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1. Hintergrund Das Schädel-Hirn-Trauma (SHT) stellt eine der häufigsten Todesursachen und Begleitverletzungen bei nichtnatürlichen Todesfällen dar und ist damit Gegenstand der Routine-Untersuchungen in der Rechtsmedizin. Eine Abschätzung der Überlebenszeit (ÜLZ, d.h. der Zeitraum zwischen der Verletzungsentstehung und dem Todeseintritt) ist für die Chronologie eines Tatablaufs und Überprüfung von Zeugenaussagen / Alibiangaben von großer Bedeutung. Primär werden hierfür postmortal pathomorphologische und klassische histologische Befunde herangezogen. Immunhistochemische Untersuchungen haben bisher kaum Eingang in die Überlebenszeitdiagnostik gefunden, könnten aber zur Konkretisierung der bisher gängigen Methoden beitragen. Häufig untersuchte Proteine im Gehirn sind das S100-Protein (S100) und die Neuronenspezifische Enolase (NSE). Die Spiegel beider Marker werden im klinischen Alltag vielfach zur Abschätzung der Schwere und der Prognose eines SHT im Blut und Liquor gemessen. Immunhistochemisch wurden beide Proteine bisher vor allem auf deren allgemeines Vorhandensein und Verteilung im Zusammenhang mit SHT untersucht. Nur eine Studie beschäftigte sich bisher mit einer möglichen zeitlichen Dynamik. 2. Fragestellungen Folgende Fragen sollten durch vorliegende Arbeit beantwortet werden:  Existiert eine Korrelation zwischen dem Anteil positiv auf S100 gefärbter Gliazellen (Astroglia und Oligodendroglia) und der Überlebenszeit?  Ist eine Korrelation zwischen dem Anteil positiv auf NSE gefärbter Neuronen und der Überlebenszeit möglich?  Welche lokalisationsspezifische Veränderungen in den untersuchten Hirnregionen (Umgebung der Kontusion, Hippocampus, Kleinhirn) in Bezug auf die Überlebenszeit existieren?  Gibt es signifikante Unterschiede zwischen Fall- und Kontrollgruppe? 3. Material und Methoden Für diese Untersuchung wurden Hirngewebeproben aus 57 gerichtlich angeordneten Sektionen verwendet. Davon wiesen 47 ein tödliches SHT und ÜLZ zwischen wenigen Sekunden und 34 Tagen auf. Zehn Fälle mit kardiovaskulären Todesursachen wurden als Kontrolle herangezogen. Die Überlebenszeiten der Fälle mit tödlichem SHT wurden in Übereinstimmung mit bisherigen Studien in folgende Kategorien eingeteilt: Akuter Todeseintritt nach SHT (ÜLZ bis 2 Stunden), subakuter Todeseintritt nach SHT (ÜLZ 2 Stunden bis 4 Tage) und verzögerter Todeseintritt nach SHT (ÜLZ über 4 Tage). Die zur Untersuchung gelangten Proben wurden spätestens 6 Tage nach dem Versterben der Personen entnommen (Mittelwert 2,7 Tage). In allen Fällen wurde die Umgebung der Kontusion, bei 35 dieser Fälle der Hippocampus und bei 31 der Fälle auch das Kleinhirn untersucht. Die verschiedenen Regionen wurden jeweils gesondert für Rinde und Mark bzw. im Hippocampus für Stratum pyramidale und radiatum beurteilt. Die immunhistochemische Färbung auf S100 und NSE wurde mit der indirekten Dextran-Polymer-Methode (DakoCytomation), die Gegenfärbung mit Hämatoxylin nach Mayer durchgeführt. Verwendet wurden ein polyklonaler S100-Kaninchen-Antikörper sowie ein monoklonaler NSE-Maus-Antikörper (beide DakoCytomation). Für die semiquantitative Evaluation wurden gefärbte und ungefärbte Neuronen, Oligodendrozyten sowie Astrozyten in jeweils 20 High Power Fields gezählt. So konnte für jede Region und Zellart ein Prozentsatz positiver Zellen ermittelt werden. Für die statistische Auswertung wurde SPSS Statistics (Version 21, 2012 IBM) und OpenOffice Calc (Version 3.4.1, 2012 Apache Software Foundation) verwendet, es kamen der Mann-Whitney-Wilcoxon-Test (nicht-parametrisch), die Spearman-Korrelation und die Benjamini-Hochberg-Prozedur zum Einsatz. Eine Zustimmung zu dem der Promotionsschrift zugrunde liegendem Forschungsvorhaben wurde durch die Ethik-Kommission der Medizinischen Fakultät der Universität Leipzig erteilt (Nr. 117-12-23012012). 