Laboratoire d'hépato-neuro du Dr Christopher Rose

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• 1.2.1: Qui sommes-nous?
• 1.2.2: Projets de recherche
• 1.2.3: Dr Rose
• 1.2.4: Équipe
• 1.2.5: Publications
  • 1.2.5.1: Publications
  • 1.2.5.2: Bibliographie
  • 1.2.5.3: Résumés/Conférences
• 1.2.6: Techniques
• 1.2.7: 24h de science

Vous pouvez trouver les profils de Dr Rose sur les sites suivants :

2012

1. Bosoi, C. R.; Yang, X.; Huynh, J.; Parent-Robitaille, C.; Jiang, W.; Tremblay, M. & Rose, C. F. (2012). Systemic oxidative stress is implicated in the pathogenesis of brain edema in rats with chronic liver failure. Free Radic Biol Med 52:1228-1235. doi:10.1016/j.freeradbiomed.2012.01.006
   

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   - [↓Résumé↓]

   Chronic liver failure leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy (HE); however, a correlation between blood ammonia levels and HE severity remains controversial. It is believed oxidative stress plays a role in modulating the effects of hyperammonemia. This study aimed to determine the relationship between chronic hyperammonemia, oxidative stress, and brain edema (BE) in two rat models of HE: portacaval anastomosis (PCA) and bile-duct ligation (BDL). Ammonia and reactive oxygen species (ROS) levels, BE, oxidant and antioxidant enzyme activities, as well as lipid peroxidation were assessed both systemically and centrally in these two different animal models. Then, the effects of allopurinol (xanthine oxidase inhibitor, 100mg/kg for 10days) on ROS and BE and the temporal resolution of ammonia, ROS, and BE were evaluated only in BDL rats. Similar arterial and cerebrospinal fluid ammonia levels were found in PCA and BDL rats, both significantly higher compared to their respective sham-operated controls (p<0.05). BE was detected in BDL rats (p<0.05) but not in PCA rats. Evidence of oxidative stress was found systemically but not centrally in BDL rats: increased levels of ROS, increased activity of xanthine oxidase (oxidant enzyme), enhanced oxidative modifications on lipids, as well as decreased antioxidant defense. In PCA rats, a preserved oxidant/antioxidant balance was demonstrated. Treatment with allopurinol in BDL rats attenuated both ROS and BE, suggesting systemic oxidative stress is implicated in the pathogenesis of BE. Analysis of ROS and ammonia temporal resolution in the plasma of BDL rats suggests systemic oxidative stress might be an important "first hit", which, followed by increases in ammonia, leads to BE in chronic liver failure. In conclusion, chronic hyperammonemia and oxidative stress in combination lead to the onset of BE in rats with chronic liver failure.
2. Sharma, V.; Ten Have, G. A. M.; Ytrebo, L.; Sen, S.; Rose, C. F.; Dalton, R. N.; Turner, C.; Revhaug, A.; Van-Eijk, H. M. H.; Deutz, N. E. P.; Jalan, R.; Mookerjee, R. P. & Davies, N. A. (2012). Nitric Oxide and L-Arginine Metabolism in a Devascularized Porcine Model of Acute Liver Failure Am J Physiol Gastrointest Liver Physiol doi:10.1152/ajpgi.00268.2011
   

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   In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of over production of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations, (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early phase ALF, we systematically assessed its interorgan transport and metabolism; and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30Kg) were randomized to SHAM (N=8) or hepatic devascularisation ALF (N=8) procedure and followed for 6 hours. Measurements: plasma arginine, citrulline, ornithine levels; arginase activity; NO, ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labelled amino-acids. Plasma arginine decreased >85% of the basal level at T=6 hrs (p<0.001), whereas citrulline and ornithine progressively increased in ALF (p<0.001 and p<0.001, vs. SHAM respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (p<0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (p<0.001). In this model of early phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which stimulated de novo arginine production.

2011

1. Bosoi, C. R.; Parent-Robitaille, C.; Anderson, K.; Tremblay, M. & Rose, C. F. (2011). AST-120 (spherical carbon adsorbent) lowers ammonia levels and attenuates brain edema in bile duct-ligated rats. Hepatology 53:1995-2002. doi:10.1002/hep.24273
   

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   The pathogenesis of hepatic encephalopathy is multifactorial, involving gut-derived toxins such as ammonia, which has been demonstrated to induce oxidative stress. Therefore, a primary hepatic encephalopathy treatment target is reducing ammonia production in the gastrointestinal tract. AST-120, an oral adsorbent of engineered activated carbon microspheres with surface areas exceeding 1600 m(2) /g, acts as a sink for neurotoxins and hepatotoxins present in the gut. We evaluated the capacity of AST-120 to adsorb ammonia in vitro and to lower blood ammonia, oxidative stress and brain edema in cirrhotic rats. Cirrhosis was induced in rats by bile duct ligation for 6 weeks. AST-120 was administered by gavage preventively for 6 weeks (0.1, 1, and 4 g/kg/day). In addition, AST-120 was evaluated as a short-term treatment for 2 weeks and 3 days (1 g/kg/day) and as a sink to adsorb intravenously infused ammonium acetate. In vitro, AST-120 efficiently adsorbed ammonia. Ammonia levels significantly decreased in a dose-dependent manner for all AST-120-treated bile duct-ligated rats (nontreated: 177.3 ± 30.8 μM; AST-120, 0.1 g/kg/day: 121.9 ± 13.8 μM; AST-120, 1 g/kg/day: 80.9 ± 30.0 μM; AST-120, 4 g/kg/day: 48.8 ± 19.6 μM) and significantly correlated with doses of AST-120 (r = -0.6603). Brain water content and locomotor activity normalized after AST-120 treatments, whereas arterial reactive oxygen species levels remained unchanged. Furthermore, AST-120 significantly attenuated a rise in arterial ammonia after ammonium acetate administration (intravenously). Conclusion:AST-120 treatment decreased arterial ammonia levels, normalized brain water content and locomotor activity but did not demonstrate an effect on systemic oxidative stress. Also, AST-120 acts as an ammonia sink, efficiently removing blood-derived ammonia. Additional studies are warranted to evaluate the effects of AST-120 on hepatic encephalopathy in patients with advanced liver disease. (HEPATOLOGY 2011;).

2010

1. Kristiansen, R. G.; Lindal, S.; Myreng, K.; Revhaug, A.; Ytrebø, L. M. & Rose, C. F. (2010). Neuropathological changes in the brain of pigs with acute liver failure. Scand J Gastroenterol 45:935-943. doi:10.3109/00365521003675047
   

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   Abstract Objective. Cerebral edema is a serious complication of acute liver failure (ALF), which may lead to intracranial hypertension and death. An accepted tenet has been that the blood-brain barrier is intact and that brain edema is primarily caused by a cytotoxic etiology due to hyperammonemia. However, the neuropathological changes in ALF have been poorly studied. Using a well characterized porcine model we aimed to investigate ultrastructural changes in the brain from pigs suffering from ALF. Materials and methods. Sixteen female Norwegian Landrace pigs weighing 27-35 kg were randomised into two groups: ALF (n = 8) and sham operated controls (n = 8). ALF was induced with an end-to-side portacaval shunt followed by ligation of the hepatic arteries. Biopsies were harvested from three different areas of the brain (frontal lobe, cerebellum, and brain stem) following eight hours of ALF and analyzed using electron microscopy. Results. Profound perivascular and interstitial edema were found in all three areas. Disruption of pericytic and astrocytic processes were seen, reflecting breakdown/lesion of the blood-brain barrier in animals suffering from ALF. Furthermore, neurons and axons were edematous and surrounded by vesicles. Severe damage to Purkinje neuron (necrosis) and damaged myelin were seen in the cerebellum and brain stem, respectively. Biopsies from sham operated animals were normal. Conclusions. Our data support the concept that vasogenic brain edema plays an important role in the development of intracranial hypertension in pigs with ALF.
2. Rose, C. F. (2010). Increase brain lactate in hepatic encephalopathy: Cause or consequence? Neurochem Int 57:389-394. doi:10.1016/j.neuint.2010.06.012
   

