The effect of rifaximin and lactulose treatments to chronic hepatic encephalopathy rats: An [18F]PBR146 in-vivo neuroinflammation imaging study.

INTRODUCTION: Hepatic encephalopathy (HE) is a severe neuropsychiatric complication of liver diseases characterized by neuroinflammation. The efficacies of nonabsorbable rifaximin (RIF) and lactulose (LAC) have been well documented in the treatment of HE. [18F]PBR146 is a translocator protein (TSPO) radiotracer used for in vivo neuroinflammation imaging. This study investigated anti-neuroinflammation effect of RIF or/and LAC in chronic HE rats by [18F]PBR146 micro-PET/CT. METHODS: Bile duct ligation (BDL) operation induced chronic HE models, and this study included Sham+normal saline (NS), BDL+NS, BDL+RIF, BDL+LAC, and BDL+RIF+LAC groups. Behavioral assessment was performed to analyze the motor function, and fecal samples were collected after successfully established the chronic HE model (more than 28 days post-surgery). In addition, fecal samples collection and micro-PET/CT scans were performed sequentially. And we also collected the blood plasma, liver, intestinal, and brain samples after sacrificing the rats for further biochemical and pathological analyses. RESULTS: The RIF- and/or LAC-treated BDL rats showed similar behavioral results with Sham+NS group, while the treatment could not reverse the biliary obstruction resulting in sustained liver injury. The RIF or/and LAC treatments can inhibit IFN-γ and IL-10 productions. The global brain uptake values of [18F]PBR146 in BDL+NS group was significantly higher than other groups (p < .0001). The brain regions analysis showed that the basal ganglia, hippocampus, and cingulate cortex had radiotracer uptake differences among groups (all p < .05), which were consistent with the brain immunohistochemistry results. Sham+NS group was mainly enriched in Christensenella, Coprobacillus, and Pseudoflavonifractor. BDL+NS group was mainly enriched in Barnesiella, Alloprevotella, Enterococcus, and Enterorhabdus. BDL+RIF+LAC group was enriched in Parabacteroides, Bacteroides, Allobaculum, Bifidobacterium, and Parasutterella. CONCLUSIONS: RIF or/and LAC had anti-neuroinflammation in BDL-induced chronic HE rats with gut microbiota alterations. The [18F]PBR146 could be used for monitoring RIF or/and LAC treatment efficacy of chronic HE rats.

Sialyltransferase ST3GAL6 silencing reduces α2,3-sialylated glycans to regulate autophagy by decreasing HSPB8-BAG3 in the brain with hepatic encephalopathy. / å”¾æ¶²é ¸ç³–åŸºè½¬ç§»é ¶ST3GAL6的沉默可通过减少α2,3-å”¾æ¶²é ¸åŒ–èšç³–é™ä½ŽHSPB8-BAG3è¡¨è¾¾ä»Žè€Œè°ƒæŽ§è‚æ€§è„‘ç— å°é¼ å¤§è„‘ä¸­çš„è‡ªå™¬.

End-stage liver diseases, such as cirrhosis and liver cancer caused by hepatitis B, are often combined with hepatic encephalopathy (HE); ammonia poisoning is posited as one of its main pathogenesis mechanisms. Ammonia is closely related to autophagy, but the molecular mechanism of ammonia's regulatory effect on autophagy in HE remains unclear. Sialylation is an essential form of glycosylation. In the nervous system, abnormal sialylation affects various physiological processes, such as neural development and synapse formation. ST3 ß|-galactoside α2,|3-sialyltransferase 6 (ST3GAL6) is one of the significant glycosyltransferases responsible for adding α2,3-linked sialic acid to substrates and generating glycan structures. We found that the expression of ST3GAL6 was upregulated in the brains of mice with HE and in astrocytes after ammonia induction, and the expression levels of α2,3-sialylated glycans and autophagy-related proteins microtubule-associated protein light chain 3 (LC3) and Beclin-1 were upregulated in ammonia-induced astrocytes. These findings suggest that ST3GAL6 is related to autophagy in HE. Therefore, we aimed to determine the regulatory relationship between ST3GAL6 and autophagy. We found that silencing ST3GAL6 and blocking or degrading α2,3-sialylated glycans by way of Maackia amurensis lectin-II (MAL-II) and neuraminidase can inhibit autophagy. In addition, silencing the expression of ST3GAL6 can downregulate the expression of heat shock protein ß8 (HSPB8) and Bcl2-associated athanogene 3 (BAG3). Notably, the overexpression of HSPB8 partially restored the reduced autophagy levels caused by silencing ST3GAL6 expression. Our results indicate that ST3GAL6 regulates autophagy through the HSPB8-BAG3 complex.

