Golexanolone improves fatigue, motor incoordination and gait and memory in rats with bile duct ligation.

BACKGROUND AND AIMS: Many patients with the chronic cholestatic liver disease primary biliary cholangitis (PBC) show fatigue and cognitive impairment that reduces their quality of life. Likewise, rats with bile duct ligation (BDL) are a model of cholestatic liver disease. Current PBC treatments do not improve symptomatic alterations such as fatigue or cognitive impairment and new, more effective treatments are therefore required. Golexanolone reduces the potentiation of GABAA receptors activation by neurosteroids. Golexanolone reduces peripheral inflammation and neuroinflammation and improves cognitive and motor function in rats with chronic hyperammonemia. The aims of the present study were to assess if golexanolone treatment improves fatigue and cognitive and motor function in cholestatic BDL rats and if this is associated with improvement of peripheral inflammation, neuroinflammation, and GABAergic neurotransmission in the cerebellum. METHODS: Rats were subjected to bile duct ligation. One week after surgery, oral golexanolone was administered daily to BDL and sham-operated controls. Fatigue was analysed in the treadmill, motor coordination in the motorater, locomotor gait in the Catwalk, and short-term memory in the Y-maze. We also analysed peripheral inflammation, neuroinflammation, and GABAergic neurotransmission markers by immunohistochemistry and Western blot. RESULTS: BDL induces fatigue, impairs memory and motor coordination, and alters locomotor gait in cholestatic rats. Golexanolone improves these alterations, and this was associated with improvement of peripheral inflammation, neuroinflammation, and GABAergic neurotransmission in the cerebellum. CONCLUSION: Golexanolone may have beneficial effects to treat fatigue, and motor and cognitive impairment in patients with the chronic cholestatic liver disease PBC.

Mild Cognitive Impairment Is Associated with Enhanced Activation of Th17 Lymphocytes in Non-Alcoholic Fatty Liver Disease.

Patients with nonalcoholic fatty liver disease (NAFLD) may show mild cognitive impairment (MCI). The mechanisms involved remain unclear. The plasma concentrations of several cytokines and chemokines were measured in 71 NAFLD patients (20 with and 51 without MCI) and 61 controls. Characterization and activation of leukocyte populations and CD4+ sub-populations were carried out and analyzed by flow cytometry. We analyzed the cytokines released from CD4+ cell cultures and the mRNA expression of transcription factors and receptors in peripheral blood mononuclear cells. The appearance of MCI in NAFLD patients was associated with increased activation of CD4+ T lymphocytes, mainly of the Th17 subtype, increased plasma levels of pro-inflammatory and anti-inflammatory cytokines such as IL-17A, IL-23, IL-21, IL-22, IL-6, INF-γ, and IL-13, and higher expression of the CCR2 receptor. Constitutive expression of IL-17 was found in cultures of CD4+ cells from MCI patients, reflecting Th17 activation. High IL-13 plasma levels were predictive of MCI and could reflect a compensatory anti-inflammatory response to the increased expression of pro-inflammatory cytokines. This study identified some specific alterations of the immune system associated with the appearance of neurological alterations in MCI patients with NAFLD that could be the basis to improve and restore cognitive functions and quality of life in these patients.

Selective improvement by rifaximin of changes in the immunophenotype in patients who improve minimal hepatic encephalopathy.

