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Das Ziel dieser Arbeit war die Charakterisierung des epileptischen Phänotyps von Emx1Cre-Lrp1 knock-out Mausmutanten während der Entwicklung. Die angewandten Methoden des Monitorings, der Histologie sowie EEG Analysen haben gezeigt, dass diese Mutanten epileptische Anfälle, Vergrößerungen des ventrikulären Volumens, Änderungen in der Morphologie der Blut-Hirn Schranke und der Myelinscheiden-Struktur aufzeigen. Die Änderungen in der neuronalen Nachkommenschaft, gezeigt durch die Reduktion von PSD-95, reichen allerdings nicht aus, um das Auftreten der Anfälle zu erklären. Die detaillierte Analyse zeigt die Erhöhung von GFAP während der Entwicklung, reduzierte Zelladhäsion, Veränderungen der Glutamatrezeptormenge und verminderte tPA Aufnahme der Astrozyten. Die vorliegende Arbeit zeigt die Signifikanz von Lrp1 in radialen Gliazellen für die Funktion von Neuronen und Gliazellen und unterstreicht somit die Wichtigkeit dieses Proteins für die zukünftige Epilepsieforschung., The aim of this thesis was to characterise the epileptic phenotype of developing Emx1Cre-Lrp1 knock-out mice. The implemented monitoring, histology and EEG analysis revealed that knock-out mice suffer from generalised seizure episodes and exhibit ventricular volume enlargement, blood-brain barrier and vessel morphology alterations. The lack of Lrp1 in neuronal cells is shown to result in a prominent downregulation of PSD-95. Changes in the neuronal progeny are however not sufficient to explain the emergence of seizures in this mutant. Detailed analysis revealed a developmental increase in GFAP, decreased adhesion capacities, altered glutamate transporter levels and impaired tPA uptake in astrocytic cells. A developmental effect of Lrp1 loss on myelin sheath structure was unveiled. The current work underlines the importance of Lrp1 in radial glia cells for neuronal and glial functioning and presents this receptor as a novel protein at the core of epilepsy research.

Orientadores: Fabio Rogério, Carlos Amilcar Parada Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A injeção de ácido quinolínico (AQ), um agonista glutamatérgico do receptor N-metil-D-aspartato, no estriado de roedores induz morte seletiva de neurônios espinhosos médios, gliose reativa e neurogênese na zona subventricular, acompanhada da migração dos neurônios recém-gerados para o estriado lesado. Tais achados são também descritos na doença de Huntington (DH). Há indícios de que a via de sinalização JAK/STAT esteja envolvida no mecanismo de ação do AQ, bem como na patogênese da DH. A interação das citocinas da família da IL-6 com seus receptores desencadeia a ativação de enzimas da família das Janus-Quinases (JAKs), que por sua vez permitem o recrutamento e a ativação de fatores de transcrição da família das proteínas transdutoras de sinais e ativadoras da transcrição (STATs). Embora as principais características da DH sejam a presença da coréia e déficits na execução de movimentos voluntários, poucos testes são realizados abordando o comportamento locomotor dos animais no modelo de lesão por AQ. Neste trabalho, estudamos o efeito do AG490, um inibidor da JAK2, na gliose, perda neuronal e neurogênese no estriado de camundongos adultos C57BL/6J após a administração estereotáxica unilateral de AQ (30nmol). Imediatamente após a lesão, os animais receberam uma injeção subcutânea de AG490 (10mg/kg) ou veículo (PBS+DMSO), e injeções diárias por 6 dias adicionais. Além disso, investigamos o possível efeito da lesão por AQ na atividade física voluntária diária (AFVD) em rodas de atividade. A distância percorrida pelos camundongos foi monitorada por 28 dias após a injeção unilateral de QA (30nmol) ou PBS no estriado. Cortes coronais do cérebro (40?m) obtidos em criostato foram utilizados para quantificação de neurônios por estereologia e para a análise de expressão protéica, através de imunoistoquímica e Western Blotting para GFAP e doublecortina, marcadores