Rats with minimal hepatic encephalopathy show reduced cGMP-dependent protein kinase activity in hypothalamus correlating with circadian rhythms alterations.

Patients with liver cirrhosis show disturbances in sleep and in its circadian rhythms which are an early sign of minimal hepatic encephalopathy (MHE). The mechanisms of these disturbances are poorly understood. Rats with porta-caval shunt (PCS), a model of MHE, show sleep disturbances reproducing those of cirrhotic patients. The aims of this work were to characterize the alterations in circadian rhythms in PCS rats and analyze the underlying mechanisms. To reach these aims, we analyzed in control and PCS rats: (a) daily rhythms of spontaneous and rewarding activity and of temperature, (b) timing of the onset of activity following turning-off the light, (c) synchronization to light after a phase advance and (d) the molecular mechanisms contributing to these alterations in circadian rhythms. PCS rats show altered circadian rhythms of spontaneous and rewarding activities (wheel running). PCS rats show more rest bouts during the active phase, more errors in the onset of motor activity and need less time to re-synchronize after a phase advance than control rats. Circadian rhythm of body temperature is also slightly altered in PCS rats. The internal period length (tau) of circadian rhythm of motor activity is longer in PCS rats. We analyzed some mechanisms by which hypothalamus modulate circadian rhythms. PCS rats show increased content of cGMP in hypothalamus while the activity of cGMP-dependent protein kinase was reduced by 41% compared to control rats. Altered cGMP-PKG pathway in hypothalamus would contribute to altered circadian rhythms and synchronization to light.

Blocking NMDA receptors delays death in rats with acute liver failure by dual protective mechanisms in kidney and brain.

Treatment of patients with acute liver failure (ALF) is unsatisfactory and mortality remains unacceptably high. Blocking NMDA receptors delays or prevents death of rats with ALF. The underlying mechanisms remain unclear. Clarifying these mechanisms will help to design more efficient treatments to increase patient's survival. The aim of this work was to shed light on the mechanisms by which blocking NMDA receptors delays rat's death in ALF. ALF was induced by galactosamine injection. NMDA receptors were blocked by continuous MK-801 administration. Edema and cerebral blood flow were assessed by magnetic resonance. The time course of ammonia levels in brain, muscle, blood, and urine; of glutamine, lactate, and water content in brain; of glomerular filtration rate and kidney damage; and of hepatic encephalopathy (HE) and intracranial pressure was assessed. ALF reduces kidney glomerular filtration rate (GFR) as reflected by reduced inulin clearance. GFR reduction is due to both reduced renal perfusion and kidney tubular damage as reflected by increased Kim-1 in urine and histological analysis. Blocking NMDA receptors delays kidney damage, allowing transient increased GFR and ammonia elimination which delays hyperammonemia and associated changes in brain. Blocking NMDA receptors does not prevent cerebral edema or blood-brain barrier permeability but reduces or prevents changes in cerebral blood flow and brain lactate. The data show that dual protective effects of MK-801 in kidney and brain delay cerebral alterations, HE, intracranial pressure increase and death. NMDA receptors antagonists may increase survival of patients with ALF by providing additional time for liver transplantation or regeneration.

Impaired release of corticosterone from adrenals contributes to impairment of circadian rhythms of activity in hyperammonemic rats.

Patients with liver cirrhosis may present impaired sleep-wake and circadian rhythms, relative adrenal insufficiency and altered hypothalamus-pituitary-adrenal gland (HPA) axis. The underlying mechanisms remain unclear. Circadian rhythms are modulated by corticosteroids which secretion is regulated by HPA axis. Hyperammonemia alters circadian rhythms of activity and corticosterone in rats. The aims were: (1) assessing whether corticosterone alterations are responsible for altered circadian rhythm in hyperammonemia: (2) to shed light on the mechanism by which corticosterone circadian rhythm is altered in hyperammonemia. The effects of daily corticosterone injection at ZT10 on circadian rhythms of activity, plasma corticosterone, adreno-corticotropic hormone (ACTH) and hypothalamic corticotropic releasing hormone (CRH) were assessed in control and hyperammonemic rats. ACTH-induced corticosterone release was analyzed in cultured adrenal cells. Corticosterone injection restores the corticosterone peak in hyperammonemic rats and their activity and circadian rhythm. Plasma ACTH and CRH in hypothalamus are increased in hyperammonemic rats. Corticosterone injection normalizes ACTH. Chronic hyperammonemia impairs adrenal function, reduces corticosterone content and ACTH-induced corticosterone release in adrenals, leading to reduced feedback modulation of HPA axis by corticosterone which contributes to impair circadian rhythms of activity. Impaired circadian rhythms and motor activity may be corrected in hyperammonemia and hepatic encephalopathy by corticosterone treatment.

