Os astrócitos são células gliais do sistema nervoso central (SNC) que, entre diversas funções, tem um importante papel na manutenção do ambiente extracelular, removendo neurotransmissores e íons os quais podem ser tóxicos em altas concentrações. Estas células estão envolvidas em diversas doenças neurodegenerativas e representam um importante alvo de estudo para o desenvolvimento de estratégias terapêuticas. A técnica de cultura primária (CP) de astrócitos de ratos é amplamente utilizada como modelo para o estudo de características mecânicas e bioquímicas dessa célula em um sistema isolado. Alternativamente, astrócitos imortalizados por transfecção (AI) e a linhagem C6 de rato (C6) são usados como modelo astrocítico, uma vez que são células mais homogêneas, além de serem de fácil e rápida manipulação. No entanto, apesar de resultados divergentes serem encontrados na literatura, existe uma carência de estudos comparativos entre estes três modelos experimentais. A escolha de um modelo adequado para o estudo destas células nos permitiria estudar de forma mais precisa as funções extracelulares de proteínas como a S100B. Essa proteína pode ser liberada ou secretada principalmente por astrócitos no SNC e, apesar de possuir efeitos tróficos, exerce funções tóxicas, quando em altas concentrações no meio extracelular. Apesar disso, pouco se sabe sobre o processo de remoção da S100B extracelular. Em vista disso, o objetivo desta tese foi comparar e caracterizar funcionalmente a CP, AI e C6 no que diz respeito a diferenças morfológicas, marcadores proteicos e funções clássicas astrocíticas, bem como estudar a capacidade destas células de internalizar a proteína S100B, avaliando-se o mecanismo deste processo. Além disso, foi realizada a padronização de um ensaio colorimétrico utilizando a atividade glutamil-transferase da glutamina sintetase (GS), visto a carência deste ensaio em cultura de astrócitos e em diferentes estruturas cerebrais. Esta enzima é considerada um marcador astrocítico e sua disfunção pode comprometer o ciclo glutamina-glutamato, processo relacionado ao quadro de excitotoxicidade glutamatérgica, observado em doenças neurodegenerativas. O ensaio mostrou-se preciso, sensível, linear, específico e com alta aplicabilidade no SNC e nos modelos de cultura celular. Nossos resultados mostraram que as duas linhagens celulares (AI e C6) foram positivas para proteínas características de astrócitos, bem como funcionais no metabolismo glutamatérgico e energético. No entanto, estes parâmetros mostraram-se reduzidos em estado basal em comparação com a CP. Frente a estímulos específicos, os AI não se mostraram como um modelo reprodutível das funções astrocíticas clássicas, ao contrário da linhagem C6, a qual apresentou resposta similar aos astrócitos de CP. De forma geral a linhagem C6 representou um modelo válido para estudo das características astrocíticas estudadas, porém sempre com valores basais reduzidos quando comparados com a CP que, em nossa avaliação, representou um melhor modelo para o estudo da internalização da proteína S100B. Estas células foram capazes de endocitar a S100B extracelular por um mecanismo dependente de RAGE e dinamina. Uma vez internalizada, a S100B seguiu pela via endocítica, colocalizando com importantes marcadores, como Dextran e Lysotracker. As vesículas de S100B mostraram um aumento de direcionalidade ao longo do tempo de incubação, afetado pela variação de cálcio intracelular. Estes dados mostram a importância da escolha de um modelo in vitro adequado de cultura de astrócitos, bem como evidenciam o importante papel destas células na remoção de concentrações tóxicas da proteína S100B do meio extracelular, auxiliando na compreensão dos mecanismos de sinalização desta proteína. / Astrocytes are glial cells that participate in a variety of function of central nervous system (CNS) such as the maintenance of the extracellular environment, removing toxic concentration of ions and neurotransmitters. These cells are involved in several neurodegenerative diseases and are important targets for the development of therapeutic strategies. Astrocyte primary culture (PC) is a potent instrument to study these cells in an isolated system, allowing the elucidation of mechanistic features specific for this type of cell. An alternative to this model is the use of cell lines, such as immortalized astrocytes (IA) or C6 glioma cells (C6). These linages have the advantage to present more homogeneous features, are easily, faster and lower cost to manipulate. Despite these facilities, cell lines present highly proliferative features and some different characteristics in comparison to PC, which put into question the validity of them as an astrocytic model. The selection of a suitable model to the study of astrocytes would allow us to evaluate protein extracellular effects, such as S100B with greater reliability. This protein is released and secreted mainly by astrocytes in