de gliose e neuroblastos, respectivamente. A área total de células doublecortina-positivas (ACDP) e o número de neurônios (NN) no lado lesado (L) e contralateral à lesão (CL) foram avaliados. O Índice de Neurogênese (IN=ACDP(L)/ACDP(CL)) e o Índice de Sobrevivência Neuronal (ISN=NN(L)/NN(CL)) foram calculados. Após a administração de AQ, o estriado ipsilateral apresentou intensa gliose e células doublecortina positivas com características de células migratórias. O Western Blotting para GFAP mostrou uma redução ipsilateral de 19% nos animais tratados com AG490, em comparação aos animais do grupo tratado apenas com veículo (0.82±0.05; 1.010±0.06, n=9, p0.05). Portanto, nossos resultados suportam um papel para a JAK2 na morte neuronal, gliose, e neurogênese estriatais após lesão com AQ. O tratamento com o inibidor AG490 causou neuroproteção e diminuição da gliose, sugerindo que a reação astrocitária pode prejudicar a sobrevivência neuronal neste modelo experimental Abstract: Injection of quinolinic acid (QA), a N-methyl-D-aspartate receptor agonist, in murine striatum induces death of medium spiny neurons, gliosis and neurogenesis in the subventricular zone with migration of newly synthesized neurons to damaged striatum. Such findings are also described in Huntington's disease (HD). The Janus-kinase (JAK) pathway would take part in QA mechanism of action and HD pathogenesis as well. The interaction of interleukin-6 family of cytokines with its receptor triggers the activation of enzymes of the family of JAKs, which in turn allow the recruitment and activation of transcription factors, known as signal transducers and activators of transcription (STATs). Although the main features of HD are the presence of chorea and deficits in performing voluntary movements, few tests are realized regarding locomotor behavioral on QA model. We studied the effect of AG490, an inhibitor of JAK isoform 2 (JAK2), on gliosis, neuronal loss and neurogenesis in the striatum of adult C57BL/6J mice after unilateral estereotaxic administration of QA (30 nmol). Immediately after injury, animals received a subcutaneous injection of AG490 (10 mg/kg) or vehicle (PBS + DMSO), and then once daily injections for 6 days. Furthermore, in a parallel experiment, we investigated the possible effect of the lesion by AQ on the voluntary daily physical activity (VDPA) in running wheels. The distance traveled by mice was monitored daily for 28 days after unilateral injection of QA (30 nmol) or PBS into the striatum. Frozen brain sections (40?m) were used for neuronal stereological quantification and immunohistochemical and Western Blotting analyses for GFAP and doublecortin, markers of gliosis and neuroblasts, respectively. The total area of doublecortin-positive cells (ADPC) and the number of neurons (NN) in the lesioned (L) and contralateral (CL) sides were evaluated. Neurogenesis index (NI = ADPC in L/ ADPC in CL) and neuronal survival ratio (NSR = NN in L/ NN in CL) were calculated. After QA administration, ipsilateral striatum showed intense gliosis and doublecortin-positive cells with few processes and ovoid bodies, morphological features corroborating a migratory activity. Western Blotting for GFAP showed an ipsilateral decrease of 19% in AG490- vs vehicle-treated animals (0.82 ± 0.05 vs 1.010 ± 0.06; n=9, p 0.05). In conclusion, our results support a role for JAK2 in striatal neuronal death, gliosis and neurogenesis determined by QA. AG490 caused neuroprotection and reduced gliosis suggesting that astrocytic reaction may impair neuronal survival in the present experimental model Mestrado Fisiologia Mestre em Biologia Funcional e Molecular