4. Ergebnisse Äußere Einflüsse. Es konnte keine Korrelation zwischen dem Anteil positiver Zellen und der Leichenliegezeit (rs= -0.27 bis 0.15, p = 0.1 bis 0.96) oder dem Geschlecht (p = 0.07 bis 0.98) festgestellt werden. Aufgrund des häufigeren Auftretens verzögerter Todeseintritte bei älteren Personen (rs = 0,33, p < 0.05) konnte keine sinnvolle Korrelation zwischen Alter und Zellpositivität durchgeführt werden. Zellzahlen insgesamt. Zur Qualitätssicherung und zur Vergleichbarkeit mit anderen Studien, wurden die Zellzahlen insgesamt erfasst. Hierbei wurden keine signifikanten Unterschiede in den unterschiedlichen ÜLZ-Kategorien festgestellt. Die Zellzahlen in den SHT-Fällen waren hingegen signifikant niedriger als in den Kontrollfällen. Unterschiede in den Kategorien der Überlebenszeit. Die Anteile S100-positiver Oligodendrozyten waren in Kontusionsumgebung signifikant niedriger in der Gruppe mit subakutem Todeseintritt als in der Gruppe mit akutem Todeseintritt (p < 0,05) sowie der Kontrollgruppe (p < 0,05). Im Hippocampus waren die Anteile S100-positiv gefärbter Oligodendrozyten in der Gruppe der akuten sowie subakuten Todeseintritte niedriger als in der Kontrollgruppe (jeweils p < 0,05). Im Vergleich mit der Kontrollgruppe waren die Anteile NSE-positiver Neuronen sowohl im Hippocampus als auch in der Kontusionsumgebung in der Gruppe der akuten Todeseintritte (jeweils p < 0,05) höher. Die Anteile NSE-positiver Neuronen im Hippocampus sanken in der Gruppe der subakuten im Vergleich zur Gruppe der akuten Todeseintritte ab (p < 0,05). Astrozyten zeigten bei dieser Studie keine signifikanten Unterschiede in ihrem Färbeverhalten in Bezug auf die ÜLZ. Überraschenderweise zeigten sich in den Gruppen mit subakutem und verzögertem Todeseintritt auch S100-positive Neuronen im Hippocampus und der Kontusionsumgebung. Diese Beobachtung konnte in der Akutphase nach Traumatisierung und in der Kontrollgruppe nicht gemacht werden. Im Hippocampus war eher eine diffuse neuroplasmatische, in der Kontusionsumgebung eine eher juxtanukleäre Färbung zu finden. In beiden Regionen war die Verteilung der S100-positiven Neuronen unsystematisch oft in räumlicher Nähe zu S100-positiven Gliazellen zu finden.

Introduction: Recent findings and better understanding to the pathogenesis of rheumatic diseases contributed to the development of biological therapies targeting cytokines and immune cells. Several S100 proteins exert cytokine-like effects and participate in the regulation of the inflammatory process. The aim of this work was to study the role of selected S100 proteins in the activity and in the pathogenesis of the rheumatic diseases. Results: Our data show for the first time an association of S100A4 proteinwith RA disease activity and decrease of the bioactive form, but not the total amount of S100A4, after aplication of tumour necrosis factor (TNF) blocking biologic therapy in patients with RA. We demonstrated that in vitro S100A4 acts as a potent pro-inflammatory mediator inducing production of TNFα, interleukin (IL)-1β and IL-6 in PBMCs via Toll-like receptor 4 (TLR-4), transcription factor NFκB and tyrosine kinases erk1/2 and p38. Moreover, S100A4 can play an important role in the pathogenesis of inflammatory myopathies. S100A4 is present in the inflammatory infiltrate of the affected muscles and in the regenerating muscles and may act as a cytokine-like factor indirectly promoting muscle fiber damage by stimulating mononuclear cells to increase the synthesis of pro-inflammatory cytokines. We...