the CNS, exerting trophic and toxic effect in a RAGE dependent manner. S100B toxic effects are shown in high extracellular concentrations, although the clearance of this protein from the extracellular space is poorly understood. In view of this, the goal of this research was to show a comparative analysis of PC, IA and C6 regarding morphological features, protein profile and classical astrocytic functions. Furthermore, we evaluated the mechanism of S100B internalization, characterization of internalized vesicles and intracellular dynamics in astrocytes. In addition, the standardization of a colorimetric assay to measure the glutamyl-transferase activity of glutamine synthetase (GS) was performed, considering the lack of this study in culture of astrocytes and different brain structures. This enzyme is considered an astrocytic marker and any dysfunction may compromise the glutamine-glutamate cycle, which is observed in many neurodegenerative diseases. The assay was accurate, sensitive, linear, specific and with high applicability in the CNS. Our results showed that the two cell lines (IA and C6) were positive for characteristic proteins of astrocytes, although expressing less quantities then PC. Similarly, the glutamatergic and energetic metabolism and cellular communication by gap junction also showed to be reduced in lineages cells. IA did not reveal a reproducible model of classic astrocytic functions, in opposite to C6 cells that presented a similar response to PC when submitted to the same stimulus. Nevertheless, PC showed pronounced astrocytic characteristics, representing the best model for the study of S100B protein internalization. The results in this study revealed that cultured astrocytes efficiently remove fluorescently labeled extracellular S100B in a time-dependent manner by vesicle endocytosis. The internalization was RAGE and dynamine dependent. In time, vesicles capturing S100B exhibit directional mobility, meaning that vesicles get functionally attached to the cytoskeleton. Moreover, the mobility of vesicles capturing S100B was affected by changes in [Ca2+]. These data support to the understanding of the mechanism of extracellular signaling of S100B protein, as well as the role of astrocytes in this regulation.

A epilepsia do lobo temporal (ELT) é a um dos casos mais frequente epilepsia em humanos e de maior refratariedade nos pacientes. A maioria dos fármacos antiepilépticos são moduladores da atividade neuronal e atuam sobre canais iônicos do receptor GABAA. Estudos vêm demonstrando o papel das células gliais e da neuroinflamação na epileptogênese e a modulação desta resposta pode ser um alvo potencial para drogas adjuvantes aos fármacos anti-epilépticos. Astrócitos são células gliais participantes da sinapse tripartite, moduladores da atividade neuronal. Os astrócitos são capazes de promover a homeostase de íons e de neurotransmissores, são responsáveis pelo metabolismo energético e da produção de fatores neurotróficos, glutationa, glutamina, S100B e citocinas. Neste trabalho, induzimos status epilepticus (SE) em ratos jovens (PN28) através do modelo lítio-pilocarpina que mimetiza alterações neuronais, bioquímicas e morfológicas similares à ELT em humanos. Os animais foram divididos nos tempos 1, 14 e 56 dias após a indução de status epilepticus (SE). Estes períodos são caracterizados respectivamente como a fase aguda, latente e crônica da epilepsia. Inicialmente, analisamos as mudanças neuroquímicas e astrocitárias ao longo do tempo. Foi observada neuroinflamação inicial e transitória que promove morte neuronal e mudanças ao longo do tempo de astrogliose e disfunção astrocitária. Também foi observado que a proteína S100B, proteína ligante de cálcio, predominantemente astrocitária, pode ser considerado um marcador da disfunção neuronal e astrocitária promovida neste modelo de epilepsia. Em seguida, demonstramos que a modulação da secreção de S100B pelo anti-inflamatório dexametasona um dia após indução de SE reverte a neuroinflamação, astrogliose e disfunção astrocitária à curto e à longo prazo. Por conseguinte, observamos que a modulação do receptor GABAA através de agonistas e antagonistas GABAérgicos altera a secreção de S100B em fatias hipocampais agudas e em cultura de astrócitos. Portanto, pode-se sugerir que as alterações astrogliais e a neuroinflamação dependentes do tempo podem estar ligadas à excitabilidade neuronal e/ou à morte neuronal em ratos jovens em modelo de epilepsia; que a proteína S100B pode ser considerada um marcador deste modelo de epilepsia e que a modulação da sua secreção pode ser um possível alvo farmacológico no tratamento da epilepsia. / Temporal lobe epilepsy (TLE) is the most frequent type of epilepsy in humans and is more associated to refractory to anti-epileptic drugs (AED) in patients. The most AEDs are modulators of neuronal activity and act on ion channels, such as GABAA receptor. Studies have been demonstrating