Estudo dos mecanismos envolvidos no efeito neuroprotetor do pré-condicionamento com N-Metil-D-Aspartato (NMDA)

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2014.O pré-condicionamento cerebral é caracterizado por um estado de tolerância transitória do tecido nervoso a um estímulo letal e/ou tóxico evocado por um dano moderado prévio. Estudos têm demonstrado que este estado de tolerância cerebral pode ser obtido através do pré-condicionamento químico com N-metil-D-aspartato (NMDA), o agonista sintético seletivo dos receptores NMDA e pode ser promovido in vivo pela administração de dose subtóxica deste agente. Contudo, as vias de sinalização e receptores ou canais iônicos associados ao processo de neuroproteção ativado pelo pré-condicionamento químico ainda precisam ser elucidados. Desta forma, investigou-se o envolvimento dos receptores de adenosina (A1R e A2AR) e as vias de sinalização celular no mecanismo de neuroproteção pelo pré-condicionamento com NMDA in vivo (em camundongos) e in vitro (em cultura primária de neurônios e em fatias hipocampais). Os resultados demonstraram que o pré-condicionamento com NMDA aumenta a afinidade de união do A1R ao ligante, sem alterar seus níveis proteicos no hipocampo de camundongos. Além disso, a ativação do A1R reverte o efeito antinociceptivo mediado pelo pré-condicionamento com NMDA, sugerindo que a pós-ativação do A1R pode interferir com o pré-condicionamento. Ainda o pré-condicionamento com NMDA não altera a funcionalidade do A1R no teste do condicionamento ao medo contextual em camundongos, mas parece promover uma dessensibilização de A2AR. E a ativação de ambos A1R e A2AR reverte o aumento da captação de glutamato em fatias de hipocampo, mediado pelo pré-condicionamento com NMDA. Em relação às vias de sinalização celular analisadas, observou-se o pré-condicionamento com NMDA depende da via da PI3K para a proteção contra as convulsões induzidas por ácido quinolínico (AQ) em camundongos, embora a inibição desta via não parece estar diretamente relacionada com a proteção contra a morte celular. Em estudos in vitro, o pré-condicionamento com NMDA também foi eficaz na proteção contra o dano induzido por AQ em cultura primária de neurônios hipocampais e em fatias hipocampais de camundongos contra o dano induzido por glutamato. Os mecanismos envolvidos na neuroproteção pelo pré-condicionamento em fatias, envolve a participação de A1R e parcialmente dos canais de potássio ativados por cálcio (BKCa). Além disso, o pré-condicionamento com NMDA é mais eficaz com a ativação de receptores que usam a glicina como co-agonista preferencial. Desta forma, este trabalho contribui para demonstrar alguns mecanismos celulares evocados por uma possível estratégia neuroprotetora, o pré-condicionamento químico.Abstract : Brain preconditioning is characterized by a state of transient tolerance in the nervous tissue to a lethal and/or toxic stimulus evoked by prior moderate damage. Studies have demonstrated that this state of brain tolerance can be obtained by chemical preconditioning with N-methyl-D-aspartate (NMDA), the synthetic selective agonist of NMDA receptors, and can be promoted by in vivo administration of subtoxic doses of this agent. However, the signaling pathways and receptors or ion channels associated with the neuroprotection process activated by chemical preconditioning remain to be elucidated. Thus, we investigated the involvement of adenosine receptors (A1R and A2AR) and cell signaling pathways in the mechanism of neuroprotection evoked by NMDA preconditioning in vivo (in mice) and in vitro (in primary cultured neurons and hippocampal slices). NMDA preconditioning increases binding affinity of the A1R to ligand, without changing its protein levels in hippocampus of mice. Furthermore, activation of A1R reverses the antinociceptive effect mediated by NMDA preconditioning, suggesting that post-activation of A1R can interfere with preconditioning. NMDA preconditioning does not alter the functionality of A1R in contextual fear conditioning test in mice, although it seems to promote a desensitization of A2AR response. The activation of both A1R and A2AR reversed the increase in glutamate uptake into hippocampal slices mediated by NMDA preconditioning. Regarding the signaling pathways analyzed, we observed that NMDA preconditioning is dependent on PI3K pathway for protection against quinolinic acid (QA)-induced seizures in mice, although inhibition of this signaling pathway is not directly related to protection against cell damage. In in vitro studies, NMDA preconditioning was also effective in protecting against QA-induced damage in primary cultured hippocampal neurons and against glutamate-induced damage in mice hippocampal slices. The mechanisms of neuroprotection exerted by preconditioning in slices, involve the participation of A1R and partially the calcium-activated potassium channels (BKCa). Moreover, NMDA preconditioning is more effective when glycine is used as the preferential co-agonist of NMDA receptor. Therefore, this study contributes to demonstrate cellular mechanisms evoked by a putative neuroprotective strategy, the chemical preconditioning

