Dysfunction In Hepatic Encephalopathy Research Articles

Portosystemic shunt placement reveals blood signatures for the development of hepatic encephalopathy through mass spectrometry

Elective transjugular intrahepatic portosystemic shunt (TIPS) placement can worsen cognitive dysfunction in hepatic encephalopathy (HE) patients due to toxins, including possible microbial metabolites, entering the systemic circulation. We conducted untargeted metabolomics on a prospective cohort of 22 patients with cirrhosis undergoing elective TIPS placement and followed them up to one year post TIPS for HE development. Here we suggest that pre-existing intrahepatic shunting predicts HE severity post-TIPS. Bile acid levels decrease in the peripheral vein post-TIPS, and the abundances of three specific conjugated di- and tri-hydroxylated bile acids are inversely correlated with HE grade. Bilirubins and glycerophosphocholines undergo chemical modifications pre- to post-TIPS and based on HE grade. Our results suggest that TIPS-induced metabolome changes can impact HE development, and that pre-existing intrahepatic shunting could be used to predict HE severity post-TIPS.

Mitochondrial Changes in Rat Brain Endothelial Cells Associated with Hepatic Encephalopathy: Relation to the Blood–Brain Barrier Dysfunction

The mechanisms underlying cerebral vascular dysfunction and edema during hepatic encephalopathy (HE) are unclear. Blood–brain barrier (BBB) impairment, resulting from increased vascular permeability, has been reported in acute and chronic HE. Mitochondrial dysfunction is a well-documented result of HE mainly affecting astrocytes, but much less so in the BBB-forming endothelial cells. Here we review literature reports and own experimental data obtained in HE models emphasizing alterations in mitochondrial dynamics and function as a possible contributor to the status of brain endothelial cell mitochondria in HE. Own studies on the expression of the mitochondrial fusion-fission controlling genes rendered HE animal model-dependent effects: increase of mitochondrial fusion controlling genes opa1, mfn1 in cerebral vessels in ammonium acetate-induced hyperammonemia, but a decrease of the two former genes and increase of fis1 in vessels in thioacetamide-induced HE. In endothelial cell line (RBE4) after 24 h ammonia and/or TNFα treatment, conditions mimicking crucial aspects of HE in vivo, we observed altered expression of mitochondrial fission/fusion genes: a decrease of opa1, mfn1, and, increase of the fission related fis1 gene. The effect in vitro was paralleled by the generation of reactive oxygen species, decreased total antioxidant capacity, decreased mitochondrial membrane potential, as well as increased permeability of RBE4 cell monolayer to fluorescein isothiocyanate dextran. Electron microscopy documented enlarged mitochondria in the brain endothelial cells of rats in both in vivo models. Collectively, the here observed alterations of cerebral endothelial mitochondria are indicative of their fission, and decreased potential of endothelial mitochondria are likely to contribute to BBB dysfunction in HE.

Hyperammonaemia induces mitochondrial dysfunction and neuronal cell death

Background & AimsIn cirrhosis, astrocytic swelling is believed to be the principal mechanism of ammonia neurotoxicity leading to hepatic encephalopathy (HE). The role of neuronal dysfunction in HE is not clear. We aimed to explore the impact of hyperammonaemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging.MethodsTo primary cocultures of astrocytes and neurons, low concentrations (1 and 5 μM) of NH4Cl were applied. In rats with bile duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonaemia and minimal HE, acute brain slices were studied. One group of BDL rats was treated twice daily with the ammonia scavenger ornithine phenylacetate (OP; 0.3 g/kg). Fluorescence measurements of changes in mitochondrial membrane potential (Δψm), cytosolic and mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LP) rates, and cell viability were performed using confocal microscopy.ResultsNeuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction, and reduced cell viability (27.8 ± 2.3% and 41.5 ± 3.7%, respectively) compared with untreated cultures (15.7 ± 1.0%, both p <0.0001). BDL led to increased cerebral LP (p = 0.0003) and cytosolic ROS generation (p <0.0001), which was restored by OP (both p <0.0001). Mitochondrial function was severely compromised in BDL, resulting in hyperpolarisation of Δψm with consequent overconsumption of adenosine triphosphate and augmentation of mitochondrial ROS production. Administration of OP restored Δψm. In BDL animals, neuronal loss was observed in hippocampal areas, which was partially prevented by OP.ConclusionsOur results elucidate that low-grade hyperammonaemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonaemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development.Lay summaryThe impact of hyperammonaemia, a common finding in patients with liver cirrhosis, on brain mitochondrial function was investigated in this study. The results show that ammonia in concentrations commonly seen in patients induces severe mitochondrial dysfunction, overproduction of damaging oxygen molecules, and profound injury and death of neurons in rat brain cells. These findings point towards a novel mechanism of ammonia-induced brain injury in liver failure and potential novel therapeutic targets.