BACKGROUND: Minimal hepatic encephalopathy (MHE) in cirrhotic patients is associated with specific changes in parameters of the immune system reflecting a more pro-inflammatory environment than in patients without MHE. The aims of this work were to assess the effects of rifaximin treatment of cirrhotic patients with MHE on: (1) MHE; (2) intermediate (CD14++CD16+) pro-inflammatory monocytes; (3) expression of early activation marker CD69 in T lymphocytes; (4) autoreactive CD4+CD28- T lymphocytes; (5) differentiation of CD4+ T lymphocytes to Th follicular and Th22; (6) serum IgG levels; and (7) levels of some pro-inflammatory cytokines. METHODS: These parameters were measured by immunophenotyping and cytokine profile analysis in 30 controls without liver disease, 30 cirrhotic patients without MHE and 22 patients with MHE. Patients with MHE were treated with rifaximin and the same parameters were measured at 3 and 6 months of treatment. We assessed if changes in these parameters are different in patients who improve MHE (responders) and those who remain in MHE (non-responders). RESULTS: Rifaximin improved MHE in 59% of patients with MHE. In these responder patients rifaximin normalized all alterations in the immune system measured while in non-responders it normalizes only IL-6, CCL20, and differentiation of T lymphocytes to Th22. Non-responder patients do not show increased expression of CD69 before treatment. CONCLUSIONS: Rifaximin normalizes changes in the immune system in patients who improve MHE but not in non-responders. Some alterations before treatment are different in responders and non-responders. Understanding these differences may identify predictors of the response of MHE to rifaximin.

Altered postural control and stability in cirrhotic patients with minimal hepatic encephalopathy correlate with cognitive deficits.

BACKGROUND & AIMS: Cognitive dysfunction in cirrhotic patients with minimal hepatic encephalopathy (MHE) is associated with falls. Alterations in postural control and stability could contribute to increase falls risk in these patients. We aimed to assess whether postural control and direction-specific limits of stability are altered in cirrhotic patients with MHE compared to patients without MHE and controls. We also assessed if alterations in postural control correlate with neurological impairment and/or blood biomarkers. METHODS: Posturography analysis, attention Stroop test and bimanual and visuo-motor coordination tests were performed in 18 controls, 19 patients with cirrhosis without MHE and 17 with MHE, diagnosed by PHES. Posturography was assessed by NedSVE® /IBV system under four sensory conditions. Limits of stability and rhythmic weight-shifting tests were also performed. Blood ammonia and serum interleukins were also measured. Falls were assessed after 12-24 months follow-up. RESULTS: MHE patients show impaired balance, mainly on unstable surface with eyes open, with longer reaction and confinement times and lower success in Limits of Stability test compared to patients without MHE. Performance in attention and motor coordination tests correlated with most posturography parameters alterations. Logistic regression analysis shows that posturography parameters and bimanual coordination test are good predictors of falls. CONCLUSION: Balance patterns and limits of stability in MHE patients are impaired compared to patients without MHE and controls. This seems to contribute to a higher falls risk. Attention and motor coordination deficits could contribute to balance impairment in patients with MHE.

Rats with mild bile duct ligation show hepatic encephalopathy with cognitive and motor impairment in the absence of cirrhosis: effects of alcohol ingestion.

Studies in animal models allow identifying mechanisms and treatments for cognitive and motor alterations in hepatic encephalopathy (HE). Liver diseases leading to HE in humans have different aetiologies (alcoholic, viral, etc.). The International Society for Hepatic Encephalopathy points out that satisfactory model for HE resulting from alcoholic cirrhosis are lacking. This work aimed to develop and characterize an animal model for HE in alcoholic liver cirrhosis. To potentiate the effects of alcohol on liver we administered it (5, 8 or 10% in drinking water) to rats showing mild liver damage induced by "mild" bile duct ligation (MBDL), obtained by sectioning 3 out of 5 bile ducts. MBDL rats show increased markers of cholestasis and liver damage, hyperammonemia and inflammation. MBDL rats also show motor in-coordination, hypokinesia, impaired learning ability in a Y maze and reduced spatial memory in the Morris water maze. Ingesting 10% ethanol does not induce relevant liver damage in control rats but potentiates liver damage in MBDL rats. In contrast, ethanol did not enhance the biochemical or neurological alterations in MBDL rats. This supports that the combination of certain levels of hyperammonemia and inflammation is enough to induce mild cognitive impairment, even in the absence of liver cirrhosis. Rats with MBDL and MBDL-OH survived more than 3 months, allowing performing long-term studies on cognitive and motor alterations and on underlying mechanisms. MBDL-OH rats are a good model to study the mechanisms of ethanol-induced liver cirrhosis and the factors making the liver susceptible to ethanol damage.

