Submitted by Hellen Luz (hellencrisluz@gmail.com) on 2017-09-20T18:48:02Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_NeurogeneseEndogenaInduzida.pdf: 2761742 bytes, checksum: 4810f02f3d3c3d4aee4b8df72ba646b1 (MD5) / Rejected by Edisangela Bastos (edisangela@ufpa.br), reason: on 2017-10-10T17:00:23Z (GMT) / Submitted by Hellen Luz (hellencrisluz@gmail.com) on 2017-10-17T18:20:30Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_NeurogeneseEndogenaInduzida.pdf: 2761742 bytes, checksum: 4810f02f3d3c3d4aee4b8df72ba646b1 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-11-23T17:48:06Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_NeurogeneseEndogenaInduzida.pdf: 2761742 bytes, checksum: 4810f02f3d3c3d4aee4b8df72ba646b1 (MD5) / Made available in DSpace on 2017-11-23T17:48:06Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_NeurogeneseEndogenaInduzida.pdf: 2761742 bytes, checksum: 4810f02f3d3c3d4aee4b8df72ba646b1 (MD5) Previous issue date: 2011-05-16 / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / O acidente vascular encefálico (AVE) resulta de redução permanente ou transitória do fluxo sanguíneo para áreas encefálicas. Pode ser classificado como hemorrágico ou isquêmico. Sendo que o último é responsável por cerca de 87% dos casos. Esta desordem neuronal é a segunda maior causa de mortalidade e incapacidade no mundo e a principal causa de mortes no Brasil. Demonstrou-se que após a oclusão da artéria cerebral média (MCAO), os neuroblastos migram para o estriado isquêmico e substituem parcialmente os neurônios perdidos durante a isquemia. Além disso, a maioria dos neuroblastos migratórios morre na primeira semana após MCAO e eventos inflamatórios, principalmente a ativação microglial, pode desencadear na morte desses neuroblastos. Neste estudo, investigaram-se os efeitos do anti-inflamatório não esteróide, indometacina, nas ativação microglial, preservação neuronal e neurogênese adulta MCAO experimental de ratos adultos. Os animais sofreram MCAO por microinjeções de endotelina-1 e foram tratados (i.p) com indometacina ( N=8) ou solução salina estéril (N=8). Analisou-se através da imunoistoquímica, neurônios maduros (NeuN), ativação microglial/macrofágica (Iba1 e ED1) e migração de neuroblastos (DCX) 8 e 14 dias. O número de células NeuN, ED1 e DCX positivas por campo foram contadas no estriado isquêmico ou zona subventricular. O tratamento com indometacina reduziu a ativação microglial em geral e o número de células ED1+ em 8 e 14 dias após a lesão (médias de ±6,9 e ±3,0 células respectivamente), comparado ao controle (±7,9 ou ±6,5 células, p<0.001, ANOVA-Tukey). Aumentou o número de células DCX+ em ambas SVZ e estriado, em todos os tempos avaliados. Não houve diferença entre o número de células NeuN+ entre grupos. Os resultados mostram que o tratamento com indometacina inibe a ativação microglial concomitante com o aumento da proliferação e migração de neuroblastos após MCAO. Isto é resultado promissor, considerando que a indometacina é também usada em tratamentos de doenças humanas não neurais e que a neurogênese adulta pode levar à recuperação funcional após o AVE. / Stroke results from the transitory or permanent reduction of cerebral blood flow. It can be classified as hemorrhagic or ischemic. Ischemic stroke is responsible for around 87% of all cases. This acute neural disorder is the second cause of mortality and disability around the world and the main cause of death in Brazil. It has been shown that neuroblasts migrate to the ischemic striatum following middle cerebral artery occlusion (MCAO) and partially replace neurons lost during ischemia. Nevertheless, most of the migrating neuroblasts die in the first weeks following MCAO and inflammatory events, mainly microglia activation, may underlie neuroblast death. In this study, we investigated the effects of the nonsteroidal anti-inflammatory indomethacin on microglial activation, neuronal preservation and adult neurogenesis following experimental MCAO in adult rats. Animals were submitted to endothelin-1 induced- MCAO and treated (i.p) with indomethacin (N=8) or sterile saline (N=8) for 7 days and perfused at 8 or 14 days. Immunohistochemistry was performed to assess neuronal loss (anti-NeuN), microglial activation (anti-Iba1 and ED1) and migrating neuroblasts (anti-DCX). The numbers of NeuN, ED1 and DCX positive cells per field were counted in the ischemic striatum or subventricular zone. Indomethacin treatment reduced microglial activation in general and the number of ED1+ cells at both 8 and 14 days (±6,9 and ±3,0 cells respectively) postinjury, compared to control (±7,9 or ±6,5 cells, p<0.001, ANOVA-Tukey). There was an increase in the number of DCX+ cells in both subventricular zone (SVZ) and striatum at the same survival times. There was no difference in the number of NeuN positive cells between groups in all investigated survival times. The results show that indomethacin treatment induces inhibition of microglial activation concomitant with increased neuroblast proliferation and migration following MCAO. This is a promising outcome, considering that indomethacin is already used in non-neural human diseases and that adult neurogenesis may underlie functional recovery following stroke.