Branched-Chain Amino Acids in Liver Diseases: Complexity and Controversy.

Branched-chain amino acids (BCAAs), as essential amino acids, engage in various physiological processes, such as protein synthesis, energy supply, and cellular signaling. The liver is a crucial site for BCAA metabolism, linking the changes in BCAA homeostasis with the pathogenesis of a variety of liver diseases and their complications. Peripheral circulating BCAA levels show complex trends in different liver diseases. This review delineates the alterations of BCAAs in conditions including non-alcoholic fatty liver disease, hepatocellular carcinoma, cirrhosis, hepatic encephalopathy, hepatitis C virus infection, and acute liver failure, as well as the potential mechanisms underlying these changes. A significant amount of clinical research has utilized BCAA supplements in the treatment of patients with cirrhosis and liver cancer. However, the efficacy of BCAA supplementation in clinical practice remains uncertain and controversial due to the heterogeneity of studies. This review delves into the complicated relationship between BCAAs and liver diseases and tries to untangle what role BCAAs play in the occurrence, development, and outcomes of liver diseases.

Elucidating hippocampal proteome dynamics in moderate hepatic encephalopathy rats: insights from high-resolution mass spectrometry.

Hepatic encephalopathy (HE) is a debilitating neurological disorder associated with liver failure and characterized by impaired brain function. Decade-long studies have led to significant advances in our understanding of HE; however, effective therapeutic management of HE is lacking, and HE continues to be a significant cause of morbidity and mortality in patients, underscoring the need for continued research into its pathophysiology and treatment. Accordingly, the present study provides a comprehensive overview aimed at elucidating the molecular underpinnings of HE and identifying potential therapeutic targets. A moderate-grade HE model was induced in rats using thioacetamide, which simulates the liver damage observed in patients, and its impact on cognitive function, neuronal arborization, and cellular morphology was also evaluated. We employed label-free LC-MS/MS proteomics to quantitatively profile hippocampal proteins to explore the molecular mechanism of HE pathogenesis; 2175 proteins were identified, 47 of which exhibited significant alterations in moderate-grade HE. The expression of several significantly upregulated proteins, such as FAK1, CD9 and Tspan2, was further validated at the transcript and protein levels, confirming the mass spectrometry results. These proteins have not been previously reported in HE. Utilizing Metascape, a tool for gene annotation and analysis, we further studied the biological pathways integral to brain function, including gliogenesis, the role of erythrocytes in maintaining blood-brain barrier integrity, the modulation of chemical synaptic transmission, astrocyte differentiation, the regulation of organ growth, the response to cAMP, myelination, and synaptic function, which were disrupted during HE. The STRING database further elucidated the protein‒protein interaction patterns among the differentially expressed proteins. This study provides novel insights into the molecular mechanisms driving HE and paves the way for identifying novel therapeutic targets for improved disease management.

Neuroprotective and cognitive enhancing effects of herbecetin against thioacetamide induced hepatic encephalopathy in rats via upregulation of AMPK and SIRT1 signaling pathways.

Acute liver injury, there is a risky neurological condition known as hepatic encephalopathy (HE). Herbacetin is a glycosylated flavonoid with many pharmacological characteristics. The purpose of this study was to assess the ability of herbacetin to protect against the cognitive deficits associated with thioacetamide (TAA) rat model and delineate the underlying behavioral and pharmacological mechanisms. Rats were pretreated with herbacetin (20 and 40 mg/kg) for 30days. On 30th day, the rats were injected with TAA (i.p. 350 mg/kg) in a single dose. In addition to a histpathological studies, ultra-structural architecture of the brain, liver functions, oxidative stress biomarkers, and behavioral tests were evaluated. Compared to the TAA-intoxicated group, herbacetin improved the locomotor and cognitive deficits, serum hepatotoxicity indices and ammonia levels. Herbacetin reduced brain levels of malodialdeyde, glutamine synthetase (GS), tumor necrosis factor- alpha (TNF-α), interleukin 1 B (IL-1ß), annexin v, and increased brain GSH, Sirtuin 1 (SIRT1), and AMP-activated kinase (AMPK) expression levels. Also, herbacetin improve the histopathological changes and ultra- structure of brain tissue via attenuating the number of inflammatory and apoptotic cells. Herbacetin treatment significantly reduced the toxicity caused by TAA. These findings suggest that herbacetin might be taken into account as a possible neuroprotective and cognitive enhancing agent due to its ability to reduce oxidative stress, inflammation and apoptosis associated with TAA.