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A epilepsia do lobo temporal (ELT) é o tipo de epilepsia focal mais frequente em indivíduos adultos. Por estar comumente associada à farmacorresistência, trata-se de uma doença com grande impacto econômico e social. A esclerose hipocampal (EH) é a principal lesão encontrada nos fragmentos hipocampais ressecados de pacientes farmacorresistentes. Histopatologicamente, a EH é caracterizada por perda neuronal e gliose ao longo da região de Cornu Ammonis (CA) do hipocampo. A EH está relacionada a diversas comorbidades da ELT, dentre as principais, destaca-se o declínio da memória episódica, principalmente dos domínios visual e verbal. No presente estudo, foi feita uma caracterização clínico-patológica da população estudada, pacientes com diagnóstico de ELT associada à EH (provenientes do Hospital das Clínicas da Universidade Federal de Minas Gerais – UFMG – e do Hospital Felício Rocho), e avaliou-se marcadores de gliose e apoptose, buscando associações e/ou correlações com aspectos clínicos, principalmente com impactos na memória verbal e visual. Os marcadores foram avaliados por reação imuno-histoquímica e análise morfométrica. Nesse sentido, demonstrou-se que a razão Bcl-2/Bax está aumentada em hipocampos escleróticos (com resultados similares entre EH tipo 1 e tipo 2), comparada à razão encontrada em hipocampos post-mortem de indivíduos não epilépticos. Apesar desse sinal anti-apoptótico, a expressão de caspase-3 clivada foi visualizada em neurônios granulares e células gliais na região de EH. Demonstrou-se também que a expressão de proteína glial ácida fibrilar (GFAP) e antígeno leucocitário humano-DR (HLA-DR), indicadores de astrogliose e microgliose, respectivamente, está aumentada em hipocampos escleróticos em comparação a hipocampos controles. Não houve diferença na expressão dessas proteínas entre EH tipo 1 e EH tipo 2. O aumento da microgliose foi associado a um pior desempenho no teste de Reprodução Visual antes do procedimento cirúrgico em pacientes com ELT. Os resultados desse estudo sugerem que a ativação de mecanismos anti-apoptóticos de via intrínseca não previnem morte de neurônios granulares e de células gliais. Além disso, demonstrou-se que a microgliose, ao contrário da astrogliose, está associada ao declínio da memória visual em pacientes com ELT. Temporal lobe epilepsy (TLE) is the focal epilepsy most frequent in adults and is commonly associated with drug resistance. Therefore, TLE promotes important economic and social impacts. Hippocampal sclerosis (HS) is the main tissue injury found in hippocampal sections of patients with drug resistant TLE. HS is characterized by neuronal loss and gliosis in Cornu Ammonis (CA) area. HS has been related with several cognitive comorbidities in TLE, such as decline of episodic memory, especially in the verbal and visual subfields. In the current study, we performed a clinicopathologic evaluation of the subjects with TLE-HS diagnosis (these subjects were provided by UFMG University Hospital and Felício Rocho Hospital, both in Belo Horizonte, Minas Gerais, Brazil). Markers for gliosis and apoptosis were analyzed and associated/correlated with clinical variables, especially verbal and visual memory performance. These markers were evaluated by immunohistochemistry and morphometric analyses. We demonstrated that Bcl-2/Bax ratio is increased in sclerotic hippocampi in comparison with post-mortem hippocampi of no-epileptic subjects. However, granular neurons and glial cells, located in HS region, express active caspase-3. We also demonstrated that the glial fibrillar acid protein (GFAP) and the human leukocyte antigen-DR (HLA-DR) are higher in sclerotic hippocampi than in control hippocampi. GFAP and HLA-DR indicates astrogliosis and microgliosis, respectively. HS type 1 and type 2 present similar expression of the both proteins. The microgliosis increase was associated with a worse performance in the Visual Reproduction test in patients with TLE, before the surgery. The results of the current study indicate that anti-apoptotic mechanisms of intrinsic pathway do not prevent apoptosis of glia and granular neurons in TLE-HS. Furthermore, we demonstrated that microgliosis, but not astrogliosis, is associated with visual memory decline in patients with TLE.