the role of glial cells and neuroinflammation in epileptogenesis. The modulation of this response may be a potential target for adjunctive drugs to anti-epileptic drugs. Astrocytes are glial cells that participated in the tripartite synapse and modulated neuronal activity. Astrocytes are able to promote homeostasis of ions and neurotransmitters, are responsible for energy metabolism and the production of neurotrophic factors, glutathione, glutamine, S100B and cytokines. In this work, we induced status epilepticus (SE) in young rats (PN28) through the lithiumpilocarpine model that mimics neuronal, biochemical and morphological alterations similar to ELT in humans. The animals were divided at times 1, 14 and 56 days after the induction of SE. These periods are characterized respectively as the acute, latent and chronic phase of epilepsy. Initially, we analyzed neurochemical and astrocytic changes over time. Initial and transient neuroinflammation was observed and promoted over time neuronal death, astrogliosis and astrocytic dysfunction. It has also been observed that the protein S100B, a calcium-binding protein, predominantly astrocytic, can be considered a marker of neuronal and astrocytic dysfunction promoted by this model of epilepsy. Next, we demonstrate that the modulation of S100B secretion by the antiinflammatory dexamethasone one day after SE induction reverses neuroinflammation, astrogliosis and astrocytic dysfunction in the acute and chronic time. Therefore, we analyzed that modulation of the GABAA receptor through GABAergics agonists and antagonists alters the secretion of S100B in acute hippocampal slices and in astrocyte culture. Therefore, it may be suggested that astroglial changes and time dependent neuroinflammation may be related to neuronal excitability and/or neuronal death in young rats in this epilepsy model; that S100B protein can be considered a marker of this epilepsy model and that the modulation of its secretion may be a possible pharmacological target in the treatment of epilepsy.

Purpose: Excessive day time sleepiness, sleep disorders and neurobehavior changes are common clinical observations in the patients with epilepsy. From literature review, they were highly related with epilepsy risk characteristics (age of onset, types or numbers of drugs, seizure frequency), co-morbidities or neuronal network changes. The serological biomarkers have been reported to reflect the phenomenon of seizure, while their correlations with neurobehavior changes were still not concluded. There were two purposes of this thesis. (1) To understand the relationship between sleep disturbance with day time performances (2) To understand the relationships between serological biomarkers, neurobehavior performances and neuronal networks in patients with temporal lobe epilepsy. Material and Methods: The study enrolled patients from epilepsy outpatient clinic. By using self-appreciated questionnaire (The Pittsburgh Sleep Quality Index, The Epworth Sleepiness Scale, Euroqol Quality of Life Scale-5D), we collected the characteristics of sleep related behavior and life quality changes and explored the relationship with epilepsy risk characteristics. In patients with temporal lobe epilepsy, we assessed the neurobehavior performances, measured the serological biomarkers (heat shock protein 70, S100£]protein, neuron specific enolase, brain derived neurotrophic factor, plasma and mitochondrial DNA) and brain magnetic resonance imaging. In statistical analysis, we compared the differences with age matched controls or performed correlation analysis among the parameters Result: One hundred and seventeen patients with epilepsy completed the sleep quality questionnaires. The results showed that 20 percent of patients had day time sleepiness, while the sleep disorder was prolong sleep latency and impaired sleep efficiency. In epilepsy characteristics, patients with complex partial seizure, intractable seizure or with multi-pharmacy were related with poor sleep quality. A total of 34 patients completed the serological, neurobehavior and brain magnetic resonance analysis. The results showed that patients with temporal lobe epilepsy had higher heat shock protein 70 and S100£]protein levels, while those with attacks more than twice per month had significant higher heat shock protein 70, S100£]protein and neuron specific enolase levels. Compared with the matched controls, the regions showing atrophy included hippocampus and parahippocampus, putamen, thalamus and supplementary motor areas. In correlation study, only heat shock protein 70 showed an inverse correlation with hippocampal volume (R square = 0.22, p = 0.007) after controlling for the effect of age. Conclusion: The study suggested that epilepsy risk characteristics, serological biomarkers, brain atrophic regions were important factors for day time sleepiness, sleep disturbances and neurobehavior changes in patients with epilepsy.