Increased levels and activation of the IL-17 receptor in microglia contribute to enhanced neuroinflammation in cerebellum of hyperammonemic rats.

BACKGROUND: Patients with liver cirrhosis may show minimal hepatic encephalopathy (MHE) with mild cognitive impairment and motor incoordination. Rats with chronic hyperammonemia reproduce these alterations. Motor incoordination in hyperammonemic rats is due to increased GABAergic neurotransmission in cerebellum, induced by neuroinflammation, which enhances TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway activation. The initial events by which hyperammonemia triggers activation of this pathway remain unclear. MHE in cirrhotic patients is triggered by a shift in inflammation with increased IL-17. The aims of this work were: (1) assess if hyperammonemia increases IL-17 content and membrane expression of its receptor in cerebellum of hyperammonemic rats; (2) identify the cell types in which IL-17 receptor is expressed and IL-17 increases in hyperammonemia; (3) assess if blocking IL-17 signaling with anti-IL-17 ex-vivo reverses activation of glia and of the TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway. RESULTS: IL-17 levels and membrane expression of the IL-17 receptor are increased in cerebellum of rats with hyperammonemia and MHE, leading to increased activation of IL-17 receptor in microglia, which triggers activation of STAT3 and NF-kB, increasing IL-17 and TNFα levels, respectively. TNFα released from microglia activates TNFR1 in Purkinje neurons, leading to activation of NF-kB and increased IL-17 and TNFα also in these cells. Enhanced TNFR1 activation also enhances activation of the TNFR1-S1PR2-CCL2-BDNF-TrkB pathway which mediates microglia and astrocytes activation. CONCLUSIONS: All these steps are triggered by enhanced activation of IL-17 receptor in microglia and are prevented by ex-vivo treatment with anti-IL-17. IL-17 and IL-17 receptor in microglia would be therapeutic targets to treat neurological impairment in patients with MHE.

Excessive intragastric alcohol administration exacerbates hepatic encephalopathy and provokes neuronal cell death in male rats with chronic liver disease.

Hepatic encephalopathy (HE) is defined as decline in neurological function during chronic liver disease (CLD). Alcohol is a major etiological factor in the pathogenesis of fibrosis/cirrhosis and has also been documented to directly impact the brain. However, the role of alcohol in the development of HE in CLD remains unclear. Here, we investigated the impact of excessive alcohol administration on neurological deterioration in rats with CLD. Starting day 7 post-BDL surgery, rats were administered alcohol twice daily (51% v/v ethanol, 3 g/kg, via gavage) for 4 weeks. Motor coordination was assessed weekly using rotarod and anxiety-like behavior was evaluated with open field and elevated plus maze at 5 weeks. Upon sacrifice, brains were collected for western blot and immunohistochemical analyses to investigate neuronal integrity and oxidative stress status. Alcohol worsened motor coordination performance and increased anxiety-like behavior in BDL rats. Impairments were associated with decreased neuronal markers of NeuN and SMI311, increased apoptotic markers of cleaved/pro-caspase-3 and Bax/Bcl2, increased necroptosis markers of pRIP3 and pMLKL, decreased total antioxidant capacity (TAC), and increased 4-hydroxynonenal (4-HNE)modified proteins in the cerebellum of BDL-alcohol rats when compared to respective controls. Immunofluorescence confirmed the colocalization of cleaved caspase-3 and pMLKL in the granular neurons of the cerebellum of BDL-alcohol rats. Excessive alcohol consumption exacerbates HE which leads to associated apoptotic and necroptotic neuronal loss in the cerebellum of BDL-alcohol rats. Additionally, higher levels of 4-HNE and decreased TAC in the cerebellum of BDL-alcohol rats suggest oxidative stress is the triggering factor of apoptotic and necroptotic neuronal loss/injury.

