Portacaval Shunted Rats Research Articles

Portacaval shunting causes differential mitochondrial superoxide production in brain regions

The portacaval shunting (PCS) prevents portal hypertension and recurrent bleeding of esophageal varices. On the other hand, it can induce chronic hyperammonemia and is considered to be the best model of mild hepatic encephalopathy (HE). Pathogenic mechanisms of HE and dysfunction of the brain in hyperammonemia are not fully elucidated, but it was originally suggested that the pathogenetic defect causes destruction of antioxidant defense which leads to an increase in the production of reactive oxygen species (ROS) and the occurrence of oxidative stress. In order to gain insight into the pathogenic mechanisms of HE in the brain tissue, we investigated the effects of PCS in rats on free radicals production and activity levels of antioxidant and prooxidant enzymes in mitochondria isolated from different brain areas. We found that O2·− production, activities of Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GT), nitric oxide synthase (NOS), and levels of carbonylated proteins differed between the four brain regions both in the amount and response to PCS. In PCS rats, Mn-SOD activity in the cerebellum was significantly decreased, and remained unchanged in the neocortex, hippocampus and striatum compared with that in sham-operated animals. Among the four brain regions in control rats, the levels of the carbonyl groups in mitochondrial proteins were maximal in the cerebellum. 4 weeks after PCS, the content of carbonylated proteins were higher only in mitochondria of this brain region. Under control conditions, O2·− production by submitochondrial particles in the cerebellum was significantly higher than in other brain regions, but was significantly increased in each brain region from PCS animals. Indeed, the production of O2·− by submitochondrial particles correlated with mitochondrial ammonia levels in the four brain regions of control and PCS-animals. These findings are the first to suggest that in vivo levels of ammonia in the brain directly affect the rate of mitochondrial O2·− production.

Sildenafil reduces neuroinflammation in cerebellum, restores GABAergic tone, and improves motor in-coordination in rats with hepatic encephalopathy.

Patients with liver disease may develop hepatic encephalopathy (HE), with cognitive impairment and motor in-coordination. Rats with HE due to portacaval shunts (PCS) show motor in-coordination. We hypothesized that in PCS rats: (i) Motor in-coordination would be due to enhanced GABAergic tone in cerebellum; (ii) increased GABAergic tone would be due to neuroinflammation; (iii) increasing cGMP would reduce neuroinflammation and GABAergic tone and restore motor coordination. To assess these hypotheses, we assessed if (i) treatment with sildenafil reduces neuroinflammation; (ii) reduced neuroinflammation is associated with reduced GABAergic tone and restored motor coordination. Rats were treated with sildenafil to increase cGMP. Microglia and astrocytes activation were analyzed by immunohistochemistry, extracellular GABA by microdialysis, and motor coordination in the beam walking. PCS rats show neuroinflammation in cerebellum, with microglia and astrocytes activation, increased IL-1b and TNF-a and reduced YM-1 and IL-4. Membrane expression of the GABA transporter GAT1 is reduced, while GAT3 is increased. Extracellular GABA and motor in-coordination are increased. Sildenafil treatment eliminates neuroinflammation, microglia and astrocytes activation; changes in membrane expression of GABA transporters; and restores motor coordination. This study supports an interplay between cGMP-neuroinflammation and GABAergic neurotransmission in impairing motor coordination in PCS rats.

Organ Distribution of 13N Following Intravenous Injection of [13N]Ammonia into Portacaval-Shunted Rats.