Brain cholinergic impairment in liver failure.

The cholinergic system is involved in specific behavioural responses and cognitive processes. Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure. An increase (~30%) in the activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE) is observed in the brain cortex from patients deceased from hepatic coma, while the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, remains unaffected. In agreement with the human data, AChE activity in brain cortical extracts of bile duct ligated (BDL) rats was increased (~20%) compared to controls. A hyperammonemic diet did not result in any further increase of AChE levels in the BDL model, and no change was observed in hyperammonemic diet rats without liver disease. Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes. A selective increase of tetrameric AChE, the major AChE species involved in hydrolysis of acetylcholine in the brain, was detected in both cirrhotic humans and BDL rats. Histological examination of BDL and non-ligated rat brains shows that the subcellular localization of both AChE and choline acetyltransferase, and thus the accessibility to their substrates, appears unaltered by the pathological condition. The BDL-induced increase in AChE activity was not parallelled by an increase in mRNA levels. Increased AChE in BDL cirrhotic rats leads to a pronounced decrease (~50-60%) in the levels of acetylcholine. Finally, we demonstrate that the AChE inhibitor rivastigmine is able to improve memory deficits in BDL rats. One week treatment with rivastigmine (0.6 mg/kg; once a day, orally, for a week) resulted in a 25% of inhibition in the enzymatic activity of AChE with no change in protein composition, as assessed by sucrose density gradient fractionation and western blotting analysis. In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy.

NMDA receptors in hyperammonemia and hepatic encephalopathy.

The NMDA type of glutamate receptors modulates learning and memory. Excessive activation of NMDA receptors leads to neuronal degeneration and death. Hyperammonemia and liver failure alter the function of NMDA receptors and of some associated signal transduction pathways. The alterations are different in acute and chronic hyperammonemia and liver failure. Acute intoxication with large doses of ammonia (and probably acute liver failure) leads to excessive NMDA receptors activation, which is responsible for ammonia-induced death. In contrast, chronic hyperammonemia induces adaptive responses resulting in impairment of signal transduction associated to NMDA receptors. The function of the glutamate-nitric oxide-cGMP pathway is impaired in brain in vivo in animal models of chronic liver failure or hyperammonemia and in homogenates from brains of patients died in hepatic encephalopathy. The impairment of this pathway leads to reduced cGMP and contributes to impaired cognitive function in hepatic encephalopathy. Learning ability is reduced in animal models of chronic liver failure and hyperammonemia and is restored by pharmacological manipulation of brain cGMP by administering phosphodiesterase inhibitors (zaprinast or sildenafil) or cGMP itself. NMDA receptors are therefore involved both in death induced by acute ammonia toxicity (and likely by acute liver failure) and in cognitive impairment in hepatic encephalopathy.

Hyperammonaemia alters the mechanisms by which metabotropic glutamate receptors in nucleus accumbens modulate motor function.

Activation of metabotropic glutamate receptors by injecting (S)3,5-dihydroxyphenylglycine (DHPG) in nucleus accumbens (NAcc) increases motor activity by different mechanisms in control rats and in rats with chronic liver failure due to portacaval shunt. In control rats DHPG increases extracellular dopamine in NAcc and induces locomotion by activating the 'normal' circuit: NAcc-->ventral pallidum-->medial-dorsal thalamus-->prefrontal cortex, which is not activated in portacaval shunt rats. In these rats, DHPG activates an 'alternative' circuit: NAcc-->substantia nigra pars reticulata-->ventro-medial thalamus-->prefrontal cortex, which is not activated in control rats. The reasons by which liver failure leads to activation of this 'alternative' circuit remain unclear. The aim of this work was to assess whether hyperammonaemia could be responsible for the alterations found in chronic liver failure. We injected DHPG in NAcc of control or hyperammonaemic rats and analysed, by in vivo brain microdialysis, the neurochemical responses of the 'normal' and 'alternative' circuits. In hyperammonaemic rats DHPG injection in NAcc activates both the 'normal' and 'alternative' circuits. In hyperammonaemia, activation of the 'alternative' circuit and increased motor response following metabotropic glutamate receptors activation in NAcc seem due to an increase in extracellular glutamate which activates AMPA receptors.