Cholesterol is a lipid found in every animal cell and is necessary for its survival. Among its multiple roles in the body, it is a component of cell membranes that is crucial for the maintenance of their structure and fluidity and is thus implicated in the modulation of many signalling pathways. Neurons are especially dependant on cholesterol input since the proper composition of their plasma membrane is required for vesicular exocytosis of neurotransmitters and transduction of the post-synaptic signal. It has been shown that both an excess and a lack of cholesterol is neurotoxic. Moreover, many neurodegenerative diseases, such as Alzheimer’s or Huntington’s disease, have been associated with dysregulation of cholesterol homeostasis, highlighting the need for a fine regulation of its metabolism in nervous tissues. Coordinated actions of neurons and glia, that exhibit complementary functions, are mandatory in that respect. In the brain, astrocytes, the main macroglial cells, may be the major source of cholesterol biosynthesis and export. Neurons, acting as consumers, may be specifically in charge of cholesterol elimination via conversion into 24S-OHC by the CYP46A1 enzyme. This metabolite is likely not only an elimination product of cholesterol but also a signalling molecule in glial cells. It might enable glial cholesterol metabolism to adjust as needed and avoid an overload in brain tissues. In the retina, cholesterol metabolism and its regulation under physiological and pathological conditions remain largely unknown. Müller cells, the major macroglial cells of the retina, could participate in cholesterol synthesis in this tissue even though evidence is still needed. Regarding 24S-OHC, its synthesis is mainly restricted to retinal ganglion cells, neurons responsible for nervous signal transmission from the retina to the brain. Studies performed in our laboratory suggest that CYP46A1 and its product could be implicated in the physiopathology of glaucoma, a disease characterized by ganglion cell degeneration.The present project aims to provide a better understanding of cholesterol metabolism in Müller cells and its potential regulation by 24S-OHC. The goal is also to unveil changes in cholesterol metabolism during the progression of glaucoma by characterizing, at different time points, the molecular players implicated in its biosynthesis, transport and elimination. Our experiments performed on primary Müller cell cultures indicate that these cells possess the molecular machinery to synthesize and export cholesterol and could therefore actively participate in its metabolism in the retina. We also reported a strong hypocholesterolemic effect of 24S-OHC in Müller cells, reinforcing the hypothesis that this compound could be a regulator of cholesterol metabolism in the retina. In an experimental glaucoma model, induced by an elevation in intraocular pressure in rats, we observed major changes in cholesterol metabolism. At the earliest time points, genes implicated in cholesterol biosynthesis and uptake in the retina were upregulated, and cholesterol precursor levels were consecutively and transiently elevated. An important component of our work was to demonstrate that counter-regulatory mechanisms were activated in response to these initial dysregulations, that enabled the maintenance of cholesterol homeostasis in the retina and likely participated in ganglion cell survival., Le cholestérol est un lipide présent dans toutes les cellules animales et indispensable à leur survie. Parmi les rôles multiples qu’il joue dans l’organisme, celui de composant des membranes cellulaires est essentiel pour le maintien de leur structure, de leur fluidité et donc clé dans la modulation de nombreuses voies de signalisation. A ce titre, les neurones sont particulièrement dépendants de l’apport en cholestérol car l’intégrité de la composition de leur membrane est requise pour l’exocytose vésiculaire des neurotransmetteurs et la transduction du signal post-synaptique. Ainsi, il a été montré qu’un excès ou un défaut de ce composé est neurotoxique. De nombreuses maladies neurodégénératives, comme la maladie d’Alzheimer, ont d’ailleurs été associées à des perturbations de l’homéostasie du cholestérol, soulignant la nécessité d’une régulation fine de son métabolisme dans les tissus nerveux. Dans ce cadre, l’existence de mécanismes de coordination entre les actions des neurones et de la glie, dont les fonctions sont complémentaires, semble indispensable. Dans le cerveau, les astrocytes, cellules macrogliales majoritaires, seraient le principal lieu de synthèse et d’export du cholestérol. Les neurones, plutôt consommateurs, seraient spécifiquement en charge de son élimination, via sa conversion en 24(S)-hydroxycholestérol (24S-OHC) par l’enzyme CYP46A1. Ce métabolite ne serait pas seulement un produit d’excrétion du cholestérol, mais jouerait également le rôle de molécule signal dans les cellules gliales. Il permettrait d’adapter le métabolisme de la glie aux besoins en cholestérol, afin d’éviter toute surcharge dans les tissus cérébraux. Dans la rétine, le métabolisme du cholestérol et sa régulation, en condition physiologique et pathologique, sont encore peu décrits. Les cellules de Müller, macroglie majoritaire de la rétine, pourraient participer à la synthèse de cholestérol dans ce tissu, mais les arguments dans ce sens méritent d’être étoffés. Concernant le 