Ammonia is neurotoxic, and chronic hyperammonemia is thought to be a major contributing factor to hepatic encephalopathy in patients with liver disease. Portacaval shunting of rats is used as an animal model to study the detrimental metabolic effects of elevated ammonia levels on body tissues, particularly brain and testes that are deleteriously targeted by high blood ammonia. In normal adult rats, the initial uptake of label (expressed as relative concentration) in these organs was relatively low following a bolus intravenous injection of [13N]ammonia compared with lungs, kidneys, liver, and some other organs. The objective of the present study was to determine the distribution of label following intravenous administration of [13N]ammonia among 14 organs in portacaval-shunted rats at 12 weeks after shunt construction. At an early time point (12s) following administration of [13N]ammonia the relative concentration of label was highest in lung with lower, but still appreciable relative concentrations in kidney and heart. Clearance of 13N from blood and kidney tended to be slower in portacaval-shunted rats versus normal rats during the 2-10min interval after the injection. At later times post injection, brain and testes tended to have higher-than-normal 13N levels, whereas many other tissues had similar levels in both groups. Thus, reduced removal of ammonia from circulating blood by the liver diverts more ammonia to extrahepatic tissues, including brain and testes, and alters the nitrogen homeostasis in these tissues. These results emphasize the importance of treatment paradigms designed to reduce blood ammonia levels in patients with liver disease.

Reducing Peripheral Inflammation with Infliximab Reduces Neuroinflammation and Improves Cognition in Rats with Hepatic Encephalopathy.

Inflammation contributes to cognitive impairment in patients with hepatic encephalopathy (HE). However, the process by which peripheral inflammation results in cognitive impairment remains unclear. In animal models, neuroinflammation and altered neurotransmission mediate cognitive impairment. Taking into account these data, we hypothesized that in rats with HE: (1) peripheral inflammation is a main contributor to neuroinflammation; (2) neuroinflammation in hippocampus impairs spatial learning by altering AMPA and/or NMDA receptors membrane expression; (3) reducing peripheral inflammation with infliximab (anti-TNF-a) would improve spatial learning; (4) this would be associated with reduced neuroinflammation and normalization of the membrane expression of glutamate receptors. The aims of this work were to assess these hypotheses. We analyzed in rats with portacaval shunt (PCS) and control rats, treated or not with infliximab: (a) peripheral inflammation by measuring prostaglandin E2, IL10, IL-17, and IL-6; (b) neuroinflammation in hippocampus by analyzing microglial activation and the content of TNF-a and IL-1b; (c) AMPA and NMDA receptors membrane expression in hippocampus; and (d) spatial learning in the Radial and Morris water mazes. We assessed the effects of treatment with infliximab on peripheral inflammation, on neuroinflammation and AMPA and NMDA receptors membrane expression in hippocampus and on spatial learning and memory. PCS rats show increased serum prostaglandin E2, IL-17, and IL-6 and reduced IL-10 levels, indicating increased peripheral inflammation. PCS rats also show microglial activation and increased nuclear NF-kB and expression of TNF-a and IL-1b in hippocampus. This was associated with altered AMPA and NMDA receptors membrane expression in hippocampus and impaired spatial learning and memory in the radial and Morris water maze. Treatment with infliximab reduces peripheral inflammation in PCS rats, normalizing prostaglandin E2, IL-17, IL-6, and IL-10 levels in serum. Infliximab also prevents neuroinflammation, reduces microglial activation, translocates NF-kB into nucleoli and normalizes TNF-a and IL-1b content in hippocampus. This was associated with normalization of AMPA receptors membrane expression in hippocampus and of spatial learning and memory. The results suggest that peripheral inflammation contributes to spatial learning impairment in PCS rats. Treatment with anti-TNF-a could be a new therapeutic approach to improve cognitive function in patients with HE.

Infliximab reduces peripheral inflammation, neuroinflammation, and extracellular GABA in the cerebellum and improves learning and motor coordination in rats with hepatic encephalopathy.