Les précurseurs neuraux adultes possèdent une plasticité cellulaire suggérant un rôle dans l'apparition de pathologies mais aussi un potentiel curatif inespéré. Cependant, l'emploi clinique de ces cellules nécessite une connaissance des mécanismes biologiques contrôlant leur prolifération, maturation ou spécification cellulaire. Dans cette thèse, nous avons étudié l'expression des protéines S100 A1 et B dans les cellules progénitrices d'oligodendrocytes (OPC) et les cellules souches astrocytaires. Nous avons démontré que <br />1) toutes les cellules gliales expriment précocement la S100A1 alors que la S100B est liée à leur maturation <br />2) la S100B régule la maturation des OPC <br />3) les cellules souches astrocytaires adultes sont maintenues dans un stade de développement immature (S100B-) grâce à l'EGF, afin de conserver leurs propriétés germinales.<br />Ces résultats démontrent un lien entre les protéines S100A1/B, la maturation des cellules gliales et leurs propriétés de différenciation cellulaire.

Controlled release formulations offer many advantages over conventional dosage forms. These include reduced plasma fluctuations and improved patient comp1i:nce. Complex controlled release formulations such as those with enteric release properties, often require additional steps in the production phase. The costs and economic impact associated with these complex controlled release dosage formulations often outweigh the immediate benefits. Thus the development of an economic method to produce controlled release particles is of great importance especially in third world countries. In controlled release formulations, the drug is generally dispersed throughout a polymer matrix. The rate of drug release is often determined by the viscosity or complexity of the polymer matrix through which the drug needs to diffuse in order to be released. With enteric release the polymer coating, insoluble in an acidic environment is often applied in the final phase of production. Chitosan is a versatile polymer of natural origin with many favourable characteristics. These include its safety, biocompatibility, and biodegradability. Simple methods can be applied and modified to produce controlled release particles form chitosan. The effect of modern controlled release polymers such as Aqoat AS-HF, Eudragit SlOO and Kollidon SR was investigated. Chitosan beads and chitosan-polymer beads, as well as chitosan granules and chitosan-polymer granules, were prepared and investigated as possible controlled release formulations. Ketoprofen was chosen as the model drug. Chitosan beads and chitosan-polymer beads were prepared by inotropic gelation in tripolyphosphate. Chitosan granules and chitosan-polymer matrix granules were prepared by binding chitosan with an acetic acid solution as a granulating system. The beads and granules appeared differed in appearance as well as in the results obtained from various experiments. Granules prepared in the study did not appear to be effective with regards to enteric and controlled release. Beads prepared form Kollidon SR appeared to be effective with regards to enteric and controlled release, with Kollidon 1% and 5% w/v chitosan beads achieving good drug loading of up to 73.13% and releasing less than 15 % of the total drug content in 0.1 M HCI after 60 minutes. Drug release continued steadily for up to 360 minutes in pH 7.2. It was concluded that Kollidon SR loaded chitosan beads nay be a viable controlled release dosage form with enteric release properties, and that future experiments, possibly with lower polymer concentrations, are worthwhile / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

Controlled release formulations offer many advantages over conventional dosage forms. These include reduced plasma fluctuations and improved patient comp1i:nce. Complex controlled release formulations such as those with enteric release properties, often require additional steps in the production phase. The costs and economic impact associated with these complex controlled release dosage formulations often outweigh the immediate benefits. Thus the development of an economic method to produce controlled release particles is of great importance especially in third world countries. In controlled release formulations, the drug is generally dispersed throughout a polymer matrix. The rate of drug release is often determined by the viscosity or complexity of the polymer matrix through which the drug needs to diffuse in order to be released. With enteric release the polymer