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1.
J Neurochem ; : e14941, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31840253

RESUMO

Minimal hepatic encephalopathy (MHE) is a neuropsychiatric syndrome produced by central nervous system dysfunction subsequent to liver disease. Hyperammonemia and inflammation act synergistically to alter neurotransmission, leading to the cognitive and motor alterations in MHE, which are reproduced in rat models of chronic hyperammonemia. Patients with MHE show altered functional connectivity in different neural networks and a reduced response in the cognitive potential mismatch negativity (MMN), which correlates with attention deficits. The mechanisms by which MMN is altered in MHE remain unknown. The objectives of this work are as follows: To assess if rats with chronic hyperammonemia reproduce the reduced response in the MMN found in patients with MHE. Analyze the functional connectivity between the areas (CA1 area of the dorsal hippocampus, prelimbic cortex, primary auditory cortex, and central inferior colliculus) involved in the generation of the MMN and its possible alterations in hyperammonemia. Granger causality analysis has been applied to detect the net flow of information between the population neuronal activities recorded from a local field potential approach. Analyze if altered MMN response in hyperammonemia is associated with alterations in glutamatergic and GABAergic neurotransmission. Extracellular levels of the neurotransmitters and/or membrane expression of their receptors have been analyzed after the tissue isolation of the four target sites. The results show that rats with chronic hyperammonemia show reduced MMN response in hippocampus, mimicking the reduced MMN response of patients with MHE. This is associated with altered functional connectivity between the areas involved in the generation of the MMN. Hyperammonemia also alters membrane expression of glutamate and GABA receptors in hippocampus and reduces the changes in extracellular GABA and glutamate induced by the MMN paradigm of auditory stimulus in hippocampus of control rats. The changes in glutamatergic and GABAergic neurotransmission and in functional connectivity between the brain areas analyzed would contribute to the impairment of the MMN response in rats with hyperammonemia and, likely, also in patients with MHE.

2.
ACS Chem Neurosci ; 10(10): 4264-4279, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31464424

RESUMO

Exposure to low levels of environmental contaminants, including pesticides, induces neurodevelopmental toxicity. Environmental and food contaminants can reach the brain of the fetus, affecting brain development and leading to neurological dysfunction. The pesticide endosulfan is a persistent pollutant, and significant levels still remain detectable in the environment although its use is banned in some countries. In rats, endosulfan exposure during brain development alters motor activity, coordination, learning, and memory, even several months after uptake, and does so in a sex-dependent way. However, the molecular mechanisms driving these effects have not been studied in detail. In this work, we performed a multiomics study in cerebellum from rats exposed to endosulfan during embryonic development. Pregnant rats were orally exposed to a low dose (0.5 mg/kg) of endosulfan, daily, from gestational day 7 to postnatal day 21. The progeny was evaluated for cognitive and motor functions at adulthood. Expression of messenger RNA and microRNA genes, as well as protein and metabolite levels, were measured on cerebellar samples from males and females. An integrative analysis was conducted to identify altered processes under endosulfan effect. Effects between males and females were compared. Pathways significantly altered by endosulfan exposure included the phosphatidylinositol signaling system, calcium signaling, the cGMP-PKG pathway, the inflammatory and immune system, protein processing in the endoplasmic reticulum, and GABA and taurine metabolism. Sex-dependent effects of endosulfan in the omics results that matched sex differences in cognitive and motor tests were found. These results shed light on the molecular basis of impaired neurodevelopment and contribute to the identification of new biomarkers of neurotoxicity.

3.
Acta Physiol (Oxf) ; 226(2): e13270, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30830722

RESUMO

Several million patients with liver cirrhosis suffer minimal hepatic encephalopathy (MHE), with mild cognitive and coordination impairments that reduce their quality of life and life span. Hyperammonaemia and peripheral inflammation act synergistically to induce these neurological alterations. We propose that MHE appearance is because of the changes in peripheral immune system, which are transmitted to brain, leading to neuroinflammation that alters neurotransmission leading to cognitive and motor alterations. We summarize studies showing that MHE in cirrhotic patients is associated with alterations in the immune system and that patients died with HE show neuroinflammation in cerebellum, with microglial and astrocytic activation and Purkinje cell loss. We also summarize studies in animal models of MHE on the role of peripheral inflammation in neuroinflammation induction, how neuroinflammation alters neurotransmission and how this leads to cognitive and motor alterations. These studies identify therapeutic targets and treatments that improve cognitive and motor function. Rats with MHE show neuroinflammation in hippocampus and altered NMDA and AMPA receptor membrane expression, which impairs spatial learning and memory. Neuroinflammation in cerebellum is associated with altered GABA transporters and extracellular GABA, which impair motor coordination and learning in a Y maze. These alterations are reversed by treatments that reduce peripheral inflammation (anti-TNFα, ibuprofen), neuroinflammation (sulphoraphane, p38 inhibitors), GABAergic tone (bicuculline, pregnenolone sulphate) or increase extracellular cGMP (sildenafil or cGMP). The mechanisms identified would also occur in other chronic diseases associated with inflammation, aging and some mental and neurodegenerative diseases. Treatments that improve MHE may also be beneficial to treat these pathologies.