24S-OHC, sa synthèse est essentiellement restreinte aux cellules ganglionnaires, neurones chargés de transmettre l’influx nerveux de la rétine au cerveau. Les résultats de quelques études menées au laboratoire suggèrent d’ailleurs que l’enzyme CYP46A1 et son métabolite seraient impliqués dans la physiopathologie du glaucome, maladie liée à la dégénérescence des cellules ganglionnaires.Le but de ce projet de thèse est de contribuer à une meilleure compréhension du métabolisme du cholestérol dans les cellules de Müller et son éventuelle régulation par le 24S-OHC. L’objectif était également de mettre en évidence de potentielles altérations du métabolisme du cholestérol au cours du glaucome en caractérisant, à différents points de temps, les acteurs moléculaires impliqués dans sa synthèse, son transport et son élimination.Nos expériences menées sur des cultures primaires de cellules de Müller indiquent que ces cellules possèdent la machinerie moléculaire pour synthétiser et exporter le cholestérol, et pourraient donc participer activement à son métabolisme dans la rétine. Nous avons également décrit un effet hypocholestérolémiant du 24S-OHC dans les cellules de Müller, ce qui renforce l’hypothèse selon laquelle ce composé pourrait intervenir dans la régulation du métabolisme du cholestérol dans la rétine. Dans un modèle de glaucome expérimental, par hyperpression oculaire chez le rat, nous avons observé des modifications majeures du métabolisme du cholestérol. De manière très précoce, il s’agit d’une surexpression des gènes impliqués dans la synthèse et la captation de cholestérol dans la rétine, puis d’une élévation transitoire des taux de ses précurseurs. Un élément important de notre travail a été de montrer que ces perturbations initiales sont suivies de l’activation de mécanismes de contre-régulation coordonnés, permettant de maintenir l’homéostasie du cholestérol dans la rétine et participant donc sans doute positivement à la survie des cellules ganglionnaires.

Activation of astrocytes or astrogliosis is a prominent component of the inflammatory response and an indicator of injury in the brain. These astrocytes produce a large array of inflammatory mediators, growth and neuroprotective factors. This study was an investigation from astrocyte (GFAP) response and TGF- ? 1 involvement during VSV acute encephalitis using immunohistochemistry to verify relation between astrocytes and TGF- ? 1. Animals developed symptoms around 6 th day after VSV inoculation. Viral proteins were mainly detected at olfactory bulb, ventricular cell layer and disseminated to hippocampus, mesencephalon and diecephalon areas and brain stem. Also at 6 th day post inoculation GFAP and TGF- ? 1 staining was observed in good association with virus-detected areas of brain. However, in mice with severe symptoms we observed reduction in the intensity of GFAP labeling at the same areas where TGF- ? 1 upregulation was observed. These areas show correlation with areas of necrosis and where are astrocytes with degenerative aspect. We observed TGF- ? 1 staining in damaged astrocytes, suggesting an effort of those cells in controlling inflammation in acute phase of VSV encephalitis.

Recent studies have demonstrated that curcumin (Cur) has antioxidant, anti-inflammatory and anti-fibrotic effects. Ethidium bromide (EB) injections into the central nervous system (CNS) are known to induce local oligodendroglial and astrocytic loss, resulting in primary demyelination and neuroinflammation. Peripheral astrogliosis is seen around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). This investigation aimed to evaluate the effect of Cur administration on astrocytic response following gliotoxic injury. Wistar rats were injected with EB into the cisterna pontis and treated, or not, with Cur (100 mg/kg/day, intraperitoneal route) during the experimental period. Brainstem sections were collected at 15, 21 and 31 days after EB injection and processed for GFAP immunohistochemical staining. Astrocytic reactivity was measured in a computerized system for image analysis. In Cur-treated rats, the GFAP-stained area around the lesion was significantly smaller in all periods after EB injection compared to untreated animals, showing that Cur reduces glial scar development following injury. RESUMO Estudos recentes têm demonstrado que a curcumina (Cur) possui efeitos antioxidantes, anti-inflamatórios e antifibróticos. Sabe-se que a injeção de brometo de etídio (EB) no sistema nervoso central induz a perda oligodendroglial e astrocitária, resultando em desmielinização primária e neuroinflamação. Astrogliose periférica é observada ao redor da lesão com aumento da imunorreatividade à proteína glial fibrilar ácida (GFAP). A presente investigação objetivou avaliar o efeito da Cur sobre a resposta astrocitária após injúria gliotóxica. Ratos Wistar foram injetados com EB na cisterna basal e tratados ou não com Cur (100 mg/kg/dia, via intraperitoneal) durante o período experimental. Amostras do tronco encefálico foram coletadas aos 15, 21 e 31 dias pós-injeção de EB e processadas para estudo imuno-histoquímico para a GFAP. A reatividade astrocitária foi medida em um sistema computadorizado para análise de imagem. Nos ratos tratados com Cur, a área marcada para GFAP foi significantemente menor em todos os períodos pós-injeção de EB, indicando que a Cur reduz o desenvolvimento da cicatriz glial após injúria.