Urinary KIM-1, NGAL and L-FABP for the diagnosis of AKI in patients with acute coronary syndrome or heart failure undergoing coronary angiography.

Acute kidney injury (AKI) is a common complication after coronary angiography. Early biomarkers of this disease are needed since increase in serum creatinine levels is a late marker. To assess the usefulness of urinary kidney injury molecule-1 (uKIM-1), neutrophil gelatinase-associated lipocalin (uNGAL) and liver-type fatty acid-binding protein (uL-FABP) for early detection of AKI in these patients, comparing their performance with another group of cardiac surgery patients. Biomarkers were measured in 193 patients, 12 h after intervention. In the ROC analysis, AUC for KIM-1, NGAL and L-FABP was 0.713, 0.958 and 0.642, respectively, in the coronary angiography group, and 0.716, 0.916 and 0.743 in the cardiac surgery group. Urinary KIM-1 12 h after intervention is predictive of AKI in adult patients undergoing coronary angiography, but NGAL shows higher sensitivity and specificity. L-FABP provides inferior discrimination for AKI than KIM-1 or NGAL in contrast to its performance after cardiac surgery. This is the first study showing the predictive capacity of KIM-1 for AKI after coronary angiography. Further studies are still needed to answer relevant questions about the clinical utility of biomarkers for AKI in different clinical settings.

Potentiation of the transient receptor potential vanilloid 1 channel contributes to pruritogenesis in a rat model of liver disease.

Persistent pruritus is a common disabling dermatologic symptom associated with different etiologic factors. These include primary skin conditions, as well as neuropathic, psychogenic, or systemic disorders like chronic liver disease. Defective clearance of potential pruritogenic substances that activate itch-specific neurons innervating the skin is thought to contribute to cholestatic pruritus. However, because the underlying disease-specific pruritogens and itch-specific neuronal pathways and mechanism(s) are unknown, symptomatic therapeutic intervention often leads to no or only limited success. In the current study, we aimed to first validate rats with bile duct ligation (BDL) as a model for hepatic pruritus and then to evaluate the contribution of inflammation, peripheral neuronal sensitization, and specific signaling pathways and subpopulations of itch-responsive neurons to scratching behavior and thermal hypersensitivity. Chronic BDL rats displayed enhanced scratching behavior and thermal hyperalgesia indicative of peripheral neuroinflammation. BDL-induced itch and hypersensitivity involved a minor contribution of histaminergic/serotonergic receptors, but significant activation of protein-activated receptor 2 (PAR2) receptors, prostaglandin PGE2 formation, and potentiation of transient receptor potential vanilloid 1 (TRPV1) channel activity. The sensitization of dorsal root ganglion nociceptors in BDL rats was associated with increased surface expression of PAR2 and TRPV1 proteins and an increase in the number of PAR2- and TRPV1-expressing peptidergic neurons together with a shift of TRPV1 receptor expression to medium sized dorsal root ganglion neurons. These results suggest that pruritus and hyperalgesia in chronic cholestatic BDL rats are associated with neuroinflammation and involve PAR2-induced TRPV1 sensitization. Thus, pharmacological modulation of PAR2 and/or TRPV1 may be a valuable therapeutic approach for patients with chronic liver pruritus refractory to conventional treatments.

Patients with minimal hepatic encephalopathy show impaired mismatch negativity correlating with reduced performance in attention tests.