Inhibition of urease-mediated ammonia production by 2-octynohydroxamic acid in hepatic encephalopathy.

Hepatic encephalopathy is a neuropsychiatric complication of liver disease which is partly associated with elevated ammonemia. Urea hydrolysis by urease-producing bacteria in the colon is often mentioned as one of the main routes of ammonia production in the body, yet research on treatments targeting bacterial ureases in hepatic encephalopathy is limited. Herein we report a hydroxamate-based urease inhibitor, 2-octynohydroxamic acid, exhibiting improved in vitro potency compared to hydroxamic acids that were previously investigated for hepatic encephalopathy. 2-octynohydroxamic acid shows low cytotoxic and mutagenic potential within a micromolar concentration range as well as reduces ammonemia in rodent models of liver disease. Furthermore, 2-octynohydroxamic acid treatment decreases cerebellar glutamine, a product of ammonia metabolism, in male bile duct ligated rats. A prototype colonic formulation enables reduced systemic exposure to 2-octynohydroxamic acid in male dogs. Overall, this work suggests that urease inhibitors delivered to the colon by means of colonic formulations represent a prospective approach for the treatment of hepatic encephalopathy.

Lessons on brain edema in HE: from cellular to animal models and clinical studies.

Brain edema is considered as a common feature associated with hepatic encephalopathy (HE). However, its central role as cause or consequence of HE and its implication in the development of the neurological alterations linked to HE are still under debate. It is now well accepted that type A and type C HE are biologically and clinically different, leading to different manifestations of brain edema. As a result, the findings on brain edema/swelling in type C HE are variable and sometimes controversial. In the light of the changing natural history of liver disease, better description of the clinical trajectory of cirrhosis and understanding of molecular mechanisms of HE, and the role of brain edema as a central component in the pathogenesis of HE is revisited in the current review. Furthermore, this review highlights the main techniques to measure brain edema and their advantages/disadvantages together with an in-depth description of the main ex-vivo/in-vivo findings using cell cultures, animal models and humans with HE. These findings are instrumental in elucidating the role of brain edema in HE and also in designing new multimodal studies by performing in-vivo combined with ex-vivo experiments for a better characterization of brain edema longitudinally and of its role in HE, especially in type C HE where water content changes are small.

Flaxseed Oil (Linum usitatissimum) Prevents Cognitive and Motor Damage in Rats with Hyperammonemia.

Hyperammonemia is characterized by the excessive accumulation of ammonia in the body as a result of the loss of liver detoxification, leading to the development of hepatic encephalopathy (HE). These metabolic alterations carry cognitive and motor deficits and cause neuronal damage, with no effective treatment at present. In this study, we aimed to evaluate the effect of two subacute oral administrations of flaxseed oil (0.26 and 0.52 mL/kg) on short- and long-term memory, visuospatial memory, locomotor activity, motor coordination, and the neuronal morphology of the prefrontal cortex (PFC) via tests on Wistar rats with hyperammonemia. The goal was to identify its role in the regulation of cerebral edema, without liver damage causing cerebral failure. In contrast with an ammonium-rich diet, flaxseed oil and normal foods did not cause cognitive impairment or motor alterations, as evidenced in the short-term and visuospatial memory tests. Furthermore, the flaxseed oil treatment maintained a regular neuronal morphology of the prefrontal cortex, which represents a neuroprotective effect. We conclude that the oral administration of flaxseed oil prevents cognitive and motor impairments as well as neuronal alterations in rats with hyperammonemia, which supports the potential use of this oil to ameliorate the changes that occur in hepatic encephalopathy.

TNF-α blockage by etanercept restores spatial learning and reduces cellular degeneration in the hippocampus during liver cirrhosis.