Im Rahmen meiner Promotionsarbeit habe ich die Rolle der Gliazellen im experimentellen autoimmunen Glaukom-Modell untersucht. Hierzu wurden Ratten mit ONA, einem Sehnervenhomogenat, und S100B, einem Kalzium-bindenden Protein, immunisiert. Durch eine immunhistologische Färbung von Brn3a wurde die Ganglienzellzahl bestimmt. Ebenfalls wurden Apoptose assoziierte Moleküle (Caspase 3, TNF-\(\alpha\)) untersucht. Anschließend folgten Analysen der Mikro- (Iba1, ED1) und Makrogliazellen (GFAP, Vimentin) in der Retina und im Sehnerv nach 14 und 28 Tagen. Anhand meiner Arbeit konnte gezeigt werden, dass eine Immunisierung mit ONA und S100B einen Untergang der Ganglienzellen auslöst. Apoptotische Prozesse scheinen hier vor allem zu frühen Zeitpunkten eine Rolle zu spielen. Der Zelltod geht mit einer frühzeitig erhöhten Anzahl und Aktivität von Mikrogliazellen einher, wobei die astrozytäre Zellantwort antigenabhängig erscheint.

Propentofylline is a xanthine derivative that depresses activation of glial cells, whose responses contribute to neural tissue damage during inflammation. Ethidium bromide injection into the central nervous system induces local oligodendroglial and astrocytic loss, resulting in primary demyelination, neuroinflammation and blood-brain barrier disruption. Surviving astrocytes present a vigorous reaction around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). Objective This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. Method Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. Results and Conclusion Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury. RESUMO A propentofilina é uma xantina que deprime a ativação das células gliais, cujas respostas contribuem para o dano neural durante inflamação. A injeção de brometo de etídio no sistema nervoso central induz a perda oligodendroglial e astrocitária, resultando em desmielinização, neuroinflamação e ruptura da barreira hematoencefálica. Os astrócitos sobreviventes apresentam vigorosa reação ao redor da lesão com aumento da imunorreatividade à proteína glial fibrilar ácida (GFAP). Objetivo Este estudo objetivou avaliar o efeito da propentofilina sobre a resposta astrocitária após injúria gliotóxica. Método Ratos Wistar foram injetados com brometo de etídio na cisterna basal e tratados ou não com propentofilina (12.5mg/kg/dia, intraperitoneal). Amostras do tronco encefálico foram coletadas dos 15 aos 31 dias pós-injeção do gliotóxico e processadas para estudo ultraestrutural e imuno-histoquímico para GFAP. Resultados e Conclusão Os resultados demonstram que a propentofilina reduziu a ativação astrocitária até o 21o dia, sugerindo que essa droga pode atuar na redução da cicatriz glial após injúria.