4.
Front Pharmacol ; 10: 132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30858801

RESUMO

Patients with liver cirrhosis may develop minimal hepatic encephalopathy (MHE) with mild cognitive impairment. Hyperammonemia is a main contributor to cognitive impairment in MHE, which is mediated by neuroinflammation. GABAergic neurotransmission is altered in hyperammonemic rats. We hypothesized that, in hyperammonemic rats, (a) enhanced GABAergic tone would contribute to induce neuroinflammation, which would be improved by reducing GABAergic tone by chronic bicuculline treatment; (b) this would improve spatial learning and memory impairment; and (c) modulation of glutamatergic neurotransmission would mediate this cognitive improvement. The aim of this work was to assess the above hypotheses. Bicuculline was administrated intraperitoneally once a day for 4 weeks to control and hyperammonemic rats. The effects of bicuculline on microglia and astrocyte activation, IL-1ß content, on membrane expression of AMPA and NMDA glutamate receptors subunits in the hippocampus and on spatial learning and memory as well as anxiety were assessed. Treatment with bicuculline reduces astrocyte activation and IL-1ß but not microglia activation in the hippocampus of hyperammonemic rats. Bicuculline reverses the changes in membrane expression of AMPA receptor subunits GluA1 and GluA2 and of the NR2B (but not NR1 and NR2A) subunit of NMDA receptors. Bicuculline improves spatial learning and working memory and decreases anxiety in hyperammonemic rats. In hyperammonemia, enhanced activation of GABAA receptors in the hippocampus contributes to some but not all aspects of neuroinflammation, to altered glutamatergic neurotransmission and to impairment of spatial learning and memory as well as anxiety, all of which are reversed by reducing activation of GABAA receptors with bicuculline.

5.
J Hepatol ; 2019 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-30654069

RESUMO

BACKGROUND & AIMS: Chronic hyperammonemia induces neuroinflammation which mediates cognitive impairment. How hyperammonemia induces neuroinflammation remains unclear. We propose the hypothesis that chronic hyperammonemia would induce peripheral inflammation that would induce neuroinflammation and cognitive impairment, which would be prevented by reducing peripheral inflammation. The aims of this work were to assess whether: 1) chronic hyperammonemia induces peripheral inflammation; 2) peripheral inflammation contributes to hyperammonemia-induced neuroinflammation, altered neurotransmission and impaired spatial learning; 3) hyperammonemia-induced inflammation and neurpoinflammation are reversible following hyperammonemia elimination; 4) reducing peripheral inflammation with anti-TNFa reduces neuroinflammation and improves neurotransmission and cognitive impairment in hyperammonemic rats. METHODS: Chronic hyperammonemia was induced by feeding rats an ammonia-containing diet. Peripheral inflammation was analyzed by measuring prostaglandin E2 (PGE2), TNFa, IL-6 and IL-10. We tested whether chronic anti-TNFa treatment improves peripheral inflammation, neuroinflammation, membrane expression of glutamate receptors in hippocampus and spatial learning. RESULTS: Hyperammonemic rats show a rapid and reversible induction of peripheral inflammation, with increased pro-inflammatory PGE2, TNFa and IL-6, followed at around 10 days by reduced anti-inflammatory IL-10. Peripheral anti-TNFa treatment prevents peripheral inflammation induction and the increase in IL-1b and TNFa and microglia activation in hippocampus of the rats, which remain hyperammonemic. This is associated with prevention of the altered membrane expression of glutamate receptors and of the impairment of spatial memory assessed in the radial and Morris water mazes. CONCLUSIONS: This report unveils a new mechanism by which chronic hyperammonemia induces neurological alterations: induction of peripheral inflammation. This supports that reducing peripheral inflammation by safe procedures would improve cognitive function in patients with minimal hepatic encephalopathy. LAY SUMMARY: This article unveils a new mechanism by which chronic hyperammonemia induces cognitive impairment in rats: chronic hyperammonemia per se induces peripheral inflammation, which mediates many of its effects on brain, including induction of neuroinflammation, which alters neurotransmission, leading to cognitive impairment. It is also shown that reducing peripheral inflammation by treating rats with anti-TNFa, which does not cross the blood-brain barrier, prevents hyperammonemia-induced neuroinflammation, alterations in neurotransmission and cognitive impairment. This new view may have an important impact on the understanding of the mechanisms involved in minimal hepatic encephalopathy in cirrhotic patients and may allow developing new therapeutic approaches.