coating, insoluble in an acidic environment is often applied in the final phase of production. Chitosan is a versatile polymer of natural origin with many favourable characteristics. These include its safety, biocompatibility, and biodegradability. Simple methods can be applied and modified to produce controlled release particles form chitosan. The effect of modern controlled release polymers such as Aqoat AS-HF, Eudragit SlOO and Kollidon SR was investigated. Chitosan beads and chitosan-polymer beads, as well as chitosan granules and chitosan-polymer granules, were prepared and investigated as possible controlled release formulations. Ketoprofen was chosen as the model drug. Chitosan beads and chitosan-polymer beads were prepared by inotropic gelation in tripolyphosphate. Chitosan granules and chitosan-polymer matrix granules were prepared by binding chitosan with an acetic acid solution as a granulating system. The beads and granules appeared differed in appearance as well as in the results obtained from various experiments. Granules prepared in the study did not appear to be effective with regards to enteric and controlled release. Beads prepared form Kollidon SR appeared to be effective with regards to enteric and controlled release, with Kollidon 1% and 5% w/v chitosan beads achieving good drug loading of up to 73.13% and releasing less than 15 % of the total drug content in 0.1 M HCI after 60 minutes. Drug release continued steadily for up to 360 minutes in pH 7.2. It was concluded that Kollidon SR loaded chitosan beads nay be a viable controlled release dosage form with enteric release properties, and that future experiments, possibly with lower polymer concentrations, are worthwhile / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

Diss. (sammanfattning) Stockholm : Karol. inst., 2007. / Härtill 4 uppsatser.

Os astrócitos são células conhecidas por sua capacidade dinâmica e versatilidade, participando não somente da manutenção da homeostase cerebral em condições fisiológicas, mas também respondendo em condições patológicas, como por exemplo, na encefalopatia hepática (EH). Esta patologia neurológica está associada principalmente à hiperamonemia, decorrente de uma falência hepática aguda ou crônica. A toxicidade da amônia no sistema nervoso central (SNC) é mediada por uma série de alterações celulares e metabólicas, principalmente nas células astrogliais. Nesse sentido, a busca por moléculas com potencial terapêutico no SNC é extremamente relevante. Trabalhos do nosso grupo mostraram que o resveratrol e o ácido lipoico, duas moléculas conhecidas pelos seus efeitos antioxidantes, modulam importantes funções gliais, relacionadas principalmente ao metabolismo glutamatérgico, à defesa antioxidante e à resposta inflamatória. Neste sentido, esta tese teve como objetivo avaliar os efeitos do resveratrol e do ácido lipoico em células astrogliais e neuronais expostas à amônia, assim como seus possíveis mecanismos protetores. Primeiramente, nós observamos que o resveratrol e o ácido lipoico foram capazes de prevenir as alterações induzidas pela amônia em células astrogliais C6, sobre parâmetros do metabolismo glutamatérgico, dentre os quais podemos destacar a captação de glutamato, a atividade da enzima glutamina sintetase (GS) e o conteúdo de glutationa (GSH). Além disso, o ácido lipoico também exerceu um efeito antiinflamatório nestas células, prevenindo a liberação de citocinas pró-inflamatórias (TNFα, IL- 1β, IL-6, IL-18) e da proteína S100B, através da diminuição da ativação do fator de transcrição NFκB. Nós também verificamos que a maioria dos efeitos protetores do resveratrol e do ácido lipoico foram dependentes da heme oxigenase 1 (HO1), uma enzima associada com a defesa celular em situações de estresse. Por fim, foram avaliados os efeitos do resveratrol e do ácido lipoico sobre células neuronais expostas à amônia. Novamente, ambos apresentaram um efeito benéfico, prevenindo tanto o aumento da produção de espécies reativas de oxigênio (ERO) quanto a diminuição do conteúdo de GSH induzidos pela amônia. Nas células neuronais, a proteína HO1 também participou dos efeitos protetores do resveratrol e do ácido lipoico. Em conjunto, esses resultados nos mostram que o resveratrol e o ácido lipoico são capazes de modular positivamente o funcionamento tanto de células astrogliais quanto de células neuronais em situações de hiperamonemia, compartilhando também alguns mecanismos de ação, como a indução da HO1. Este estudo in vitro sugere que o resveratrol e o ácido lipoico são