La MA est caractérisée par une altération progressive des fonctions cognitives, et définie par les "dégénérescences neurofibrillaires", les "plaques séniles" et une neuroinflammation impliquant les cellules microgliales. L’immunothérapie constitue une approche thérapeutique prometteuse dans la MA. Un premier essai clinique de vaccination anti-Aβ a été arrêté après la survenue de 6% de cas de méningoencéphalites imputés à l’activation de réponses T anti-Aβ pro-inflammatoires. Cependant, des études récentes suggèrent un effet bénéfique de certaines populations de cellules T CD4+ anti-Aβ. L'ensemble de ces données suggère des rôles complexes de différentes réponses lymphocytaires T au cours de la MA, et soulignent la nécessité de mieux comprendre leur implication dans la maladie et les mécanismes de leur régulation. L’objectif de ce travail a été d’étudier l’impact des Tregs sur la progression de la maladie dans un modèle murin APPPS1. Les résultats montrent que la déplétion transitoire des Tregs accélère l’apparition des troubles cognitifs, sans altérer la pathologie amyloïde. Ces observations sont corrélées avec une réduction du recrutement des cellules microgliales autour des plaques Aβ et une altération du profil d’expression, dans le cerveau des souris, de certains gènes impliqués dans la maladie. A l’inverse, l’amplification sélective des réponses Tregs entraine une augmentation du recrutement des cellules microgliales autour des plaques et une amélioration des fonctions cognitives. L’ensemble de ces résultats suggère un rôle bénéfique des Tregs au cours de la maladie d’Alzheimer et encourage le développement d’approches d’immunothérapie basées sur leur modulation. / Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by progressive loss of memory and cognitive functions. Accumulation of Aβ peptide is considered the initiating cause of pathogenic lesions, and immunotherapy strategies targeting Aβ represent promising therapeutic approaches. Vaccination against Aβ provided encouraging results in experimental mouse models and, to a lesser extent, in a subsequent clinical trial (AN1792). Although the AN1792 trial had to be interrupted due to meningoencephalitis attributed to pro-inflammatory T cell responses in 6% of the patients, preclinical murine models did not show evidence of T cell-related side effects. In addition, several reports suggest that Aβ-specific CD4+ T cells may be implicated in the natural course of AD and could have a strong therapeutic potential as well, pointing out the need for better understanding the role and regulation of T cell responses to Aβ. We previously showed that regulatory T cells (Tregs) control Aβ-specific CD4+ T cell responses in physiological and pathological settings upon vaccination. The aim of this work was to analyze the impact of Tregs on the natural course of the disease progression in a mouse model of AD. Early transient depletion of Tregs accelerated the onset of cognitive deficits in APPPS1 mice. This result was correlated with less recruitment of microglia towards amyloid deposits and altered disease-related gene expression profile. Conversely, Tregs selectively amplifying resulted in higher numbers of plaque-associated microglia and improved cognitive functions in APPPS1 mice. These data suggest a beneficial role of Tregs in the pathophysiology of AD by modulating microglial response. Our study highlithts the therapeutic potential of Treg-based immunotherapies in AD.