Long Noncoding RNAs Regulate Hyperammonemia-Induced Neuronal Damage in Hepatic Encephalopathy

Background Hyperammonemia can result in various neuropathologies, including sleep disturbance, memory loss, and motor dysfunction in hepatic encephalopathy. Long noncoding RNA (lncRNA) as a group of noncoding RNA longer than 200 nucleotides is emerging as a promising therapeutic target to treat diverse diseases. Although lncRNAs have been linked to the pathogenesis of various diseases, their function in hepatic encephalopathy has not yet been elucidated. Research Design and Methods. To identify the roles of lncRNAs in hepatic encephalopathy brain, we used a bile duct ligation (BDL) mouse model and examined the alteration of neuronal cell death markers and neuronal structure-related proteins in BDL mouse cortex tissue. Furthermore, analysis of the transcriptome of BDL mouse brain cortex tissues revealed several lncRNAs critical to the apoptosis and neuronal structural changes associated with hepatic encephalopathy. Results We confirmed the roles of the lncRNAs, ZFAS1, and GAS5 as strong candidate lncRNAs to regulate neuropathologies in hepatic encephalopathy. Our data revealed the roles of lncRNAs, ZFAS1, and GAS5, on neuronal cell death and neural structure in hyperammonemia in in vivo and in vitro conditions. Conclusion Thus, we suggest that the modulation of these lncRNAs may be beneficial for the treatment of hepatic encephalopathy.

Hyperammonemia-induced changes in the cerebral transcriptome and proteome

Molecular alterations underlying cerebral impairment in hyperammonemic disorders such as in hepatic encephalopathy (HE) are only poorly understood. Using transcriptomics and proteomics on brains of mice with systemic hyperammonemia resulting from knockout of hepatic glutamine synthetase (LGS-KO) we identified up to 214 genes and 34 proteins whose expressions were altered in brains of LGS-KO mice in a brain region-specific way. Differentially expressed genes were enriched for those related to oxidative stress, cell proliferation, heme metabolism and others. Due to their particularly high expression changes, coactivator associated arginine methyltransferase 1 (CARM1), TROVE2 and Lipocalin-2 (LCN2) were selected for further analyses. All selected candidates were expressed by astrocytes in rodent brain and challenging cultured astrocytes with NH4Cl changed their protein and mRNA levels similar to what was found in brains of LGS-KO mice. Further functional analyses suggested a role of CARM1 for senescence, TROVE2 for RNA quality control and LCN2 for disturbed iron homeostasis in ammonia-exposed astrocytes. LCN2 protein and Trove2 mRNA were also elevated in cerebral cortex of ammonium acetate-challenged rats and in post mortem brain tissue from patients with liver cirrhosis and HE, respectively. This study identified new molecular players potentially relevant for cerebral dysfunction in HE.

Association between serum albumin and cognitive dysfunction in hepatic encephalopathy: An exploratory data analysis.