BackgroundPeripheral inflammation contributes to the neurological alterations in hepatic encephalopathy (HE). Neuroinflammation and altered GABAergic neurotransmission mediate cognitive and motor alterations in rats with HE. It remains unclear (a) if neuroinflammation and neurological impairment in HE are a consequence of peripheral inflammation and (b) how neuroinflammation impairs GABAergic neurotransmission. The aims were to assess in rats with HE whether reducing peripheral inflammation with anti-TNF-α (1) prevents cognitive impairment and motor in-coordination, (2) normalizes neuroinflammation and extracellular GABA in the cerebellum and also (3) advances the understanding of mechanisms linking neuroinflammation and increased extracellular GABA.MethodsRats with HE due to portacaval shunt (PCS) were treated with infliximab. Astrocytes and microglia activation and TNF-α and IL-1β were analyzed by immunohistochemistry. Membrane expression of the GABA transporters GAT-3 and GAT-1 was analyzed by cross-linking with BS3. Extracellular GABA was analyzed by microdialysis. Motor coordination was tested using the beam walking and learning ability using the Y maze task.ResultsPCS rats show peripheral inflammation, activated astrocytes, and microglia and increased levels of TNF-α and IL-1β. Membrane expression of GAT-3 and extracellular GABA are increased, leading to impaired motor coordination and learning ability. Infliximab reduces peripheral inflammation, microglia, and astrocyte activation and neuroinflammation and normalizes GABAergic neurotransmission, motor coordination, and learning ability.ConclusionsNeuroinflammation is associated with altered GABAergic neurotransmission and increased GAT-3 membrane expression and extracellular GABA (a); peripheral inflammation is a main contributor to the impairment of motor coordination and of the ability to learn the Y maze task in PCS rats (b); and reducing peripheral inflammation using safe procedures could be a new therapeutic approach to improve cognitive and motor function in patients with HE (c).

Sildenafil Treatment Eliminates Pruritogenesis and Thermal Hyperalgesia in Rats with Portacaval Shunts.

Pruritus is a common symptom in chronic liver diseases, which may also alter thermal sensitivity. The underlying mechanisms remain unclear and treatments are not satisfactory. Portal-systemic shunting has been proposed to alter thermal sensitivity in cirrhotics. Inflammation-induced enhanced activity of the Transient Receptor Potential Vanilloid 1 (TRPV1) may contribute to pruritus and thermal hyperalgesia. Sildenafil reduces neuroinflammation in portacaval shunt (PCS) rats. The aims were to assess whether: (1) PCS rats show enhanced scratching or thermal sensitivity; (2) TRPV1 activity is enhanced in PCS rats; (3) treatment with sildenafil reduces TRPV1 activation, scratching and thermal hyperalgesia. Rats were treated with sildenafil beginning 3weeks after surgery. The number of scratches performed were counted. Thermal hyperalgesia was analyzed using the Hargreaves' Plantar Test. TRPV1 activation by measuring the increase in Ca2+ induced by capsaicin in dorsal root ganglia neurons. PCS rats show enhanced scratching behavior, reaching 66 ± 5 scratches/h (p < 0.01) at 21days after surgery, while controls show 37 ± 2 scratches/h. PCS rats show thermal hyperalgesia. Paw withdrawal latency was reduced (p < 0.05) to 10 ± 1s compared to controls (21 ± 2s). Capsaicin-induced calcium increase was higher in dorsal root ganglia cultures from PCS rats, indicating TRPV1functional increase. PCS rats show enhanced scratching behavior and thermal sensitivity and are a good model to study these alterations in chronic liver diseases. Enhanced sensitivity and activity of TRPV1 channel underlies these alterations. Treatment with sildenafil reduces TRPV1 channel sensitivity and activity and normalizes scratching behavior and thermal sensitivity.

Sildenafil reduces neuroinflammation and restores spatial learning in rats with hepatic encephalopathy: underlying mechanisms.