Hepatic encephalopathy (HE) is one of the most debilitating cerebral complications of liver cirrhosis. The one-year survival of patients with liver cirrhosis and severe encephalopathy is less than 50%. Recent studies have indicated that neuroinflammation is a new player in the pathogenesis of HE, which seems to be involved in the development of cognitive impairment. In this study, we demonstrated neurobehavioral and neuropathological consequences of liver cirrhosis and tested the therapeutic potential of the tumor necrosis factor-α (TNF-α) inhibitor, etanercept. Sixty male adult Wistar albino rats (120-190 g) were allocated into four groups, where groups I and IV served as controls. Thioacetamide (TAA; 300 mg/kg) was intraperitoneally injected twice a week for five months to induce liver cirrhosis in group II (n = 20). Both TAA and etanercept (2 mg/kg) were administered to group III (n = 20). At the end of the experiment, spatial learning was assessed using Morris water maze. TNF-α was detected in both serum and hippocampus. The excised brains were also immunohistochemically stained with glial fibrillary acidic protein (GFAP) to estimate both the number and integrity of hippocampal astrocytes. Ultrastructural changes in the hippocampus were characterized by transmission electron microscopy. The results showed that blocking TNF-α by etanercept was accompanied by a lower TNF-α expression and a higher number of GFAP-positive astrocytes in the hippocampus. Etanercept intervention alleviated the neuronal and glial degenerative changes and impeded the deterioration of spatial learning ability. In conclusion, TNF-α is strongly involved in the development of liver cirrhosis and the associated encephalopathy. TNF-α blockers may be a promising approach for management of hepatic cirrhosis and its cerebral complications.

Impaired Enzymatic Antioxidant Defense in Erythrocytes of Rats with Ammonia-Induced Encephalopathy: Role of NMDA Receptors.

Hepatic encephalopathy (HE), a neuropsychiatric disorder developing in patients with severe hepatic dysfunction, has been known for more than a century. However, pathogenetic mechanisms of cerebral dysfunction associated with liver disease are still poorly understood. There is a consensus that the primary cause of HE is accumulation of ammonia in the brain as a result of impaired liver detoxification capacity or the portosystemic shunt. Current evidence suggests that ammonia toxicity is mediated by hyperactivation of glutamate receptors, mainly N-methyl-D-aspartate receptors (NMDARs), and affects brain aerobic metabolism, which provides energy for multiple specific functions and neuronal viability. Recent reports on the presence of functional NMDARs in erythrocytes and the data on the deviations of blood parameters from their normal ranges indicate impaired hemodynamics and reduced oxygen-carrying capacity of erythrocytes in most patients with HE, thus suggesting a relationship between erythrocyte damage and cerebral dysfunction. In order to understand how hyperammonemia (HA)-induced disturbances in the energy metabolism in the brain (which needs a constant supply of large amounts of oxygen in the blood) lead to encephalopathy, it is necessary to reveal ammonia-induced impairments in the energy metabolism and antioxidant defense system of erythrocytes and to explore a potential role of ammonia in reduced brain oxygenation. To identify the said missing link, the activities of antioxidant enzymes and concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), and H2O2 were measured in the erythrocytes of rats with HA that were injected with the noncompetitive NMDAR antagonist MK-801. We found that in rats with HA, ammonia was accumulated in erythrocytes (cells lacking ammonia removal enzymes), which made them more susceptible to the prooxidant environment created during oxidative stress. This effect was completely or partially inhibited by MK-801. The data obtained might help to identify the risk factors in cognitive disorders and facilitate prediction of unfavorable outcomes of hypoperfusion in patients with a blood elevated ammonia concentration.

Low-dose ketamine improves animals' locomotor activity and decreases brain oxidative stress and inflammation in ammonia-induced neurotoxicity.

Ammonium ion (NH4 + ) is the major suspected molecule responsible for neurological complications of hepatic encephalopathy (HE). No specific pharmacological action for NH4 + -induced brain injury exists so far. Excitotoxicity is a well-known phenomenon in the brain of hyperammonemic cases. The hyperactivation of the N-Methyl- d-aspartate (NMDA) receptors by agents such as glutamate, an NH4 + metabolite, could cause excitotoxicity. Excitotoxicity is connected with events such as oxidative stress and neuroinflammation. Hence, utilizing NMDA receptor antagonists could prevent neurological complications of NH4 + neurotoxicity. In the current study, C57BL6/J mice received acetaminophen (APAP; 800 mg/kg, i.p) to induce HE. Hyperammonemic animals were treated with ketamine (0.25, 0.5, and 1 mg/kg, s.c) as an NMDA receptor antagonist. Animals' brain and plasma levels of NH4 + were dramatically high, and animals' locomotor activities were disturbed. Moreover, several markers of oxidative stress were significantly increased in the brain. A significant increase in brain tissue levels of TNF-α, IL-6, and IL-1ß was also detected in hyperammonemic animals. It was found that ketamine significantly normalized animals' locomotor activity, improved biomarkers of oxidative stress, and decreased proinflammatory cytokines. The effects of ketamine on oxidative stress biomarkers and inflammation seem to play a key role in its neuroprotective mechanisms in the current study.