6.
Biochim Biophys Acta Mol Basis Dis ; 1864(1): 286-295, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29107806

RESUMO

Hyperammonemia contributes to altered neurotransmission and cognition in patients with hepatic encephalopathy. Hyperammonemia in rats affects differently high- and low-affinity AMPA receptors (AMPARs) in cerebellum. We hypothesized that hyperammonemia would alter differently membrane expression of AMPARs GluA1 and GluA2 subunits by altering its phosphorylation. This work aims were: 1) assess if hyperammonemia alters GluA1 and GluA2 subunits membrane expression in cerebellum and 2) analyze the underlying mechanisms. Hyperammonemia reduces membrane expression of GluA2 and enhances membrane expression of GluA1 in vivo. We show that changes in GluA2 and GluA1 membrane expression in hyperammonemia would be due to enhanced NMDA receptors activation which reduces cGMP levels and phosphodiesterase 2 (PDE2) activity, resulting in increased cAMP levels. This leads to increased protein kinase A (PKA) activity which activates phospholipase C (PLC) and protein kinase C (PKC) thus increasing phosphorylation of GluA2 in Ser880, which reduces GluA2 membrane expression, and phosphorylation of GluA1 in Ser831, which increases GluA1 membrane expression. Blocking NMDA receptors or inhibiting PKA, PLC or PKC normalizes GluA2 and GluA1 phosphorylation and membrane expression in hyperammonemic rats. Altered GluA2 and GluA1 membrane expression would alter signal transduction which may contribute to cognitive and motor alterations in hyperammonemia and hepatic encephalopathy.


Assuntos
Membrana Celular/metabolismo , Hiperamonemia/genética , Receptores de AMPA/genética , Animais , Membrana Celular/patologia , Doença Crônica , Encefalopatia Hepática/genética , Encefalopatia Hepática/metabolismo , Encefalopatia Hepática/patologia , Hiperamonemia/metabolismo , Hiperamonemia/patologia , Masculino , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Transdução de Sinais/genética , Transmissão Sináptica/genética
7.
ACS Chem Neurosci ; 9(2): 369-380, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29094921

RESUMO

Exposure to pesticides has been associated with neurodevelopmental toxicity. Usually people are exposed to mixtures of pesticides. However, most studies analyze the effects of individual pesticides. Developmental exposure to mixtures of pesticides may result in additive effects or in antagonistic or synergistic effects. The aim of this work was to compare the effects of developmental exposure of rats to cypermethrin or endosulfan with the effects of its mixture on cognitive and motor function and on some underlying mechanisms. Exposure to individual pesticides or the mixture was from gestational day 7 to postnatal day 21. We analyzed the effects, in males and females, on spatial learning and memory, associative learning, anxiety, motor coordination, and spontaneous motor activity. We also analyzed neuroinflammation and NMDA receptor subunits in hippocampus and extracellular GABA in cerebellum. Exposure to the mixture, but not to individual pesticides, impaired spatial memory in males, associative learning in females, and increased motor activity in males and females. This indicates a synergistic effect of cypermethrin and endolsufan exposure on these end points. In contrast, motor coordination was impaired by individual exposure to endosulfan or cypermethrin, associated with increased extracellular GABA in cerebellum, but these effects were prevented in rats exposed to the mixture, indicating an antagonistic effect of cypermethrin and endolsufan exposure on these end points. The results show different interaction modes (synergism or antagonism) of the pesticides, depending on the end point analyzed and the sex of the rats.


Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Endossulfano/toxicidade , Praguicidas/toxicidade , Piretrinas/toxicidade , Animais , Ansiedade/induzido quimicamente , Ansiedade/metabolismo , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Antagonismo de Drogas , Sinergismo Farmacológico , Endossulfano/antagonistas & inibidores , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Feminino , Aprendizagem/efeitos dos fármacos , Aprendizagem/fisiologia , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Praguicidas/antagonistas & inibidores , Piretrinas/antagonistas & inibidores , Ratos Wistar , Caracteres Sexuais , Memória Espacial/efeitos dos fármacos , Memória Espacial/fisiologia , Ácido gama-Aminobutírico/metabolismo
8.
Brain Behav Immun ; 69: 386-398, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29288802

RESUMO

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.


Assuntos
Cerebelo/metabolismo , GMP Cíclico/farmacologia , Hiperamonemia/metabolismo , Inflamação/metabolismo , Destreza Motora/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Astrócitos/metabolismo , Bicuculina/farmacologia , Cerebelo/efeitos dos fármacos , Antagonistas de Receptores de GABA-A/farmacologia , Glutaminase/metabolismo , Masculino , Microglia/metabolismo , Destreza Motora/efeitos dos fármacos , Ratos , Ratos Wistar
9.
Mol Neurobiol ; 55(6): 5047-5058, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28815510

RESUMO

γ-Secretase inhibitors (GSIs) are potential therapeutic agents for Alzheimer's disease (AD); however, trials have proven disappointing. We addressed the possibility that γ-secretase inhibition can provoke a rebound effect, elevating the levels of the catalytic γ-secretase subunit, presenilin-1 (PS1). Acute treatment of SH-SY5Y cells with the GSI LY-374973 (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, DAPT) augments PS1, in parallel with increases in other γ-secretase subunits nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1, yet with no increase in messenger RNA expression. Over-expression of the C-terminal fragment (CTF) of APP, C99, also triggered an increase in PS1. Similar increases in PS1 were evident in primary neurons treated repeatedly (4 days) with DAPT or with the GSI BMS-708163 (avagacestat). Likewise, rats examined after 21 days administered with avagacestat (40 mg/kg/day) had more brain PS1. Sustained γ-secretase inhibition did not exert a long-term effect on PS1 activity, evident through the decrease in CTFs of APP and ApoER2. Prolonged avagacestat treatment of rats produced a subtle impairment in anxiety-like behavior. The rebound increase in PS1 in response to GSIs must be taken into consideration for future drug development.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Presenilina-1/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Comportamento Animal , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Humanos , Masculino , Camundongos , Neuroblastoma/patologia , Neurônios/metabolismo , Oxidiazóis/farmacologia , Ratos , Ratos Wistar , Especificidade por Substrato/efeitos dos fármacos , Sulfonamidas/farmacologia
10.
Anal Bioanal Chem ; 409(25): 6015-6026, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28799107

RESUMO

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 ß-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. Graphical abstract Optimized workflow for the determination of selected insecticides in small amounts of animal tissue including newly developed mini-extractor.


Assuntos
Inseticidas/análise , Neurotoxinas/análise , Extração em Fase Sólida/instrumentação , Tecido Adiposo/química , Animais , Química Encefálica , Cromatografia em Gel/instrumentação , Cromatografia em Gel/métodos , Desenho de Equipamento , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Inseticidas/isolamento & purificação , Inseticidas/farmacocinética , Limite de Detecção , Fígado/química , Neurotoxinas/isolamento & purificação , Neurotoxinas/farmacocinética , Ratos , Extração em Fase Sólida/métodos , Sonicação/instrumentação , Sonicação/métodos
11.
CNS Neurosci Ther ; 23(5): 386-394, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28296282

RESUMO

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.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Cerebelo/efeitos dos fármacos , Encefalopatia Hepática/tratamento farmacológico , Destreza Motora/efeitos dos fármacos , Citrato de Sildenafila/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Astrócitos/fisiologia , Cerebelo/imunologia , Cerebelo/patologia , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Encefalopatia Hepática/patologia , Encefalopatia Hepática/fisiopatologia , Interleucina-1beta/metabolismo , Interleucina-4/metabolismo , Masculino , Microglia/efeitos dos fármacos , Microglia/patologia , Microglia/fisiologia , Destreza Motora/fisiologia , Neuroimunomodulação/efeitos dos fármacos , Neuroimunomodulação/fisiologia , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismo , Ácido gama-Aminobutírico/metabolismo
12.
Neurochem Res ; 42(3): 788-794, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27321307

RESUMO

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.