Background and AimBecause covert hepatic encephalopathy (CHE) has been shown to affect the prognosis of cirrhotic patients, early diagnosis of hepatic encephalopathy (HE) is a prerequisite for the preservation of patients' quality of life and for prophylaxis of overt HE. The aim of this study was to identify a clinical parameter to predict impairment of cognitive function in cirrhotic patients with early‐stage HE.MethodsWe investigated the data from 172 patients with cirrhotic or idiopathic portosystemic shunt (PSS) in phase II/III trials of rifaximin in Japan. Classification and regression trees (CARTs) were constructed to identify clinical profiles related to cognitive dysfunction as indicated by the prolongation of time required for the Number Connection Test (NCT‐B).ResultsCART analysis detected age 65 years as the variable for the initial split, and serum albumin level was selected as the variable for the second split among patients aged ≤65 years. In 27 cirrhotic patients aged ≤65 years without PSS, receiver operating characteristic curve analysis revealed that the optimal albumin level cutoff point was 3.05 g/dL, and the area under the curve was 0.80 for the prolongation of NCT‐B time, which was higher than that of the branched‐chain amino acids‐to‐tyrosine ratio (0.46), the prothrombin time–international normalized ratio (PT‐INR) (0.68), serum ammonia (0.61), and total bilirubin (0.69).ConclusionsLower serum albumin level as a clinical biomarker associated with impaired cognitive function may be available as a screening examination for early‐stage HE in cirrhotic patients aged ≤65 years without PSS before undergoing neuropsychological tests.

Contributions of mitochondrial dysfunction to baroreflex and cardiac dysregulation in hepatic encephalopathy

Hepatic encephalopathy (HE) is a common neurocognitive complication of acute and chronic liver failure and is associated with 50–90% mortality without liver transplantation. Despite a highly challenging clinical problem, systematic evaluations of the cellular mechanisms of HE‐related mortality are still lacking. Clinical studies showed that the degree of baroreflex dysregulation is related to the severity of HE. At the cellular level, oxidative stress resulting from mitochondrial dysfunction in brain stem nuclei in the baroreflex circuit, including nucleus tractus solitarii (NTS) and rostral ventrolateral medulla (RVLM), and in the heart are known to result baroreflex dysregulation and cardiac dysfunction. This study investigated whether the same mechanism underlies the high mortality in HE. An azoxymethane (AOM)‐induced acute liver failure model of HE employing C57BL/6 mouse was used. Diffusion tensor imaging (DTI) or magnetic resonance spectroscopy (MRS) of the brain stem was performed, together with blood pressure, heart rate and indices of baroreflex recorded by radiotelemetry. Animals died within 24–30 h after AOM (100 μg/g, ip) injection. DTI further revealed that the connectivity between the NTS and nucleus ambiguus (NA), the origin of the vagal innervation of the heart, was progressively disrupted though sustained, concurrent with impaired but persistent cardiac vagal baroreflex. On the other hand, the connectivity between NTS and RVLM was progressively disrupted until its disappearance, coincidental with the abolition of baroreflex‐mediated sympathetic vasomotor tone that signifies brain death clinically. Results from 31P MRS obtained from mouse brain stem showed reduced ratio between phosphocreatine and γATP, indicating mitochondrial dysfunction in HE. Furthermore, there was a decrease in cardiac contractility, alongside an elevation of reactive oxygen species and necrotic cell death and a reduction in ATP level in the NTS or RVLM. Our results showed that impairment of baroreflex and cardiac function takes place during the progression towards death in HE, and bioenergetic failure, oxidative stress and necrotic cell death are the culprits.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cognitive reserve is a resilience factor for cognitive dysfunction in hepatic encephalopathy.

