Effects of dexamethasone on the Li-pilocarpine model of epilepsy : protection against hippocampal inflammation and astrogliosis

Background: Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy and is accompanied, in one third of cases, by resistance to antiepileptic drugs (AED). Most AED target neuronal activity modulated by ionic channels, and the steroid sensitivity of these channels has supported the use of corticosteroids as adjunctives to AED. Assuming the importance of astrocytes in neuronal activity, we investigated inflammatory and astroglial markers in the hippocampus, a key structure affected in TLE and in the Li-pilocarpine model of epilepsy. Methods: Initially, hippocampal slices were obtained from sham rats and rats subjected to the Li-pilocarpine model of epilepsy, at 1, 14, and 56 days after status epilepticus (SE), which correspond to the acute, silent, and chronic phases. Dexamethasone was added to the incubation medium to evaluate the secretion of S100B, an astrocyte-derived protein widely used as a marker of brain injury. In the second set of experiments, we evaluated the in vivo effect of dexamethasone, administrated at 2 days after SE, on hippocampal inflammatory (COX-1/2, PGE2, and cytokines) and astroglial parameters: GFAP, S100B, glutamine synthetase (GS) and water (AQP-4), and K+ (Kir 4.1) channels. Results: Basal S100B secretion and S100B secretion in high-K+ medium did not differ at 1, 14, and 56 days for the hippocampal slices from epileptic rats, in contrast to sham animal slices, where high-K+ medium decreased S100B secretion. Dexamethasone addition to the incubation medium per se induced a decrease in S100B secretion in sham and epileptic rats (1 and 56 days after SE induction). Following in vivo dexamethasone administration, inflammatory improvements were observed, astrogliosis was prevented (based on GFAP and S100B content), and astroglial dysfunction was partially abrogated (based on Kir 4.1 protein and GSH content). The GS decrease was not prevented by dexamethasone, and AQP-4 was not altered in this epileptic model. Conclusions: Changes in astroglial parameters emphasize the importance of these cells for understanding alterations and mechanisms of epileptic disorders in this model. In vivo dexamethasone administration prevented most of the parameters analyzed, reinforcing the importance of anti-inflammatory steroid therapy in the Li-pilocarpine model and possibly in other epileptic conditions in which neuroinflammation is present

Methylglyoxal Induces Changes in the Glyoxalase System and Impairs Glutamate Uptake Activity in Primary Astrocytes

The impairment of astrocyte functions is associated with diabetes mellitus and other neurodegenerative diseases. Astrocytes have been proposed to be essential cells for neuroprotection against elevated levels of methylglyoxal (MG), a highly reactive aldehyde derived from the glycolytic pathway. MG exposure impairs primary astrocyte viability, as evaluated by different assays, and these cells respond to MG elevation by increasing glyoxalase 1 activity and glutathione levels, which improve cell viability and survival. However, C6 glioma cells have shown strong signs of resistance against MG, without significant changes in the glyoxalase system. Results for aminoguanidine coincubation support the idea that MG toxicity is mediated by glycation. We found a significant decrease in glutamate uptake by astrocytes, without changes in the expression of the major transporters. Carbenoxolone, a nonspecific inhibitor of gap junctions, prevented the cytotoxicity induced by MG in astrocyte cultures. Thus, our data reinforce the idea that astrocyte viability depends on gap junctions and that the impairment induced by MG involves glutamate excitotoxicity. The astrocyte susceptibility to MG emphasizes the importance of this compound in neurodegenerative diseases, where the neuronal damage induced by MG may be aggravated by the commitment of the cells charged with MG clearance

Liberação de S100B in vitro modulada por adrenalina

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Boletín oficial de la provincia de Santander: Año XXXIV Número 135 - 1970 Noviembre 11