Orientadores: Gil Guerra-Junior, Lício Augusto Velloso Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Na obesidade experimental induzida por dieta, o hipotálamo é afetado por uma resposta inflamatória que altera a função de neurônios envolvidos na regulação da ingestão de calorias e do gasto energético. Em adultos obesos, estudos com ressonância magnética funcional (RNMf) mostraram a existência de uma resposta hipotalâmica alterada para nutrientes e exposição ao frio. Além disso, estudos revelaram por meio de análise de ressonância magnética estrutural a existência de gliose no núcleo médio-basal hipotalâmico (MBH) que aumenta em proporção direta ao índice de massa corporal (IMC). Portanto, avaliações por diferentes abordagens de neuroimagem demonstram a presença de anormalidades no hipotálamo de adultos obesos e em modelos animais. A obesidade infantil é um importante fator de risco para a obesidade e suas comorbidades na idade adulta. No entanto, nenhum estudo anterior avaliou a presença de alterações hipotalâmicas em crianças obesas. Aqui, foram utilizados métodos de neuroimagem funcional e estrutural para estudar o hipotálamo de doze crianças obesas e onze com peso normal. Também foram avaliadas a composição corporal e as dosagens de hormônios da saciedade e marcadores inflamatórios. Nas crianças obesas o MBH apresentou sinais radiológicos sugestivos de gliose, que está diretamente correlacionada com a adiposidade corporal e, particularmente, com a adiposidade visceral. Foi encontrada também correlação entre os sinais de gliose e os níveis séricos de leptina, porém não com os níveis de insulina. Além disso, em estudos funcionais, o hipotálamo das crianças obesas apresentou uma resposta diminuída à ingestão oral de glicose, além de uma menor conectividade entre o hipotálamo e o restante do cérebro, em comparação com os indivíduos eutróficos. Foi encontrada correlação entre os menores sinais funcionais do hipotálamo e a maior presença de gliose no MBH. Este é o primeiro estudo que demonstra que o hipotálamo pode ser funcionalmente e estruturalmente afetado na obesidade infantil Abstract: In experimental diet-induced obesity, the hypothalamus is affected by an inflammatory response that disturbs the function of key neurons involved in the regulation of caloric intake and energy expenditure. In adult obese humans, functional magnetic resonance imaging (fMRI) studies have shown a defective hypothalamic response to nutrients and to cold exposure; moreover, structural MRI analysis provide evidence for the existence of hypothalamic gliosis in the medium basal hypothalamus (MBH) that increases in direct proportion to body mass index. Therefore, hypothalamic abnormalities, as assessed by different approaches, are present in the hypothalamus of obese adults and experimental animals. Childhood obesity is a major risk factor for obesity and its comorbidities during adulthood. However, no previous study has evaluated such abnormalities among obese children. Here, we employed functional and structural neuroimaging methods to study the hypothalamus of twelve obese and eleven lean children. Body composition and satiety hormones and inflammatory markers were also evaluated. Obese children presented radiological signs of hypothalamic gliosis, which is directly correlated with adiposity and particularly with visceral adiposity. Signs of gliosis in the MBH were correlated to plasma leptin concentration, but not to insulin. Moreover, in functional studies, the hypothalamus of obese children showed a decreased response to oral glucose intake, and a lower connectivity with the whole brain gray matter when compared to lean participants. In addition, the decreased hypothalamic functional response to glucose was correlated to increased signs of gliosis in the MBH. This is the first study demonstrating that the hypothalamus can be functionally and structurally affected in childhood obesity Doutorado Saúde da Criança e do Adolescente Doutora em Ciências FAPESP 2009/50809-5 CAPES 3881/15-6

Temporal lobe epilepsy (TLE), the most frequent type of focal epilepsy in adults, is characterized by resistance to pharmacological treatment, progressive hipocampal neuronal loss and impairment of cognitive and neuropsychological performances. Studies with human specimens and experimental animal models have demonstrated exuberant and dynamic neuronal plasticity phenomena in the hipocampal formation associated with TLE. The study of these phenomena is fundamental for the definition of the genesis and progression mechanisms of epilepsy and for the development of better therapeutic strategies than those current available. In this article, we review neuronal and glial plastic alterations as neuronal death and birth, dendritic spine formation, axonal sprouting, gliosis and gap junction establishment and their role in the pathophysiology of TLE.