BackgroundThere are no specific treatments for the neurological alterations of cirrhotic patients with minimal hepatic encephalopathy (MHE). Rats with MHE due to portacaval shunt (PCS) show impaired spatial learning. The underlying mechanisms remain unknown. The aims of this work were to assess: (a) whether PCS rats show neuroinflammation in hippocampus, (b) whether treatment with sildenafil reduces neuroinflammation and restores spatial learning in PCS rats, and (c) analyze the underlying mechanisms.MethodsNeuroinflammation was assessed by determining inflammatory markers by Western blot. Phosphorylation of MAP-kinase p38 was assessed by immunohistochemistry. Membrane expression of GABA and glutamate receptors was analyzed using BS3 cross-linker. Spatial learning was analyzed using the radial and Morris water mazes. To assess if sildenafil reverses the alterations, rats were treated with sildenafil in the drinking water.ResultsPCS rats show increased IL-1β and TNF-α levels and phosphorylation (activity) of p38 in hippocampus. Membrane expression of subunits α1 of GABAA receptor and GluR2 of AMPA receptor are increased in PCS rats, while subunits GluR1 of AMPA receptors and NR1 and NR2a of NMDA receptors are reduced. PCS rats show reduced spatial learning in the radial and Morris water mazes. Sildenafil treatment normalizes IL-1β and TNF-α levels, p38 phosphorylation, and membrane expression of GABAA, AMPA, and NMDA receptors and restores spatial learning.ConclusionsIncreased IL-1β alters GABAergic and glutamatergic neurotransmission in hippocampus and impairs spatial learning in rats with MHE. Sildenafil reduces neuroinflammation and restores learning. Phosphodiesterase-5 inhibitors may be useful to improve cognitive function in patients with MHE.

The expression levels of prolyl oligopeptidase responds not only to neuroinflammation but also to systemic inflammation upon liver failure in rat models and cirrhotic patients.

BackgroundLiver failure in experimental animals or in human cirrhosis elicits neuroinflammation. Prolyl oligopeptidase (PREP) has been implicated in neuroinflammatory events in neurodegenerative diseases: PREP protein levels are increased in brain glial cells upon neuroinflammatory insults, but the circulating PREP activity levels are decreased in multiple sclerosis patients in a process probably mediated by bioactive peptides. In this work, we studied the variation of PREP levels upon liver failure and correlated it with several inflammatory markers to conclude on the relation of PREP with systemic and/or neuroinflammation.MethodsPREP enzymatic activity and protein levels measured with immunological techniques were determined in the brain and plasma of rats with portacaval shunt (PCS) and after treatment with ibuprofen. Those results were compared with the levels of PREP measured in plasma from cirrhotic patients with or without minimal hepatic encephalopathy (MHE). Levels of several pro-inflammatory cytokines and those of NO/cGMP homeostasis metabolites were measured in PCS rats and cirrhotic patients to conclude on the role of PREP in inflammation.ResultsIn PCA rats, we found that PREP levels are significantly increased in the hippocampus, striatum and cerebellum, that in the cerebellum the PREP increase was significantly found in the extracellular space and that the levels were restored to those measured in control rats after administration of an anti-inflammatory agent, ibuprofen. In cirrhotic patients, circulatory PREP activity was found to correlate to systemic and neuroinflammatory markers and had a negative correlation with the severity of the disease, although no clear relation to MHE.ConclusionsThese results support the idea that PREP levels could be used as indicators of cirrhosis severity in humans, and using other markers, it might contribute to assessing the level of neuroinflammation in those patients. This work reports, for the first time, that PREP is secreted to the extracellular space in the cerebellum most probably due to glial activation and supports the role of the peptidase in the inflammatory response.

GR3027 antagonizes GABAA receptor-potentiating neurosteroids and restores spatial learning and motor coordination in rats with chronic hyperammonemia and hepatic encephalopathy.

