Showing papers by "Vicente Felipo published in 2017"

Sildenafil reduces neuroinflammation in cerebellum, restores GABAergic tone, and improves motor in‐coordination in rats with hepatic encephalopathy

Abstract: Aims Patients with liver disease may develop hepatic encephalopathy (HE), with cognitive impairment and motor in-coordination. Rats with HE due to portacaval shunts (PCS) show motor in-coordination. We hypothesized that in PCS rats: (i) Motor in-coordination would be due to enhanced GABAergic tone in cerebellum; (ii) increased GABAergic tone would be due to neuroinflammation; (iii) increasing cGMP would reduce neuroinflammation and GABAergic tone and restore motor coordination. To assess these hypotheses, we assessed if (i) treatment with sildenafil reduces neuroinflammation; (ii) reduced neuroinflammation is associated with reduced GABAergic tone and restored motor coordination. Methods Rats were treated with sildenafil to increase cGMP. Microglia and astrocytes activation were analyzed by immunohistochemistry, extracellular GABA by microdialysis, and motor coordination in the beam walking. Results PCS rats show neuroinflammation in cerebellum, with microglia and astrocytes activation, increased IL-1b and TNF-a and reduced YM-1 and IL-4. Membrane expression of the GABA transporter GAT1 is reduced, while GAT3 is increased. Extracellular GABA and motor in-coordination are increased. Sildenafil treatment eliminates neuroinflammation, microglia and astrocytes activation; changes in membrane expression of GABA transporters; and restores motor coordination. Conclusions This study supports an interplay between cGMP-neuroinflammation and GABAergic neurotransmission in impairing motor coordination in PCS rats.

Increasing extracellular cGMP in cerebellum in vivo reduces neuroinflammation, GABAergic tone and motor in-coordination in hyperammonemic rats.

Abstract: Hyperammonemia is a main contributor to cognitive impairment and motor in-coordination in patients with hepatic encephalopathy. Hyperammonemia-induced neuroinflammation mediates the neurological alterations in hepatic encephalopathy. Intracerebral administration of extracellular cGMP restores some but not all types of cognitive impairment. Motor in-coordination, is mainly due to increased GABAergic tone in cerebellum. We hypothesized that extracellular cGMP would restore motor coordination in hyperammonemic rats by normalizing GABAergic tone in cerebellum and that this would be mediated by reduction of neuroinflammation. The aims of this work were to assess whether chronic intracerebral administration of cGMP to hyperammonemic rats: 1) restores motor coordination; 2) reduces neuroinflammation in cerebellum; 3) reduces extracellular GABA levels and GABAergic tone in cerebellum; and also 4) to provide some advance in the understanding on the molecular mechanisms involved. The results reported show that rats with chronic hyperammonemia show neuroinflammation in cerebellum, including microglia and astrocytes activation and increased levels of IL-1b and TNFa and increased membrane expression of the TNFa receptor. This is associated with increased glutaminase expression and extracellular glutamate, increased amount of the GABA transporter GAT-3 in activated astrocytes, increased extracellular GABA in cerebellum and motor in-coordination. Chronic intracerebral administration of extracellular cGMP to rats with chronic hyperammonemia reduces neuroinflammation, including microglia and astrocytes activation and membrane expression of the TNFa receptor. This is associated with reduced nuclear NF-κB, glutaminase expression and extracellular glutamate, reduced amount of the GABA transporter GAT-3 in activated astrocytes and reduced extracellular GABA in cerebellum and restoration of motor coordination. The data support that extracellular cGMP restores motor coordination in hyperammonemic rats by reducing microglia activation and neuroinflammation, leading to normalization of extracellular glutamate and GABA levels in cerebellum and of motor coordination.

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

Abstract: 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.

Minimal hepatic encephalopathy is associated with expansion and activation of CD 4+ CD28 − , Th22 and Tfh and B lymphocytes

Abstract: Peripheral inflammation acts synergistically with hyperammonemia in inducing neurological alterations in cirrhotic patients with minimal hepatic encephalopathy (MHE). We hypothesized that appearance of MHE would be associated to some specific qualitative change in peripheral inflammation. The aim of this work was to characterize the changes in peripheral inflammation associated to appearance of MHE. We analyzed it by immunophenotyping and cytokine profile analysis, in cirrhotic patients without or with MHE and controls. The main alterations associated specifically with MHE are: 1) increased activation of all subtypes of CD4+ T-lymphocytes, with the increased expression of CD69; 2) increased amount of CD4+CD28− T lymphocytes, associated with increased levels of CX3CL1 and of IL-15; 3) increased differentiation of CD4+ T lymphocytes to Th follicular and Th22; 4) increased activation of B lymphocytes and serum IgG. This study has identified some specific alterations of the immune system associated with appearance of the neurological alterations in MHE patients.