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   Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome which develops as a result of liver failure or disease. Increased concentrations of brain lactate (microdialysate, cerebrospinal fluid, tissue) are commonly measured in patients with HE induced by either acute or chronic liver failure. Whether an increase in brain lactate is a cause or a consequence of HE remains undetermined. A rise in cerebral lactate may occur due to (1) blood-borne lactate (hyperlactataemia) crossing the blood-brain barrier, (2) increased glycolysis due to energy failure or impairment and (3) increased lactate production/release or decreased lactate utilization/uptake. This review explores the different reasons for lactate accumulation in the brain during liver failure and describes the possible roles of lactate in the pathogenesis of HE.
3. Yang, X.; Bosoi, C. R.; Jiang, W.; Tremblay, M. & Rose, C. F. (2010). Portacaval anastomosis-induced hyperammonemia does not lead to oxidative stress. Metab Brain Dis 25:11-15. doi:10.1007/s11011-010-9174-1
   

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   Ammonia is neurotoxic and believed to play a major role in the pathogenesis of hepatic encephalopathy (HE). It has been demonstrated, in vitro and in vivo, that acute and high ammonia treatment induces oxidative stress. Reactive oxygen species (ROS) are highly reactive and can lead to oxidization of proteins resulting in protein damage. The present study was aimed to assess oxidative status of proteins in plasma and brain (frontal cortex) of rats with 4-week portacaval anastomosis (PCA). Markers of oxidative stress, 4-hydroxy-2-nonenal (HNE) and carbonylation were evaluated by immunoblotting in plasma and frontal cortex. Western blot analysis did not demonstrate a significant difference in either HNE-linked or carbonyl derivatives on proteins between PCA and sham-operated control rats in both plasma and frontal cortex. The present study suggests PCA-induced hyperammonemia does not lead to systemic or central oxidative stress.

2009

1. Bosoi, C. R. & Rose, C. F. (2009). Identifying the direct effects of ammonia on the brain. Metab Brain Dis 24:95-102. doi:10.1007/s11011-008-9112-7
   

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   Elevated concentrations of ammonia in the brain as a result of hyperammonemia leads to cerebral dysfunction involving a spectrum of neuropsychiatric and neurological symptoms (impaired memory, shortened attention span, sleep-wake inversions, brain edema, intracranial hypertension, seizures, ataxia and coma). Many studies have demonstrated ammonia as a major player involved in the neuropathophysiology associated with liver failure and inherited urea cycle enzyme disorders. Ammonia in solution is composed of a gas (NH(3)) and an ionic (NH(4) (+)) component which are both capable of crossing plasma membranes through diffusion, channels and transport mechanisms and as a result have a direct effect on pH. Furthermore, NH(4) (+) has similar properties as K(+) and, therefore, competes with K(+) on K(+) transporters and channels resulting in a direct effect on membrane potential. Ammonia is also a product as well as a substrate for many different biochemical reactions and consequently, an increase in brain ammonia accompanies disturbances in cerebral metabolism. These direct effects of elevated ammonia concentrations on the brain will lead to a cascade of secondary effects and encephalopathy.
2. Ytrebø, L. M.; Kristiansen, R. G.; Maehre, H.; Fuskevåg, O. M.; Kalstad, T.; Revhaug, A.; Cobos, M. J.; Jalan, R. & Rose, C. F. (2009). L-ornithine phenylacetate attenuates increased arterial and extracellular brain ammonia and prevents intracranial hypertension in pigs with acute liver failure. Hepatology 50:165-174. doi:10.1002/hep.22917
   

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   Hyperammonemia is a feature of acute liver failure (ALF), which is associated with increased intracranial pressure (ICP) and brain herniation. We hypothesized that a combination of L-ornithine and phenylacetate (OP) would synergistically reduce toxic levels of ammonia by (1) L-ornithine increasing glutamine production (ammonia removal) through muscle glutamine synthetase and (2) phenylacetate conjugating with the ornithine-derived glutamine to form phenylacetylglutamine, which is excreted into the urine. The aims of this study were to determine the effect of OP on arterial and extracellular brain ammonia concentrations as well as ICP in pigs with ALF (induced by liver devascularization). ALF pigs were treated with OP (L-ornithine 0.07 g/kg/hour intravenously; phenylbutyrate, prodrug for phenylacetate; 0.05 g/kg/hour intraduodenally) for 8 hours following ALF induction. ICP was monitored throughout, and arterial and extracellular brain ammonia were measured along with phenylacetylglutamine in the urine. Compared with ALF + saline pigs, treatment with OP significantly attenuated concentrations of arterial ammonia (589.6 +/- 56.7 versus 365.2 +/- 60.4 mumol/L [mean +/- SEM], P= 0.002) and extracellular brain ammonia (P= 0.01). The ALF-induced increase in ICP was prevented in ALF + OP-treated pigs (18.3 +/- 1.3 mmHg in ALF + saline versus 10.3 +/- 1.1 mmHg in ALF + OP-treated pigs;P= 0.001). The value of ICP significantly correlated with the concentration of extracellular brain ammonia (r(2) = 0.36,P< 0.001). Urine phenylacetylglutamine levels increased to 4.9 +/- 0.6 micromol/L in ALF + OP-treated pigs versus 0.5 +/- 0.04 micromol/L in ALF + saline-treated pigs (P< 0.001).Conclusion:L-Ornithine and phenylacetate act synergistically to successfully attenuate increases in arterial ammonia, which is accompanied by a significant decrease in extracellular brain ammonia and prevention of intracranial hypertension in pigs with ALF.

2007

1. Rose, C.; Ytrebø, L. M.; Davies, N. A.; Sen, S.; Nedredal, G. I.; Belanger, M.; Revhaug, A. & Jalan, R. (2007). Association of reduced extracellular brain ammonia, lactate, and intracranial pressure in pigs with acute liver failure. Hepatology 46:1883-1892. doi:10.1002/hep.21877
   

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   We previously demonstrated in pigs with acute liver failure (ALF) that albumin dialysis using the molecular adsorbents recirculating system (MARS) attenuated a rise in intracranial pressure (ICP). This was independent of changes in arterial ammonia, cerebral blood flow and inflammation, allowing alternative hypotheses to be tested. The aims of the present study were to determine whether changes in cerebral extracellular ammonia, lactate, glutamine, glutamate, and energy metabolites were associated with the beneficial effects of MARS on ICP. Three randomized groups [sham, ALF (induced by portacaval anastomosis and hepatic artery ligation), and ALF+MARS] were studied over a 6-hour period with a 4-hour MARS treatment given beginning 2 hours after devascularization. Using cerebral microdialysis, the ALF-induced increase in extracellular brain ammonia, lactate, and glutamate was significantly attenuated in the ALF+MARS group as well as the increases in extracellular lactate/pyruvate and lactate/glucose ratios. The percent change in extracellular brain ammonia correlated with the percent change in ICP (r(2) = 0.511). Increases in brain lactate dehydrogenase activity and mitochondrial complex activity for complex IV were found in ALF compared with those in the sham, which was unaffected by MARS treatment. Brain oxygen consumption did not differ among the study groups. Conclusion: The observation that brain oxygen consumption and mitochondrial complex enzyme activity changed in parallel in both ALF- and MARS-treated animals indicates that the attenuation of increased extracellular brain ammonia (and extracellular brain glutamate) in the MARS-treated animals reduces energy demand and increases supply, resulting in attenuation of increased extracellular brain lactate. The mechanism of how MARS reduces extracellular brain ammonia requires further investigation.