Rheumatic diseases are common, usually chronic, painful and to some extent invalidating medical conditions. Understanding of the disease pathogenesis is still very fragmentary. Hyperreactivity of the immune system and defect of autotolerance are probably contributed by local factors, which helps to explain, why some joints/muscles are more affected than others. All this results from a complex net of interactions between immune cells, synovial fibroblasts, chondrocytes, osteocytes, myocytes and other cells. In the submitted PhD thesis I have focused on three groups of molecules: regulatory RNAs, S100 proteins and autoantibodies. In the theoretical part, I sum up the current knowledge on their biogenesis, function and the role in rheumatology. In the investigative part, I present six original publications and one review on the role of those molecules in development of rheumatoid arthritis (RA) and idiopathic inflammatory myositis (IIM). One of the main studies was focused on expression of PIWI-interacting RNAs (piRNAs) in RA synovial fibroblasts (SF). piRNAs are small regulatory RNAs which in complex with PIWIL proteins regulate gene expression and silence transpozoms. piRNA expression was considered to be limited to germline and cancer cells. We have found 267 PIWI-interacting RNAs to be expressed...

Rheumatic diseases are common, usually chronic, painful and to some extent invalidating medical conditions. Understanding of the disease pathogenesis is still very fragmentary. Hyperreactivity of the immune system and defect of autotolerance are probably contributed by local factors, which helps to explain, why some joints/muscles are more affected than others. All this results from a complex net of interactions between immune cells, synovial fibroblasts, chondrocytes, osteocytes, myocytes and other cells. In the submitted PhD thesis I have focused on three groups of molecules: regulatory RNAs, S100 proteins and autoantibodies. In the theoretical part, I sum up the current knowledge on their biogenesis, function and the role in rheumatology. In the investigative part, I present six original publications and one review on the role of those molecules in development of rheumatoid arthritis (RA) and idiopathic inflammatory myositis (IIM). One of the main studies was focused on expression of PIWI-interacting RNAs (piRNAs) in RA synovial fibroblasts (SF). piRNAs are small regulatory RNAs which in complex with PIWIL proteins regulate gene expression and silence transpozoms. piRNA expression was considered to be limited to germline and cancer cells. We have found 267 PIWI-interacting RNAs to be expressed...

Epidemiological studies report that infarct size is decreased and stroke outcomes are improved in young females when compared to males. However, mechanistic insight is lacking. We posit that sex-specific differences in glial cell functions occurring immediately after ischemic stroke are a source of dichotomous outcomes. In this study we assessed astrocyte Ca2+ dynamics, aquaporin 4 (AQP4) polarity, Sloop expression pattern, as well as, microglia morphology and phagocytic marker CD11b in male and female mice following 60 min of middle cerebral artery (MCA) occlusion. We reveal sex differences in the frequency of intracellular astrocyte Ca2+ elevations (F-(1,F-86) = 8.19, P = 0.005) and microglia volume (F-(1,F-40) = 12.47, P = 0.009) immediately following MCA occlusion in acute brain slices. Measured in fixed tissue, AQP4 polarity was disrupted (F-(5,F-86) = 3.30, P = 0.009) and the area of non-S100 beta immunoreactivity increased in ipsilateral brain regions after 60 min of MCA occlusion (F-(5,F-86) = 4.72, P = 0.007). However, astrocyte changes were robust in male mice when compared to females. Additional sex differences were discovered regarding microglia phagocytic receptor CD11b. In sham mice, constitutively high CD11b immunofluorescence was observed in females when compared to males (P = 0.03). When compared to sham, only male mice exhibited an increase in CD11b immunoreactivity after MCA occlusion (P = 0.006). We posit that a sex difference in the presence of constitutive CD11b has a role in determining male and female microglia phagocytic responses to ischemia. Taken together, these findings are critical to understanding potential sex differences in glial physiology as well as stroke pathobiology which are foundational for the development of future sex-specific stroke therapies. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Embora a inflamação seja um processo de defesa fisiológica, neuroinflamação desequilibrada tem sido associada com a fisiopatologia de doenças do SNC. Além disso, a exposição pré-natal à infecção bacteriana e/ou viral tem sido implicada em vários estudos como um importante fator ambiental capaz de afetar prejudicialmente o neurodesenvolvimento, aumentando assim o risco de esquizofrenia. Resposta imune materna, em vez de infecção direta do feto, poderia ser responsável pelo desenvolvimento da doença. Glia ativada libera uma variedade de citocinas pró-inflamatórias que contribuem para a disfunção neuronal. Além disso, as alterações de marcadores astrogliais parecem estar intimamente relacionadas à patologia da esquizofrenia, por exemplo, níveis elevados de S100B, uma proteína derivada da glia, foram observados no soro e LCR de pacientes com esquizofrenia. Nesta