La maladie d'Alexander est une maladie neurodégénérative due à des mutations hétérozygotes du gène GFAP codant le principal filament intermédiaire des astrocytes matures. Nous avons étudié l'effet des mutations GFAP dans l'hippocampe d'un patient avec AxD infantile et de deux souris knockin, l'une portant une mutation dans le rod domain (p.R85C) et l'autre dans le tail domain (p.T409I). Chez le patient, nous décrivons pour la première fois: (i) des changements morphologiques sévères des cellules GFAP+ dans la zone subgranulaire du gyrus denté, qui ont perdu la plupart de leurs processus radiaux; (ii) une réactivité microgliale; (iii) et un déficit de la neurogénèse hippocampique postnatale. Nous avons trouvé des anomalies similaires dans les deux souris knockin, plus sévères chez les homozygotes. La comparaison de ces modèles a montré que ces anomalies prédominent chez les souris GFAPT409I, tandis que l’accumulation de GFAP est supérieure chez les souris GFAPR85C. La comparaison des deux modèles de souris a montré que les conséquences pathologiques dépendent la localisation de la mutation dans la GFAP. Ces résultats suggèrent qu'en plus du gain évident de fonction, d'autres dysfonctions astrocytaires dans peuvent être dues à une perte de fonction. De plus, nous avons traité les souris mutantes avec de la ceftriaxone, connu pour son effet neuroprotecteur, mais nous n'avons observé aucun effet significatif. Enfin, la mégalencéphalie étant fréquente chez les patients AxD, nous avons mesuré la quantité d'eau cérébrale chez les souris mutantes GFAP. Nous avons trouvé une augmentation significative de la teneur en eau chez les souris GFAPR85C/R85C âgées d'un an. Nous avons observé une localisation anormale de l'AQP4 dans les astrocytes des asouris mutées, pouvant participer au déséquilibre hydrique cérébral. / Alexander disease is a neurodegenerative disorder caused by heterozygous mutations of GFAP gene coding the major intermediate filament of mature astrocytes. We studied the effect of GFAP mutation in the hippocampus of infantile onset AxD patient and two novel knockin mouse models, one bearing a mutation located in the rod domain (p.R85C), and the other bearing a mutation located in the tail domain (p.T409I) of mouse Gfap. In the AxD patient, we describe for the first time: (i) obvious morphological changes of GFAP+ cells in the subgranular zone of the dentate gyrus, which have lost most of their radial processes; (ii) microglial reactivity; (iii) and deficit in postnatal hippocampal neurogenesis. We found similar abnormalities in the two knockin mouse lines, more obvious in homozygous mice. A comparison of these mouse models showed that pathological findings predominated in the GFAPT409I mice, whereas GFAP accumulated in larger amounts in the GFAPR85C mice. The comparison of the two mouse models showed that their pathological consequences depend on the location of the mutated residues in GFAP. These findings suggest that in addition to the evident gain of GFAP function, other astrocyte dysfunctions in this disease may be due to a loss of function of GFAP. In addition, we treated the mice mutants with ceftriaxone, which has been reported to have a neuroprotective effect, but we observe no significant effect. Finally, AxD patients have often megalencephaly, therefore we measured the brain water content in AxD mouse models. We found a significant increase in brain water content in the one year old GFAPR85C/R85C mice vs controls. We observed mislocalization of AQP4 in mutant mice astrocytes that can participated to water imbalance in brain.

The nervous system and immune system have broad and overlapping distributions in the body, and interactions of these ubiquitous systems are central to the field of neuroimmunology. Over the past two decades, there has been explosive growth in our understanding of neuroanatomical, cellular, and molecular mechanisms that mediate central modulation of immune functions through the autonomic nervous system. A major catalyst for growth in this field was the discovery that vagal nerve stimulation (VNS) caused a prominent attenuation of the systemic inflammatory response evoked by endotoxin in experimental animals. This effect was mediated by acetylcholine (ACh) stimulation of nicotinic receptors on splenic macrophages. Hence, the circuit was dubbed the “cholinergic anti-inflammatory pathway”. Subsequent work identified the α7 nicotinic ACh receptor (α7nAChR) as the crucial target for attenuation of pro-inflammatory cytokine release from macrophages and dendritic cells. Further investigation made the important discovery that cholinergic T cells within the spleen and not cholinergic nerve cells were the source of ACh that stimulated α7 receptors on splenic macrophages. Given the important role that inflammation plays in numerous disease processes, cholinergic anti-inflammatory mechanisms are under intensive investigation from a basic science perspective and in translational studies of animal models of diseases such as inflammatory bowel disease and rheumatoid arthritis. This basic work has already fostered several clinical trials examining the efficacy of VNS and cholinergic therapeutics in human inflammatory diseases. This review provides an overview of basic and translational aspects of the cholinergic anti-inflammatory response and relevant pharmacology of drugs acting at the α7nAChR.