UNLABELLED: Attention deficit is an early event in the cognitive impairment of patients with minimal hepatic encephalopathy (MHE). The underlying mechanisms remain unclear. Mismatch negativity (MMN) is an auditory event-related potential that reflects an attentional trigger. Patients with schizophrenia show impaired attention and cognitive function, which are reflected in altered MMN. We hypothesized that patients with MHE, similarly to those with schizophrenia, should show MMN alterations related with attention deficits. The aims of this work were to assess whether (1) MMN is altered in cirrhotic patients with MHE, compared to those without MHE, (2) MMN changes in parallel with performance in attention tests and/or MHE in a longitudinal study, and (3) MMN predicts performance in attention tests and/or in the Psychometric Hepatic Encephalopathy Score (PHES). We performed MMN analysis as well as attention and coordination tests in 34 control subjects and in 37 patients with liver cirrhosis without MHE and 23 with MHE. Patients with MHE show reduced performance in selective and sustained attention tests and in visuomotor and bimanual coordination tests. The MMN wave area was reduced in patients with MHE, but not in those without MHE. In the longitudinal study, MMN area improved in parallel with performance in attention tests and PHES in 4 patients and worsened in parallel in another 4. Logistic regression analyses showed that MMN area predicts performance in attention tests and in PHES, but not in other tests or critical flicker frequency. Receiver operating characteristic curve analyses showed that MMN area predicts attention deficits in the number connection tests A and B, Stroop tasks, and MHE, with sensitivities of 75%-90% and specificities of 76%-83%. CONCLUSION: MMN area is useful to diagnose attention deficits and MHE in patients with liver cirrhosis.

Serum nitrotyrosine and psychometric tests as indicators of impaired fitness to drive in cirrhotic patients with minimal hepatic encephalopathy.

BACKGROUND & AIMS: Cirrhotic patients with minimal hepatic encephalopathy (MHE) show impaired driving ability and increased vehicle accidents. The neurological deficits contributing to impair driving and the underlying mechanisms are poorly understood. Early detection of driving impairment would help to reduce traffic accidents in MHE patients. It would be therefore useful to have psychometric or biochemical parameters reflecting driving impairment. The aims of this work were as follows: (i) to shed light on the neurological deficits contributing to impair driving; (ii) to assess whether some psychometric test or biochemical parameter is a good indicator of driving impairment. METHODS: We assessed in 22 controls, 36 cirrhotic patients without and 15 with MHE, driving performance using a driving simulator (SIMUVEG) and Driver Test. MHE was diagnosed using the psychometric hepatic encephalopathy score (PHES). Psychometric tests assessing different neurological functions (mental processing speed, attention, visuo-spatial and bimanual coordination) were performed. Blood ammonia and parameters related with nitric oxide-cGMP metabolism, IL-6, IL-18 and 3-nitrotyrosine were measured. RESULTS: Patients with MHE showed impaired driving ability correlating with MHE grade, with impaired vehicle lateral control in spite of reduced driving speed. Patients with MHE show psychomotor slowing, longer reaction times, impaired bimanual and visuo-spatial coordination and concentrated attention and slowed speed of anticipation and increased blood ammonia, cGMP, IL-6, IL-18 and 3-nitrotyrosine. CONCLUSIONS: Impaired mental processing speed, attention and alterations in visuo-spatial and motor coordination seem main contributors to impaired driving ability in patients with MHE. Increased serum 3-nitrotyrosine is associated with impaired driving ability.

Chronic hyperammonemia, glutamatergic neurotransmission and neurological alterations.

This mini-review focus on our studies on alterations in glutamatergic neurotransmission and their role in neurological alterations in rat models of chronic hyperammonemia and hepatic encephalopathy (HE). Hyperammonemia impairs the glutamate-nitric oxide (NO)-cGMP pathway in cerebellum, which is responsible for reduced learning ability. We studied the underlying mechanisms and designed treatments to restore the pathway and learning. This was achieved by treatment with: phosphodiesterase 5 inhibitors, cGMP, anti-inflammatories (ibuprofen), p38 inhibitors or GABAA receptor antagonists (bicuculline). Hyperammonemia alters signal transduction associated to metabotropic glutamate receptors (mGluRs). Hypokinesia in hyperammonemia and HE is due to increased extracellular glutamate and mGluR1 activation in substantia nigra; blocking this receptor restores motor activity. The motor responses to mGluRs activation in nucleus accumbens (NAcc) are altered in hyperammonemia and HE, with reduced dopamine and increased glutamate release. This leads to activation of different neuronal circuits and enhanced motor responses. These studies show that altered responses to activation of NMDA receptors and mGluRs play essential roles in cognitive and motor alterations in hyperammonemia and HE and provide new treatments restoring cognitive and motor function.