Assuntos
Hiperalgesia/tratamento farmacológico , Inibidores da Fosfodiesterase 5/farmacologia , Derivação Portocava Cirúrgica , Prurido/tratamento farmacológico , Citrato de Sildenafila/farmacologia , Animais , Cálcio/metabolismo , Gânglios Espinais/citologia , Temperatura Alta , Hiperalgesia/fisiopatologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Inibidores da Fosfodiesterase 5/uso terapêutico , Prurido/fisiopatologia , Ratos Wistar , Citrato de Sildenafila/uso terapêutico , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/metabolismo
13.
Food Chem Toxicol ; 99: 135-148, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27908700

RESUMO

The use of pesticides has been associated with impaired neurodevelopment in children. The aims of this work were to assess: 1) the effects on spatial learning of developmental exposure to pesticides 2) if the effects are sex-dependent and 3) if hippocampal neuroinflammation is associated with the impairment of spatial learning. We analyzed the effects of developmental exposure to four pesticides: chlorpyrifos, carbaryl, endosulfan and cypermethrin. Exposure was from gestational day 7 to post-natal day 21 and spatial learning and memory was assessed when the rats were young adults. The effects of pesticides on spatial learning were pesticide and gender-dependent. Carbaryl did not affect spatial learning in males or females. Endosulfan and chlorpyrifos impaired learning in males but not in females. Cypermethrin improved spatial learning in the Morris water maze both in males and females while impaired learning in the radial maze only in males. Spatial learning ability was lower in control female rats than in males. All pesticides induced neuroinflammation, increasing IL-1b content in the hippocampus and there is a negative correlation between IL-1b levels in the hippocampus and spatial learning. Neuroinflammation would contribute to the effects of pesticides on spatial learning.


Assuntos
Hipocampo/patologia , Inflamação/induzido quimicamente , Aprendizagem em Labirinto/efeitos dos fármacos , Praguicidas/toxicidade , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Aprendizagem Espacial/efeitos dos fármacos , Animais , Citocinas/metabolismo , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/crescimento & desenvolvimento , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Masculino , Memória/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Fatores Sexuais
14.
Front Mol Neurosci ; 9: 106, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27853420

RESUMO

Inflammation contributes to cognitive impairment in patients with hepatic encephalopathy (HE). However, the process by which peripheral inflammation results in cognitive impairment remains unclear. In animal models, neuroinflammation and altered neurotransmission mediate cognitive impairment. Taking into account these data, we hypothesized that in rats with HE: (1) peripheral inflammation is a main contributor to neuroinflammation; (2) neuroinflammation in hippocampus impairs spatial learning by altering AMPA and/or NMDA receptors membrane expression; (3) reducing peripheral inflammation with infliximab (anti-TNF-a) would improve spatial learning; (4) this would be associated with reduced neuroinflammation and normalization of the membrane expression of glutamate receptors. The aims of this work were to assess these hypotheses. We analyzed in rats with portacaval shunt (PCS) and control rats, treated or not with infliximab: (a) peripheral inflammation by measuring prostaglandin E2, IL10, IL-17, and IL-6; (b) neuroinflammation in hippocampus by analyzing microglial activation and the content of TNF-a and IL-1b; (c) AMPA and NMDA receptors membrane expression in hippocampus; and (d) spatial learning in the Radial and Morris water mazes. We assessed the effects of treatment with infliximab on peripheral inflammation, on neuroinflammation and AMPA and NMDA receptors membrane expression in hippocampus and on spatial learning and memory. PCS rats show increased serum prostaglandin E2, IL-17, and IL-6 and reduced IL-10 levels, indicating increased peripheral inflammation. PCS rats also show microglial activation and increased nuclear NF-kB and expression of TNF-a and IL-1b in hippocampus. This was associated with altered AMPA and NMDA receptors membrane expression in hippocampus and impaired spatial learning and memory in the radial and Morris water maze. Treatment with infliximab reduces peripheral inflammation in PCS rats, normalizing prostaglandin E2, IL-17, IL-6, and IL-10 levels in serum. Infliximab also prevents neuroinflammation, reduces microglial activation, translocates NF-kB into nucleoli and normalizes TNF-a and IL-1b content in hippocampus. This was associated with normalization of AMPA receptors membrane expression in hippocampus and of spatial learning and memory. The results suggest that peripheral inflammation contributes to spatial learning impairment in PCS rats. Treatment with anti-TNF-a could be a new therapeutic approach to improve cognitive function in patients with HE.