Submitted by Irvana Coutinho (irvana@ufpa.br) on 2012-10-30T13:25:06Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AnaliseComparativaPadroes.pdf: 2406192 bytes, checksum: 6858ce99ece9ddda05ed401668b6c9c9 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2012-10-30T16:10:19Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AnaliseComparativaPadroes.pdf: 2406192 bytes, checksum: 6858ce99ece9ddda05ed401668b6c9c9 (MD5) / Made available in DSpace on 2012-10-30T16:10:19Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AnaliseComparativaPadroes.pdf: 2406192 bytes, checksum: 6858ce99ece9ddda05ed401668b6c9c9 (MD5) Previous issue date: 2012 / O acidente vascular encefálico (AVE) pode ocorrer em qual região do Sistema Nervoso Central (SNC), sendo o córtex cerebral é uma das regiões mais frequentemente afetadas por essa desordem neural aguda, embora inexistam investigações que tenham comparado o padrão lesivo em diferentes regiões corticais após isquemia focal de mesma intensidade. O objetivo desta investigação foi avaliar o padrão degenerativo de diferentes áreas corticais após lesão isquêmica focal. Para isso, induziu-se isquemia focal por microinjeções estereotáxicas de endotelina-1 (ET-1) nos córtices somestésico, motor e de associação de ratos adultos (N=45). Nos animais controle injetou-se o mesmo volume de solução salina estéril (N=27). Os animais foram perfundidos 1, 3, e 7 dias após o evento isquêmico. O encéfalo foi removido, pós-fixado, crioprotegido e seccionado em criostato. A histopatologia geral foi avaliada em secções de 50 coradas pela violeta de cresila. Secções de 20μm foram submetidas à imunoistoquímica para marcação de astrócitos (anti-GFAP), micróglia/macrófagos ativados (anti-ED1) e microglia em geral (anti-Iba1). Avaliou-se os padrões lesivos qualitativamente (por inspeção em microscópio óptico) e quantitativamente (pela contagem do número de células nos lados ipsi e contralateral à lesão), pela estatística descritiva e comparações intra e intergrupos com análise de variância com correção a posteriori de Tukey. Os animais isquêmicos apresentaram conspícua perda tecidual, ativação microglial e astrocitose entre 3 e 7 dias após a indução isquêmica, o que não foi observado nos animais controle. A perda tecidual e a ativação de células gliais foram mais intensas no córtex somestésico, depois no córtex motor, com intensidade reduzida na área de associação, o que foi confirmado por análise quantitativa. Os resultados sugerem que uma lesão isquêmica de mesma intensidade induz um padrão diferencial de perda tecidual e neuroinflamação, dependendo da área cortical, e que as áreas sensoriais primárias e motoras são mais susceptíveis ao processo isquêmico do que áreas de associação. / Stroke can occur in any region of the central nervous system (CNS). The cerebral cortex is one of the most often affected areaby this acute neural disorder, but there are no studies that have compared the damaging pattern in different cortical regions after acomparable focal ischemia. The aim of this investigation was to evaluate the degenerative pattern of different cortical areas after focal ischemic injury. Focal ischemia was induced by stereotaxic microinjections of endothelin-1 (ET-1) into the somatosensory, motor and association cortices of adult rats (N = 45). The control animals were injected with the same volume of sterile saline (N = 27). The animals were perfused 1, 3 and 7 days after the ischemic event. The brain was removed, postfixed, cryoprotected, and sectioned in a cryostat. The general histopathology was evaluated in 50μm sections stained with cresyl violet. 20μm sections were submitted to immunohistochemistry for astrocytes (anti-GFAP), activated microglia / macrophages (anti-ED1) and overall microglial population (anti-Iba1). The damaging patterns werequalitatively evaluated under optical microscopy and quantitatively by counting the number of cells in the ipsilateral and contralateral sides to injury.Descriptive statistics and comparisons within and between groups were performed using analysis of variance with Tukey post-hoc test. Conspicuous ischemic tissue loss, microglial activation and astrocytosis were observed mainly 3 and 7 days after ischemia, which was not observed in control animals. The tissue loss and activation of glial cells were more intense in the somatosensory cortex, followed by the motor cortex. The association cortex displayed less damage compared to other cortical areas, which was confirmed by quantitative analysis. The results suggest that an ischemic lesion of the same intensity induces a differential pattern of tissue loss and neuroinflammation, depending on the cortical area, and that the primary sensory and motor areas are more susceptible to ischemia than association areas.