Golexanolone, a GABAA receptor modulating steroid antagonist, restores motor coordination and cognitive function in hyperammonemic rats by dual effects on peripheral inflammation and neuroinflammation.

AIMS: Hyperammonemic rats show peripheral inflammation, increased GABAergic neurotransmission and neuroinflammation in cerebellum and hippocampus which induce motor incoordination and cognitive impairment. Neuroinflammation enhances GABAergic neurotransmission in cerebellum by enhancing the TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways. Golexanolone reduces GABAA receptors potentiation by allopregnanolone. This work aimed to assess if treatment of hyperammonemic rats with golexanolone reduces peripheral inflammation and neuroinflammation and restores cognitive and motor function and to analyze underlying mechanisms. METHODS: Rats were treated with golexanolone and effects on peripheral inflammation, neuroinflammation, TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways, and cognitive and motor function were analyzed. RESULTS: Hyperammonemic rats show increased TNFα and reduced IL-10 in plasma, microglia and astrocytes activation in cerebellum and hippocampus, and impaired motor coordination and spatial and short-term memories. Treating hyperammonemic rats with golexanolone reversed changes in peripheral inflammation, microglia and astrocytes activation and restored motor coordination and spatial and short-term memory. This was associated with reversal of the hyperammonemia-enhanced activation in cerebellum of the TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways. CONCLUSION: Reducing GABAA receptors activation with golexanolone reduces peripheral inflammation and neuroinflammation and improves cognitive and motor function in hyperammonemic rats. The effects identified would also occur in patients with hepatic encephalopathy and, likely, in other pathologies associated with neuroinflammation.

Metabolic syndrome is associated with poor response to rifaximin in minimal hepatic encephalopathy.

Patients with cirrhosis may show minimal hepatic encephalopathy (MHE), for which rifaximin is effective. Metabolic syndrome may be associated with cognitive impairment. Our aims were to evaluate the influence of metabolic syndrome features on response to rifaximin for neurological and inflammatory alterations in MHE. A prospective cohort study was conducted in 63 cirrhotic patients and 30 controls from two tertiary centres recruited between 2015 and 2019. Metabolic syndrome was defined according to the Adult Treatment Panel-III. Patients were classified into 31 without and 32 with MHE according to the Psychometric Hepatic Encephalopathy Score (PHES). All participants performed specific psychometric tests, and inflammatory parameters were studied. Patients with MHE received rifaximin (400 mg/8 h). Response was evaluated by PHES at 3 and 6 months. Response according to metabolic syndrome manifestations was compared. The response rate was 66%. Older age (p = 0.012) and all metabolic syndrome diseases (p < 0.05) were associated with non-response, plus an increase in risk as the number of manifestations rose (p < 0.001). Patients with metabolic manifestations exhibited worse processing speed (p = 0.011), working memory (p = 0.005), visual coordination (p = 0.013) and lower proportion of activated CD4+ lymphocytes (p = 0.039) at baseline, as well as worse concentration (p = 0.030), bimanual coordination (p = 0.004) and higher levels of intermediate monocytes (p = 0.026), CX3CL1 (p < 0.05), IL-17 (p = 0.022), AHR (p = 0.010) and IgG (p < 0.05) at 3 and/or 6 months of rifaximin. Patients with clinical signs of metabolic syndrome have poor response to rifaximin for MHE, with a higher proportion of neurological alterations associated with a pro-inflammatory environment.