Cognitive Reserve (CR) modulates symptoms of brain disease. The aim of this study was: to evaluate the effect of CR on cognition in cirrhosis and on the mismatch between cognitive and neurophysiologic assessment of hepatic encephalopathy (HE). Eighty-two outpatient patients with cirrhosis without overt HE were studied [73% males; age: 62 (54-68) (median, interq. range) yrs.; education: 8 (6-13) yrs.]. The Psychometric Hepatic Encephalopathy Score (PHES) was used as cognitive measure of HE. The spectral analysis of the electroencephalogram (EEG) was used as neurophysiologic measure of HE. The CR was assessed by the CR Index (CRI), which was measured by the CRI questionnaire (CRIq) ( http://cri.psy.unipd.it ). The PHES was altered in 28% of patients and the EEG in 41%. Altered PHES was related to the severity of cirrhosis as assessed by Child-Pugh classification (R=0.31, p<0.005). Patients with maintained PHES had higher CRI than those with altered PHES (CRI=100±20 vs. 88±12 vs., p<0.01), but not the ones with normal EEG compared to those with abnormal EEG (CRI=96±17 vs. 98±17 vs. p: n.s.).The PHES, but not the EEG, was found to be related to the CRI (r=0.35, p<0.01). The mismatch between cognitive and neurophysiologic evaluation of non-overt HE (the ratio between PHES and the mean dominant frequency -MDF- of the EEG i.e., cognitive performance normalized by EEG speed) was found to be correlated to the CRI (r=0.36, p<0.005). CR is a resilience factor for cognitive dysfunction in cirrhosis, and is easily measurable by CRIq.

Ammonia mediates cortical hemichannel dysfunction in rodent models of chronic liver disease.

The pathogenesis of hepatic encephalopathy (HE) in cirrhosis is multifactorial and ammonia is thought to play a key role. Astroglial dysfunction is known to be present in HE. Astrocytes are extensively connected by gap junctions formed of connexins, which also exist as functional hemichannels allowing exchange of molecules between the cytoplasm and the extracellular milieu. The astrocyte‐neuron lactate shuttle hypothesis suggests that neuronal activity is fueled (at least in part) by lactate provided by neighboring astrocytes. We hypothesized that in HE, astroglial dysfunction could impair metabolic communication between astrocytes and neurons. In this study, we determined whether hyperammonemia leads to hemichannel dysfunction and impairs lactate transport in the cerebral cortex using rat models of HE (bile duct ligation [BDL] and induced hyperammonemia) and also evaluated the effect of ammonia‐lowering treatment (ornithine phenylacetate [OP]). Plasma ammonia concentration in BDL rats was significantly reduced by OP treatment. Biosensor recordings demonstrated that HE is associated with a significant reduction in both tonic and hypoxia‐induced lactate release in the cerebral cortex, which was normalized by OP treatment. Cortical dye loading experiments revealed hemichannel dysfunction in HE with improvement following OP treatment, while the expression of key connexins was unaffected. Conclusion: The results of the present study demonstrate that HE is associated with central nervous system hemichannel dysfunction, with ammonia playing a key role. The data provide evidence of a potential neuronal energy deficit due to impaired hemichannel‐mediated lactate transport between astrocytes and neurons as a possible mechanism underlying pathogenesis of HE. (Hepatology 2017;65:1306‐1318)

Ammonia Mediates Cortical Hemichannel Dysfunction in Rodent Models of Chronic Liver Disease

The pathogenesis of hepatic encephalopathy (HE) in cirrhosis is multifactorial and ammonia is thought to play a key role. Astroglial dysfunction is known to be present in HE. Astrocytes are extensively connected by gap junctions formed of connexins, which also exist as functional hemichannels allowing exchange of molecules between the cytoplasm and the extracellular milieu. The astrocyte-neuron lactate shuttle hypothesis suggests that neuronal activity is fuelled (at least in part) by lactate provided by neighbouring astrocytes. We hypothesised that in HE, astroglial dysfunction could impair metabolic communication between astrocytes and neurons. In this study we determined whether hyperammonemia leads to hemichannel dysfunction and impairs lactate transport in the cerebral cortex using rat models of HE (bile duct ligation [BDL] and induced-hyperammonemia [HA]) and also evaluated the effect of ammonia-lowering treatment (ornithine phenylacetate, OP). Plasma ammonia concentration in BDL rats was indeed significantly reduced by OP treatment. Biosensor recordings demonstrated that HE is associated with a significant reduction in both tonic and hypoxia-induced lactate release in the cerebral cortex, which was normalized by OP treatment. Cortical dye loading experiments revealed hemichannel dysfunction in HE with improvement following OP treatment, while the expression of key connexins was unaffected. Conclusion. The results of the present study demonstrate that HE is associated with CNS hemichannel dysfunction, with ammonia playing a key role. The data provide evidence of a potential neuronal energy deficit due to impaired hemichannel-mediated lactate transport between astrocytes and neurons as a possible mechanism underlying pathogenesis of HE. This article is protected by copyright. All rights reserved.