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-03-20T12:38:30Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_AcidenteVascularEncefalico.pdf: 2650147 bytes, checksum: e07a16617ef0a7ac68d8b0a7f9026e9f (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-03-24T12:24:26Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_AcidenteVascularEncefalico.pdf: 2650147 bytes, checksum: e07a16617ef0a7ac68d8b0a7f9026e9f (MD5) / Made available in DSpace on 2017-03-24T12:24:26Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_AcidenteVascularEncefalico.pdf: 2650147 bytes, checksum: e07a16617ef0a7ac68d8b0a7f9026e9f (MD5) Previous issue date: 2015-02-27 / O acidente vascular encefálico (AVE) é a segunda maior causa de morte no mundo e a principal no Brasil, sendo que 87% dos AVE ocorrem por processos isquêmicos (AVEI). O consumo crônico de etanol, que se inicia geralmente na adolescência, é reconhecido como um fator de risco independente para a elevação da morbidade e mortalidade por AVEI. Apesar de que casos que combinem as duas patologias sejam relativamente frequentes, não há dados disponíveis em modelos animais ou clínicos que demonstrem a qualidade ou mecanismos de interação entre as duas morbidades, e tão pouco suas consequências sobre a intervenção terapêutica. Considerando então os recentes estudos que propõem a minociclina como nova ferramenta terapêutica para o tratamento do AVEI, este estudo teve por objetivo investigar a interação entre a Intoxicação Alcoólica Crônica (IAC) iniciada na adolescência e o AVEI em córtex motor na fase adulta em ratos, e os efeitos do tratamento com minociclina sobre esta interação, usando parâmetros comportamentais, celulares e moleculares. Ratas Wistar (de 35 dias de idade) foram expostas cronicamente a etanol (6,5 g/kg/dia, 22,5% m/v) ou água por 55 dias. Um dia após o fim da IAC, foi induzida isquemia focal no córtex motor com endotelina- 1 (ET-1), seguindo-se sete dias de tratamento com minociclina ou salina. Ao final deste período os animais foram testados em modelos de campo aberto e rota rod. A seguir, os animais foram sacrificados e o córtex dissecado para avaliação dos níveis de nitritos e de peroxidação lipídica. De cada grupo, alguns animais foram perfundidos e o córtex motor submetidos à analise histológica, para avaliação do dano, e histopatológica, para a morte neuronal (anti-NeuN), ativação microglial/macrófagica (Anti-ED1) e astrocitária (anti-GFAP). A intoxicação por etanol a partir da puberdade até a idade adulta potencializou os danos causados pela isquemia, causando grandes perdas na capacidade de iniciar e gerir os movimentos, bem como na coordenação e força motora em comparação aos animais isquêmicos pré-tratados com água. Estas manifestações foram acompanhadas de aumento da perda neuronal, redução do número de células ED1+ e GFAP+ e maiores níveis de nitritos e peroxidação lipídica. O tratamento com minociclina foi eficiente em prevenir/reverter as perdas motoras e danos teciduais induzidos pela isquemia focal, inibindo também a elevação dos marcadores de estresse oxidativo. A IAC tanto isoladamente como sucedida pela isquemia focal, modificaram o desfecho do tratamento com minociclina. Os nossos resultados indicam que a intoxicação com álcool durante a adolescência agrava o déficit motor e danos no tecido em animais sujeitos a isquemia focal no córtex motor. Este processo parece estar associado com a ativação microglial/ astrocitária, mas principalmente com o estresse oxidativo. Mostra ainda que o histórico prévio de IAC iniciado na adolescência interfere significativamente no tratamento da isquemia cerebral com minociclina. / Stroke