A New Score Unveils a High Prevalence of Mild Cognitive Impairment in Patients with Nonalcoholic Fatty Liver Disease.

Patients with nonalcoholic fatty liver disease (NAFLD) may show mild cognitive impairment (MCI). The neurological functions affected remain unclear. The aims were to: (1) Characterize the neuropsychological alterations in NAFLD patients; (2) assess the prevalence of impairment of neurological functions evaluated; (3) develop a new score for sensitive and rapid MCI detection in NAFLD; (4) assess differences in MCI features between patients with nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH); and (5) compare neuropsychological alterations in NAFLD patients with cirrhotic patients with MCI. Fifty-nine NAFLD patients and 53 controls performed psychometric tests assessing different neurological functions: PHES (Psychometric Hepatic Encephalopathy Score) battery, d2, Stroop, Oral SDMT (Symbol Digit Modalities Test), Digit Span, number-letter test, and bimanual and visual-motor coordination tests. NAFLD patients show impairment in attention, mental concentration, psychomotor speed, cognitive flexibility, inhibitory mental control, and working memory. We developed a new, rapid, and sensitive score based on the most affected parameters in NAFLD patients, unveiling that 32% of NAFLD show MCI. Prevalence was similar in NAFL (36%) or NASH (27%) patients, but lower in NAFLD than in cirrhosis (65%). MCI prevalence is significant in NAFLD patients. Psychometric testing is warranted in these patients to unveil MCI and take appropriate measures to reverse and prevent its progression.

Patients with Minimal Hepatic Encephalopathy Show Altered Thermal Sensitivity and Autonomic Function.

Cirrhotic patients may experience alterations in the peripheral nervous system and in somatosensory perception. Impairment of the somatosensory system could contribute to cognitive and motor alterations characteristic of minimal hepatic encephalopathy (MHE), which affects up to 40% of cirrhotic patients. We assessed the relationship between MHE and alterations in thermal, vibration, and/or heat pain sensitivity in 58 cirrhotic patients (38 without and 20 with MHE according to Psychometric Hepatic Encephalopathy Score) and 39 controls. All participants underwent attention and coordination tests, a nerve conduction study, autonomic function testing, and evaluation of sensory thresholds (vibration, cooling, and heat pain detection) by electromyography and quantitative sensory testing. The detection thresholds for cold and heat pain on the foot were higher in patients with, than those without MHE. This hyposensitivity was correlated with attention deficits. Reaction times in the foot were longer in patients with, than without MHE. Patients with normal sural nerve amplitude showed altered thermal sensitivity and autonomic function, with stronger alterations in patients with, than in those without MHE. MHE patients show a general decrease in cognitive and sensory abilities. Small fibers of the autonomic nervous system and thermal sensitivity are altered early on in MHE, before large sensory fibers. Quantitative sensory testing could be used as a marker of MHE.

Cirrhotic patients with minimal hepatic encephalopathy have increased capacity to eliminate superoxide and peroxynitrite in lymphocytes, associated with cognitive impairment.

Patients with minimal hepatic encephalopathy (MHE) show increased oxidative stress in blood. We aimed to assess whether MHE patients show alterations in different types of blood cells in (a) basal reactive oxygen and nitrogen species levels; (b) capacity to metabolise these species. To assess the mechanisms involved in the altered capacity to metabolise these species we also analysed: (c) peroxynitrite formation and d) peroxynitrite reaction with biological molecules. Levels of reactive oxygen and nitrogen species were measured by flow cytometry in blood cell populations from cirrhotic patients with and without MHE and controls, under basal conditions and after adding generators of superoxide (plumbagin) or nitric oxide (NOR-1) to assess the capacity to eliminate them. Under basal conditions, MHE patients show reduced superoxide and peroxynitrite levels and increased nitric oxide (NO) and nitrotyrosine levels. In patients without MHE plumbagin strongly increases cellular superoxide, moderately peroxynitrite and reduces NO levels. In MHE patients, plumbagin increases slightly superoxide and strongly peroxynitrite levels and affects slightly NO levels. NOR-1 increases NO levels much less in patients with than without MHE. These data show that the mechanisms and the capacity to eliminate cellular superoxide, NO and peroxynitrite are enhanced in MHE patients. Superoxide elimination is enhanced through reaction with NO to form peroxynitrite which, in turn, is eliminated by enhanced reaction with biological molecules, which could contribute to cognitive impairment in MHE. The data show that basal free radical levels do not reflect the oxidative stress status in MHE.