15.
ACS Chem Neurosci ; 7(12): 1753-1759, 2016 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-27673574

RESUMO

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.


Assuntos
Cerebelo/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espaço Extracelular/metabolismo , Hiperamonemia/metabolismo , Espaço Intracelular/metabolismo , Óxido Nítrico Sintase/metabolismo , Compostos de Amônio , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , GMP Cíclico/metabolismo , Dieta , Modelos Animais de Doenças , Ácido Glutâmico/metabolismo , Masculino , Óxido Nítrico/metabolismo , Fosforilação , Ratos Wistar , Transdução de Sinais , Técnicas de Cultura de Tecidos
16.
Sci Rep ; 6: 33124, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27634333

RESUMO

It has been proposed that extracellular cGMP modulates the ability to learn a Y maze task, but the underlying mechanisms remained unknown. Here we show that extracellular cGMP, at physiological concentrations, modulates learning in the Y maze in a biphasic way by modulating the glutamate-nitric oxide-cGMP pathway in cerebellum. Extracellular cGMP reduces glycine receptors activation inducing a voltage-dependent calcium-channels-mediated increase of calcium in Purkinje neurons. This calcium increase modulates CaMKII phosphorylation in a biphasic way. When basal calcium concentration is low extracellular cGMP reduces CaMKII phosphorylation, increasing nitric oxide synthase activity, the glutamate-NO-cGMP pathway function and learning ability. When basal calcium is normal extracellular cGMP increases CaMKII phosphorylation, reducing nitric oxide synthase activity, the pathway function and learning. These data unveil new mechanisms modulating learning in the Y maze and likely other learning types which may be therapeutic targets to improve learning in pathological situations associated with altered cGMP levels.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , GMP Cíclico/metabolismo , Ácido Glutâmico/metabolismo , Óxido Nítrico/metabolismo , Receptores da Glicina/metabolismo , Animais , Cálcio/metabolismo , Cerebelo/metabolismo , Aprendizagem/fisiologia , Masculino , Óxido Nítrico Sintase/metabolismo , Fosforilação/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia
17.
J Neuroinflammation ; 13(1): 245, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27623772

RESUMO

BACKGROUND: Peripheral inflammation contributes to the neurological alterations in hepatic encephalopathy (HE). Neuroinflammation and altered GABAergic neurotransmission mediate cognitive and motor alterations in rats with HE. It remains unclear (a) if neuroinflammation and neurological impairment in HE are a consequence of peripheral inflammation and (b) how neuroinflammation impairs GABAergic neurotransmission. The aims were to assess in rats with HE whether reducing peripheral inflammation with anti-TNF-α (1) prevents cognitive impairment and motor in-coordination, (2) normalizes neuroinflammation and extracellular GABA in the cerebellum and also (3) advances the understanding of mechanisms linking neuroinflammation and increased extracellular GABA. METHODS: Rats with HE due to portacaval shunt (PCS) were treated with infliximab. Astrocytes and microglia activation and TNF-α and IL-1ß were analyzed by immunohistochemistry. Membrane expression of the GABA transporters GAT-3 and GAT-1 was analyzed by cross-linking with BS3. Extracellular GABA was analyzed by microdialysis. Motor coordination was tested using the beam walking and learning ability using the Y maze task. RESULTS: PCS rats show peripheral inflammation, activated astrocytes, and microglia and increased levels of TNF-α and IL-1ß. Membrane expression of GAT-3 and extracellular GABA are increased, leading to impaired motor coordination and learning ability. Infliximab reduces peripheral inflammation, microglia, and astrocyte activation and neuroinflammation and normalizes GABAergic neurotransmission, motor coordination, and learning ability. CONCLUSIONS: Neuroinflammation is associated with altered GABAergic neurotransmission and increased GAT-3 membrane expression and extracellular GABA (a); peripheral inflammation is a main contributor to the impairment of motor coordination and of the ability to learn the Y maze task in PCS rats (b); and reducing peripheral inflammation using safe procedures could be a new therapeutic approach to improve cognitive and motor function in patients with HE