2006

1. Chatauret, N.; Desjardins, P.; Zwingmann, C.; Rose, C.; Rao, K. V. R. & Butterworth, R. F. (2006). Direct molecular and spectroscopic evidence for increased ammonia removal capacity of skeletal muscle in acute liver failure. J Hepatol 44:1083-1088. doi:10.1016/j.jhep.2005.11.048
   

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   BACKGROUND/AIMS: It has been proposed that, in acute liver failure, skeletal muscle adapts to become the principle organ responsible for removal of blood-borne ammonia by increasing glutamine synthesis, a reaction that is catalyzed by the cytosolic ATP-dependent enzyme glutamine synthetase. To address this issue, glutamine synthetase expression and activities were measured in skeletal muscle of rats with acute liver failure resulting from hepatic devascularization. METHODS: Glutamine synthetase protein and gene expression were investigated using immunoblotting and semi-quantitative RT-PCR analysis. Glutamine synthetase activity and glutamine de novo synthesis were measured using, respectively, a standard enzymatic assay and [13C]-nuclear magnetic resonance spectroscopy. RESULTS: Glutamine synthetase protein (but not gene) expression and enzyme activities were significantly up-regulated leading to increased de novo synthesis of glutamine and increased skeletal muscle capacity for ammonia removal in acute liver failure. In contrast to skeletal muscle, expression and activities of glutamine synthetase in the brain were significantly decreased. CONCLUSIONS: These findings demonstrate that skeletal muscle adapts, through a rapid induction of glutamine synthetase, to increase its capacity for removal of blood-borne ammonia in acute liver failure. Maintenance of muscle mass together with the development of agents with the capacity to stimulate muscle glutamine synthetase could provide effective ammonia-lowering strategies in this disorder.
2. Rose, C. (2006). Effect of ammonia on astrocytic glutamate uptake/release mechanisms. J Neurochem 97 Suppl 1:11-15. doi:10.1111/j.1471-4159.2006.03796.x
   

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   Hyperammonemic disorders such as acute liver failure (ALF) or urea cycle enzymopathies are associated with hyperexcitability, seizures, brain edema and increased extracellular brain glutamate. Mechanisms responsible for increased glutamate content in the extracellular space of the brain include decreased uptake by perineuronal astrocytes and/or increased release from neurons and/or astrocytes. Exposure of astrocytes to millimolar concentrations of ammonia results in cell swelling, loss of expression of the glutamate transporters excitatory amino acid transporter (EAAT-1) and EAAT-2 and increased release of glutamate. Three distinct mechanisms are theoretically possible to explain ammonia-induced glutamate release from astrocytes namely: release due to swelling; reversal of glutamate transporters and due to Ca2+-dependent vesicular release. Recent identification of vesicular docking and fusion proteins in astrocytes together with glutamate-release (due to intracellular alkanization and mobilization of intracellular Ca2+-stores) studies implies that vesicular release is a predominant mechanism responsible for ammonia-induced release of glutamate from astrocytes.
3. Rose, C. & Jalan, R. (2006). Congenital glutamine deficiency with glutamine synthetase mutations. N Engl J Med 354:1093-4; author reply 1093-4. doi:10.1056/NEJMc053332
   

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4. Sen, S.; Rose, C.; Ytrebø, L. M.; Davies, N. A.; Nedredal, G. I.; Drevland, S. S.; Kjønnø, M.; Prinzen, F. W.; Hodges, S. J.; Deutz, N. E. P.; Williams, R.; Butterworth, R. F.; Revhaug, A. & Jalan, R. (2006). Effect of albumin dialysis on intracranial pressure increase in pigs with acute liver failure: a randomized study. Crit Care Med 34:158-164. doi:10.1097/01.CCM.0000196203.39832.3C
   

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   BACKGROUND: Increased intracranial pressure (ICP) worsens the outcome of acute liver failure (ALF). This study investigates the underlying pathophysiological mechanisms and evaluates the therapeutic effect of albumin dialysis in ALF with use of the Molecular Adsorbents Recirculating System without hemofiltration/dialysis (modified, M-MARS). METHODS: Pigs were randomized into three groups: sham, ALF, and ALF + M-MARS. ALF was induced by hepatic devascularization (time = 0). M-MARS began at time = 2 and ended with the experiment at time = 6. ICP, arterial ammonia, brain water, cerebral blood flow (CBF), and plasma inflammatory markers were measured. RESULTS: ICP and arterial ammonia increased significantly over 6 hrs in the ALF group, in comparison with the sham group. M-MARS attenuated (did not normalize) the increased ICP in the ALF group, whereas arterial ammonia was unaltered by M-MARS. Brain water in the frontal cortex (grey matter) and in the subcortical white matter at 6 hrs was significantly higher in the ALF group than in the sham group. M-MARS prevented a rise in water content, but only in white matter. CBF and inflammatory mediators remained unchanged in all groups. CONCLUSION: The initial development of cerebral edema and increased ICP occurs independently of CBF changes in this noninflammatory model of ALF. Factor(s) other than or in addition to hyperammonemia are important, however, and may be more amenable to alteration by albumin dialysis.
5. Ytrebø, L. M.; Sen, S.; Rose, C.; Davies, N. A.; Nedredal, G. I.; Fuskevaag, O.-M.; Have, G. A. M. T.; Prinzen, F. W.; Williams, R.; Deutz, N. E. P.; Jalan, R. & Revhaug, A. (2006). Systemic and regional hemodynamics in pigs with acute liver failure and the effect of albumin dialysis. Scand J Gastroenterol 41:1350-1360. doi:10.1080/00365520600714527
   

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   OBJECTIVE: Acute liver failure (ALF) is haemodynamically characterized by a hyperdynamic circulation. The aims of this study were to investigate the systemic and regional haemodynamics in ALF, to measure changes in nitric oxide metabolites (NOx) and to evaluate whether these haemodynamic disturbances could be attenuated with albumin dialysis. MATERIAL AND METHODS: Norwegian Landrace pigs (23-30 kg) were randomly allocated to groups as controls (sham-operation, n = 8), ALF (hepatic devascularization, n = 8) and ALF + albumin dialysis (n = 8). Albumin dialysis was started 2 h after ALF induction and continued for 4 h. Systemic and regional haemodynamics were monitored. Creatinine clearance, nitrite/nitrate and catecholamines were measured. A repeated measures ANOVA was used to analyse the data. RESULTS: In the ALF group, the cardiac index increased (PGT < 0.0001), while mean arterial pressure (PG = 0.02) and systemic vascular resistance decreased (PGT < 0.0001). Renal resistance (PG = 0.04) and hind-leg resistance (PGT = 0.003) decreased in ALF. There was no difference in jejunal blood flow between the groups. ALF pigs developed renal dysfunction with increased serum creatinine (PGT = 0.002) and decreased creatinine clearance (P = 0.02). Catecholamines were significantly higher in ALF, but NOx levels were not different. Albumin dialysis did not attenuate these haemodynamic or renal disturbances. CONCLUSIONS: The haemodynamic disturbances during the early phase of ALF are characterized by progressive systemic vasodilatation with no associated changes in metabolites of NO. Renal vascular resistance decreased and renal dysfunction developed independently of changes in renal blood flow. After 4 h of albumin dialysis there was no attenuation of the haemodynamic or renal disturbances.
6. Ytrebø, L. M.; Sen, S.; Rose, C.; Have, G. A. M. T.; Davies, N. A.; Hodges, S.; Nedredal, G. I.; Romero-Gomez, M.; Williams, R.; Revhaug, A.; Jalan, R. & Deutz, N. E. P. (2006). Interorgan ammonia, glutamate, and glutamine trafficking in pigs with acute liver failure. American journal of physiology. Gastrointestinal and liver physiology 291:G373-G381. doi:10.1152/ajpgi.00440.2005
   

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   Ammonia reduction is the target for therapy of hepatic encephalopathy, but lack of quantitative data about how the individual organs handle ammonia limits our ability to develop novel therapeutic strategies. The study aims were to evaluate interorgan ammonia metabolism quantitatively in a devascularized pig model of acute liver failure (ALF). Ammonia and amino acid fluxes were measured across the portal drained viscera (PDV), kidneys, hind leg, and lungs in ALF pigs. ALF pigs developed hyperammonemia and increased glutamine levels, whereas glutamate levels were decreased. PDV contributed to the hyperammonemic state mainly through increased shunting and not as a result of increased glutamine breakdown. The kidneys were quantitatively as important as PDV in systemic ammonia release, whereas muscle took up ammonia. Data suggest that the lungs are able to remove ammonia from the circulation during the initial stage of ALF. Our study provides new data supporting the concept of glutamate deficiency in a pig model of ALF. Furthermore, the kidneys are quantitatively as important as PDV in ammonia production, and the muscles play an important role in ammonia removal.