The PHES battery does not detect all cirrhotic patients with early neurological deficits, which are different in different patients.

BACKGROUND AND AIMS: The psychometric hepatic encephalopathy score (PHES) is the "gold standard" for minimal hepatic encephalopathy (MHE) diagnosis. Some reports suggest that some cirrhotic patients "without" MHE according to PHES show neurological deficits and other reports that neurological alterations are not homogeneous in all cirrhotic patients. This work aimed to assess whether: 1) a relevant proportion of cirrhotic patients show neurological deficits not detected by PHES; 2) cirrhotic patients with mild neurological deficits are a homogeneous population or may be classified in sub-groups according to specific deficits. METHODS: Cirrhotic patients "without" (n = 56) or "with" MHE (n = 41) according to PHES and controls (n = 52) performed psychometric tests assessing attention, concentration, mental processing speed, working memory and bimanual and visuomotor coordination. Heterogeneity of neurological alterations was analysed using Hierarchical Clustering Analysis. RESULTS: PHES classified as "with" MHE 42% of patients. Around 40% of patients "without" MHE according to PHES fail two psychometric tests. Oral SDMT, d2, bimanual and visuo-motor coordination tests are failed by 54, 51, 51 and 43% of patients, respectively. The earliest neurological alterations are different for different patients. Hierarchical clustering analysis shows that patients "without" MHE according to PHES may be classified in clusters according to the tests failed. In some patients coordination impairment appear before cognitive impairment while in others concentration and attention deficits appear before. CONCLUSIONS: PHES is not sensitive enough to detect early neurological alterations in a relevant proportion of cirrhotic patients. Oral SDMT, d2 and bimanual and visuo-motor coordination tests are more sensitive. The earliest neurological alterations are different in different cirrhotic patients. These data also have relevant clinical implications. Patients classified as "without MHE" by PHES belonging to clusters 3 and 4 in our study have a high risk of suffering clinical complications, including overt HE and must be diagnosed and clinically followed.

Is cognitive impairment in cirrhotic patients due to increased peroxynitrite and oxidative stress?

Cirrhotic patients may suffer minimal hepatic encephalopathy (MHE), with mild cognitive impairment. 3-Nitro-tyrosine levels are a good biomarker for diagnosis of the cognitive impairment and MHE in cirrhotic patients. This suggests that oxidative stress could be involved in the induction of cognitive and motor alterations in MHE. We have observed that patients with MHE show increased oxidative stress in blood compared with cirrhotic patients without MHE, with increased lipid peroxidation, DNA oxidation, protein carbonylation, 3-nitrotyrosine, oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio, and GSH levels. The activities of antioxidant enzymes are enhanced in erythrocytes and mononuclear cells from patients with and without MHE compared with control subjects. Only glutathione peroxidase activity was increased in MHE patients compared with patients without MHE. Oxidative stress markers in blood, especially GSSG/GSH ratio, GSH, malondialdehyde, and 3-nitrotyrosine, correlate with deficits in attention and motor coordination. The increase in antioxidant activities in patients would be an adaptive mechanism to cope with enhanced oxidative stress, although it is not effective enough to normalize it. Our observations lead to the hypothesis that oxidative stress and increased peroxynitrite formation would mediate the synergistic effects of hyperammonemia and inflammation on cognitive and motor impairment in MHE.

Urinary Klotho measured by ELISA as an early biomarker of acute kidney injury in patients after cardiac surgery or coronary angiography.