Assuntos
Cerebelo/metabolismo , Encefalopatia Hepática/patologia , Inflamação/tratamento farmacológico , Infliximab/uso terapêutico , Transtornos Psicomotores/tratamento farmacológico , Ácido gama-Aminobutírico/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , GMP Cíclico/metabolismo , Citocinas/metabolismo , Dinoprostona/metabolismo , Modelos Animais de Doenças , Líquido Extracelular/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Encefalopatia Hepática/complicações , Inflamação/etiologia , Infliximab/farmacologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos Psicomotores/etiologia , Ratos , Ratos Wistar
18.
Brain Behav Immun ; 57: 360-370, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27189036

RESUMO

Patients with hepatic encephalopathy (HE) show working memory and visuo-spatial orientation deficits. Hyperammonemia is a main contributor to cognitive impairment in HE. Hyperammonemic rats show impaired spatial learning and learning ability in the Y maze. Intracerebral administration of extracellular cGMP restores learning in the Y-maze. The underlying mechanisms remain unknown. It also remains unknown whether extracellular cGMP improves neuroinflammation or restores spatial learning in hyperammonemic rats and if it affects differently reference and working memory. The aims of this work were: Spatial working and reference memory were assessed using the radial and Morris water mazes and neuroinflammation by immunohistochemistry and Western blot. Membrane expression of NMDA and AMPA receptor subunits was analyzed using the BS3 crosslinker. Extracellular cGMP was administered intracerebrally using osmotic minipumps. Chronic hyperammonemia induces neuroinflammation in hippocampus, with astrocytes activation and increased IL-1ß, which are associated with increased NMDA receptors membrane expression and impaired working memory. This process is not affected by extracellular cGMP. Hyperammonemia also activates microglia and increases TNF-α, alters membrane expression of AMPA receptor subunits (increased GluA1 and reduced GluA2) and impairs reference memory. All these changes are reversed by extracellular cGMP. These results show that extracellular cGMP modulates spatial reference memory but not working memory. This would be mediated by modulation of TNF-α levels and of membrane expression of GluA1 and GluA2 subunits of AMPA receptors.


Assuntos
Disfunção Cognitiva/metabolismo , GMP Cíclico/farmacologia , Hipocampo/metabolismo , Hiperamonemia/metabolismo , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Memória de Curto Prazo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Memória Espacial , Fator de Necrose Tumoral alfa/metabolismo , Animais , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/etiologia , GMP Cíclico/administração & dosagem , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hiperamonemia/complicações , Hiperamonemia/tratamento farmacológico , Inflamação/tratamento farmacológico , Inflamação/etiologia , Interleucina-1beta/efeitos dos fármacos , Masculino , Memória de Curto Prazo/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de AMPA/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Memória Espacial/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacos
19.
J Neuroinflammation ; 13(1): 83, 2016 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-27090509

RESUMO

BACKGROUND: Hyperammonemia induces neuroinflammation and increases GABAergic tone in the cerebellum which contributes to cognitive and motor impairment in hepatic encephalopathy (HE). The link between neuroinflammation and GABAergic tone remains unknown. New treatments reducing neuroinflammation and GABAergic tone could improve neurological impairment. The aims were, in hyperammonemic rats, to assess whether: (a) Enhancing endogenous anti-inflammatory mechanisms by sulforaphane treatment reduces neuroinflammation and restores learning and motor coordination. (b) Reduction of neuroinflammation by sulforaphane normalizes extracellular GABA and glutamate-NO-cGMP pathway and identify underlying mechanisms. (c) Identify steps by which hyperammonemia-induced microglial activation impairs cognitive and motor function and how sulforaphane restores them. METHODS: We analyzed in control and hyperammonemic rats, treated or not with sulforaphane, (a) learning in the Y maze; (b) motor coordination in the beam walking; (c) glutamate-NO-cGMP pathway and extracellular GABA by microdialysis; (d) microglial activation, by analyzing by immunohistochemistry or Western blot markers of pro-inflammatory (M1) (IL-1b, Iba-1) and anti-inflammatory (M2) microglia (Iba1, IL-4, IL-10, Arg1, YM-1); and (e) membrane expression of the GABA transporter GAT-3. RESULTS: Hyperammonemia induces activation of astrocytes and microglia in the cerebellum as assessed by immunohistochemistry. Hyperammonemia-induced neuroinflammation is associated with increased membrane expression of the GABA transporter GAT-3, mainly in activated astrocytes. This is also associated with increased extracellular GABA in the cerebellum and with motor in-coordination and impaired learning ability in the Y maze. Sulforaphane promotes polarization of microglia from the M1 to the M2 phenotype, reducing IL-1b and increasing IL-4, IL-10, Arg1, and YM-1 in the cerebellum. This is associated with astrocytes deactivation and normalization of GAT-3 membrane expression, extracellular GABA, glutamate-nitric oxide-cGMP pathway, and learning and motor coordination. CONCLUSIONS: Neuroinflammation increases GABAergic tone in the cerebellum by increasing GAT-3 membrane expression. This impairs motor coordination and learning in the Y maze. Sulforaphane could be a new therapeutic approach to improve cognitive and motor function in hyperammonemia, hepatic encephalopathy, and other pathologies associated with neuroinflammation by promoting microglia differentiation from M1 to M2.