is the second largest cause of death in the world and the leading in Brazil, with 87% of strokes due to ischemic processes. Chronic ethanol consumption, usually beginning in adolescence, is recognized as an independent risk factor for increased morbidity and mortality by stroke. Although cases combining the two diseases are relatively common, there is no data in animals or clinical models demonstrating the quality or mechanisms of interaction between the two morbidities, nor its impact on therapeutic intervention. Considering the recent studies proposing minocycline as a new therapeutic tool for the treatment of stroke, this study aimed to investigate the interaction between the Chronic Alcoholic Intoxication (CAI) started in adolescence and the stroke in motor cortex of adult rats, and the effects of treatment with minocycline on this interaction, using behavioral, cellular and molecular parameters. Female Wistar rats (35 days-old) were chronically exposed to ethanol (6.5 g/kg/day, 22.5% w/v) or water for 55 days. One day after the end of the CAI focal ischemia was induced in motor cortex with the endothelin-1 (ET-1), followed by seven-day treatment with minocycline or saline. After this period, the animals were assayed with open field and rota rod tests. Immediately, animals were sacrificed and cortex was dissected for evaluation of nitrite and lipid peroxidation levels. In all groups, some animals were perfused and the motor cortex subjected to histological analysis to assess the damage, and immunohistochemical labeling to neuronal death (anti-NeuN), microglial/macrophage (anti-ED1) and astrocytes (anti-GFAP) activation. The ethanol intoxication from puberty to adulthood potentiated the damage caused by stroke, causing major losses in capacity to start and running movements as well as the strength and motor coordination compared to ischemic animals pretreated with water. These manifestations were accompanied by increased neuronal loss, reduced ED-1+ and GFAP+ cells and higher levels of nitrite and lipid peroxidation. Treatment with minocycline was effective in preventing/reverse motor deficits and tissue damage induced by focal ischemia, also inhibiting the increase in oxidative stress markers. The CAI either alone with succeeded by focal ischemia, harmed the outcome of treatment with minocycline. Our results indicate that heavy alcohol intoxication during adolescence exacerbates the motor deficit and tissue damage in animals subjected to focal ischemia. This process appears to be associated with microglia/astroglial activation, but mainly with oxidative stress. It also shows that the previous history of CAI started adolescence interferes significantly in the treatment of cerebral ischemia with minocycline.

In der vorliegenden Arbeit wurde die Auswirkung der Behandlung muriner Mikrogliazellen mit Agonisten von TLR2, TLR4 und TLR9 untersucht. Zu diesem Zweck wurden murine Mikrogliazellkulturen angelegt. Als TLR - Agonisten dienten Pam3Cys und HKAL (TLR2), Pneumolysin und LPS (TLR4) sowie CPG (TLR9). Die Stimulation muriner Mikrogliazellen mit den verschiedenen TLR - Agonisten führte zur Freisetzung von NO und TNF-α. Durch den Einsatz unterschiedlicher Konzentrationen der TLR - Agonisten konnten Dosis - Wirkungs - Kurven für die Freisetzung von NO und TNF-α erstellt werden. Anhand der EC50 wurde die Potenz der TLR - Liganden beurteilt. Für die Freisetzung von NO wies LPS die höchste stimulatorische Potenz auf, gefolgt von Pneumolysin, CpG und Pam3Cys. Für die TNF-α - Freisetzung besaß ebenfalls LPS die höchste stimulatorische Potenz, auch hier folgten Pneumolysin und CpG. Das verwendete Pam3Cys löste sich nicht optimal, vermutlich aus diesem Grund wurde durch die Pam3Cys - Gabe keine maximale Stimulation erreicht. Darum konnte die EC50 für die TNF-α - Freisetzung fur Pam3Cys nicht ermittelt werden. Die EC50 für die TNF-a - Freisetzung war jeweils höher als die entsprechende EC50 für die Freisetzung von NO. Die Behandlung mit HKAL führte zur starken NO - und TNF-a - Freisetzung. Ein direkter Vergleich der Potenz von HKAL mit der der anderen Liganden ist nicht möglich, da die Konzentration von HKAL in Zellzahl pro ml gemessen wird. Die Konzentrationen von Pam3Cys, Pneumolysin und LPS werden jedoch in µg/l gemessen.  