tese avaliou-se se a secreção de S100B (em células de glioma C6 e fatias de hipocampo de ratos Wistar) poderia ser diretamente modulada por citocinas inflamatórias alteradas na esquizofrenia, assim como o possível envolvimento da via da proteína cinase ativada por mítógeno (MAPK) nestas respostas. Os efeitos dos antipsicóticos típicos e atípicos na secreção de S100B induzida por citocinas foram analisadas. Nós também avaliamos se a administração de LPS em ratas prenhas no final da gestação afeta alguns aspectos comportamentais da prole em sua vida adulta, e investigamos os efeitos deste tratamento sobre parâmetros gliais usando preparação ex vivo dos descendentes, além de observar se o tratamento pré-natal afeta marcadores gliais (S100B e GFAP) da prole adulta. Usamos a prole aos 30 e 60 dias para avaliar as diferenças entre os ratos jovens e adultos e também investigar a existência de diferenças entre os descendentes do sexo masculino e feminino. Avaliamos também os parâmetros de estresse oxidativo NO e GSH. Nós observamos que secreção de S100B foi aumentada pelas citocinas pró-inflamatórias (IL-1, TNF-, IL-6 e IL-8) em glioma C6 e em fatias hipocampais através da via MAPK, e estresse oxidativo pode ser um componente desta modulação. Além disso, IL-6 foi capaz de induzir um aumento no conteúdo de S100B e GFAP em glioma C6 e os antipsicóticos, haloperidol e risperidona, foram capazes de inibir a secreção de S100B estimulada por IL-6. Observamos também que o modelo de alteração imune maternal provocou alterações comportamentais na prole adulta, especialmente no comportamento social dos ratos afetados, além de modificar o padrão de secreção da proteína S100B e da captação de glutamato em fatias ex vivo da prole adulta. Constatamos que conteúdo de glutationa e NO são modificados de forma dependente de idade/gênero. Foi observada também uma pronunciada astrogliose particularmente hipocampal, com GFAP apresentando-se aumentada em ratos juvenis e adultos enquanto que conteúdo de S100B apresentou diminuição na prole juvenil e aumento em ratos adultos em resposta a exposição imune maternal. Aparentemente, infecção pré-natal parece levar a anormalidades comportamentais e neuroquímicas, incluindo modificação de marcadores gliais, na vida pós-natal, possivelmente via citocinas. / Although inflammation may be a physiological defense process, imbalanced neuroinflammation has been associated with the pathophysiology of brain disorders. Furthermore, prenatal exposure to bacterial and viral infection has been implicated by several studies indicating that such exposure is an important environmental factor out that may detrimentally affect neurodevelopment, increasing the risk of schizophrenia. Maternal immune response, rather than direct infection of the fetus, could be responsible for disease development. Activated glia releases a variety of pro-inflammatory cytokines that contribute to neuronal dysfunction. Moreover, changes in astrogliais markers seem to be closely related pathology of schizophrenia, for example, elevated levels of S100B, a glia derived protein, have been observed in the serum and CSF of schizophrenic patients. We evaluated whether S100B secretion (in C6 glioma cells and hippocampal slices in Wistar rats) could be directly modulated by the main inflammatory cytokines altered in schizophrenia, as well as the possible involvement of mitogen-activated protein kinase (MAPK) pathways in these responses. The effects of typical and atypical antipsychotic drugs on glial cytokine-induced S100B release were analyzing. We also evaluated whether administration of LPS to rats dams in late gestation affects some aspects of the offspring behavior in their adult life, and investigated the effects of this treatment on glial parameters using ex vivo preparation of offsprings, and whether it affects astroglial markers (S100B and GFAP) of the offspring in later life. We used the offspring at 30 and 60 days to evaluate the differences between juvenile and adult rats and also investigate the existence of differences between male and female offspring. We also investigated the oxidative stress parameters NO and GSH. We observed that S100B secretion was increased by the pro-inflammatory cytokines (IL-1 , TNF-, IL-6 and IL-8) in C6 glioma and in hippocampal slices via the MAPK, and oxidative stress may be a component this modulation. Furthermore, IL-6 was capable of inducing an increase in content GFAP and S100B in C6 glioma and antipsychotic, haloperidol and risperidone have been capable of inhibiting S100B secretion stimulated by IL-6. We also note that the model of maternal immune changes caused behavioral changes in the adult offspring, especially in the social behavior of rat affected and modified the pattern of secretion of S100B protein and glutamate uptake in ex vivo slices of adult offspring. We found that content of glutathione and NO are modified so age / gender dependent. There was also a pronounced hippocampal astrogliosis, with GFAP presenting increased in juvenile and adult rats while S100B content was reduced in juvenile and increased in adult offspring rats in response to exposure to maternal immune. Apparently, prenatal infection appears to lead to behavioral and neurochemical abnormalities, including modification of glial markers in the postnatal life, possibly via cytokines.