potenciais agentes terapêuticos para doenças do SNC, as quais envolvam produção de espécies reativas e resposta inflamatória, como a EH. / Astrocytes are dynamic and versatile cells which participate in the maintenance of brain homeostasis in physiological conditions and also in response to pathological conditions, such as hepatic encephalopathy. This neurological disease is mainly associated with hyperamonnemia, resulting from acute or chronic liver failure. Ammonia toxicity in the central nervous system (CNS) is mediated by several cellular and metabolic alterations, primarily in astroglial cells. In this sense, the search for molecules with therapeutical potential for CNS becomes highly relevant. Our group has shown that resveratrol and lipoic acid, two molecules known for their antioxidant activities, are able to modulate important glial functions mainly related to glutamate metabolism, antioxidant defense and inflammatory response. In this sense, this study aimed to evaluate the effects of resveratrol and lipoic acid in astroglial and neuronal cells exposed to ammonia, as well as their possible protective mechanisms. Firstly, we observed that resveratrol and lipoic acid were able to prevent ammonia-induced alterations in C6 astroglial cells functioning, such as glutamate uptake, glutamine synthetase (GS) activity and intracellular GSH content. Moreover, lipoic acid also exerted an anti-inflammatory effect in C6 astroglial cells, preventing the ammonia-stimulated release of pro-inflammatory cytokines (TNF, IL-1β, IL-6, IL-18) and S100B protein, by decreasing the activation of the transcription factor NFκB. We also verified that the most protective effects of resveratrol and lipoic acid involved the heme oxygenase 1 (HO1), an enzyme associated with protection against stressful conditions. Finally, we evaluated the effects of resveratrol and lipoic acid on neuronal cells exposed to ammonia. Again, both showed beneficial roles, preventing the increase of reactive oxygen species (ROS) and decrease of GSH content induced by ammonia. In neuronal cells, HO1 also mediated the protective effects of resveratrol and lipoic acid. Taken together, these results show that resveratrol and lipoic acid are able to positively modulate the functioning of both astroglial and neuronal cells in hyperamonemmia conditions, sharing some mechanisms of action, such as HO1. This study in vitro suggests that resveratrol and lipoic acid may represent potential therapeutic agents for CNS, during oxidative and inflammatory damages, as the induced by ammonia.

As respostas inflamatórias no cérebro são mediadas principalmente pela microglia, mas evidências crescentes sugerem uma importância crucial dos astrócitos. A S100B, uma proteína ligante de cálcio e secretada por astrócitos, tem propriedades neurotróficas e de citocina inflamatória. No entanto, não se sabe se sinais primários que ocorrem durante a indução de uma resposta inflamatória como, por exemplo, lipopolissacarídeo (LPS) modulam diretamente a S100B. A neuroinflamação está implicada na patogênese ou na progressão de uma variedade de distúrbios neurodegenerativos e muitos estudos procuram uma conexão entre S100B e doenças degenerativas, incluindo a doença de Alzheimer e esquizofrenia. O uso terapêutico de fármacos anti-inflamatórios não-esteroidais (AINEs) para estas doenças tem aumentado. No entanto, existem poucos estudos sobre o efeito desses fármacos em relação à proteína S100B. Neste trabalho, nós avaliamos se os níveis de S100B no líquido cefalorraquidiano (LCR) e soro de ratos Wistar são afetados por injeção de LPS administrado por via intraperitoneal (IP) ou intracerebroventricular (ICV), bem como se as culturas primárias de astrócitos respondem diretamente ao LPS. Além disso, nós avaliamos o conteúdo e a secreção de S100B medido por ELISA (bem como o conteúdo de GFAP e secreção de TNF-α) em culturas primárias de astrócitos expostos a dexametasona e quatro classes químicas diferentes de AINEs (ácido acetilsalicílico, ibuprofeno, diclofenaco e nimesulida) durante 24 h. Os nossos dados sugerem que a secreção de S100B no tecido cerebral é estimulada rapidamente e persistentemente (durante pelo menos 24 h) por administração ICV de LPS. Este aumento da S100B no LCR foi transitório quando o LPS foi administrado IP. Em contraste com estes resultados de S100B, observou-se um aumento nos níveis de TNF-α no soro, mas não no LCR, após a administração IP de LPS. Em astrócitos isolados e em fatias de hipocampo frescas, observou-se uma estimulação direta da