Ebselen prevents chronic alcohol-induced rat hippocampal stress and functional impairment.

BACKGROUND: Most of the previously published data suggest a role for oxidative or nitrosative stress in ethanol-induced nervous system damage. Moreover, ethanol is able to impair learning abilities in adult mammalian brain, a process suggested to be directly related to hippocampal neurogenesis. Ebselen, a synthetic compound with antioxidant properties, is able to prevent ethanol-induced impairment of neurogenesis in adult rats. The aim of the present work was to further demonstrate the ability of ebselen to prevent biochemical alterations, and preserve long-term potentiation (LTP) and learning abilities, in the hippocampus of chronic alcoholic adult rats. METHODS: Biochemical markers of oxidative stress (glutathione and malondialdehyde) were assayed in hippocampi of control rats and animals fed a liquid alcoholic diet (Lieber-De Carli) supplemented or not with ebselen. Long-term potentiation and hippocampal-dependent tests were studied in all animal groups. RESULTS: The hippocampal concentrations of glutathione and malondialdehyde were decreased and increased, respectively, in alcohol-treated animals, and did not differ from those of the control and the alcohol+ebselen groups. Long-term potentiation in hippocampal slices from ethanol-treated animals was prevented, when compared with controls, and occurred with a similar profile in control animals and in the alcohol+ebselen groups. Learning ability was tested with the Morris water maze test. Escape latencies were higher in ethanol-treated rats than in control animals or the ones treated with ethanol+ebselen. CONCLUSIONS: The results herein strongly suggest that oxidative mechanisms may underlie the hippocampal effects of ethanol in adult rats, in view of the protective effect of ebselen.

Hypolocomotion in rats with chronic liver failure is due to increased glutamate and activation of metabotropic glutamate receptors in substantia nigra.

BACKGROUND/AIMS: Patients with hepatic encephalopathy show altered motor function, psychomotor slowing and hypokinesia. The underlying mechanisms remain unclear. This work's aims were: (1) to analyse in rats with chronic liver failure due to portacaval shunt (PCS) the neurochemical alterations in the basal ganglia-thalamus-cortex circuits; (2) to correlate these alterations with those in motor function and (3) to normalize motor activity of PCS rats by pharmacological means. METHODS: Extracellular neurotransmitters levels were analysed by in vivo brain microdialysis. Motor activity was determined by counting crossings in open field. RESULTS: Extracellular glutamate is increased in substantia nigra pars reticulata (SNr) of PCS rats. Blocking metabotropic receptor 1 (mGluR1) in SNr normalizes motor activity in PCS rats. In ventro-medial thalamus of PCS rats GABA is increased and it is normalized by blocking mGluR1 in SNr. Blocking mGluR1 in SNr increases and mGluR1 activation reduces glutamate in motor cortex and motor activity. CONCLUSIONS: Increased extracellular glutamate and activation of mGluR1 in SNr are responsible for reduced motor activity in rats with chronic liver failure. Blocking mGluR1 in SNr normalizes motor activity in PCS rats, suggesting that, under appropriate conditions, similar treatments could be useful to treat the psychomotor slowing and hypokinesia in patients with hepatic encephalopathy.

Synthesis of new 2-arylamino-6-trifluoromethylpyridine-3-carboxylic acid derivatives and investigation of their analgesic activity.

A new series of 2-arylamino-6-trifluoromethyl-3-carboxylic acid derivatives was synthesized and assayed in vivo for their analgesic properties by means of writhing test in rats. When compared to aspirin, ibuprofen and flufenamic acid some of the new compounds exhibited a comparable or improved analgesic activity and a lower ulcerogenic effect.

Trialkylglycines: a new family of compounds with in vivo neuroprotective activity.