Convulsões prolongadas, status epilepticus (SE), durante o desenvolvimento cerebral podem afetar a estrutura e a função neuronal, levando a morte celular e alterações comportamentais na idade adulta. Nesta tese, investigamos os efeitos neuroquímicos e comportamentais, a curto e longo prazo, do SE induzido por LiCl-pilocarpina em animais jovens. O SE induzido por LiCl-pilocarpina reduz em 50% a atividade da Na+/K+ ATPase 1,5 h após a indução do insulto. Doze e vinte e quatro horas após a administração de pilocarpina, a atividade da Na+/K+ ATPase retornou aos níveis do controle. A diminuição da atividade da Na+/K+ ATPase foi acompanhada por uma diminuição significativa na captação de glutamato em concentrações fisiológicas (1 μM). No entanto, em concentrações elevadas (100 μM) não observou-se alteração da captação de glutamato. Nos animais tratados com LiCl-pilocarpina observou-se uma diminuição dos níveis de fosforilação das proteínas PKB e GKS-3β de 35,84% e 38,79%, respectivamente, 1,5 h após a indução do SE na região CA1 do hipocampo. No entanto, 24 h após a indução do SE, a fosforilação da PKB e GKS-3β retornaram aos níveis do controle. No giro denteado, observou-se uma redução de aproximadamente 30% nos níveis de fosforilação tanto da PKB e GKS-3β em 1,5 e 24 h após a indução do SE. Um elevado número de neurônios em degeneração foi observado 24 h após a indução do SE nas regiões CA1 e hilo do hipocampo. Na idade adulta, os animais tratados com LiCl-pilocapina apresentaram um severo déficit de memória-aprendizado na tarefa de esquiva inibitória, sendo que os animais que apresentaram menor latência para descida da plataforma apresentaram um maior escore para brotamento de fibras musgosas no hipocampo. No teste do claro-escuro, os animais submetidos ao SE retornaram menos e permaneceram menos tempo no compartimento claro quando comparados aos animais controles. Além disso, os animais tratados com LiCl-pilocarpina apresentaram elevados níveis da proteína S100B no líquor, bem como apresentaram uma correlação positiva entre o escore para brotamento de fibras musgosas e o imunoconteúdo de GFAP na região CA1. Portanto, nossos resultados mostraram que o SE induzido por LiCl-pilocarpina diminui a atividade da Na+/K+ ATPase, bem como a captação de glutamato em hipocampo 1,5 h após a indução do insulto. Estes efeitos foram acompanhados por uma diminuição da fosforilação das proteínas PKB e GKS-3β nas regiões CA1 e giro denteado, os quais podem estar relacionados com a morte neuronal observada 24 h após a indução do SE. Além disso, na idade adulta, o SE induzido aos 14 dias pós-natal induziu alterações no processo de memória-aprendizado e no comportamento emocional. As alterações comportamentais parecem estar relacionadas ao brotamento de fibras musgosas, visto que os animais que apresentaram déficit de memória na tarefa de esquiva inibitória e elevados níveis de ansiedade no teste claro-escuro apresentaram maior escore para brotamento de fibras musgosas no hipocampo. Além disso, os animais tratados com LiCl-pilocarpina apresentaram elevados níveis da proteína S100B no líquor bem como uma correlação positiva entre o imunoconteúdo de GFAP na região CA1 e o escore para brotamento de fibras musgosas, sugerindo uma resposta glial ao dano neuronal. Estes dados indicam que o status epilepticus induzido em períodos iniciais do desenvolvimento cerebral é danoso ao sistema nervoso central, alterando sua maturação e levando a alteração neuroquímicas, morfológicas e comportamentais na idade adulta. / Prolonged seizure activity, i.e. status epilepticus (SE), or repeated, brief seizures affect neuronal