2005

1. Bélanger, M.; Desjardins, P.; Chatauret, N.; Rose, C. & Butterworth, R. F. (2005). Mild hypothermia prevents brain edema and attenuates up-regulation of the astrocytic benzodiazepine receptor in experimental acute liver failure. J Hepatol 42:694-699. doi:10.1016/j.jhep.2004.12.029
   

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   BACKGROUND/AIMS: Mild hypothermia has proven useful in the clinical management of patients with acute liver failure. Acute liver failure in experimental animals results in alterations in the expression of genes coding for astrocytic proteins including the "peripheral-type" (astrocytic) benzodiazepine receptor (PTBR), a mitochondrial complex associated with neurosteroid synthesis. To gain further insight into the mechanisms whereby hypothermia attenuates the neurological complications of acute liver failure, we investigated PTBR expression in the brains of hepatic devascularized rats under normothermic (37 degrees C) and hypothermic (35 degrees C) conditions. METHODS: PTBR mRNA was measured using semi-quantitative RT-PCR in cerebral cortical extracts and densities of PTBR sites were measured by quantitative receptor autoradiagraphy. Brain pregnenolone content was measured by radioimmunoassay. RESULTS: At coma stages of encephalopathy, animals with acute liver failure manifested a significant increase of PTBR mRNA levels. Brain pregnenolone content and [(3)H]PK 11195 binding site densities were concomitantly increased. Mild hypothermia prevented brain edema and significantly attenuated the increased receptor expression and pregnenolone content. CONCLUSIONS: These findings suggest that an attenuation of PTBR up-regulation resulting in the prevention of increased brain neurosteroid content represents one of the mechanisms by which mild hypothermia exerts its protective effects in ALF.
2. Rose, C. & Felipo, V. (2005). Limited capacity for ammonia removal by brain in chronic liver failure: potential role of nitric oxide. Metab Brain Dis 20:275-283. doi:10.1007/s11011-005-7906-4
   

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   Chronic liver failure leads to hyperammonemia and consequently increased brain ammonia concentrations, resulting in hepatic encephalopathy. When the liver fails to regulate ammonia concentrations, the brain, devoid of a urea cycle, relies solely on the amidation of glutamate to glutamine through glutamine synthetase, to efficiently clear ammonia. Surprisingly, under hyperammonemic conditions, the brain is not capable of increasing its capacity to remove ammonia, which even decreases in some regions of the brain. This non-induction of glutamine synthetase in astrocytes could result from possible limiting substrates or cofactors for the enzyme, or an indirect effect of ammonia on glutamine synthetase expression. In addition, there is evidence that nitration of the enzyme resulting from exposure to nitric oxide could also be implicated. The present review summarizes these possible factors involved in limiting the increase in capacity of glutamine synthetase in brain, in chronic liver failure.
3. Rose, C.; Kresse, W. & Kettenmann, H. (2005). Acute insult of ammonia leads to calcium-dependent glutamate release from cultured astrocytes, an effect of pH. J Biol Chem 280:20937-20944. doi:10.1074/jbc.M412448200
   

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   Hyperammonemia is a key factor in the pathogenesis of hepatic encephalopathy (HE) as well as other metabolic encephalopathies, such as those associated with inherited disorders of urea cycle enzymes and in Reye's syndrome. Acute HE results in increased brain ammonia (up to 5 mM), astrocytic swelling, and altered glutamatergic function. In the present study, using fluorescence imaging techniques, acute exposure (10 min) of ammonia (NH4+/NH3) to cultured astrocytes resulted in a concentration-dependent, transient increase in [Ca2+]i. This calcium transient was due to release from intracellular calcium stores, since the response was thapsigargin-sensitive and was still observed in calcium-free buffer. Using an enzyme-linked fluorescence assay, glutamate release was measured indirectly via the production of NADH (a naturally fluorescent product when excited with UV light). NH4+/NH3 (5 mM) stimulated a calcium-dependent glutamate release from cultured astrocytes, which was inhibited after preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester but unaffected after preincubation with glutamate transport inhibitors dihydrokainate and DL-threo-beta-benzyloxyaspartate. NH4+/NH3 (5 mM) also induced a transient intracellular alkaline shift. To investigate whether the effects of NH4+/NH3 were mediated by an increase in pH(i), we applied trimethylamine (TMA+/TMA) as another weak base. TMA+/TMA (5 mM) induced a similar transient increase in both pH(i) and [Ca2+]i (mobilization from intracellular calcium stores) and resulted in calcium-dependent release of glutamate. These results indicate that an acute exposure to ammonia, resulting in cytosolic alkalinization, leads to calcium-dependent glutamate release from astrocytes. A deregulation of glutamate release from astrocytes by ammonia could contribute to glutamate dysfunction consistently observed in acute HE.
4. Vaquero, J.; Rose, C. & Butterworth, R. F. (2005). Keeping cool in acute liver failure: rationale for the use of mild hypothermia. J Hepatol 43:1067-1077. doi:10.1016/j.jhep.2005.05.039
   

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   - [↓Résumé↓]

   Encephalopathy, brain edema and intracranial hypertension are neurological complications responsible for substantial morbidity/mortality in patients with acute liver failure (ALF), where, aside from liver transplantation, there is currently a paucity of effective therapies. Mirroring its cerebro-protective effects in other clinical conditions, the induction of mild hypothermia may provide a potential therapeutic approach to the management of ALF. A solid mechanistic rationale for the use of mild hypothermia is provided by clinical and experimental studies showing its beneficial effects in relation to many of the key factors that determine the development of brain edema and intracranial hypertension in ALF, namely the delivery of ammonia to the brain, the disturbances of brain organic osmolytes and brain extracellular amino acids, cerebro-vascular haemodynamics, brain glucose metabolism, inflammation, subclinical seizure activity and alterations of gene expression. Initial uncontrolled clinical studies of mild hypothermia in patients with ALF suggest that it is an effective, feasible and safe approach. Randomized controlled clinical trials are now needed to adequately assess its efficacy, safety, clinical impact on global outcomes and to provide the guidelines for its use in ALF.

2004

1. Jalan, R. & Rose, C. (2004). Hypothermia in acute liver failure. Metab Brain Dis 19:215-221. doi:10.1023/B:MEBR.0000043971.72170.a2
   

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   The development of encephalopathy in patients with acute liver injury defines the occurrence of liver failure. The encephalopathy of acute liver failure is characterized by brain edema which manifests clinically as increased intracranial pressure. Despite the best available medical therapies a significant proportion of patients with acute liver failure die due to brain herniation. The present review explores the experimental and clinical data to define the role of hypothermia as a treatment modality for increased intracranial pressure in patients with acute liver failure.
2. Rose, C. & Jalan, R. (2004). Is minimal hepatic encephalopathy completely reversible following liver transplantation? Liver Transpl 10:84-87. doi:10.1002/lt.20030
   

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3. Sen, S.; Ytrebø, L. M.; Rose, C.; Fuskevaag, O.-M.; Davies, N. A.; Nedredal, G. I.; Williams, R.; Revhaug, A. & Jalan, R. (2004). Albumin dialysis: a new therapeutic strategy for intoxication from protein-bound drugs. Intensive Care Med 30:496-501. doi:10.1007/s00134-003-2141-0
   

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   - [↓Résumé↓]

   OBJECTIVE: Although water-soluble drugs can be removed by haemofiltration/haemodialysis, morbidity and mortality from intoxication with protein-bound drugs remains high. The present study investigates whether albumin dialysis in the form of the Molecular Adsorbents Recirculating System (MARS) is effective in removal of protein-bound drugs. DESIGN: Prospective animal study. SETTING: Surgical research laboratory in a university hospital. SUBJECTS: Seven female Norwegian Landrace pigs. INTERVENTION: We studied whether midazolam (97% albumin-bound) and fentanyl (85% alpha-1-acid glycoprotein-bound), administered as anaesthetics to pigs with induced acute liver failure, could be removed by MARS dialysis lasting for 4 h. MEASUREMENTS: After 4 h of dialysis, total and free anaesthetic concentrations were measured in the blood and dialysate from different segments of the MARS circuit. MAIN RESULTS: Midazolam: total plasma concentrations fell by 47.1+/-2.1% (in 4 h) across the MARS filter ( p<0.01). The charcoal component of the system reduced the total dialysate drug concentration by 16.4+/-2.2% ( p<0.05). Free midazolam removal followed a similar pattern. Fentanyl: total plasma concentrations fell by 56.1+/-2.4% (in 4 h) across the MARS filter ( p<0.01). Clearance of fentanyl from the dialysate by the charcoal was 70+/-0.7% at 4 h ( p<0.001). CONCLUSIONS: The results of the study show that MARS can remove both albumin and other protein-bound drugs efficiently from the plasma, and it may have a place for the treatment of patients suffering from intoxication with this class of compounds.
4. Ytrebø, L. M.; Ekse, S.; Sen, S.; Rose, C.; Nedredal, G. I.; Fuskevåg, O.-M.; Jalan, R. & Revhaug, A. (2004). Contractile response of femoral arteries in pigs with acute liver failure. Scand J Gastroenterol 39:1000-1004. doi:10.1080/00365520410003254
   