Die Stimulation von Mikrogliazellen über verschiedene TLR hatte eine relativ gleich starke Sezernierung von NO und TNF-a zur Folge. Die Costimulation der Mikrogliazellen mit Konzentrationen von zwei unterschiedlichen TLR - Agonisten, welche allein jeweils zur maximalen NO - Produktion geführt hatten, resultierte nicht in einer weiteren Erhöhung der NO - Freisetzung.  Niedrig dosiert zeigte die Pneumolysinbehandlung einen immunstimulatorischen Effekt. Das Maximum an Stimulation, gemessen an der Zunahme der NO - Produktion, wurde bei einer Pneumolysin - Konzentration von 0,3 µg/ml (6nM) beobachtet. Eindeutige zytotoxische Effekte anhand der signifikant geringeren NO - Freisetzung waren bei Konzentrationen von 3 µg/ml bzw. 10 µg/ml (60 nM bzw. 200 nM) nachweisbar. Durch die Isolectin-B4 - Färbung wurden bei diesen Konzentrationen pneumolysinbedingte Zellschäden dargestellt. Bei den anderen Substanzen wurde in hohen Konzentrationen keine Zytotoxität beobachtet Die Behandlung TLR4 - defizienter Mikrogliazellen mit den spezifischen TLR4 - Agonisten Pneumolysin und LPS führte zu einer signifikant geringeren Freisetzung von NO im Vergleich zu Wildtypzellen. Schlussfolgernd kann gesagt werden: Die Stimulation von Mikrogliazellen über unterschiedliche TLR resultiert in einer relativ einheitlichen Freisetzung von NO und TNF-α. Die gleichzeitige Stimulation mit zwei jeweils niedrigdosierten TLR - Agonisten führt zu einem additiven oder supraadditiven Effekt. Nicht nur bakterielle Substanzen, sondern auch endogene Stoffe sind Agonisten an TLR - Rezeptoren. Der additive Effekt durch die simultane Stimulation mehrerer TLR erhöht nicht nur die Sensitivität von Mikroglia während Infektionen, sondern kann ebenfalls Wechselwirkungen zwischen exogenen und endogenen Agonisten von TLR zur Folge haben. Dies kann ein Grund für die Exazerbation oder Induktion autoimmuner Krankheiten durch Infektionen sein.

<p>Transforming growth factor β-activated kinase 1 (TAK1), a hub kinase at the convergence of multiple signaling pathways, is critical to the development of the central nervous system and has been found to play a role in cell death and apoptosis. TAK1 may have the potential to elucidate mechanisms of cell cycle and neurodegeneration. The Belecky-Adams laboratory has aimed to study TAK1 and its potential roles in cell cycle by studying its role in chick retinal development as well as its possible implication in the progression of diabetic retinopathy (DR). Chapter 3 includes studies that explore TAK1 in a study in chick retinal development and TAK1 in in vitro studies in retinal microglia. Using the embryonic chick, immunohistochemistry for the activated form of TAK1 (pTAK1) showed localization of pTAK1 in differentiated and progenitor cells of the retina. Using an inhibitor or TAK1 activite, (5Z)-7-Oxozeaenol, in chick eye development showed an increase in progenitor cells and a decrease in differentiated cells. This study in chick suggests TAK1 may be a critical player in the regulation of the cell cycle during retinal development. Results from experimentation in chick led to studying the potential role of TAK1 in inflammation and neurodegeneration. TAK1 has previously been implicated in cell death and apoptosis suggesting that TAK1 may be a critical player in inflammatory pathways. TAK1 has been implicated in the regulation of inflammatory factors in different parts of the CNS but has not yet been studied specifically in retina or in specific retinal cells [3, 4]. Chapter 2 includes studies from the Belecky-Adams laboratory of in vitro work with retinal microglia. Retinal microglia were treated with activators and the translocation to the nucleus of a downstream factor of TAK1 was determined: NF-kB. Treatment of retinal microglia in the presence of activators with TAKinib, an inhibitor of TAK1 activation, revealed that TAK1 inhibition reduces the activation of downstream NF-kB. Together this data suggests that TAK1 may be implicated in various systems of the body and further studies on its mechanisms may help elucidate potential therapeutic roles of the kinase.</p>

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