Avaliação de polimorfonucleares (PMNs) séricos e peroxidação lipídica de tabagistas e não tabagistas

Introduction: Smoking is considered a worldwide epidemic and, according to the World Health Organization (WHO), about five million people die each year as a result of tobacco-related illnesses. Tobacco, in addition to several harmful effects, is related to the increase of defense cells. Objective: To evaluate the viability of polymorphonuclear cells (PMNs) and oxidative damage in the blood of smokers and nonsmokers through the formation of thiobarbituric acid reactive species (TBARS). Methodology: We evaluated 22 volunteers (both sexes): 11 smokers and 11 non-smokers. After responding to a questionnaire on tobacco dependence, blood samples were obtained by venipuncture, part of which was destined for analysis under the light microscope to evaluate the viability of PMNs and part for the analysis of TBARS formation. Results: Data showed that 18% have low degree of dependence on nicotine, 27% of smokers have a medium degree of dependency and 18% consider high nicotine addiction. Regarding the viability of the PMNs and the formation of TBARS, no significant difference was observed between the smoking and non-smoking groups, although the smokers group presented a decrease in the percentage of the viability of the PMNs. Conclusion: The data showed that the highest percentage of participants had an average degree of nicotine dependence. The viability of the PMNs and the formation of TBARS did not change between the smoking and non-smoking groups, although the smokers group presented a decrease in the percentage of feasibility of the PMNs. Keywords: Tobacco. Imune system. Polymorphonuclear cells. TBARS.Introdução: O tabagismo é considerado uma epidemia mundial e, segundo a Organização Mundial de Saúde (OMS), cerca de cinco milhões de pessoas morre a cada ano em consequência das doenças relacionadas ao tabaco. O tabaco, além de vários efeitos nocivos, está relacionado com aumento das células de defesa. Objetivo: Avaliar a viabilidade de células polimorfonucleares (PMNs) e o dano oxidativo no sangue de indivíduos fumantes e não fumantes através da formação de espécies reativas ao ácido tiobarbitúrico (TBARS). Metodologia: Foram avaliados 22 voluntários (ambos os sexos): 11 fumantes e 11 não fumantes. Após responderem um questionário sobre dependência do tabaco, amostras de sangue foram obtidas por punção venosa, sendo parte das mesmas destinadas para análise no microscópio de luz para avaliar a viabilidade dos PMNs e parte para análise da formação de TBARS. Resultados: Os dados demonstraram que 18% possuem grau de dependência à nicotina baixo, 27% dos tabagistas possuem grau de dependência médio e 18% considerados com nível de dependência à nicotina elevada. Com relação à viabilidade dos PMNs e a formação de TBARS, nenhuma diferença significativa foi observada entre os grupos tabagistas e não tabagistas, embora o grupo tabagistas apresentou uma diminuição na porcentagem da viabilidade dos PMNs. Conclusão: Os dados demonstraram que a maior percentagem dos participantes possui um grau médio de dependência à nicotina, A viabilidade dos PMNs e a formação de TBARS não foi alterada entre os grupos tabagistas e não tabagistas, embora o grupo tabagistas apresentou uma diminuição na porcentagem da viabilidade dos PMNs. Palavras-chave: Tabaco. Sistema imune. Polimorfonucleares. TBARS