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   - [↓Résumé↓]

   BACKGROUND: Acute liver failure (ALF) is characterized haemodynamically by a progressive hyperdynamic circulation. The pathophysiological mechanism is unknown, but impaired contractility of vascular smooth muscle may play an important role. The aim of this study was to evaluate the vascular response to stimulation with norepinephrine and angiotensin II in endothelium-denuded femoral artery rings. METHODS: Norwegian Landrace pigs weighing 27.1 +/- 0.5 kg (mean +/- sx (standard error of the mean)) were used. ALF was induced by performing a portacaval shunt followed by ligation of the hepatic arteries (n = 6). Sham-operated animals served as controls (n = 5). Cumulative isometric concentration contraction curves were obtained after in vitro stimulation of the femoral artery rings with either angiotensin II (10(-13) - 10(-5) mol/L) or norepinephrine (10(-13) - 10(-3) mol/L). RESULTS: Pigs suffering from ALF developed a hyperdynamic circulation with an increased ca^rdiac index (P = 0.017) and decreased systemic vascular resistance index (P = 0.015). Studies of the hind leg revealed a decreased vascular resistance index and increased blood flow compared to sham-operated controls (P = 0.003 and P = 0.01, respectively). Angiotensin II caused a concentration-dependent contraction of the arterial segments, with no significant differences in vascular responses between the two groups. Maximum force generated did not differ (55 +/- 7 versus 56 +/- 7 mN, P = 0.95). Furthermore, there were no differences for norepinephrine in the cumulative concentration-response curves and the maximum contractile force was not significantly different (87 +/- 8 versus 93 +/- 16 mN, P = 0.55). CONCLUSIONS: This study documents for the first time that there are no signs of endothelium-independent peripheral vascular hyporesponsiveness to angiotensin II and norepinephrine in pigs with ALF.
5. Zwingmann, C.; Chatauret, N.; Rose, C.; Leibfritz, D. & Butterworth, R. F. (2004). Selective alterations of brain osmolytes in acute liver failure: protective effect of mild hypothermia. Brain Res 999:118-123. doi:10.1016/j.brainres.2003.11.048
   

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   - [↓Résumé↓]

   The principal cause of mortality in patients with acute liver failure (ALF) is brain herniation resulting from intracranial hypertension caused by a progressive increase of brain water. In the present study, ex vivo high-resolution 1H-NMR spectroscopy was used to investigate the effects of ALF, with or without superimposed hypothermia, on brain organic osmolyte concentrations in relation to the severity of encephalopathy and brain edema in rats with ALF due to hepatic devascularization. In normothermic ALF rats, glutamine concentrations in frontal cortex increased more than fourfold at precoma stages, i.e. prior to the onset of severe encephalopathy, but showed no further increase at coma stages. In parallel with glutamine accumulation, the brain organic osmolytes myo-inositol and taurine were significantly decreased in frontal cortex to 63% and 67% of control values, respectively, at precoma stages (p<0.01), and to 58% and 67 respectively, at coma stages of encephalopathy (p<0.01). Hypothermia, which prevented brain edema and encephalopathy in ALF rats, significantly attenuated the depletion of myo-inositol and taurine. Brain glutamine concentrations, on the other hand, did not respond to hypothermia. These findings demonstrate that experimental ALF results in selective changes in brain organic osmolytes as a function of the degree of encephalopathy which are associated with brain edema, and provides a further rationale for the continued use of hypothermia in the management of this condition.

2003

1. Chatauret, N.; Zwingmann, C.; Rose, C.; Leibfritz, D. & Butterworth, R. F. (2003). Effects of hypothermia on brain glucose metabolism in acute liver failure: a H/C-nuclear magnetic resonance study. Gastroenterology 125:815-824. doi:10.1016/S0016-5085(03)01054-0
   

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   - [↓Résumé↓]

   BACKGROUND & AIMS: Mild hypothermia has a protective effect on brain edema and encephalopathy in both experimental and human acute liver failure. The goals of the present study were to examine the effects of mild hypothermia (35 degrees C) on brain metabolic pathways using combined (1)H and (13)C-Nuclear Magnetic Resonance (NMR) spectroscopy, a technique which allows the study not only of metabolite concentrations but also their de novo synthesis via cell-specific pathways in the brain. METHODS: (1)H and (13)C NMR spectroscopy using [1-(13)C] glucose was performed on extracts of frontal cortex obtained from groups of rats with acute liver failure induced by hepatic devascularization whose body temperature was maintained either at 37 degrees C (normothermic) or 35 degrees C (hypothermic), and appropriate sham-operated controls. RESULTS: At coma stages of encephalopathy in the normothermic acute liver failure animals, glutamine concentrations in frontal cortex increased 3.5-fold compared to sham-operated controls (P < 0.001). Comparable increases of brain glutamine were observed in hypothermic animals despite the absence of severe encephalopathy (coma). Brain glutamate and aspartate concentrations were respectively decreased to 60.9% +/- 7.7% and 42.2% +/- 5.9% (P < 0.01) in normothermic animals with acute liver failure compared to control and were restored to normal values by mild hypothermia. Concentrations of lactate and alanine in frontal cortex were increased to 169.2% +/- 15.6% and 267.3% +/- 34.0% (P < 0.01) respectively in normothermic rats compared to controls. Furthermore, de novo synthesis of lactate and alanine increased to 446.5% +/- 48.7% and 707.9% +/- 65.7% (P < 0.001), of control respectively, resulting in increased fractional (13)C-enrichments in these cytosolic metabolites. Again, these changes of lactate and alanine concentrations were prevented by mild hypothermia. CONCLUSIONS: Mild hypothermia (35 degrees C) prevents the encephalopathy and brain edema resulting from hepatic devascularization, selectively normalizes lactate and alanine synthesis from glucose, and prevents the impairment of oxidative metabolism associated with this model of ALF, but has no significant effect on brain glutamine. These findings suggest that a deficit in brain glucose metabolism rather than glutamine accumulation is the major cause of the cerebral complications of acute liver failure.
2. Desjardins, P.; Sauvageau, A.; Bouthillier, A.; Navarro, D.; Hazell, A. S.; Rose, C. & Butterworth, R. F. (2003). Induction of astrocytic cyclooxygenase-2 in epileptic patients with hippocampal sclerosis. Neurochem Int 42:299-303. doi:10.1016/S0197-0186(02)00101-8
   

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   - [↓Résumé↓]

   Induction of cyclooxygenase-2 (COX-2) has been described in a wide range of neurological diseases including animal models of epilepsy. The present study was undertaken to assess COX-2 expression in hippocampal biopsies from patients with therapy-refractive temporal lobe epilepsy (TLE). For this purpose, hippocampal CA1 subfield was dissected from epileptic patients with (n=5) or without (n=2) hippocampal sclerosis (HS). COX-2 expression was investigated using immunohistochemistry and semi-quantitative RT-PCR. COX-2 immunoreactivity in TLE patient material in the absence of HS was restricted to a few neurons of the hippocampus. In the presence of HS, on the other hand, a significant induction of astrocytic COX-2 immunoreactivity associated with a concomitant increase in the steady-state level of COX-2 mRNA was observed in the CA1 subfield. These findings suggest that induction of astrocytic COX-2 is implicated in the pathogenesis of HS in TLE and is consistent with the previous findings of increased concentrations of prostaglandins in the cerebrospinal fluid of these patients.