secreção de S100B por LPS numa concentração de 10 ug/ml. Um envolvimento de TLR4 foi confirmado pelo uso de antagonistas específicos deste receptor. No entanto, baixas concentrações de LPS em culturas de astrócitos foram capazes de induzir uma diminuição na secreção de S100B após 24 h, sem alteração significativa no conteúdo intracelular de S100B. Além disso, após 24 horas de exposição ao LPS, observou-se um decréscimo na glutationa e um aumento na proteína ácida fibrilar glial. Também foi observado que os AINEs apresentam diferentes efeitos sobre parâmetros gliais. O ácido acetilsalicílico e o diclofenaco foram capazes de aumentar a GFAP, enquanto que a nimesulida, um inibidor seletivo de COX-2, e a dexametasona diminuiram a secreção de S100B. No entanto, todos os AINEs reduziram os níveis de PGE2. Juntos, esses dados contribuem para a compreensão dos efeitos de LPS em astrócitos, especialmente sobre a secreção de S100B, e nos ajuda a interpretar mudanças nesta proteína no LCR e soro em doenças neuroinflamatórias. Além disso, tecidos periféricos que expressam S100B talvez devam ser regulados diferentemente, uma vez que a administração IP de LPS não foi capaz de aumentar os níveis séricos de S100B. Em relação aos AINEs, a PGE2 parece estar envolvida no mecanismo de secreção de S100B, mas vias adicionais, não claras neste momento, necessitam de uma maior caracterização. O papel inflamatório de S100B em doenças degenerativas, onde também são observados níveis elevados da COX-2 e PGE2, poderia ser atenuado por inibidores de COX-2. / Inflammatory responses in brain are primarily mediated by microglia, but growing evidence suggests a crucial importance of astrocytes. S100B, a calciumbinding protein secreted by astrocytes, may act as a neurotrophic or an inflammatory cytokine. However, it is not known whether primary signals occurring during induction of an inflammatory response (e.g. lipopolysaccharide, LPS) directly modulate S100B. Neuroinflammation has been implicated in the pathogenesis or progression of a variety of neurodegenerative disorders and several studies have looked for a connection of S100B, and degenerative diseases including Alzheimer’s disease and schizophrenia. The therapeutic use of non-steroid anti-inflammatory drugs (NSAID) to these diseases has growth up. However, there are few reports about the effect of these drugs on S100B. In this work, we evaluated whether S100B levels in cerebrospinal fluid (CSF) and serum of Wistar rats are affected by LPS administered by intraperitoneal (IP) or intracerebroventricular (ICV) injection, as well as whether primary astrocyte cultures respond directly to lipopolysaccharide. Moreover we evaluated S100B content and secretion measured by ELISA (as well as GFAP content and TNF-α secretion) in primary astrocyte cultures exposed to dexamethasone and 4 different chemical classes of NSAID (acetyl salicylic acid, ibuprofen, diclofenac and nimesulide) for 24 h. Our data suggest that S100B secretion in brain tissue is stimulated rapidly and persistently (for at least 24 h) by ICV LPS administration. This increase in CSF S100B was transient when LPS was IP administered. In contrast to these S100B results, we observed an increase in in TNFα levels in serum, but not in CSF, after IP administration of LPS. In isolated astrocytes and in acute hippocampal slices, we observed a direct stimulation of S100B secretion by LPS at a concentration of 10 μg/mL. An involvement of TLR4 was confirmed by use of specific inhibitors. However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B. In addition, after 24 h exposure to LPS, we observed a decrease in astrocytic glutathione and an increase in astrocytic glial fibrillary acidic protein. We also observe that NSAIDs have distinct effects on glial parameters. ASA and diclofenac are able to increase GFAP, while nimesulide, a selective COX-2 inhibitor, and dexamethasone were able to decrease S100B secretion. However, all anti-inflammatories were able to reduce levels of PGE2. Together, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases. Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100. With respect to NSAIDs, PGE2 is possibly involved in the mechanism of S100B secretion but additional pathways, unclear at this moment, demand further characterization. The inflammatory role of S100B in degenerative diseases, where also is observed elevated levels of COX-2 and PGE2, could be attenuated by COX-2 inhibitors in which elevated levels of COX-2.

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