Assuntos
Proteínas da Membrana Plasmática de Transporte de GABA/biossíntese , Encefalopatia Hepática/metabolismo , Hiperamonemia/metabolismo , Microglia/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Western Blotting , Membrana Celular/metabolismo , Cerebelo , Modelos Animais de Doenças , Encefalopatia Hepática/complicações , Hiperamonemia/etiologia , Hiperamonemia/fisiopatologia , Imuno-Histoquímica , Inflamação/metabolismo , Isotiocianatos/farmacologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Microdiálise , Microglia/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/metabolismo
20.
J Neuroinflammation ; 13: 41, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26883214

RESUMO

BACKGROUND: Patients with liver cirrhosis and minimal hepatic encephalopathy (MHE) show mild cognitive impairment and spatial learning dysfunction. Hyperammonemia acts synergistically with inflammation to induce cognitive impairment in MHE. Hyperammonemia-induced neuroinflammation in hippocampus could contribute to spatial learning impairment in MHE. Two main aims of this work were: (1) to assess whether chronic hyperammonemia increases inflammatory factors in the hippocampus and if this is associated with microglia and/or astrocytes activation and (2) to assess whether hyperammonemia-induced neuroinflammation in the hippocampus is associated with altered membrane expression of glutamate and GABA receptors and spatial learning impairment. There are no specific treatments for cognitive alterations in patients with MHE. A third aim was to assess whether treatment with sulforaphane enhances endogenous the anti-inflammatory system, reduces neuroinflammation in the hippocampus of hyperammonemic rats, and restores spatial learning and if normalization of receptor membrane expression is associated with learning improvement. METHODS: We analyzed the following in control and hyperammonemic rats, treated or not with sulforaphane: (1) microglia and astrocytes activation by immunohistochemistry, (2) markers of pro-inflammatory (M1) (IL-1ß, IL-6) and anti-inflammatory (M2) microglia (Arg1, YM-1) by Western blot, (3) membrane expression of GABA, AMPA, and NMDA receptors using the BS3 cross-linker, and (4) spatial learning using the radial maze. RESULTS: The results reported show that hyperammonemia induces astrocytes and microglia activation in the hippocampus, increasing pro-inflammatory cytokines IL-1ß and IL-6. This is associated with altered membrane expression of AMPA, NMDA, and GABA receptors which would be responsible for altered neurotransmission and impairment of spatial learning in the radial maze. Treatment with sulforaphane promotes microglia differentiation from pro-inflammatory M1 to anti-inflammatory M2 phenotype and reduces activation of astrocytes in hyperammonemic rats. This reduces neuroinflammation, normalizes membrane expression of glutamate and GABA receptors, and restores spatial learning in hyperammonemic rats. CONCLUSIONS: Hyperammonemia-induced neuroinflammation impairs glutamatergic and GABAergic neurotransmission by altering membrane expression of glutamate and GABA receptors, resulting in impaired spatial learning. Sulforaphane reverses all these effects. Treatment with sulforaphane could be useful to improve cognitive function in cirrhotic patients with minimal or clinical hepatic encephalopathy.


Assuntos
Anti-Inflamatórios/uso terapêutico , Encefalite/etiologia , Hipocampo/metabolismo , Hiperamonemia/complicações , Isotiocianatos/uso terapêutico , Receptores de Neurotransmissores/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Peso Corporal/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalite/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/patologia , Hiperamonemia/patologia , Técnicas In Vitro , Isotiocianatos/farmacologia , /etiologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Ratos , Ratos Wistar , Aprendizagem Espacial/efeitos dos fármacos , Aprendizagem Espacial/fisiologia
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