structure and function in the developing nervous system leading to neuronal death and behavioral impairment at adulthood. In this study, we investigated the short- and long-term effects of early-life LiCl-pilocarpine-induced SE on neurochemical and behavioral parameters. LiClpilocarpine- induced SE decreased Na+/K+ ATPase activity by 42% in hippocampal plasma membranes 1.5 h after SE induction when compared with control group. However, at 12 and 24 h after SE induction the activity of pump returned to control levels. At physiologic concentration of glutamate (1 μM), LiCl-pilocarpine treated animals showed decreased levels of glutamate uptake 1.5 h after SE induction. However, LiCl-pilocarpine-induced SE did not alter the glutamate uptake 12 and 24 h after SE induction. At higher concentration of extracellular glutamate (100 μM), no alterations were observed in glutamate uptake between control and LiCl-pilocarpine-induced SE groups in all times tested. LiCl-pilocarpine-induced SE decreased the phosphorylation of PKB by 35.84% and GSK-3β by 38.79% 1.5 h after SE induction in CA1 subfield. However, 24 h after LiCl-pilocarpine-induced SE the phosphorylation of PKB and GSK-3β returned to control levels. At dentate gyrus, the phosphorylation of PKB was reduced by 35.18% and 31.19% at 1.5 and 24 h after SE induction, respectively. GSK-3β phosphorylation was reduced by 38.05% and 33.12% at 1.5 and 24 h, respectively, after LiCl-pilocarpine administration. Twenty four h after SE, it was observed an increased number of degenerating neurons in the CA1 subfield and in the hilus. At adulthood, SE group presented an aversive memory deficit in an inhibitory avoidance task and the animals that presented lower latency to the step down showed a higher score for mossy fiber sprouting. In the light-dark exploration task, SE rats returned less and spent less time in the light compartment and present an increased number of risk assessment behavior (RA). There was a negative correlation between the time spent in the light compartment and the score for mossy fiber sprouting and a positive correlation between score for mossy fiber sprouting and number of RA. LiCl-pilocarpine-treated animals showed higher levels of S100B immunocontent in the CSF as well as a positive correlation between the score for sprouting and the GFAP immunocontent in the CA1 subfield, suggesting an astrocytic response to neuronal injury. Our results showed that early-life LiCl-pilocapine-induced SE reduce hippocampal Na+/K+ ATPase as well as glutamate uptake 1.5 h after SE induction. These effects were accompanied by a reduced phosphorylation of Akt and GSK- 3β at CA1 and dentate gyrus, which may be related to neuronal death observed 24 h after SE induction. Moreover, at adulthood the animals submitted to LiCl-pilocapine induced SE displayed memory impairment and increased anxiety-like behavior. The behavioral alterations appear to be associated with the mossy fiber sprouting, since the animals that presented impairment in the performance of inhibitory avoidance task and higher levels of anxiety in adulthood showed higher scores for mossy fiber sprouting in the hippocampus. Moreover, LiCl-pilocarpine-treated animals showed higher levels of S100B immunocontent in the CSF as well as a positive correlation between the score for mossy fiber sprouting and the GFAP immunocontent in the CA1 subfield, suggesting an astrocytic response to neuronal injury. These data indicate that LiCl-pilocarpine-induced SE early in life might harmfully affect brain maturation, leading to behavioral, morphological and neurochemical alterations in adulthood.