A S100B, uma proteína ligante de cálcio de 21 kDa, possui muitos alvos intracelulares envolvidos no ciclo celular e na regulação do citoesqueleto em astrócitos. Além disso, essa proteína é secretada e possui efeitos autócrinos e parácrinos na glia, plasticidade sináptica e microglia. A expressão de S100B, particularmente a S100B extracelular, é usada como um parâmetro de ativação e/ou morte glial em diversas situações de dano cerebral. Existem muitos imunoensaios para a dosagem de S100B, que diferem em relação à especificidade, sensibilidade, aplicação das amostras e custo. Nós padronizamos dois protocolos para a dosagem de S100B (variando em sensibilidade de 1,9 pg a 10 ng/mL) em amostras de humanos e de ratos, de tecido cerebral, tecido adiposo, soro, líquor, urina e amostras de cultura de células. Muitos secretagogos de S100B já foram identificados, mas o mecanismo de secreção dessa proteína ainda é desconhecido e envolve um mecanismo do tipo não-clássico de secreção. Nós investigamos o papel do Ca2+ na secreção de S100B em cultura primária de astrócitos. Nossos resultados mostraram que o DMSO é um potente secretagogo da S100B. A secreção de S100B induzida por DMSO foi dependente da mobilização de cálcio do retículo endoplasmático, mas não de reorganização do citoesqueleto. A propriedade do DMSO em induzir a secreção de S100B deve ser adicionada à lista de aplicações terapêuticas desse composto, especialmente considerando os efeitos neuroprotetores que têm sido observados da S100B em situações de dano cerebral agudo. Além disso, a utilidade do DMSO como uma ferramenta para investigar a mobilização de cálcio intracelular deve ser levada em consideração. Sabe-se que os astrócitos sentem, interagem e respondem a estímulos gerados pelos neurônios ou pelo dano neuronal e essa resposta envolve a comunicação por junção gap. A vulnerabilidade neuronal a danos é aumentada quando co-culturas de astrócitos e neurônios são expostas a inibidores de junção gap. Entretanto, a inibição das junções gap pode limitar a extensão de uma lesão. Nós investigamos uma possível relação entre a comunicação por junção gap e a secreção de S100B. Nossos dados indicam que o bloqueio das junções gap estimula a secreção de S100B em cultura de astrócitos, bem como em fatias agudas hipocampais. A secreção de S100B foi observada com o uso de diferentes tipos de bloqueadores de junção gap e o resultado foi dependente do tempo, da natureza do inibidor, de seu possível alvo intracelular e/ou do tipo de preparação celular utilizada. Fisiologicamente, um bloqueio local da comunicação por junção gap associado com a liberação de S100B em uma situação de dano favorece a idéia da existência de um mecanismo comum para limitar a extensão da lesão e, simultaneamente, aumentar as chances de sobrevivência celular.S100B, a calcium-binding protein of 21 kDa, has many putative intracellular targets involved in cell cycle and cytoskeleton regulation in astrocytes. In addition, this protein is also secreted and has autocrine and paracrine effects on glia, synaptic plasticity and microglia. S100B expression, particularly extracellular S100B, is used as a parameter of glial activation and/or death in several situations of brain injury. Several immunoassays for S100B measurement are available, which differ with regard to specificity, sensitivity, sample application, and, of course, economic costs. We standardized two protocols for S100B measurement (range between 1,9 pg and 10 ng/mL) in human and rat samples from brain and adipose tissues, blood serum, cerebrospinal fluid, urine and cell culture. Many S100B secretagogues have been identified, but the underlying mechanism of secretion remains unknown and involves a non-classic export. Herein, we investigate the role of Ca2+ in S100B secretion in primary cultured astrocytes. Results indicate that DMSO is a powerful S100B secretagogue. DMSO induced S100B secretion was dependent on increased intracellular Ca2+ mobilization from endoplasmatic reticulum and independent of cytoskeleton reorganization. Furthermore, the S100B-secreting property of DMSO should be added to the list of therapeutic applications of this compound, particularly considering the neuroprotective effects of S100B that have been observed in acute brain damage. In addition, the usefulness of DMSO per se as a tool to investigate intracellular calcium mobilization should be taken into consideration. Astrocytes sense, integrate, and respond to stimuli generated by neurons or neural injury; this response involves gap junction (GJ) communication. Neuronal vulnerability to injury increased when cocultures of astrocytes and neurons were exposed to GJ inhibitors. However, GJ uncoupling could limit the extension of a lesion. We investigated a possible link between GJ communication and S100B secretion. Our data indicate that GJ blocking stimulates S100B secretion in astrocyte cultures and acute hippocampal slices. S100B secretion was observed with different types of GJ inhibitors; the resulting event was dependent on time, the nature of the inhibitor, its putative molecular target of GJ blocking, and/or the cell preparation used. Physiologically, a local GJ closure associated with release of S100B in injury conditions favors the idea of a common mechanism available to limit the extension of lesion and increase the chances of cell survival