Hyperammonemia induced gut microbiota dysbiosis and motor coordination disturbances in mice: new insight into gut­brain axis involvement in hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric hepatic­induced syndrome in which several factors are involved in promoting brain perturbations, with ammonia being the primary factor. Motor impairment, incoordination, and gut dysbiosis are some of the well­known symptoms of HE. Nevertheless, the link between the direct effect of hyperammonemia and associated gut dysbiosis in the pathogenesis of HE is not well established. Thus, this work aimed to assess motor function in hyperammonemia and gut dysbiosis in mice. Twenty­eight Swiss mice were distributed into three groups: two­week and four­week hyperammonemia groups were fed with an ammonia­rich diet (20% w/w), and the control group was pair­fed with a standard diet. Motor performance in the three groups was measured through a battery of motor tests, namely the rotarod, parallel bars, beam walk, and static bars. Microbial analysis was then carried out on the intestine of the studied mice. The result showed motor impairments in both hyperammonemia groups. Qualitative and quantitative microbiological analysis revealed decreased bacterial load, diversity, and ratios of both aerobic and facultative anaerobic bacteria, following two and four weeks of ammonia supplementation. Moreover, the Shannon diversity index revealed a time­dependent cutback of gut bacterial diversity in a treatment­time­dependent manner, with the presence of only Enterobacteriaceae, Streptococcaceae, and Enterococcaceaeat at four weeks. The data showed that ammonia­induced motor coordination deficits may develop through direct and indirect pathways acting on the gut­brain axis.

The pathogenesis of gut microbiota in hepatic encephalopathy by the gut-liver-brain axis.

Hepatic encephalopathy (HE) is a neurological disease occurring in patients with hepatic insufficiency and/or portal-systemic blood shunting based on cirrhosis. The pathogenesis is not completely clear till now, but it is believed that hyperammonemia is the core of HE. Hyperammonemia caused by increased sources of ammonia and decreased metabolism further causes mental problems through the gut-liver-brain axis. The vagal pathway also plays a bidirectional role in the axis. Intestinal microorganisms play an important role in the pathogenesis of HE through the gut-liver-brain axis. With the progression of cirrhosis to HE, intestinal microbial composition changes gradually. It shows the decrease of potential beneficial taxa and the overgrowth of potential pathogenic taxa. Changes in gut microbiota may lead to a variety of effects, such as reduced production of short-chain fatty acids (SCFAs), reduced production of bile acids, increased intestinal barrier permeability, and bacterial translocation. The treatment aim of HE is to decrease intestinal ammonia production and intestinal absorption of ammonia. Prebiotics, probiotics, antibiotics, and fecal microbiota transplantation (FMT) can be used to manipulate the gut microbiome to improve hyperammonemia and endotoxemia. Especially the application of FMT, it has become a new treated approach to target microbial composition and function. Therefore, restoring intestinal microbial homeostasis can improve the cognitive impairment of HE, which is a potential treatment method.

Neurometabolic changes in a rat pup model of type C hepatic encephalopathy depend on age at liver disease onset.