BACKGROUND: Acute kidney injury (AKI) is a common complication after cardiac surgery and percutaneous coronary interventions which markedly worsens prognosis. In recent years, new early biomarkers of AKI have been identified, but many important aspects still remain to be solved. Klotho is a pleiotropic protein that acts as a paracrine and endocrine factor in multiple organs. Reduced renal Klotho levels have been show in several animal models of AKI. No study has been published in which Klotho was tested in humans as an early marker of AKI. The aim of this work is to assess the usefulness of measuring urinary Klotho for the early diagnosis of AKI in patients with acute coronary syndrome or heart failure undergoing cardiac surgery or coronary angiography. METHODS: Urinary Klotho was measured 12 hours after intervention in 60 patients admitted to the Intensive Care Unit with acute coronary syndrome or heart failure secondary to coronary or valvular conditions, who underwent coronary angiography (30 patients) or cardiac bypass surgery or heart valve replacement (30 patients). The primary endpoint used was the onset of AKI according to the RIFLE classification system. Human Klotho levels were measured using an ELISA assay. RESULTS: We found no differences in urinary Klotho levels between AKI patients and those who did not develop AKI. Moreover, there was not significant correlation between urinary Klotho levels and the presence of AKI. CONCLUSION: Urinary Klotho measured by ELISA does not seem to be a good candidate to be used as an early biomarker of AKI.

Non invasive blood flow measurement in cerebellum detects minimal hepatic encephalopathy earlier than psychometric tests.

AIM: To assess whether non invasive blood flow measurement by arterial spin labeling in several brain regions detects minimal hepatic encephalopathy. METHODS: Blood flow (BF) was analyzed by arterial spin labeling (ASL) in different brain areas of 14 controls, 24 cirrhotic patients without and 16 cirrhotic patients with minimal hepatic encephalopathy (MHE). Images were collected using a 3 Tesla MR scanner (Achieva 3T-TX, Philips, Netherlands). Pulsed ASL was performed. Patients showing MHE were detected using the battery Psychometric Hepatic Encephalopathy Score (PHES) consisting of five tests. Different cognitive and motor functions were also assessed: alterations in selective attention were evaluated using the Stroop test. Patients and controls also performed visuo-motor and bimanual coordination tests. Several biochemical parameters were measured: serum pro-inflammatory interleukins (IL-6 and IL-18), 3-nitrotyrosine, cGMP and nitrates+nitrites in plasma, and blood ammonia. Bivariate correlations were evaluated. RESULTS: In patients with MHE, BF was increased in cerebellar hemisphere (P = 0.03) and vermis (P = 0.012) and reduced in occipital lobe (P = 0.017). BF in cerebellar hemisphere was also increased in patients without MHE (P = 0.02). Bimanual coordination was impaired in patients without MHE (P = 0.05) and much more in patients with MHE (P < 0.0001). Visuo-motor coordination was impaired only in patients with MHE (P < 0.0001). Attention was slightly affected in patients without MHE and more strongly in patients with MHE (P < 0.0001). BF in cerebellar hemisphere and vermis correlated with performance in most tests of PHES [(number connection tests A (NCT-A), B (NCT-B)and line tracing test] and in the congruent task of Stroop test. BF in frontal lobe correlated with NCT-A. Performance in bimanual and visuomotor coordination tests correlated only with BF in cerebellar hemisphere. BF in occipital lobe correlates with performance in the PHES battery and with CFF. BF in cerebellar hemisphere correlates with plasma cGMP and nitric oxide (NO) metabolites. BF in vermis cerebellar also correlates with NO metabolites and with 3-nitrotyrosine. IL-18 in plasma correlates with BF in thalamus and occipital lobe. CONCLUSION: Non invasive BF determination in cerebellum using ASL may detect MHE earlier than the PHES. Altered NO-cGMP pathway seems to be associated to altered BF in cerebellum.