A amônia é uma neurotoxina implicada em desordens metabólicas cerebrais associadas com hiperamonemia. A neurotoxicidade aguda da amônia pode ser mediada por mecanismos excitotóxicos envolvendo o sistema glutamatérgico, incluindo a ativação do receptor NMDA e o subsequente aumento na concentração de Ca2+. O estresse oxidativo está relacionado à neurotoxicidade da amônia e o óxido nítrico parece estar envolvido nesta condição. Os astrócitos desempenham um papel essencial na proteção dos neurônios contra excitotoxicidade por captar o excesso de amônia e glutamato e convertê-los em glutamina, usando a enzima glutamina sintetase, e também protegendo contra o estresse oxidativo. A proteína S100B, particularmente a S100B extracelular, é usada como parâmetro de ativação ou comprometimento em várias situações de dano cerebral, incluindo hiperamonemia. Antioxidantes, como o resveratrol, apresentam muitos efeitos biológicos, incluindo a modulação de parâmetros gliais como a captação de glutamato, a atividade da glutamina sintetase e a secreção de S100B. Neste estudo, foi investigado o efeito de antioxidantes sobre a secreção de S100B induzida pela amônia em células astrogliais. O resveratrol foi capaz de prevenir o aiumento da secreção de S100B, após 24 h de exposição à amônia, provavelmente via inibição de óxido nítrico e proteína cinase A (PKA). Então, o resveratrol pode ser um possível agente protetor contra a neurotoxicidade induzida pela amônia. / Ammonia is a neurotoxin implicated in brain metabolic disorders associated with hyperammonemia. Acute ammonia neurotoxicity can be mediated by excitotoxic mechanism involving glutamatergic system, including NMDA receptor activation and subsequent increase in intracellular Ca2+ concentration. Oxidative stress is related to ammonia neurotoxicity and nitric oxide can be involved in this condition. Astrocytes play an essential role in protecting neurons against excitotoxicity uptake excess ammonia and glutamate and converting it into glutamine, using enzyme glutamine synthetase and also protected against oxidative stress. S100B protein, particularly extracellular S100B, is used as a parameter of glial activation or commitment in several situations of brain injury, including hyperammonemia. Antioxidants, such as resveratrol, showed many biological effects, including modulation of glial parameters as glutamate uptake, glutamine synthetase activity and S100B secretion. In this study, we investigated the effect of antioxidants on S100B secretion induced by ammonia in astroglial cells. Resveratrol was able to prevent the increase of S100B secretion, after 24 h ammonia exposure, probably via nitric oxide and protein kinase A (PKA) inhibition. Then, resveratrol may be a possible protective agent against neurotoxicity induced by ammonia.

A ingestão de ácidos graxos na forma de gordura proveniente da dieta tem aumentado nos últimos anos, principalmente na forma de gordura saturada. Tem sido observado que ácidos graxos saturados podem causar a liberação de citocinas em diversos tecidos e células. Além disso, em indivíduos obesos, tem se observado que há uma inflamação crônica e de baixo grau, que poderia ter entre seus causadores os ácidos graxos provenientes da dieta. Já os ácidos graxos poli-insaturados têm sido citados como sendo neuroprotetores devido a suas propriedades anti-inflamatórias e anti-apoptóticas. A barreira hematoencefálica (BHE) é um dos principais fatores que regulam a captação dos ácidos graxos no sistema nervoso central (SNC). A captação de ácidos graxos pelo cérebro é seletiva, priorizando a entrada de ácidos graxos poli-insaturados e suprimindo a entrada de ácidos graxos saturados. Além disso, a quantidade relativa de ácidos graxos no sangue pode afetar o grau de penetração desses no SNC. Existem diversas condições onde há aumento na permeabilidade da BHE e, nessas situações, o SNC pode ficar mais exposto a substâncias que normalmente não estão presentes ou estão em baixas concentrações, como os ácidos graxos saturados de cadeia longa. Dessa forma, é importante que se analise os efeitos dessas substâncias em células cerebrais. Os astrócitos são células presentes no SNC caracterizadas por expressarem e secretarem a proteína S100B e por expressarem a proteína glial fibrilar ácida (GFAP). Os astrócitos captam glutamato e o metaboliza através da enzima glutamina sintetase (GS), formando glutamina, um processo importante para evitar o acúmulo de glutamato e excitotoxicidade. Essas células são muito resistentes ao estresse oxidativo devido ao seu alto conteúdo de glutationa reduzida (GSH). Em condições patológicas, os astrócitos liberam o fator de necrose tumoral alfa (TNF-α), e a produção dessa citocina é um importante componente da resposta neuroinflamatória que está associada com muitas desordens neurológicas. Os astrócitos possuem o receptor para produtos finais de glicação avançada (RAGE), que tem entre seus ligantes a S100B e sua ativação está envolvida na resposta inflamatória imediata. O receptor tipo toll 4 (TLR4) também está presente nessas células e ligado à resposta inflamatória. A ciclooxigenase 2 (COX-2) é uma enzima associada à inflamação e sua