Hyperammonemia induces mitochondrial dysfunction and neuronal cell death

BACKGROUND & AIMS: In liver 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 hyperammonemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging. METHODS: To primary co-cultures of astrocytes and neurons, low concentrations (1 and 5μM) of NH4Cl were applied. In rats with bile-duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonemia and minimal HE, acute brain slices were studied. One group of BDL rats were treated twice daily with the ammonia scavenger ornithine phenylacetate (OP, 0.3g/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. RESULTS: Neuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction and reduced cell viability (27.8±2.3% and 41.5±3.7%, respectively) compared to 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 hyperpolarization of ΔΨm with consequent overconsumption of ATP 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. CONCLUSIONS: Our results elucidate that low-grade hyperammonemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development. LAY SUMMARY: The impact of hyperammonemia, 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

Abnormal brain oxygen homeostasis in an animal model of liver disease

Background & Aims: Increased plasma ammonia concentration and consequent disruption of brain energy metabolism could underpin the pathogenesis of hepatic encephalopathy (HE). Brain energy homeostasis relies on effective maintenance of brain oxygenation, and dysregulation impairs neuronal function leading to cognitive dysfunction. We hypothesise that HE is associated with reduced brain oxygenation and explored the potential role of ammonia as an underlying pathophysiological factor. / Methods: In a rat model of chronic liver disease with minimal HE (mHE; bile duct ligation [BDL]), brain tissue oxygen measurement and proton magnetic resonance spectroscopy were used to investigate how hyperammonemia impacts oxygenation and metabolic substrate availability in the CNS. Ornithine phenylacetate (OP, OCR-002; Ocera Therapeutics, USA) was used as an experimental treatment to reduce ammonia concentration in plasma. / Results: In BDL animals, glucose, lactate and tissue oxygen concentrations in the cerebral cortex were significantly lower compared to sham-operated controls. OP treatment corrected the hyperammonemia and restored brain tissue oxygen. While BDL animals were hypotensive, cortical tissue oxygen was significantly improved by treatments which increased arterial blood pressure. Cerebrovascular reactivity to exogenously applied CO2 was found to be normal in BDL animals. / Conclusions: These data suggest that hyperammonemia significantly decreases cortical oxygenation, potentially compromising brain energy metabolism. These findings have potential clinical implications for the treatment of patients with mHE aiming to restore normal brain blood flow alongside ammonia-reducing strategies

Combination of G-CSF and a TLR4 inhibitor reduce inflammation and promote regeneration in a mouse model of ACLF

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is an unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with Granulocyte-Colony Stimulating Factor (G-CSF) targets inflammation whilst enhancing liver regeneration. METHODS: Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride followed by either lipopolysaccharide (LPS) or galactosamine (GalN) as extrahepatic or hepatic insults, respectively. G-CSF and/or TLR4-antagonist, TAK-242, were administered daily. The treatment duration was 24h and 5d in the LPS model and 48h in the GalN model, respectively. RESULTS: In a LPS-induced ACLF mouse model treatment with G-CSF was associated with a significant mortality of 66% after 48 hours compared with 0% without G-CSF. Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterized by p21 over-expression suggesting hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase of markers of hepatocyte regeneration. CONCLUSION: TLR4 inhibition with TAK-242 rescued G-CSF-driven cell death, inflammation, enhanced tissue repair, and significantly induced regeneration thus suggesting that the combination of G-CSF and TAK-242 is a novel approach for the treatment of ACLF. LAY SUMMARY: The combinatorial therapy of Granulocyte-Colony Stimulating Factor and TAK-242, a Toll-like Receptor-4 inhibitor, achieves the dual aim of reducing hepatic inflammation and inducing liver regeneration for the treatment of acute-on-chronic liver failure