Hepatic encephalopathy (HE) is one of the primary complications of liver cirrhosis. Current treatments for HE, mainly directed to reduction of ammonia levels, are not effective enough because they cannot completely eliminate hyperammonemia and inflammation, which induce the neurological alterations. Studies in animal models show that overactivation of GABAA receptors is involved in cognitive and motor impairment in HE and that reducing this activation restores these functions. We have developed a new compound, GR3027, that selectively antagonizes the enhanced activation of GABAA receptors by neurosteroids such as allopregnanolone and 3α,21-dihydroxy-5α-pregnan-20-one (THDOC). This work aimed to assess whether GR3027 improves motor incoordination, spatial learning, and circadian rhythms of activity in rats with HE. GR3027 was administered subcutaneously to two main models of HE: rats with chronic hyperammonemia due to ammonia feeding and rats with portacaval shunts (PCS). Motor coordination was assessed in beam walking and spatial learning and memory in the Morris water maze and the radial maze. Circadian rhythms of ambulatory and vertical activity were also assessed. In both hyperammonemic and PCS rats, GR3027 restores motor coordination, spatial memory in the Morris water maze, and spatial learning in the radial maze. GR3027 also partially restores circadian rhythms of ambulatory and vertical activity in PCS rats. GR3027 is a novel approach to treatment of HE that would normalize neurological functions altered because of enhanced GABAergic tone, affording more complete normalization of cognitive and motor function than current treatments for HE.

P0215 : Metformin reduces hyperammonemia in portacaval shunted rats inhibiting glutaminase activity

P0215 : Metformin reduces hyperammonemia in portacaval shunted rats inhibiting glutaminase activity

Hepatocyte Transplants Improve Liver Function and Encephalopathy in Portacaval Shunted Rats

Rats with portacaval shunt (PCS) are useful experimental models of human hepatic encephalopathy in chronic liver dysfunction. We have previously shown that PCS modifies amine neurotransmitter systems in the CNS and increases voluntary alcohol intake by rats. Hepatocyte transplantation, used in acute liver failure, has recently also been applied to chronic liver diseases, which prompted us to investigate whether the altered brain amine system and the drinking behavior in long-term shunted rats could be normalized by hepatocyte transplants. Hepatocytes, isolated from syngeneic donors by collagenase digestion, were injected (3 × 10(6) cells/rat) into the pancreatic tail region, 6 months after PCS. Hepatic function was evaluated by measuring urine urea and plasma L-histidine concentrations. A free choice test with two bottles (tap water and 10% ethyl alcohol) was performed for 3 days to assess the rats' preference for alcohol. The rats were euthanized 2 months posttransplantation. Brain histamine and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured by radioenzymatic assay and by HPLC-EC, respectively, N-tele-methylhistamine by GC/MS while MAOA and MAOB activities by isotopic procedures. Portacaval shunt rats with hepatocyte transplants gave more urea than before transplantation, with lower plasma L-His levels and higher body weight versus the PCS counterparts. Also, those rats consumed less alcohol. The CNS amines and 5-HIAA concentrations, as well as MAO-B activity, being abnormally high in untreated PCS rats, significantly reduced after PCS hepatocyte treatment. The results support the therapeutic values of hepatocyte transplants in chronic liver diseases and the temporary character of PCS-exerted CNS dysfunctions.

Induction of systemic oxidative stress leads to brain oedema in portacaval shunted rats

The pathogenesis of hepatic encephalopathy (HE) is multifactorial and often associated with the development of brain oedema. In addition to ammonia playing a central role, systemic oxidative stress is believed to aggravate the neuropsychological effects of ammonia in patients with chronic liver disease (CLD). The aim of this study was to (i) induce systemic oxidative stress in hyperammonaemic portacaval anastomosed (PCA) rats by inhibiting the antioxidant glutathione using Dimethyl maleate (DEM) and (ii) investigate whether a synergistic relationship between ammonia and oxidative stress contributes to the pathogenesis of brain oedema in CLD. Four-week PCA and sham-operated rats received DEM (0.4-4 mg/kg/day) for the last 10 days before sacrifice when oxidative stress markers [reactive oxygen species (ROS) and malondialdehyde (MDA)] were assessed in blood and frontal cortex. Brain water content was measured using a specific gravimetric technique. Dimethyl maleate induced an increase in ROS and MDA in the blood, but not in the brain, of the PCA rats, compared with non-treated PCA rats. This was accompanied with an increase in brain water content (PCA+DEM: 78.45 ± 0.13% vs. PCA: 77.38 ± 0.11%, P < 0.001). Higher doses of DEM induced systemic oxidative stress in sham-operated controls, but brain oedema did not develop. Dimethyl maleate provoked systemic, not central, oxidative stress in PCA rats, resulting in the development of brain oedema. Independently, hyperammonaemia and systemic oxidative stress do not precipitate brain oedema; therefore, our findings sustain that a synergistic effect between hyperammonaemia and systemic oxidative stress is responsible for the development of brain oedema in HE.