Mechanisms of hepatic encephalopathy and thiamine deficiency.

Hepatic encephalopathy (HE) and thiamine deficiency (TD) represent two disorders in which metabolic derangements play a major role in their pathophysiology. They also define the areas of primary focus of Dr. Roger F. Butterworth, Ph.D., D.Sc. who retired from the University of Montreal in the summer of 2013. This special issue of Metabolic Brain Disease commemorates his valuable contribution to the study of these maladies. Although HE is considered a metabolic disorder, new evidence is emerging to show that it has a molecular basis as well. Vemuganti et al reports altered cerebral profiles of microRNAs after acute liver failure (ALF) that likely contribute in a significant way to gene expression changes associated with the ensuing HE. Disrupted ammonia detoxification is considered a major precipitator of neurological dysfunction in HE, and current understanding of the contribution of ammonia in liver diseases is discussed in an article by Ott and Vilstrup, while the effects of hyperammonemia on cerebral metabolic function are focussed on in an article by Schousboe and colleagues. In addition, impaired brain energy metabolism is a serious complicating feature of liver failure, with lactate production being a major consequence of this problem. In relation to this, the role of increased lactate accumulation and its pathogenetic basis in HE is discussed by Bosoi and Rose. Also, a major and often lethal complication of ALF is brain edema, and the significance of this edema and its underlying basis in ALF-induced HE is examined by Rama Rao et al, while the usefulness of magnetic resonance imaging as a tool in both acute and chronic liver failure is considered by Chavarria and Cordoba. Minimal hepatic encephalopathy (MHE) represents the mildest of the spectrum of HE forms in which patients do not display clinical symptoms but have mild cognitive and psychomotor deficits. It is also associated with poor survival rates. Two papers examine aspects of MHE, in which cognitive impairment and the issue of cognitive variability as a contributing factor in these patients is a focus of attention in Bisiacchi et al, and in an experimental model of chronic liver failure in which focal increases in the binding of translocator (18kD) protein in brain is reported by Agusti et al. Increasingly, the phenomenon of acute-on-chronic liver failure, in which acute deterioration of liver function in patients with cirrhosis is a consequence, is being considered a distinct clinical entity. The subject is discussed in Wright et al. In addition, considerable evidence has shown that astrocyte dysfunction plays a major role in HE. Karabara and colleagues explore the potential role of GlcNAcylation in these cells, while the protective effect of inhibition of glutamine synthesis is examined in neurological diseases, including HE, by Jeitner and Cooper. Development of well-defined biomarkers and improved therapeutic targets represents an important subject regarding HE. In this regard, the paper by Cooper and Kuhara shows that α-ketoglutaramate is a good marker of HE, and the paper by Mondal and Trigun demonstrates that pannexin-1 can be a marker as well as a target for HE. Although the mechanism(s) underlying the beneficial effects of rifaximin in HE remain largely unknown, the topic is tackled using multi-modal MRI in Ahluwalia et al. In addition, the subject of microglial proliferation and its significance in alcoholics with HE is explored experimentally in Dennis et al. Dienel and Cruz address the subject of decreased proteolysis as a result of hyperammonemia due to liver failure, and the effects of thioacetamide-induced ALF on citrulline uptake is examined in a study by Zielinska et al. Finally, Roger’s interest in the pathophysiology of TD has been long and enduring. A number of papers in this special issue deal with different aspects of TD. Afadlal et al shows that astrocytes play a significant role in TD-induced encephalopathy, while Bettendorff explores the role of thiamine triphosphate in cells. In addition, since mitochondrial dysfunction is an important aspect of both TD and Alzheimer’s disease in which thiamine-dependent enzyme activity is reduced, Huang et al explores how cellular calcium stores are affected in a fibroblast cell line, an important first step towards the eventual determination of how this process comes about in the AD patient and development of protective/treatment strategies targeting this mechanism, which may also have relevance for the future treatment of TD.