Influência da amônia sobre o conteúdo e secreção de S100B em cultura de astrócitos

A hiperamonemia é o principal elemento na patogênese da encefalopatia hepática e a neurotoxicidade da amônia envolve um efeito no sistema de neurotransmissão glutamatérgica. Os astrócitos são intimamente relacionados com a transmissão glutamatérgica e, de fato, muitas alterações gliais específicas têm sido relatadas devido à exposição à amônia. A proteína S100B, particularmente a S100B extracelular, é usada como um parâmetro de ativação glial em diversas situações de injúria cerebral. Entretanto, existe pouca informação sobre essa proteína na toxicidade da amônia e nada se sabe sobre a sua secreção por astrócitos durante uma exposição à amônia. Nesse trabalho, nós investigamos a secreção de S100B em astrócitos corticais de ratos expostos de forma aguda à amônia, bem como a morfologia astrocítica, o imunoconteúdo da proteína fibrilar ácida glial (GFAP) e a atividade da enzima glutamina sintetase (GS). Além disso, investigamos um possível efeito da creatina nesses parâmetros gliais, devido a esse composto ter um suposto papel contra a toxicidade da amônia em culturas celulares. Encontramos um aumento da secreção de S100B em astrócitos expostos por 24 h à amônia, acompanhado de uma redução do imunoconteúdo de GFAP e da atividade da GS. Como elevados e persistentes aumentos extracelulares de S100B têm um efeito tóxico em células neuronais, a secreção alterada de S100B induzida pela amônia pode contribuir para o dano cerebral observado na encefalopatia hepática. A adição de creatina não impediu esse aumento na secreção de S100B, mas foi capaz de impedir a redução da concentração de GFAP e da atividade da GS induzidas pela exposição à amônia.Hyperammonemia is a major element in the pathogenesis of hepatic encephalopathy (HE) and ammonia neurotoxicity involves an effect on the glutamatergic neurotransmitter system. Astrocytes are intimately related to glutamatergic neurotransmission and, in fact, many specific glial alterations have been reported due to ammonia exposure. S100B protein, particularly extracellular S100B, is used as a parameter of glial activation or commitment in several situations of brain injury. However, there is little information about this protein in ammonia toxicity and none about its secretion in astrocytes under ammonia exposure. In this study we investigated S100B secretion in rat cortical astrocytes acutely exposed to ammonia, as well astrocyte morphology, glial fibrillary acidic protein (GFAP) content and glutamine synthetase (GS) activity. Moreover, we studied a possible effect of creatine on these glial parameters, since that this compound has a putative role against ammonia toxicity in cell cultures. We found an increase in S100B secretion by astrocytes exposed to ammonia for 24 h, accompanied by a decrease in GFAP content and GS activity. Since elevated and persistent extracellular S100B plays a toxic effect on neural cells, altered extracellular content of S100B induced by ammonia could contribute to the brain impairment observed in HE. Creatine addition did not prevent this increment in S100B secretion, but was able to prevent the decrease in GFAP content and GS activity induced by ammonia exposure