Rats with minimal hepatic encephalopathy due to portacaval shunt show differential increase of translocator protein (18 kDa) binding in different brain areas, which is not affected by chronic MAP-kinase p38 inhibition

Neuroinflammation plays a main role in neurological deficits in rats with minimal hepatic encephalopathy (MHE) due to portacaval shunt (PCS). Treating PCS rats with SB239063, an inhibitor of MAP-kinase-p38, reduces microglial activation and brain inflammatory markers and restores cognitive and motor function. The translocator protein-(18-kDa) (TSPO) is considered a biomarker of neuroinflammation. TSPO is increased in brain of PCS rats and of cirrhotic patients that died in hepatic coma. Rats with MHE show strong microglial activation in cerebellum and milder in other areas when assessed by MHC-II immunohistochemistry. This work aims were assessing: 1) whether binding of TSPO ligands is selectively increased in cerebellum in PCS rats; 2) whether treatment with SB239063 reduces binding of TSPO ligands in PCS rats; 3) which cell type (microglia, astrocytes) increases TSPO expression. Quantitative autoradiography was used to assess TSPO-selective (3)H-(R)-PK11195 binding to different brain areas. TSPO expression increased differentially in PCS rats, reaching mild expression in striatum or thalamus and very high levels in cerebellum. TSPO was expressed in astrocytes and microglia. Treatment with SB239063 did not reduces (3)[H]-PK11195 binding in PCS rats. SB239063 reduces microglial activation and levels of inflammatory markers, but not binding of TSPO ligands. This indicates that SB239063-induced neuroinflammation reduction in PCS rats is not mediated by effects on TSPO. Also, enhanced TSPO expression is not always associated with cognitive or motor deficits. If enhanced TSPO expression plays a role in mechanisms leading to neurological alterations in MHE, SB239063 would interfere these mechanisms at a later step.

Reduced clearance of proteins labeled with diisopropylfluorophosphate in portacaval-shunted rats

Portacaval shunting is a model for hepatic encephalopathy that causes chronic hyperammonemia, disruption of metabolic, signaling, and neurotransmitter systems, and progressive morphological changes. Exposure of cultured cells to ammonia raises intralysosomal pH and inhibits proteolysis, and the present study tested the hypothesis that proteolytic capacity is diminished in portacaval-shunted rats. Proteins were labeled in vivo with tracer doses of diisopropylfluorophosphate (DFP) and clearance of label was assayed. This approach labeled proteins independent of protein synthesis, which is reported to be altered in shunted rats, and avoided complications arising from re-utilization of labeled amino acids that causes underestimation of degradation rate. Characterization of DFP labeling showed that protein labeling was fast, about 50% of the label was released during a 24 h interval, labeling by DFP metabolites was negligible, inhibition of brain acetylcholinesterase was not detectable, and labeling by [(3)H]- and [(14)C]DFP was equivalent. To assay degradative capacity, proteins were first labeled with [(3)H]DFP, followed by labeling with [(14)C]DFP that was given 24 or 72 h later. The (3)H/(14)C ratio in each animal was used as a relative measure of removal of (3)H-labeled proteins. (3)H/(14)C ratios were generally significantly higher in portacaval-shunted rats than in controls, consistent with reduced proteolytic capacity. Assays of amino acid incorporation into brain protein generally replicated literature reports, supporting the conclusion that protein synthesis unlikely to be markedly inhibited and amino acid recycling influences calculated protein synthesis rates in shunted rats. Therapeutic strategies to reduce ammonia level would help normalize lysosomal functions and protein and lipid turnover.