Glutamate neurotoxicity is involved in the pathogenesis of neurodegenerative disorders such as Huntington's, Parkinson's and Alzheimer's diseases. It plays also a major role in the neuronal damage that occurs in brain ischemia and head trauma. Finding molecules that prevent or reverse glutamate neurotoxicity (excitotoxicity) is, therefore, of great interest. Strategies aimed at this end include the screening of libraries of compounds synthesized by combinatorial chemistry to find molecules that prevent neuronal death in vitro and in vivo. A library of trialkylglycines was screened to assess whether they prevent glutamate-induced neuronal death in primary cultures of cerebellar neurons. Two types of trialkylglycines have been found that significantly reduce the incidence of glutamate-induced neuronal death. The first type includes two compounds (referred to as 6-1-2 and 6-1-10) that efficiently prevent glutamate or NMDA-induced neuronal death. They also prevent excitotoxicity in vivo as assessed by using two animal models of excitotoxicity: acute intoxication with ammonia and a model of cerebral ischemia in rats. Trialkylglycines 6-1-2 and 6-1-10 prevent ammonia-induced (NMDA receptor-mediated) death of mice and neuronal degeneration in the model of cerebral ischemia. The trialkylglycines of the second type act as open channel blockers of the NMDA receptor. The first group of trialkylglycines does not block NMDA receptor channels and does not affect the glutamate-nitric oxide-cGMP pathway. Their molecular target has not yet been identified. These two types of trialkylglycines (especially those that do not affect NMDA receptor function) might represent effective drugs for the treatment of neurodegeneration. They are likely to be well tolerated and have fewer side effects than NMDA receptor antagonists.

Glutamine synthetase activity and glutamine content in brain: modulation by NMDA receptors and nitric oxide.

Acute intoxication with large doses of ammonia leads to rapid death. The main mechanism for ammonia elimination in brain is its reaction with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase and consumes ATP. In the course of studies on the molecular mechanism of acute ammonia toxicity, we have found that glutamine synthetase activity and glutamine content in brain are modulated by NMDA receptors and nitric oxide. The main findings can be summarized as follows. Blocking NMDA receptors prevents ammonia-induced depletion of brain ATP and death of rats but not the increase in brain glutamine, indicating that ammonia toxicity is not due to increased activity of glutamine synthetase or formation of glutamine but to excessive activation of NMDA receptors. Blocking NMDA receptors in vivo increases glutamine synthetase activity and glutamine content in brain, indicating that tonic activation of NMDA receptors maintains a tonic inhibition of glutamine synthetase. Blocking NMDA receptors in vivo increases the activity of glutamine synthetase assayed in vitro, indicating that increased activity is due to a covalent modification of the enzyme. Nitric oxide inhibits glutamine synthetase, indicating that the covalent modification that inhibits glutamine synthetase is a nitrosylation or a nitration.Inhibition of nitric oxide synthase increases the activity of glutamine synthetase, indicating that the covalent modification is reversible and it must be an enzyme that denitrosylate or denitrate glutamine synthetase.NMDA mediated activation of nitric oxide synthase is responsible only for part of the tonic inhibition of glutamine synthetase. Other sources of nitric oxide are also contributing to this tonic inhibition. Glutamine synthetase is not working at maximum rate in brain and its activity may be increased pharmacologically by manipulating NMDA receptors or nitric oxide content. This may be useful, for example, to increase ammonia detoxification in brain in hyperammonemic situations.

Molecular mechanism of acute ammonia toxicity: role of NMDA receptors.

Acute administration of large doses of ammonia leads to the rapid death of animals. This article reviews the role of excessive activation of N-methyl-D-aspartate (NMDA) receptors in the mediation of ammonia-induced mortality. The studies reviewed here show that acute intoxication with large doses of ammonia leads to the activation of NMDA receptors in brain in vivo. Moreover, excessive activation of NMDA receptors is responsible for ammonia-induced death of animals, which is prevented by different antagonists of NMDA receptors. This article also reviews the studies showing that activation of NMDA receptors is also responsible for the following effects of acute ammonia intoxication: (1) depletion of brain ATP, which, in turn, leads to release of glutamate; (2) activation of calcineurin and dephosphorylation and activation of Na+/K+-ATPase in brain, thus increasing ATP consumption; (3) impairment of mitochondrial function and calcium homeostasis at different levels, thus decreasing ATP synthesis; (4) activation of calpain that degrades the microtubule-associated protein MAP-2, thus altering the microtubular network; (5) increased formation of nitric oxide (NO) formation, which, in turn, reduces the activity of glutamine synthetase, thus reducing the elimination of ammonia in brain.