Protein oxidative damage in the hippocampus in a mouse model of acute hyperammonemia

Hepatic encephalopathy (HE) is a common neuropsychiatric complication of both chronic and acute liver failure that is characterized by psychomotor, intellectual and cognitive impairment [1]. The mechanism of neuronal dysfunction in HE has not yet been conclusively investigated and possibly implies numerous pathological processes. It is widely accepted that among the factors contributing to HE, hyperammonemia, due to ammonia dysmetabolism and inflammation as a response to infection, play a major role in the pathogenesis. A growing body of evidence, e.g. RNA oxidation and protein nitrosylation, further indicate that oxidative stress occurs in neural cells as a result of ammonia intoxication and may lead to subsequent tissue damage [2,3]. Particular interest of this study was to elucidate the effect of an acute ammonia load on protein homeostasis in the hippocampus, the brain area of learning, memory formation and consolidation. To this end we analysed the accumulation of oxidized proteins, protein ubiquitination, proteasome activity and expression of immunoproteasome subunits after a single intraperitoneal injection of NH4Ac (10 mmol/kg body weight) in 6 weeks old C57B/6 mice. Following ammonia administration the animals fall into coma with a survival rate of 10%. After approximately one hour of coma, the motor ability and reflexes were restored. To analyse whether ammonia intoxication has a delayed effect on CNS tissue, animals were sacrificed 24 hours post injection, perfused with PBS and hippocampi were dissected. Western blot detection of protein carbonylation by OxiBlot technique revealed a significant accumulation of oxidized proteins in the hippocampus of animals with ammonia-precipitated encephalopathy compared to the saline-treated control group. Involvement of oxidative stress in disturbed protein homeostasis was further supported by detection of increased expression of the antioxidant enzymes hemeoxygenase 1 (HO-1) and thioredoxin 1 (Trx1). Similar to the animal model used in this study, up-regulation of HO-1 has been previously described in CNS tissue of patients with HE [4]. Moreover we found that protein oxidation was associated with elevated levels of ubiquitinated proteins and activation of proteasome (chemotrypsin-like) activity, representing the major cellular machinery for degradation of misfolded or damaged proteins. These data indicate that the ubiquitin-proteasome system may play an important role in the cellular response to ammonia-induced protein damage. To analyse whether hyperammonemia has a direct effect on neural cells, in an in vitro approach we exposed mixed glial cultures and the BV2 microglial cell line to 5 mM NH4Cl. Here, comparable to our findings in animals with ammonia-induced encephalopathy, 24 hours treatment increased the total amount of oxidized and ubiquitinated proteins, proteasome activity and expression of antioxidant enzymes. Notably, ammonia-induced protein damage was associated with a diminished functional role of microglia in terms of myelin debris clearance as it was observed using a myelin phagocytosis assay. Treatment with the antioxidant compound N-acetylcysteine rescued the effect of ammonia on impaired myelin engulfment and accumulation of ubiquitinated proteins. Taken together, our data show that ammonia intoxication induces protein oxidation in hippocampal tissue. This result corresponds to findings of other groups indicating impaired long-term potentiation in the hippocampus of animals with hyperammonemia and impaired cognitive function. We observed that an alteration of the protein homeostasis may persist longer after initial onset of neurological deterioration and thus, may contribute to a delayed effect of ammonia toxicity on neuronal activity. Upregulation of anti-oxidant enzymes in both mouse experimental hyperammonemia and patients with HE indicates that similar oxidative processes may underlie the CNS tissue damage. Induction of proteasome activity observed in this study may have a crucial role in the cellular adaptation in response to disturbed protein homeostasis. Moreover, our data indicate that hyperammonemia alone is able to affect microglia (the brain’s innate immune cells) and may have a negative impact on homeostatic and/or inflammatory function of microglia during the course of hepatic encephalopathy.