2002

1. Chatauret, N.; Rose, C. & Butterworth, R. F. (2002). Mild hypothermia in the prevention of brain edema in acute liver failure: mechanisms and clinical prospects. Metab Brain Dis 17:445-451. doi:10.1023/A:1021982523691
   

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   - [↓Résumé↓]

   Mild hypothermia (32 degrees C-35 degrees C) reduces intracranial pressure in patients with acute liver failure and may offer an effective adjunct therapy in the management of these patients. Studies in experimental animals suggest that this beneficial effect of hypothermia is the result of a decrease in blood-brain ammonia transfer resulting in improvement in brain energy metabolism and normalization of glutamatergic synaptic regulation. Improvement in brain energy metabolism by hypothermia may result from a reduction in ammonia-induced decrease of brain glucose (pyruvate) oxidation. Restoration of normal glutamatergic synaptic regulation by hypothermia may be the consequence of the removal of ammonia-induced decreases in expression of astrocytic glutamate transporters resulting in normal glutamate neurotransmitter inactivation in brain. Randomized controlled clinical trials of hypothermia are required to further evaluate its clinical impact.
2. Rose, C. (2002). Increased extracellular brain glutamate in acute liver failure: decreased uptake or increased release? Metab Brain Dis 17:251-261. doi:10.1023/A:1021945515514
   

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   - [↓Résumé↓]

   Glutamatergic dysfunction has been suggested to play an important role in the pathogenesis of hepatic encephalopathy (HE) in acute liver failure (ALF). Increased extracellular brain glutamate concentrations have consistently been described in different experimental animal models of ALF and in patients with increased intracranial pressure due to ALF. High brain ammonia levels remain the leading candidate in the pathogenesis of HE in ALF and studies have demonstrated a correlation between ammonia and increased concentrations of extracellular brain glutamate both clinically and in experimental animal models of ALE Inhibition of glutamate uptake or increased glutamate release from neurons and/or astrocytes could cause an increase in extracellular glutamate. This review analyses the effect of ammonia on glutamate release from (and uptake into) both neurons and astrocytes and how these pathophysiological mechanisms may be involved in the pathogenesis of HE in ALF.
3. Sauvageau, A.; Desjardins, P.; Lozeva, V.; Rose, C.; Hazell, A. S.; Bouthillier, A. & Butterworth, R. F. (2002). Increased expression of "peripheral-type" benzodiazepine receptors in human temporal lobe epilepsy: implications for PET imaging of hippocampal sclerosis. Metab Brain Dis 17:3-11. doi:10.1023/A:1014044128845
   

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   - [↓Résumé↓]

   Increased binding sites for "peripheral-type" benzodiazepine receptor (PTBR) ligands have been described in a wide range of neurological disorders including both human and experimental epilepsy. This study was undertaken to assess PTBR expression in relation to the presence of hippocampal sclerosis in human temporal lobe epilepsy (TLE). For this purpose, hippocampal CA1 subfields were dissected from surgical samples from patients with therapy-refractive TLE with (n = 5) or without (n = 2) hippocampal sclerosis and from age-matched nonepileptic postmortem controls (n = 5). PTBR expression was assessed by immunohistochemistry and reverse-transcription polymerase chain reaction. Receptor sites were evaluated using an in vitro binding assay and the selective PTBR ligand [3H]PK11195. Epileptic patients with hippocampal sclerosis showed increases in PTBR binding sites, immunoreactivity, and mRNA expression compared to both nonsclerotic TLE patients and postmortem nonepileptic controls. Induction of PTBR expression and binding sites were directly correlated with the presence of hippocampal sclerosis and the accompanying reactive gliosis.

2001

1. Chatauret, N.; Rose, C.; Therrien, G. & Butterworth, R. F. (2001). Mild hypothermia prevents cerebral edema and CSF lactate accumulation in acute liver failure. Metab Brain Dis 16:95-102. doi:10.1023/A:1011622830569
   

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   - [↓Résumé↓]

   Evidence from both clinical and experimental studies demonstrates that mild hypothermia prevents encephalopathy and brain edema in acute liver failure (ALF). As part of a series of studies to elucidate the mechanism(s) involved in this protective effect, groups of rats with ALF resulting from hepatic devascularization were maintained at either 37 degrees C (normothermic) or 35 C (hypothermic), and neurological status was monitored in relation to cerebrospinal fluid (CSF) concentrations of ammonia and lactate. CSF was removed via implanted cisterna magna catheters. Mild hypothermia resulted in a delay in onset of encephalopathy and prevention of brain edema, CSF concentrations of ammonia and lactate were concomitantly decreased. Blood ammonia concentrations, on the other hand, were not affected by hypothermia in ALF rats. These findings suggest that brain edema and encephalopathy in ALF are the consequence of ammonia-induced impairment of brain energy metabolism and open the way for magnetic resonance spectroscopic monitoring of cerebral function in ALF. Mild hypothermia could be beneficial in the prevention of severe encephalopathy and brain edema in patients with ALF awaiting liver transplantation.
2. Michalak, A.; Rose, C. & Butterworth, R. F. (2001). Loss of noradrenaline transporter sites in frontal cortex of rats with acute (ischemic) liver failure. Neurochem Int 38:25-30. doi:10.1016/S0197-0186(00)00048-6
   

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   - [↓Résumé↓]

   There is increasing evidence that central noradrenaline (NA) transport mechanisms are implicated in the central nervous system complications of acute liver failure. In order to assess this possibility, binding sites for the high affinity NA transporter ligand [3H]-nisoxetine were measured by quantitative receptor autoradiography in the brains of rats with acute liver failure resulting from hepatic devascularization and in appropriate controls. In vivo microdialysis was used to measure extracellular brain concentrations of NA. Severe encephalopathy resulted in a significant loss of [3H]-nisoxetine sites in frontal cortex and a concomitant increase in extracellular brain concentrations of NA in rats with acute liver failure. A loss of transporter sites was also observed in thalamus of rats with acute liver failure. This loss of NA transporter sites could result from depletion of central NA stores due to a reserpine-like effect of ammonia which is known to accumulate to millimolar concentrations in brain in ischemic liver failure. Impaired NA transport and the consequent increase in synaptic concentrations and increased stimulation of neuronal and astrocytic noradrenergic receptors could be implicated in the pathogenesis of the encephalopathy and brain edema characteristic of acute liver failure.

2000

1. Rose, C.; Michalak, A.; Pannunzio, M.; Chatauret, N.; Rambaldi, A. & Butterworth, R. F. (2000). Mild hypothermia delays the onset of coma and prevents brain edema and extracellular brain glutamate accumulation in rats with acute liver failure. Hepatology 31:872-877. doi:10.1053/he.2000.5923
   

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   - [↓Résumé↓]

   Mild hypothermia is effective in the prevention of brain edema associated with cerebral ischemia and traumatic brain injury. Brain edema is also a serious complication of acute liver failure (ALF). To assess the effectiveness of hypothermia in ALF, groups of rats were subjected to hepatic devascularization (portacaval anastomosis, followed 48 hours later by hepatic artery ligation), and body temperatures were maintained at either 35 degrees C (hypothermic) or 37 degrees C (normothermic). Mild hypothermia resulted in a significant delay in the onset of severe encephalopathy and in reduction of brain water content compared with normothermic ALF rats (control [n = 8] 80.22 ALF-37 degrees C [n = 8] 81.74 ALF-35 degrees C [n = 8] 80.48% [P <.01 compared with ALF-37 degrees C]). This protective effect was accompanied by a significant reduction of cerebrospinal fluid (CSF) (but not plasma) ammonia concentrations (CSF ammonia: control: 0.05 mg/dL; ALF-37 degrees C: 1.01 mg/dL; ALF-35 degrees C: 0.07 mg/dL, P <.01 compared with ALF-37 degrees C). In vivo cerebral microdialysis studies revealed that mild hypothermia resulted in a significant reduction of extracellular glutamate concentrations in the brains of rats with ALF (control: 1. 06 micromol/L; ALF-37 degrees C: 2.74 micromol/L; ALF-35 degrees C: 1.49 micromol/L [P <.01 compared with ALF-37 degrees C]). These findings suggest that: 1) mild hypothermia is an effective approach to the prevention of the central nervous system consequences of experimental ALF; and that 2) the beneficial effect of hypothermia is mediated via mechanisms involving reduced blood-brain transfer of ammonia and/or reduction of extracellular brain glutamate concentrations. Mild hypothermia may be an effective approach to delay the onset of brain edema in patients with ALF awaiting liver transplantation.