Oxidative variables and antioxidant enzymes activities in the mdx mouse brain

Dystrophin is a protein found at the plasmatic membrane in muscle and postsynaptic membrane of some neurons, where it plays an important role on synaptic transmission and plasticity. Its absence is associated with Duchenne`s muscular dystrophy (DMD), in which cognitive impairment is found. Oxidative stress appears to be involved in the physiopathology of DMD and its cognitive dysfunction. In this regard, the present study investigated oxidative parameters (lipid and protein peroxidation) and antioxidant enzymes activities (superoxide dismutase and catalase) in prefrontal cortex, cerebellum, hippocampus, striatum and cortex tissues from male dystrophic mdx and normal C57BL10 mice. We observed (I) reduced lipid peroxidation in striatum and protein peroxidation in cerebellum and prefrontal cortex; (2) increased superoxide dismutase activity in cerebellum, prefrontal cortex, hippocampus and striatum: and (3) reduced catalase activity in striatum. It seems by our results, that the superoxide dismutase antioxidant mechanism is playing a protective role against lipid and protein peroxidation in mdx mouse brain. (C) 2009 Elsevier Ltd. All rights reserved.CNPqConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESCFundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC)Instituto Cerebro e Mente and UNESCInstituto Cerebro e Mente and UNES

Guanosine Prevents Spatial Memory Impairment and Hippocampal Damage Following Amyloid-β1–42 Administration in Mice

Alzheimer’s disease (AD) is a progressive neurodegenerative illness responsible for cognitive impairment and dementia. Accumulation of amyloid-beta (Aβ) peptides in neurons and synapses causes cell metabolism to unbalance, and the production of reactive oxygen species (ROS), leading to neuronal death and cognitive damage. Guanosine is an endogenous nucleoside recognized as a neuroprotective agent since it prevents glutamate-induced neurotoxicity by a mechanism not yet completely elucidated. In this study, we evaluated behavioral and biochemical effects in the hippocampus caused by the intracerebroventricular (i.c.v.) infusion of Aβ1–42 peptide (400 pmol/site) in mice, and the neuroprotective effect of guanosine (8 mg/kg, i.p.). An initial evaluation on the eighth day after Aβ1–42 infusion showed no changes in the tail suspension test, although ex vivo analyses in hippocampal slices showed increased ROS production. In the second protocol, on the tenth day following Aβ1–42 infusion, no effect was observed in the sucrose splash test, but a reduction in the recognition index in the object location test showed impaired spatial memory. Analysis of hippocampal slices showed no ROS production and mitochondrial membrane potential alteration, but a tendency to increase glutamate release and a significant lactate release, pointing to a metabolic alteration. Those effects were accompanied by decreased cell viability and increased membrane damage. Guanosine treatment prevented behavioral and biochemical alterations evoked by Aβ1–42, suggesting a potential role against behavioral and biochemical damage evoked by Aβ in the hippocampus

Behavioral and Neurochemical Consequences of Pentylenetetrazol-Induced Kindling in Young and Middle-Aged Rats

(1) Objectives: Epilepsy disorder is likely to increase with aging, leading to an increased incidence of comorbidities and mortality. In spite of that, there is a lack of information regarding this issue and little knowledge of cognitive and emotional responses in aging subjects following epileptogenesis. We investigated whether and how aging distress epilepsy-related behavioral and biochemical outcomes are associated with cognition and emotion. (2) Methods: Young and middle-aged Wistar rats (3 or 12 months old) were treated with pentylenetetrazol (PTZ, 35 mg/kg) and injected on alternated days for 20 (young rats) and 32 days (middle-aged rats). Kindling was reached after two consecutive stages 4 plus one stage 5 or 6 in Racine scale. Control and kindled rats were evaluated in the elevated plus-maze (EPM) and object-recognition tests and their hippocampus was collected 24 h later for mitogen-activated protein kinases (MAPK) dosage. (3) Results: Middle-aged rats presented a higher resistance to develop kindling, with a decrease in the seizure severity index observed following the 4th and 9th PTZ injections. Middle-aged rats displayed an increased duration of the first myoclonic seizure and an increased latency to the first generalized seizure when compared to younger rats. The induction of kindling did not impair the animals’ performance (regardless of age) in the object-recognition task and the EPM test as well as it did not alter the hippocampal levels of MAPKs. (4) Significance: Our findings reveal that, despite age-related differences during epileptogenesis, middle-aged rats evaluated after kindling performed similarly during discriminative learning and emotional tasks in comparison to young animals, with no alteration of hippocampal MAPKs. Additional investigation must be carried out to explore the electrophysiological mechanisms underlying these responses, as well as the long-term effects displayed after kindling