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

Abstract: 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.

Reduced resting state connectivity and gray matter volume correlate with cognitive impairment in minimal hepatic encephalopathy.

Abstract: Background and aims Minimal hepatic encephalopathy (MHE) is associated with cognitive alterations and changes in connectivity. We assessed the relationship of the abnormalities of resting-state functional connectivity (rs-FC) and gray matter (GM) volume with different cognitive alterations and biochemical parameters associated to MHE. Methods Thirty-nine cirrhotic patients (26 without and 13 with MHE) and 24 controls were widely cognitive assessed with a battery of psychometric tests. Atrophy was determined using Voxel-Based Morphometry and rs-FC was assessed by independent component analysis. Receiver operating characteristic (ROC) curves was performed to assess the diagnostic utility of rs-FC and GM reduction for the discrimination of patients with and without MHE. Blood ammonia, cGMP, and levels of pro-inflammatory interleukins were measured. Results MHE patients showed significant decrease of GM volume and lesser degree of rs-FC in different networks related to attention and executive functions as compared to controls and patients without MHE. There is a progressive reduction in rs-FC in the default mode network with the progression of cognitive impairment. MHE patients showed GM reduction in the right frontal lobe, right insula and right cerebellum compared to patients without MHE. Alterations in GM volume and rs-FC correlated with the scores of different cognitive tests. Conclusions Decreased cognitive performance is associated by reduced rs-FC and GM atrophy in MHE patients. These changes could have predictive value for detecting MHE.

Real-time cytometric assay of nitric oxide and superoxide interaction in peripheral blood monocytes: A no-wash, no-lyse kinetic method.

Abstract: Background Nitric oxide (NO) and its related reactive nitrogen species (RNS) and reactive oxygen species (ROS) are crucial in monocyte responses against pathogens and also in inflammatory conditions. Central to both processes is the generation of the strong oxidant peroxynitrite (ONOO) by a fast reaction between NO and superoxide anion. ONOO is a biochemical junction for ROS- and RNS cytotoxicity and causes protein nitrosylation. Circulating by-products of protein nitrosylation are early biomarkers of inflammation-based conditions, including minimal hepatic encephalopathy in cirrhotic patients (Montoliu et al., Am J Gastroenterol 2011; 106:1629–1637). In this context, we have designed a novel no-wash, no-lyse real-time flow cytometry assay to detect and follow-up the NO- and superoxide-driven generation of ONOO in peripheral blood monocytes. Methods Whole blood samples were stained with CD45 and CD14 antibodies plus one of a series of fluorescent probes sensitive to RNS, ROS, or glutathione, namely 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, dihydrorhodamine 123, MitoSOX Red, dihydroethidium, and 5-chloromethylfluorescein diacetate. Samples were exposed sequentially to a NO donor and three different superoxide donors, and analyzed in real time by kinetic flow cytometry. Relevant kinetic descriptors, such as the rate of fluorescence change, were calculated from the kinetic plot. Results The generation of ONOO, which consumes both NO and superoxide, led to a decrease in the intensity of the cellular fluorescence of the probes sensitive to these molecules. Conclusion This is a fast and simple assay that may be used to monitor the intracellular generation of ONOO in physiological, pathological, and pharmacological contexts. © 2015 International Clinical Cytometry Society

Extracellular Cyclic GMP Modulates Membrane Expression of The GluA1 and GluA2 Subunits of AMPA Receptor in Cerebellum: Molecular Mechanisms Involved.