1999

1. Desjardins, P.; Rao, K. V.; Michalak, A.; Rose, C. & Butterworth, R. F. (1999). Effect of portacaval anastomosis on glutamine synthetase protein and gene expression in brain, liver and skeletal muscle. Metab Brain Dis 14:273-280. doi:10.1023/A:1020741226752
   

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   - [↓Résumé↓]

   The effects of chronic liver insufficiency resulting from end-to-side portacaval anastomosis (PCA) on glutamine synthetase (GS) activities, protein and gene expression were studied in brain, liver and skeletal muscle of male adult rats. Four weeks following PCA, activities of GS in cerebral cortex and cerebellum were reduced by 32% and 37% (p<0.05) respectively whereas GS activities in muscle were increased by 52% (p<0.05). GS activities in liver were decreased by up to 90% (p<0.01), a finding which undoubtedly reflects the loss of GS-rich perivenous hepatocytes following portal-systemic shunting. Immunoblotting techniques revealed no change in GS protein content of brain regions or muscle but a significant loss in liver of PCA rats. GS mRNA determined by semi-quantitative RT-PCR was also significantly decreased in the livers of PCA rats compared to sham-operated controls. These findings demonstrate that PCA results in a loss of GS gene expression in the liver and that brain does not show a compensatory induction of enzyme activity, rendering it particularly sensitive to increases in ammonia in chronic liver failure. The finding of a post-translational increase of GS in muscle following portacaval shunting suggests that, in chronic liver failure, muscle becomes the major organ responsible for the removal of excess blood-borne ammonia.
2. Rose, C.; Butterworth, R. F.; Zayed, J.; Normandin, L.; Todd, K.; Michalak, A.; Spahr, L.; Huet, P. M. & Pomier-Layrargues, G. (1999). Manganese deposition in basal ganglia structures results from both portal-systemic shunting and liver dysfunction. Gastroenterology 117:640-644. doi:10.1016/S0016-5085(99)70457-9
   

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   - [↓Résumé↓]

   BACKGROUND & AIMS: Manganese (Mn) deposition could be responsible for the T(1)-weighted magnetic resonance signal hyperintensities observed in cirrhotic patients. These experiments were designed to assess the regional specificity of the Mn increases as well as their relationship to portal-systemic shunting or hepatobiliary dysfunction. METHODS: Mn concentrations were measured in (1) brain samples from basal ganglia structures (pallidum, putamen, caudate nucleus) and cerebral cortical structures (frontal, occipital cortex) obtained at autopsy from 12 cirrhotic patients who died in hepatic coma and from 12 matched controls; and from (2) brain samples (caudate/putamen, globus pallidus, frontal cortex) from groups (n = 8) of rats either with end-to-side portacaval anastomosis, with biliary cirrhosis, or with fulminant hepatic failure as well as from sham-operated and normal rats. RESULTS: Mn content was significantly increased in frontal cortex (by 38, occipital cortex (by 55, pallidum (by 186, putamen (by 66, and caudate (by 54 of cirrhotic patients compared with controls. Brain Mn content did not correlate with patient age, etiology of cirrhosis, or history of chronic hepatic encephalopathy. In cirrhotic and portacaval-shunted rats, Mn content was increased in pallidum (by 27% and 57 respectively) and in caudate/putamen (by 57% and 67 respectively) compared with control groups. Mn concentration in pallidum was significantly higher in portacaval-shunted rats than in cirrhotic rats. No significant changes in brain Mn concentrations were observed in rats with acute liver failure. CONCLUSIONS: These findings suggest that brain Mn deposition results both from portal-systemic shunting and from liver dysfunction.
3. Rose, C.; Michalak, A.; Rao, K. V.; Quack, G.; Kircheis, G. & Butterworth, R. F. (1999). L-ornithine-L-aspartate lowers plasma and cerebrospinal fluid ammonia and prevents brain edema in rats with acute liver failure. Hepatology 30:636-640. doi:10.1002/hep.510300311
   

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   - [↓Résumé↓]

   Brain edema sufficient to cause intracranial hypertension and brain herniation remains a major cause of mortality in acute liver failure (ALF). Studies in experimental animal models of ALF suggest a role for ammonia in the pathogenesis of both encephalopathy and brain edema in this condition. As part of a series of studies to evaluate the therapeutic efficacy of ammonia-lowering agents, groups of rats with ALF caused by hepatic devascularization were treated with L-ornithine-L-aspartate (OA), an agent shown previously to be effective in reducing blood ammonia concentrations in both experimental and human chronic liver failure. Treatment of rats in ALF with infusions of OA (0.33 g/kg/h, intravenously) resulted in normalization of plasma ammonia concentrations and in a significant delay in onset of severe encephalopathy. More importantly, brain water content was significantly reduced in OA-treated rats with ALF. These protective effects of OA were accompanied by increased plasma concentrations of several amino acids including glutamate, gamma-aminobutyric acid (GABA), taurine, and alanine, as well as the branched-chain amino acids, leucine, isoleucine, and valine. Increased availability of glutamate following OA treatment provides the substrate for the major ammonia-removal mechanism (glutamine synthetase). Plasma (but not cerebrospinal fluid) glutamine concentrations were increased 2-fold (P <.02) in OA-treated rats, consistent with increased muscle glutamine synthesis. Direct measurement of glutamine synthetase activities revealed a 2-fold increase following OA treatment. These findings demonstrate a significant ammonia-lowering effect of OA together with a protective effect on the development of encephalopathy and brain edema in this model of ALF.

1998

1. Layrargues, G. P.; Rose, C.; Spahr, L.; Zayed, J.; Normandin, L. & Butterworth, R. F. (1998). Role of manganese in the pathogenesis of portal-systemic encephalopathy. Metab Brain Dis 13:311-317. doi:10.1023/A:1020636809063
   

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   - [↓Résumé↓]

   Amongst the potential neurotoxins implicated in the pathogenesis of hepatic encephalopathy, manganese emerges as a new candidate. In patients with chronic liver diseases, manganese accumulates in blood and brain leading to pallidal signal hyperintensity on T1-weighted Magnetic Resonance (MR) Imaging. Direct measurements in globus pallidus obtained at autopsy from cirrhotic patients who died in hepatic coma reveal 2 to 7-fold increases of manganese concentration. The intensity of pallidal MR images correlates with blood manganese and with the presence of extrapyramidal symptoms occurring in a majority of cirrhotic patients. Liver transplantation results in normalization of pallidal MR signals and disappearance of extrapyramidal symptoms whereas transjugular intrahepatic portosystemic shunting induces an increase in pallidal hyperintensity with a concomitant deterioration of neurological dysfunction. These findings suggest that the toxic effects of manganese contribute to extrapyramidal symptoms in patients with chronic liver disease. The mechanisms of manganese neurotoxicity are still speculative, but there is evidence to suggest that manganese deposition in the pallidum may lead to dopaminergic dysfunction. Future studies should be aimed at evaluating the effects of manganese chelation and/or of treatment of the dopaminergic deficit on neurological symptomatology in these patients.
2. Michalak, A.; Rose, C.; Buu, P. N. & Butterworth, R. F. (1998). Evidence for altered central noradrenergic function in experimental acute liver failure in the rat. Hepatology 27:362-368. doi:10.1002/hep.510270208
   

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   - [↓Résumé↓]

   These is increasing evidence to suggest that central noradrenergic mechanisms may contribute to the central nervous system manifestations of acute liver failure. To further elucidate this possibility, extracellular brain concentrations of the monoamines, noradrenaline (NA), dopamine (DA), and serotonin, were measured by high-performance liquid chromatography with electrochemical detection in microdialysates from the extracellular compartment of frontal cortex in rats with acute (ischemic) liver failure at various times during the progression of encephalopathy and brain edema, as well as in obligate control groups of animals. In addition, binding sites for the noradrenergic receptor subtype ligands, [3H]-prazosin (alpha1 sites), [3H]-RX821002 (alpha2 sites), and [125]I-iodopindolol (beta sites), were assessed using quantitative receptor autoradiography in regions of the brains of rats at coma stage of acute liver failure and of control groups of animals. Coma stages of encephalopathy in acute liver failure were associated with selectively increased noradrenaline concentrations (P < .05) and a concomitant selective loss of alpha1 and beta1 sites in frontal cortex and thalamus. These findings add to a growing body of evidence that central noradrenergic function is modified in acute liver failure and suggest that alpha1/beta1 receptor-mediated noradrenergic mechanisms may play a role in the pathogenesis of brain edema and encephalopathy in this condition.
3. Rose, C.; Michalak, A.; Pannunzio, P.; Therrien, G.; Quack, G.; Kircheis, G. & Butterworth, R. F. (1998). L-ornithine-L-aspartate in experimental portal-systemic encephalopathy: therapeutic efficacy and mechanism of action. Metab Brain Dis 13:147-157. doi:10.1023/A:1020613314572
   