Chronic liver disease (CLD) is a serious condition where various toxins present in the blood affect the brain leading to type C hepatic encephalopathy (HE). Both adults and children are impacted, while children may display unique vulnerabilities depending on the affected window of brain development.We aimed to use the advantages of high field proton Magnetic Resonance Spectroscopy (1H MRS) to study longitudinally the neurometabolic and behavioural effects of Bile Duct Ligation (animal model of CLD-induced type C HE) on rats at post-natal day 15 (p15) to get closer to neonatal onset liver disease. Furthermore, we compared two sets of animals (p15 and p21-previously published) to evaluate whether the brain responds differently to CLD according to age onset.We showed for the first time that when CLD was acquired at p15, the rats presented the typical signs of CLD, i.e. rise in plasma bilirubin and ammonium, and developed the characteristic brain metabolic changes associated with type C HE (e.g. glutamine increase and osmolytes decrease). When compared to rats that acquired CLD at p21, p15 rats did not show any significant difference in plasma biochemistry, but displayed a delayed increase in brain glutamine and decrease in total-choline. The changes in neurotransmitters were milder than in p21 rats. Moreover, p15 rats showed an earlier increase in brain lactate and a different antioxidant response. These findings offer tentative pointers as to which neurodevelopmental processes may be impacted and raise the question of whether similar changes might exist in humans but are missed owing to 1H MRS methodological limitations in field strength of clinical magnet.

Severe Acute Liver Dysfunction Induces Delayed Hepatocyte Swelling and Cytoplasmic Vacuolization, and Delayed Cortical Neuronal Cell Death.

Liver dysfunction is the main cause of hepatic encephalopathy. However, histopathological changes in the brain associated with hepatic encephalopathy remain unclear. Therefore, we investigated pathological changes in the liver and brain using an acute hepatic encephalopathy mouse model. After administering ammonium acetate, a transient increase in the blood ammonia level was observed, which returned to normal levels after 24 h. Consciousness and motor levels also returned to normal. It was revealed that hepatocyte swelling, and cytoplasmic vacuolization progressed over time in the liver tissue. Blood biochemistry also suggested hepatocyte dysfunction. In the brain, histopathological changes, such as perivascular astrocyte swelling, were observed 3 h after ammonium acetate administration. Abnormalities in neuronal organelles, especially mitochondria and rough endoplasmic reticulum, were also observed. Additionally, neuronal cell death was observed 24 h post-ammonia treatment when blood ammonia levels had returned to normal. Activation of reactive microglia and increased expression of inducible nitric oxide synthase (iNOS) were also observed seven days after a transient increase in blood ammonia. These results suggest that delayed neuronal atrophy could be iNOS-mediated cell death due to activation of reactive microglia. The findings also suggest that severe acute hepatic encephalopathy causes continued delayed brain cytotoxicity even after consciousness recovery.

Hyperammonemia induces microglial NLRP3 inflammasome activation via mitochondrial oxidative stress in hepatic encephalopathy.

BACKGROUND: The role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in the pathogenesis of hepatic encephalopathy (HE) is unclear. Mitochondrial reactive oxygen species (mtROS) is a signal for NLRP3 inflammasome activation. Therefore, we aimed to determine whether mtROS-dependent NLRP3 inflammasome activation is involved in HE, using in vivo and in vitro models. METHODS: Bile duct ligation (BDL) in C57/BL6 mice was used as an in vivo HE model. NLRP3 activation was assessed in the hippocampus. Immunofluorescence staining was performed to determine the cellular source of NLRP3 in the hippocampal tissue. For the in vitro experiment, BV-2 microglial cells were primed with lipopolysaccharide (LPS), followed by ammonia treatment. NLRP3 activation and mitochondrial dysfunction were measured. Mito-TEMPO was used to suppress mtROS production. RESULTS: BDL mice showed cognitive impairment with hyperammonemia. Both the priming and activation steps of NLRP3 inflammasome activation were processed in the hippocampus of BDL mice. Moreover, intracellular ROS levels increased in the hippocampus, and NLRP3 was mainly expressed in the microglia of the hippocampus. In LPS-primed BV-2 cells, ammonia treatment induced NLRP3 inflammasome activation and pyroptosis, with elevation of mtROS and altered mitochondrial membrane potential. Pretreatment with Mito-TEMPO suppressed mtROS production and the subsequent NLRP3 inflammasome activation and pyroptosis under LPS and ammonia treatment in BV-2 cells. CONCLUSIONS: Hyperammonemia in HE may be involved in mtROS overproduction and subsequent NLRP3 inflammasome activation. Further studies using NLRP3-specific inhibitor or NLRP3 knockout mice are needed to elucidate the important role of NLRP3 inflammasome in HE development.