Cerebral oedema is not responsible for motor or cognitive deficits in rats with hepatic encephalopathy

Low-grade cytotoxic oedema is considered a main contributor to the neurological (motor and cognitive) alterations in patients with hepatic encephalopathy (HE). This assumption is mainly based on studies with cultured astrocytes treated with very large ammonia concentrations or with animal models of acute liver failure with strong HE. However, the possible contribution of cerebral oedema (vasogenic or cytotoxic) to cognitive or motor alterations in chronic mild HE has not been demonstrated. The aim of this work was to assess whether cerebral oedema contributes to cognitive and/or motor alterations in rats with chronic mild HE. Motor activity and coordination and different types of learning and memory were assessed in rats with porta-caval shunts (PCS). Brain oedema was assessed by gravimetry in cerebellum and cortex and apparent diffusion coefficient (ADC) by magnetic resonance in 16 areas. Four weeks after surgery, PCS rats show reduced motor activity and coordination, impaired ability to learn a conditional discrimination task in the Y maze and reduced spatial memory in the Morris water maze. PCS rats did not show increased brain water content at 4 or 10 weeks or changes in ADC at 4 weeks. At 10 weeks, increased ADC in some areas is compatible with vasogenic but not cytotoxic oedema. Cerebral oedema is not involved in motor and cognitive alterations in rats (and likely in humans) with mild HE. Proper understanding of the mechanisms responsible for the neurological alterations in HE is necessary to design efficient treatments.

Progressive reduction of sleep time and quality in rats with hepatic encephalopathy caused by portacaval shunts

Patients with liver cirrhosis show sleep disturbances. Insight into their relationship with hepatic encephalopathy (HE) can be obtained using animal models of HE. The aims of this work were to assess (1) whether rats with portacaval shunts (PCS), a model of HE, show alterations in sleep and if they are similar to those in patients with HE; (2) Whether hyperammonemia plays a role in these sleep alterations; and (3) the time course of sleep alterations in these animal models. Rats were subjected to PCS to induce HE. Another group of rats was fed an ammonium-containing diet to induce hyperammonemia. Polysomnographic recordings were acquired for 24 h and sleep architecture was analyzed in control, PCS, and hyperammonemic rats at 4, 7, and 11 weeks after surgery or diet, respectively. PCS rats show a significant reduction in rapid eye movement (REM) and non-rapid eye movement (NREM) sleep time and increased sleep fragmentation, whereas reduced sleep occurs at 4 weeks and worsens at 7 and 11 weeks, sleep fragmentation appears at 7 weeks and worsens at 11 weeks. Hyperammonemic rats show decreased REM sleep, starting at 7 weeks and worsening at 11 weeks, with no changes in NREM sleep or sleep fragmentation. Therefore, PCS rats are a good model to study sleep alterations in HE, their mechanisms, and potential treatment. Mild hyperammonemia mainly impacts mechanisms involved in REM generation and/or maintenance but does not seem to be involved in sleep fragmentation.

P38 MAP kinase is a therapeutic target for hepatic encephalopathy in rats with portacaval shunts

ObjectiveInflammation plays a role in neurological alterations in patients with hepatic encephalopathy (HE). Animal models of HE show neuroinflammation. Treatment with ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), reduces neuroinflammation and...