Decrease of glutathione content in the prefrontal cortical mitochondria of rats with acute hepatic encephalopathy: prevention by histidine

Mitochondrial glutathione (mGSH) is a critical factor in the cell defense against oxidative and nitrosative stress (ONS), and ONS is a key pathogenic event in hepatic encephalopathy (HE). Acute HE in the thioacetamide (TAA) model caused a 54% decrease of mGSH content in the rat prefrontal cortex (pfc), but not in the striatum (str), nor did it affect the GSH content in the pfc or str homogenate. In the pfc, treatment with L- histidine (His), which is known to alleviate ONS-related symptoms in HE animals, attenuated the decrease of mGSH, and increased the GSH content in pfc and str homogenates and pfc microdialysates of control animals. His increased the expression of mRNA coding for the GSH synthesizing enzyme, glutamate cysteine ligase (GCL) and decreased that of the GSH-degrading enzyme γ-glutamyltranspeptidase (γGT). The results suggest that the decrease of mGSH may be an important contributing factor to mitochondrial dysfunction in HE, and delineate a new mechanistic aspect of the therapeutic potential of His in HE.

Down‐regulation of Kir4.1 in the cerebral cortex of rats with liver failure and in cultured astrocytes treated with glutamine: Implications for astrocytic dysfunction in hepatic encephalopathy

Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na(+) -K(+) -2Cl(-) cotransporter-1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide-induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D-[(3) H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT-1 mRNA. Both Gln and ammonia decreased hypoosmolarity-induced (86) Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down-regulation of Kir4.1 may mediate distinct aspects of Gln-induced astrocytic dysfunction in HE.

Stimulation of natriuretic peptide receptor C attenuates accumulation of reactive oxygen species and nitric oxide synthesis in ammonia‐treated astrocytes

Oxidative and nitrosative stress contribute to ammonia-induced astrocytic dysfunction in hepatic encephalopathy. Treatment of cultured astrocytes with 5 mmol/L ammonium chloride ('ammonia') increased the production of reactive oxygen species (ROS), including the toxic NADPH oxidase reaction product, •O(2)(-). Atrial natriuretic peptide (ANP), natriuretic peptide C and a selective natriuretic peptide receptor (NPR)-C ligand, cANP((4-23),) each decreased the total ROS content both in control cells and cells treated with ammonia. However, attenuation of •O(2)(-) accumulation by ANP and cANP((4-23),) was observed in ammonia-treated cells only and the effect of cANP((4-23)) was decreased when the NADPH oxidase-regulatory protein G(iα-2) was blocked with a specific anti-G(iα-2) antibody. Although in contrast to ANP, cANP((4-23)) did not elevate the cGMP content in control astrocytes, it decreased cAMP content and reduced the expression of G(iα-2), the NADPH oxidase-regulatory protein. The results show the presence of functional NPR-C in astrocytes, activation of which (i) attenuates basal ROS production, and (ii) prevents excessive accumulation of the toxic ROS species, •O(2)(-) by ammonia. Ammonia, ANP and cANP((4-23)) added separately, each stimulated formation of NO(x) (nitrates + nitrites) which was associated with up-regulation of the activity [cANP((4-23))] or/and expression (ammonia) of the endothelial isoform of nitric oxide synthase. However, the ammonia-induced increase of NO(x) was not augmented by co-addition of ANP, and was reduced to the control level by co-addition of cANP((4-23)) , indicating that activation of NPR-C may also reduce nitrosative stress. Future hepatic encephalopathy therapy might include the use of cANP((4-23)) or other NPR-C agonists to control oxidative/nitrosative stress induced by ammonia.

Attention, Memory, and Cognitive Function in Hepatic Encephalopathy

Deficits in attention and arousal play a major role in the clinical presentation of hepatic encephalopathy. Attention deficits are also the main components of minimal hepatic encephalopathy. The present paper summarizes some findings about attentional and memory dysfunction in hepatic encephalopathy, with reference to basic knowledge about normal attention and memory function and their cerebral representation.

Neurotransmitter dysfunction in hepatic encephalopathy: new approaches and new findings.