Clearance and production of ammonia quantified in humans by constant ammonia infusion - the effects of cirrhosis and ammonia-targeting treatments.

BACKGROUND & AIMS: Hyperammonaemia is a key pathological feature of liver disease and the primary driver of hepatic encephalopathy (HE). However, the relative roles of increased ammonia production and reduced clearance are poorly understood as is the action of ammonia-targeting drugs for HE. We aimed to quantify whole-body ammonia metabolism in healthy persons and patients with cirrhosis and to validate our method by examining the effects of glycerol phenylbutyrate and lactulose + rifaximin treatment. METHODS: Ten healthy men and ten male patients with cirrhosis were investigated by 90-minute constant ammonia infusion to achieve steady-state plasma ammonia. Whole-body ammonia clearance was calculated as infusion rate divided by steady-state concentration increase and ammonia production was calculated as clearance multiplied by baseline ammonia concentration. Participants were re-investigated after the ammonia-targeting interventions. RESULTS: In healthy persons, ammonia clearance was 3.5 (3.1-3.9) L/min and ammonia production was 49 (35-63) µmol/min. Phenylbutyrate increased clearance by 11% (4-19%, p = 0.009). In patients with cirrhosis, ammonia clearance was 20% lower at 2.7 (2.1-3.3) L/min (p = 0.02) and production was nearly threefold higher at 131 (102-159) µmol/min (p <0.0001). Lactulose + rifaximin reduced production by 20% (2-37%, p = 0.03). The infusion was generally well-tolerated apart from in one hyperammonaemic patient, with cirrhosis and possible bleeding unrelated to the infusion, who developed clinical HE that reverted when infusion was discontinued. CONCLUSIONS: Whole-body ammonia clearance and production may be measured separately using the described technique. This technique identified a lower clearance and a higher production of ammonia in patients with cirrhosis, and showed that phenylbutyrate increases clearance, whereas lactulose + rifaximin reduces production. IMPACT AND IMPLICATIONS: High blood ammonia plays a key role in cirrhosis-related brain dysfunction. However, the relative roles of reduced ammonia clearance and increased ammonia production are poorly understood as is the action of ammonia-targeting treatments. This study presents a relatively simple test to measure ammonia metabolism. By using this test, it was possible to show that patients with cirrhosis exhibit decreased ammonia clearance and increased ammonia production compared to healthy persons, and to quantify the unique effects of different ammonia-targeting treatments. The test described herein may be used to examine a range of questions related to normal physiology, pathophysiology and the mechanisms of action of ammonia-targeting treatments. CLINICAL TRIAL NUMBER: ClinicalTrials.gov (1-16-02-297-20).

Ferrous sulfate reverses cerebral metabolic abnormality induced by minimal hepatic encephalopathy.

Orally administered ferrous iron was previously reported to significantly improve the cognition and locomotion of patients with minimal hepatic encephalopathy (MHE). However, the metabolic mechanisms of the therapeutic effect of ferrous iron are unknown. In this study, MHE was induced in rats by partial portal vein ligation (PPVL), and was treated with ferrous sulfate. The Morris water maze was used to evaluate the cognitive condition of the rats. The metabolites observed by NMR and validated by liquid chromatography-mass spectrometry were defined as the key affected metabolites. The enzyme activities and trace element contents in the rat brains were also investigated. The Mn content was found to be increased but the ferrous iron content decreased in the cortex and striatum in MHE. Decreased oxoglutarate dehydrogenase activity and increased glutamine synthetase (GS) and pyruvate carboxylase (PC) activity were observed in the cortex of MHE rats. Decreased pyruvate dehydrogenase activity and increased GS and PC activity were observed in the striatum of MHE rats. The levels of BCAAs and taurine were significantly decreased, and the contents of GABA, lactate, arginine, aspartate, carnosine, citrulline, cysteine, glutamate, glutamine, glycine, methionine, ornithine, proline, threonine and tyrosine were significantly increased. These metabolic abnormalities described above were restored after treatment with ferrous sulfate. Pathway enrichment analysis suggested that urea cycle, aspartate metabolism, arginine and proline metabolism, glycine and serine metabolism, and glutamate metabolism were the major metabolic abnormalities in MHE rats, but these processes could be restored and cognitive impairment could be improved by ferrous sulfate administration.