Brain biomolecules oxidation in portacaval-shunted rats

Oxidative stress induced by a high ammonia concentration has been suggested to be implicated in the pathophysiology of hepatic encephalopathy (HE). Therefore, oxidative damage of brain biomolecules could contribute towards explaining the neurological and motor alterations observed in HE. Portacaval-shunted (PCS) rats (n = 5) were used as an animal model of chronic HE. Plasma and brain ammonia were measured by the l-glutamate dehydrogenase method. Reactive oxygen species was measured by the dichlorodihydrofluorescein diacetate method. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) by a colorimetric method; malondialdehyde (MDA) and 4-hydroxy-2-noneal (HNE) were measured by HPLC and an immunological method respectively. Protein oxidation (carbonylation) was measured as total carbonyl after labelling with 2,4-dinitrophenyl hydrazine (DNPH) using a spectrophotometric method. Individual protein oxidation was studied, after labelling with DNPH and its separation by one-dimensional (1D) electrophoresis, by an immunological method. Ammonia-induced oxidative stress in PCS rats was associated with increased MDA and HNE, together with increased protein oxidation, evidenced by total carbonyl quantification and by the analysis of individual protein bands separated by 1D electrophoresis. However, lipid peroxidation measured as TBARS did not show differences. Our data show an increased evidence of oxidative stress in PCS rat brain; moreover, PCS rat brain proteins are oxidized (carbonylated), some proteins being more sensitive to oxidation than others. These data also show that at least six specific brain proteins in PCS rats are highly sensitive to carbonylation. Identification of these proteins may be crucial for a better understanding of HE pathophysiology.

Portacaval shunting attenuates portal hypertension and systemic hypotension in rat anaphylactic shock

Anaphylactic shock in rats is characterized by antigen-induced hepatic venoconstriction and the resultant portal hypertension. We determined the role of portal hypertension in anaphylactic hypotension by using the side-to-side portacaval shunt- and sham-operated rats sensitized with ovalbumin (1mg). We measured the mean arterial blood pressure (MAP), portal venous pressure (PVP), and central venous pressure (CVP) under pentobarbital anesthesia and spontaneous breathing. Anaphylactic hypotension was induced by an intravenous injection of ovalbumin (0.6mg). In sham rats, the antigen caused not only an increase in PVP from 11.3cmH(2)O to the peak of 27.9cmH(2)O but also a decrease in MAP from 103mmHg to the lowest value of 41mmHg. CVP also decreased significantly after the antigen. In the portacaval shunt rats, in response to the antigen, PVP increased slightly, but significantly, to the peak of 17.5cmH(2)O, CVP did not decrease, and MAP decreased to a lesser degree with the lowest value being 60mmHg. These results suggest that the portacaval shunt attenuated anaphylactic portal hypertension and venous return decrease, partially preventing anaphylactic hypotension. In conclusion, portal hypertension is involved in rat anaphylactic hypotension presumably via splanchnic congestion resulting in decreased venous return and thus systemic arterial hypotension.

Neuroinflammation contributes to hypokinesia in rats with hepatic encephalopathy: Ibuprofen restores its motor activity

Patients with hepatic encephalopathy show altered motor function, psychomotor slowing, and hypokinesia, which are reproduced in rats with portacaval shunts (PCS). Increased extracellular glutamate in substantia nigra pars reticulata (SNr) is responsible for hypokinesia in PCS rats. The mechanisms by which liver failure leads to increased extracellular glutamate in SNr remain unclear. Inflammation seems to act synergistically with hyperammonemia to induce neurological alterations in hepatic encephalopathy. It is therefore possible that inflammation-associated alterations may contribute to motor alterations in hepatic encephalopathy. The aim of this work was to assess whether treatment with an antiinflammatory, ibuprofen, is able to normalize extracellular glutamate in SNr and/or to improve hypokinesia in PCS rats. The amounts of the glutamate transporters GLT-1 and EAAC-1 are reduced by 26% and 32%, respectively, in SNr of PCS rats. This reduction is associated with a tenfold increase in extracellular glutamate in SNr and a reduction in motor activity. Chronic treatment with 30 mg/kg ibuprofen completely normalizes the amount of GLT-1 and EAAC-1 and significantly reduces (by 53%) extracellular glutamate in SNr of PCS rats. Moreover, ibuprofen, at 15 or 30 (but not at 5) mg/kg/day, completely eliminates hypokinesia, restoring normal motor activity. This supports the idea that inflammation is a main contributor to the induction of hypokinesia in hepatic encephalopathy. Moreover, these data point to the possible therapeutic utility of decreasing inflammation, by safe procedures, in the treatment of the motor deficits in patients with hepatic encephalopathy.