Impaired brain glymphatic flow in experimental hepatic encephalopathy

Background & Aims: Neuronal function is exquisitely sensitive to alterations in the extracellular environment. In patients with hepatic encephalopathy (HE), accumulation of metabolic waste products and noxious substances in the interstitial fluid of the brain is thought to result from liver disease and may contribute to neuronal dysfunction and cognitive impairment. This study was designed to test the hypothesis that the accumulation of these substances, such as bile acids, may result from reduced clearance from the brain. Methods: In a rat model of chronic liver disease with minimal HE (the bile duct ligation [BDL] model), we used emerging dynamic contrast-enhanced MRI and mass-spectroscopy techniques to assess the efficacy of the glymphatic system, which facilitates clearance of solutes from the brain. Immunofluorescence of aquaporin-4 (AQP4) and behavioural experiments were also performed. Results: We identified discrete brain regions (olfactory bulb, prefrontal cortex and hippocampus) of altered glymphatic clearance in BDL rats, which aligned with cognitive/behavioural deficits. Reduced AQP4 expression was observed in the olfactory bulb and prefrontal cortex in HE, which could contribute to the pathophysiological mechanisms underlying the impairment in glymphatic function in BDL rats. Conclusions: This study provides the first experimental evidence of impaired glymphatic flow in HE, potentially mediated by decreased AQP4 expression in the affected regions. Lay summary: The 'glymphatic system' is a newly discovered brain-wide pathway that facilitates clearance of various sub-stances that accumulate in the brain due to its activity. This study evaluated whether the function of this system is altered in a model of brain dysfunction that occurs in cirrhosis. For the first time, we identified that the clearance of substances from the brain in cirrhosis is reduced because this clearance system is defective. This study proposes a new mechanism of brain dysfunction in patients with cirrhosis and provides new targets for therapy

The Lipopolysaccharide-Sensing Caspase(s)-4/11 Are Activated in Cirrhosis and Are Causally Associated With Progression to Multi-Organ Injury

International audienceBackground and aims: The development of multi-organ injury in cirrhosis is associated with increased intestinal permeability, translocation of gut-derived bacterial products [e.g., lipopolysaccharide (LPS)] into the circulation, and increased non-apoptotic hepatocyte cell death. Pyroptosis is a non-apoptotic, lytic form of cell death mediated by the LPS-sensing caspase(s)-4/11 (caspase-4 in humans, caspase-11 in mice), which leads to activation of the effector protein Gasdermin D (GSDMD) and subsequent formation of pores in the plasma membrane. Endoplasmic reticulum (ER) stress, a feature of cirrhosis, has been identified as a factor promoting the activation of caspase-11, thus increasing sensitivity of the cell to LPS-mediated pyroptosis. The aim of this study was to determine the role of bacterial LPS in the activation of hepatic caspase(s)-4/11 and progression of hepatic and extra-hepatic organ injury in cirrhosis.Materials and methods: Human liver samples from patients with stable cirrhosis (SC) or acutely decompensated cirrhosis (AD) were analyzed for caspase-4 activation by immunohistochemistry. Wild-type and Casp11 -/- mice underwent CCl4 treatment by gavage to induce advanced liver fibrosis, and subsequently low-dose injection of LPS to mimic bacterial translocation and induce multi-organ injury. Liver, kidney, and brain function were assessed by plasma ALT/creatinine and brain water respectively. The activity of inflammatory caspases was assessed by fluorometric assay and the occurrence of pyroptosis and overall cell death in liver tissue by GSDMD cleavage and TUNEL assay, respectively. Primary human hepatocytes were cultured according to standard techniques.Results: Human liver samples demonstrated increased caspase-4 activation in AD cirrhosis. Caspase-4 activation was associated with MELD score and circulating levels of LDH. Wild-type mice treated with CCl4 developed significant multi-organ injury (increased ALT, creatinine, and brain water) upon LPS injection, and showed increased hepatic GSDMD cleavage compared to mice treated with CCl4 alone. Primary human hepatocytes could be sensitized to pyroptosis by pre-treatment with the ER-stress inducer tunicamycin and LPS. Casp11 -/- mice treated with CCl4 + LPS were significantly protected from multi-organ injury compared to wild-type CCl4 + LPS.Conclusion: These data demonstrate for the first time a causal relationship between LPS-mediated activation of caspase(s)-4/11 and development of hepatic and extra-hepatic injury in cirrhosis