Hepatic Encephalopathy (HE) is a serious neuropsychiatric condition of both acute and chronic liver failure. Acute liver failure is characterized by rapid evolution of HE and by brain edema. Portal-Systemic encephalopathy (PSE) is particularly prevalent following treatment of portal hypertension or ascites by the TIPS procedure. Available evidence currently suggests that neurotransmission changes rather than brain energy failure are the primary cause of HE. Recent studies both in autopsied brain tissue from HE patients as well as in experimental animal models of HE reveal that liver failure results in altered expression of several genes coding for proteins having key roles in the control of neuronal excitability. Such alterations include decreased expression of the glutamate transporter GLT-1, and increased expression of monoamine oxidase (MAO-A isoform), the "peripheral-type" benzodiazepine receptor (PTBR) as well as constitutive neuronal nitric oxide synthase (nNOS). Such changes result in altered protein expression and in increased extracellular brain glutamate, increased degradation of monoamine neurotransmitters, increased synthesis of neurosteroids with inhibitory properties, and increased production of nitric oxide (respectively) in brain in chronic liver failure. In the case of GLT-1, PTBR, and nNOS, alterations in expression result from exposure to ammonia and/or manganese, two neurotoxic agents shown previously to be increased in brain in liver failure.

Astroglial dysfunction in hepatic encephalopathy.

While the pathogenesis of hepatic encephalopathy (HE) remains elusive, there is considerable evidence pointing to a key role of ammonia-induced dysfunction of astrocytes in this condition. Deficits in the ability of astrocytes to take up glutamate from the extracellular space may lead to abnormal glutamatergic neurotransmission. Furthermore, excessive stimulation of neuronal and glial glutamate receptors by elevated extracellular levels of glutamate may lead to excitotoxicity and greater glial dysfunction. Ammonia also causes upregulation of astroglial peripheral-type benzodiazepine receptors (PBRs) which is associated with increased production of neurosteroids. These neurosteroids have potent positive modulatory effects on the neuronal GABA(A) receptor which, combined with an ammonia-induced astroglial defect in GABA uptake, may result in enhanced GABAergic tone. Brain edema, associated with fulminant hepatic failure, may also result from astroglial abnormalities as the edema appears to be principally caused by swelling of these cells. Increased amounts of glutamine in astrocytes resulting from elevated brain ammonia levels may be a factor in this swelling. Other osmolytes such as glutathione may also be involved. Glial swelling may also result from NH4+ - and K+ -mediated membrane depolarization as well as by the actions of PBR agonists and neurosteroids. These findings show that an ammonia-induced gliopathy is a major factor in the pathogenesis of HE.

The “Hot nose” sign in the cerebral radionuclide angiogram

A 64-YEAR-OLD MAN came to the emergency room with altered mental status. He had a history of adenocarcinoma of the small bowel for which he had undergone surgical resection. Brain imaging was done to rule out metastases. On the anterior cerebral angiogram, increased tracer uptake in the early arterial phase was seen in the midline overlying the nasal area, which gradually diminished in the venous phase (Fig 1A) but was still quite prominent in the blood pool phase (Fig 1B). This phenomanon has been described as the hot ''1-3 sign. The static brain images did not show any evidence of brain metastases. The patient had an elevated serum ammonia of 280 ~g/dL (normal = 18 to 48 ~g/dL). Liver biopsy showed hepatic cirrhosis. The patient subsequently died of hepatic encephalopathy. The various causes for the hot nose sign are: 1. Ceased or decreased cerebral blood flow resulting in increased flow to the external carotid circulation. 1-5 These conditions include (A) functional occlusion such as cerebral death, cerebral atrophy, increased intracranial pressure of any cause 6 or (B) anatomic occlusion of one or both internal carotid arteries. Hepatocellular dysfunction in hepatic encephalopathy causes intrahepatic and extrahepatic shunting of portal venous blood into the systemic circulation, and various toxic substances, such as ammonia, accumulate in the brain and interfere with its metabolism. 7 Beside the functional cerebral changes, structural changes such as hyperplasia of protoplasmic-type astrocytes occur principally in the cortex, but they also occur in other parts of the brain. There is cerebral edema in some individuals. 8 Hence, hepatic encephalopathy certainly is one of the causes of increased intracranial pressure.