indução é responsável por desencadear muitas citocinas e mediadores inflamatórios presentes em diversos tipos celulares. Utilizando cultura primária de astrócitos de ratos Wistar, nós investigamos a ação dos ácidos graxos saturados e insaturados sobre parâmetros função de astrocitária, oxidativos e inflamatórios. Nosso estudo não encontrou alterações na viabilidade e integridade celular. Observamos um aumento dose-dependente na secreção de S100B nos astrócitos incubados apenas com o ácido palmítico. Não encontramos alterações para nenhum ácido graxo no conteúdo de GFAP, GSH ou captação de glutamato. Para o ácido linoleico observamos um aumento na atividade da GS e na oxidação do DCF. A secreção de TNF-α aumentou para os ácidos graxos saturados e não se alterou para os poli-insaturados. O conteúdo de COX-2, TLR4 e RAGE não foi alterado. Nossos resultados reforçam os dados de que ácidos graxos saturados, e não os insaturados, causam aumento na liberação de citocinas e podem contribuir para o processo neuroinflamatório encontrado em condições de aumento na permeabilidade da BHE. Além disso, verificamos que nem toda inflamação desencadeia aumento na secreção de S100B, visto que só tivemos esse comportamento para o ácido palmítico. Nossos resultados também reforçam o potencial terapêutico do ácido linoleico, que poderia desencadear adaptações nos astrócitos com possíveis benefícios para pacientes acometidos por patologias que envolvam a excitotoxicidade glutamatérgica. / The intake of fatty acids in the form of fat from the diet has increased in recent years, especially in the form of saturated fat. It has been observed that saturated fatty acids can cause the release of cytokines in various tissues and cells. Furthermore, in obese individuals, it has been observed a chronic and low-grade inflammation, which could have among their causers the diet fatty acids. Polyunsaturated fatty acids have been considered neuroprotective because of their antiinflammatory and antiapoptotic properties. The blood-brain barrier (BBB) is one of the major factors regulating the uptake of fatty acids in the central nervous system (CNS). The brain uptake of fatty acids is selective, prioritizing the entry of polyunsaturated fatty acids and suppressing the entry of saturated fatty acids. In addition, the relative amount of fatty acids in the blood can affect the degree of penetration of these into the CNS. There are several conditions where there is an increase in BBB permeability and in these situations the CNS may be more exposed to substances that are not normally present or are in low concentrations, such as saturated long chain fatty acids. Thus, it is important to analyze the effects of these substances in brain cells. Astrocytes are cells present in the CNS characterized by expressing and secreting the S100B protein and by expressing the glial fibrillary acidic protein (GFAP). Astrocytes uptake glutamate and metabolize it through the glutamine synthetase (GS) enzyme, forming glutamine, an important process to avoid the accumulation of glutamate and excitotoxicity. These cells are very resistant to oxidative stress because of their high reduced glutathione content (GSH). In pathological conditions, astrocytes release tumor necrosis factor alpha (TNF-α), and the production of this cytokine is an important component of the neuroinflammatory response that is associated with many neurological disorders. Astrocytes have the receptor for advanced glycation endproducts (RAGE), which has among its ligands S100B and its activation is involved in the immediate inflammatory response. The tolllike receptor 4 (TLR4) is another receptor present in these cells, which is also linked to the inflammatory response. The cycloxygenase-2 (COX-2) is an enzyme associated with inflammation and its induction is responsible for triggering many cytokines and inflammatory mediators in many cell types. Using astrocyte primary cultures from Wistar rats, we investigated the effect of saturated and unsaturated fatty acids on astrocytic function and oxidative and inflammatory parameters. Our study found no changes in cell viability and integrity. We observed a dose-dependent increase in S100B secretion in astrocytes incubated only with palmitic acid. We did not find alterations from fatty acids in the content of GFAP and GSH as well as uptake of glutamate. For linoleic acid, we observed an increase in GS activity and in DCF oxidation. TNF-α secretion increased to the saturated fatty acids and did not change for polyunsaturated. The contents of COX-2, TLR4 and RAGE did not change. Our results reinforce the data that saturated fatty acids, and not unsaturated, cause an increase in the release of cytokines and may contribute to the neuroinflammatory process found under conditions of increase in BBB permeability. In addition, we found that inflammation not always triggers an increase in S100B secretion, since we only had this behavior for palmitic acid. Our results also reinforce the therapeutic potential of linoleic acid, which could trigger adaptations in astrocytes with possible benefits for patients affected by pathologies involving glutamatergic excitotoxicity.