Abstract: There is increasing evidence that extracellular cGMP modulates glutamatergic neurotransmission and some forms of learning. However, the underlying mechanisms remain unknown. We proposed the hypotheses that extracellular cGMP may regulate membrane expression of AMPA receptors. To do this extracellular cGMP should act on a membrane protein and activate signal transduction pathways modulating phosphorylation of the GluA1 and/or GluA2 subunits. It has been shown that extracellular cGMP modulates glycine receptors. The aims of this work were to assess: 1) whether extracellular cGMP modulates membrane expression of GluA1 and GluA2 subunits of AMPA receptors in cerebellum in vivo; 2) whether this is mediated by glycine receptors; 3) the role of GluA1 and GluA2 phosphorylation and 4) identify steps of the intracellular pathways involved. We show that extracellular cGMP modulates membrane expression of GluA1 and GluA2 in cerebellum in vivo and unveil the mechanisms involved. Extracellular cGMP reduced glycine receptor activation, modulating cAMP, protein kinases and phosphatases, and GluA1 and GluA2 phosphorylation, resulting in increased GluA1 and reduced GluA2 membrane expression. Extracellular cGMP therefore modulates membrane expression of AMPA receptors and glutamatergic neurotransmission. The steps identified may be therapeutic targets to improve neurotransmission and neurological function in pathological situations with abnormal glutamatergic neurotransmission.

Determination of selected neurotoxic insecticides in small amounts of animal tissue utilizing a newly constructed mini-extractor

Abstract: We developed a simple analytical method for the simultaneous determination of representatives of various groups of neurotoxic insecticides (carbaryl, chlorpyrifos, cypermethrin, and α-endosulfan and β-endosulfan and their metabolite endosulfan sulfate) in limited amounts of animal tissues containing different amounts of lipids. Selected tissues (rodent fat, liver, and brain) were extracted in a special in-house-designed mini-extractor constructed on the basis of the Soxhlet and Twisselmann extractors. A dried tissue sample placed in a small cartridge was extracted, while the nascent extract was simultaneously filtered through a layer of sodium sulfate. The extraction was followed by combined clean-up, including gel permeation chromatography (in case of high lipid content), ultrasonication, and solid-phase extraction chromatography using C18 on silica and aluminum oxide. Gas chromatography coupled with high-resolution mass spectrometry was used for analyte separation, detection, and quantification. Average recoveries for individual insecticides ranged from 82 to 111%. Expanded measurement uncertainties were generally lower than 35%. The developed method was successfully applied to rat tissue samples obtained from an animal model dealing with insecticide exposure during brain development. This method may also be applied to the analytical treatment of small amounts of various types of animal and human tissue samples. A significant advantage achieved using this method is high sample throughput due to the simultaneous treatment of many samples.

Sildenafil Treatment Eliminates Pruritogenesis and Thermal Hyperalgesia in Rats with Portacaval Shunts.

Abstract: Pruritus is a common symptom in chronic liver diseases, which may also alter thermal sensitivity. The underlying mechanisms remain unclear and treatments are not satisfactory. Portal-systemic shunting has been proposed to alter thermal sensitivity in cirrhotics. Inflammation-induced enhanced activity of the Transient Receptor Potential Vanilloid 1 (TRPV1) may contribute to pruritus and thermal hyperalgesia. Sildenafil reduces neuroinflammation in portacaval shunt (PCS) rats. The aims were to assess whether: (1) PCS rats show enhanced scratching or thermal sensitivity; (2) TRPV1 activity is enhanced in PCS rats; (3) treatment with sildenafil reduces TRPV1 activation, scratching and thermal hyperalgesia. Rats were treated with sildenafil beginning 3 weeks after surgery. The number of scratches performed were counted. Thermal hyperalgesia was analyzed using the Hargreaves’ Plantar Test. TRPV1 activation by measuring the increase in Ca2+ induced by capsaicin in dorsal root ganglia neurons. PCS rats show enhanced scratching behavior, reaching 66 ± 5 scratches/h (p < 0.01) at 21 days after surgery, while controls show 37 ± 2 scratches/h. PCS rats show thermal hyperalgesia. Paw withdrawal latency was reduced (p < 0.05) to 10 ± 1 s compared to controls (21 ± 2 s). Capsaicin-induced calcium increase was higher in dorsal root ganglia cultures from PCS rats, indicating TRPV1functional increase. PCS rats show enhanced scratching behavior and thermal sensitivity and are a good model to study these alterations in chronic liver diseases. Enhanced sensitivity and activity of TRPV1 channel underlies these alterations. Treatment with sildenafil reduces TRPV1 channel sensitivity and activity and normalizes scratching behavior and thermal sensitivity.