by amino acid analysis of brain cortex homogenates, whereas changes in amino acids with neurotransmitter activity were assessed in cerebrospinal fluid; brain water content was measured by subtracting dry from wet brain weights and intracranial pressure was measured by a pressure transducer connected to a cisterna magna cannula. RESULTS Although similar increased blood and brain ammonia concentrations were obtained in both experimental groups, only AI-portacaval shunted rats developed encephalopathy, associated with a significant increase in intracranial pressure. Other significant differences were: higher concentrations of brain glutamine and aromatic amino acids, higher concentrations of cerebrospinal fluid glutamine, aromatic amino acids, glutamate and aspartate in AI-portacaval shunted rats than in AI-NORM rats. CONCLUSIONS These results indicate that hyperammonaemia alone dose not induce encephalopathy, whereas portal-systemic shunting adds an essential contribution to the pathogenesis of encephalopathy. It is hypothesised that the larger increase in brain glutamine in AI-portacaval shunted rats than in AI-NORM rats is responsible for increased brain concentrations of aromatic amino acids, for cell swelling and for extracellular release of glutamate and aspartate. This might promote encephalopathy. If cell swelling is not restricted, intracranial hypertension will develop.

Liver progenitor cell line HepaRG differentiated in a bioartificial liver effectively supplies liver support to rats with acute liver failure

A major roadblock to the application of bioartificial livers is the need for a human liver cell line that displays a high and broad level of hepatic functionality. The human bipotent liver progenitor cell line HepaRG is a promising candidate in this respect, for its potential to differentiate into hepatocytes and bile duct cells. Metabolism and synthesis of HepaRG monolayer cultures is relatively high and their drug metabolism can be enhanced upon treatment with 2% dimethyl sulfoxide (DMSO). However, their potential for bioartificial liver application has not been assessed so far. Therefore, HepaRG cells were cultured in the Academic Medical Center bioartificial liver (AMC-BAL) with and without DMSO and assessed for their hepatic functionality in vitro and in a rat model of acute liver failure. HepaRG-AMC-BALs cultured without DMSO eliminated ammonia and lactate, and produced apolipoprotein A-1 at rates comparable to freshly isolated hepatocytes. Cytochrome P450 3A4 transcript levels and activity were high with 88% and 37%, respectively, of the level of hepatocytes. DMSO treatment of HepaRG-AMC-BALs reduced the cell population and the abovementioned functions drastically. Therefore, solely HepaRG-AMC-BALs cultured without DMSO were tested for efficacy in rats with acute liver failure (n = 6). HepaRG-AMC-BAL treatment increased survival time of acute liver failure rats ∼50% compared to acellular-BAL treatment. Moreover, HepaRG-AMC-BAL treatment decreased the progression of hepatic encephalopathy, kidney failure, and ammonia accumulation. These results demonstrate that the HepaRG-AMC-BAL is a promising bioartificial liver for clinical application.

Metabolic activity of microcarrier attached liver cells after intraperitoneal transplantation during severe liver insufficiency in the rat

Short- and long-term effects of intraperitoneally transplanted microcarrier attached liver cells (MAL) have been studied in two experimental models of severe liver insufficiency in the rat: subtotal hepatectomy (HX) and acute liver ischemia. Intraperitoneal transplantation of MAL immediately after subtotal hepatectomy resulted in a significantly lower plasma ammonia level, a higher caffeine clearance, a higher urea production and a significantly smaller loss in body weight in comparison to sham transplanted control rats. Since thymidine kinase activity in the regenerating host liver was only significantly stimulated at t = 48 h it is concluded that the observed metabolic effects are mainly due to the metabolic activity of the transplanted MAL, although a small stimulative effect of MAL-TX on host liver regeneration cannot be excluded. In the course of acute liver ischemia, MAL transplantation results in delayed development of acute hepatic encephalopathy (HE), judged by clinical grading, EEG spectral analysis and Visual Evoked Response (VER) parameters. Furthermore, MAL transplantation is associated with less increased levels of plasma ammonia during acute liver ischemia.

Exhaled breath analysis with electronic nose technology for detection of acute liver failure in rats

The aim of this study was to assess the classification accuracy of an e-Nose in detecting acute liver failure (ALF) in rats. Exhaled breath from 14 rats was repeatedly sampled by e-Nose (8 sensors) and an additional external CO2 sensor at three stages: healthy period; portacaval shunt; and during the development of ALF due to surgically induced complete liver ischemia. We performed principal component analysis (PCA) on the (grouped) sensor data in each stage and the classification accuracy of the first two principal components was assessed by the leave-one-out approach. In addition we performed gas chromatography-mass spectrometry (GC-MS) analysis of the exhaled breath from three rats. The first and second principal components from the PCA analysis of e-Nose data accounted for more than 95% variance in the data. Measurements in the ALF stage were contrasted with the measurements in the control stage. Leave-one-out validation showed classification accuracy of 96%. This accuracy was reached after 3h of ALF development, and was reached already after 2h when data of an external CO2 sensor were also included. GC-MS identified 2-butanol, 2-butanone, 2-pentanone and 1-propanol to be possibly elevated in the ALF stage. This is the first study to demonstrate that ALF in rats can be detected by e-Nose data analysis of the exhaled breath. Confirmation of these results in humans will be an important step forward in the non-invasive diagnosis of ALF.