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   - [↓Résumé↓]

   Strategies aimed at the lowering of blood ammonia remain the treatment of choice in portal-systemic encephalopathy (PSE). L-ornithine-L-aspartate (OA) has recently been shown to be effective in the prevention of ammonia-precipitated coma in humans with PSE. These findings prompted the study of mechanisms of the protective effect of OA in portacaval-shunted rats in which reversible coma was precipitated by ammonium acetate administration (3.85 mmol/kg i.p.). OA infusions (300 mg/kg/h, i.v) offered complete protection in 12/12 animals compared to 0/12 saline-infused controls. This protective effect was accompanied by significant reductions of blood ammonia, concomitant increases of urea production and significant increases in blood and cerebrospinal fluid (CSF) glutamate and glutamine. Increased CSF concentrations of leucine and alanine also accompanied the protective effect of OA. These findings demonstrate the therapeutic efficacy of OA in the prevention of ammonia-precipitated coma in portacaval-shunted rats and suggest that this protective effect is both peripherally-mediated (increased urea and glutamine synthesis) and centrally-mediated (increased glutamine synthesis).

1997

1. Knecht, K.; Michalak, A.; Rose, C.; Rothstein, J. D. & Butterworth, R. F. (1997). Decreased glutamate transporter (GLT-1) expression in frontal cortex of rats with acute liver failure. Neurosci Lett 229:201-203. doi:10.1016/S0304-3940(97)00444-8
   

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   - [↓Résumé↓]

   It has been suggested that reduced astrocytic uptake of neuronally released glutamate contributes to the pathogenesis of hepatic encephalopathy in acute liver failure. In order to further address this issue, the recently cloned and sequenced astrocytic glutamate transporter GLT-1 was studied in brain preparations from rats with ischemic liver failure induced by portacaval anastomosis followed 24 h later by hepatic artery ligation and from appropriate sham-operated controls. GLT-1 expression was studied using reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of GLT-1 transcript was significantly decreased in frontal cortex at coma stages of acute liver failure. Western blotting using a polyclonal antibody to GLT-1 revealed a concomitant decrease in expression of transporter protein in the brains of rats with acute liver failure. Reduced capacity of astrocytes to reuptake neuronally released glutamate, resulting from a GLT-1 transporter deficit and the consequently compromised neuron-astrocytic trafficking of glutamate could contribute to the pathogenesis of hepatic encephalopathy and brain edema, two major complications of acute liver failure.
2. Therrien, G.; Rose, C.; Butterworth, J. & Butterworth, R. F. (1997). Protective effect of L-carnitine in ammonia-precipitated encephalopathy in the portacaval shunted rat. Hepatology 25:551-556. doi:10.1002/hep.510250310
   

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   - [↓Résumé↓]

   L-carnitine administration prevents the neurological symptoms of acute ammonia toxicity. To further evaluate its efficacy in the prevention of hepatic encephalopathy in hyperammonemic conditions, L-carnitine (16 mmol/kg, intraperitoneally [i.p.] was administered 1 hour before ammonium acetate (NH4OAc) (8.5 mmol/kg, subcutaneously) to portacaval shunted (PCS) rats. Cerebrospinal fluid (CSF) ammonia, lactate, and amino acid levels were measured in relation to deteriorating neurological status in these animals. None of 35 L-carnitine-treated animals showed neurological deterioration after NH4OAC administration compared with saline-treated controls; the latter manifested severe encephalopathy progressing through loss of righting reflex to coma. Survival rate was 100% in the L-carnitine-treated group compared with 5% in saline-treated controls. Following NH4OAC administration to PCS rats, CSF ammonia increased to 0.93 +/- 0.15 mmol/L and 1.24 +/- 0.15 mmol/L at precoma and coma stages of encephalopathy (P < .01) respectively. Treatment with L-carnitine reduced CSF ammonia at both precoma and coma stages; the time-course of this protective effect paralleled blood and CSF L-carnitine accumulation. CSF alanine and lactate increases following NH4OAC administration to PCS rats were significantly attenuated following L-carnitine treatment. However, L-carnitine treatment did not lead to significant reductions in plasma ammonia nor CSF or brain glutamine in these animals. These findings show the therapeutic efficacy of L-carnitine in ammonia-precipitated coma in PCS rats and suggest that this protective effect is centrally mediated involving improved mitochondrial respiration. L-carnitine could be of therapeutic benefit in the prevention of hepatic encephalopathy precipitated by ammoniagenic conditions in humans with chronic liver disease.
3. de Waele, J. P.; Audet, R. M.; Rose, C. & Butterworth, R. F. (1997). The portacaval-shunted rat: a new model for the study of the mechanisms controlling voluntary ethanol consumption and ethanol preference? Alcohol Clin Exp Res 21:305-310. doi:10.1111/j.1530-0277.1997.tb03765.x
   

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   - [↓Résumé↓]

   Portacaval anastomosis (PCA) is a surgical procedure whereby blood from the portal vein is shunted into the inferior vena cava. PCA in the rat results in a significant increase (from 0.77 +/- 0.26 to 3.51 +/- 0.37 g of ethanol/kg/day) in voluntary ethanol consumption in a free-choice paradigm between water and 5% ethanol solution. After PCA surgery, increased voluntary ethanol consumption starts abruptly at 6 to 7 days and is maintained for > 28 weeks. Voluntary ethanol consumption in rats after PCA results in blood ethanol levels up to 158 mg After PCA, the ethanol preference ratio (defined as the percentage of total fluid intake constituted by ethanol) increased from 19 +/- 2% to 78 +/- 2% (P < 0.001). Administration of the nonselective opioid receptor antagonist naloxone (5 mg/kg, sc) resulted in a significant 6-fold attenuation of voluntary ethanol consumption by rats with PCA, an effect that was not mediated by an effect on locomotor activity. These findings, together with previous reports of widespread alterations of the mu- and delta-opioid receptors in the brain after PCA, suggest that increased voluntary ethanol consumption and ethanol preference in PCA rats may result from activation of the endogenous opioid system. Preliminary studies suggest that rats with PCA manifest behavioral signs consistent with the development of dependence. The portacaval-shunted rat may provide a useful preparation for the study of mechanisms, in particular those involving the liver, implicated in the development of increased voluntary ethanol consumption and ethanol preference.

1996

1. Michalak, A.; Rose, C.; Butterworth, J. & Butterworth, R. F. (1996). Neuroactive amino acids and glutamate (NMDA) receptors in frontal cortex of rats with experimental acute liver failure. Hepatology 24:908-913. doi:10.1002/hep.510240425
   

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   - [↓Résumé↓]

   It has been proposed that alterations of excitatory and inhibitory amino acids play a role in the pathogenesis of hepatic encephalopathy in acute liver failure. To evaluate this possibility, in vivo cerebral microdialysis was used to sample extracellular concentrations of amino acids in the frontal cortex of unanesthetized rats at various times during the progression of encephalopathy resulting from acute liver failure. Liver failure was induced by portacaval anastomosis followed 24 hours later by hepatic artery ligation. Dialysate concentrations of amino acids were measured by high-performance liquid chromatography (HPLC) with fluorescence detection. Deterioration of neurological status was accompanied by two- to four-fold increases in extracellular glutamate, glutamine, and glycine; concentrations of gamma-aminobutyric acid (GABA) and taurine were unchanged. Densities of binding sites for the glutamate (N-methyl-D-aspartate [NMDA]) receptor ligand 3H-MK801, assessed using quantitative receptor autoradiography, however, were unchanged in the frontal cortex of rats at coma stages of ischemic liver failure. Increased extracellular glutamate concentrations were positively correlated with the severity of encephalopathy and with arterial ammonia concentrations. Such changes may result from an ammonia-induced reduction in the capacity for astrocytes to uptake glutamate. Increased extracellular glutamate in brain, together with increases in concentrations of glycine, a positive allosteric modulator of glutamate (NMDA) receptors, are consistent with increased NMDA-related glutamatergic neurotransmission in this model of acute liver failure. Increased extracellular glutamate, therefore, could contribute to the pathogenesis of hepatic encephalopathy and brain edema in acute liver failure.

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