Orientador : Prof. Dr. Luiz Cláudio Fernandes Co-orientador : Rubens Bertazolli Filho Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 19/12/2013 Inclui referências Área de concentração : Fisiologia Resumo: O mercúrio é um poluente ambiental neurotóxico. Atualmente não é conhecido o sítio inicial das lesões do sistema nervoso central (SNC) nem tão pouco as bases moleculares das lesões. Em SNC de mamíferos, os astrócitos são sítio de acumulação preferencial do metilmercúrio (MeHg). Após exposição crônica, o mercúrio se acumula em diversas estruturas oculares, particularmente na retina. Os efeitos do mercúrio na retina têm sido estudados, mas as bases moleculares das alterações observadas são pobremente conhecidas. O presente trabalho objetivou estabelecer um modelo de lesão de retinas de ratos por intoxicação de cloreto de metilmercúrio (CH3HgCl) e investigar os mecanismos celulares subjacentes ao processo de lesão ocular provocado pela intoxicação. Além disso, determinou o efeito da suplementação com óleo de peixe como possível neuroprotetor no processo de lesão supracitado. Para isso, ratos Wistar foram suplementados com óleo de peixe (ácido graxo poliinsaturado rico em n-3), na dose de 1 g/kg p.c., desde o desmame (21 dias) até a fase adulta. Ao completarem 70 dias, estes ratos receberam injeção de CH3HgCl, na dose única de 2500 ?g/kg de p.c., via administração intraperitoneal. Os efeitos da suplementação com óleo de peixe foram avaliados sobre a expressão de p ERK ½ e GFAP (marcadores de gliose) de retina dos animais intoxicados, analisados 3 e 6 dias após a exposição à droga. Expressão das proteínas COX2, caspase 3 clivada e Nrf2 também foram analisadas por ensaios de western blotting. Determinou-se a atividade da enzima catalase, glutationa e concentrações de lipoperóxidos, os quais podem refletir o estado redox celular. Os resultados obtidos demonstram que nos dias 3 e 6 após a intoxicação mercurial apresentaram maior pico de expressão de p ERK ½ e de GFAP, respectivamente, caracterizando gliose reativa no tecido retiniano. A suplementação com óleo de peixe impediu as expressões de ambas as proteínas sendo similar à do grupo controle. Não houve alteração na expressão das proteínas COX2 e caspase 3 clivada entre os grupos experimentais. A intoxicação mercurial promoveu aumento da expressão total da proteína Nrf2 e alterou o estado redox das células, uma vez que, este metal pesado foi capaz de reduzir a atividade da enzima catalase e glutationa. Com relação às concentrações de lipoperóxidos, este parâmetro foi aumentado quando exposto à droga. Nossos resultados sugerem a caracterização de gliose reativa no tecido retiniano de ratos submetidos à intoxicação por mercúrio, gerando desequilíbrio do estado redox neste modelo experimental. Notavelmente, a suplementação com óleo de peixe promoveu efeito neuroprotetor sobre o tecido retiniano lesado, levando-se em consideração as expressões proteicas de GFAP e p ERK ½. Abstract: Mercury is a higly neurotoxic environmental contaminant. Currently it is not known the initial site of injury in the central nervous system (CNS) neither the molecular basis of the lesions. In the mammalian CNS, methylmercury (MeHg) preferentially accumulates in the astrocytes. After chronic exposure mercury accumulates in several ocular structures, particularly in the retina. The effects of mercury in the retina have been studied but the molecular basis of the cell change is poorly known. This study aimed to establish an experimental model of retinal injury in rats intoxicated by methylmercury chloride (CH3HgCl) and further investigation upon the cellular mechanisms underlying the eye injury caused by the intoxication. Also to determine the effect of fish oil supplementation as a possible neuroprotector against CH3HgCl intoxication. To achieve those aims male Wistar rats were supplemented with fish oil (rich in n-3 polyunsaturated fatty) at a dose of 1 g/kg bw, from weaning (21 days) until adulthood. At 70 days, the rats received a single dose intravenously injection CH3HgCl of 2500 mg/kg bw. The effect of fish oil supplementation was evaluated by measuring the expression of p ERK ½ and GFAP (glial markers) from the retina of intoxicated animals, analyzed at 3 and 6 days after the exposure to CH3HgCl. Expression of COX2 , cleaved caspase-3 and total Nrf2 proteins were also analyzed by western blotting. The activity of catalase, glutathione levels and concentrations of lipoperoxides were analyzed, which may reflect the cellular redox state. The results show that at the 3 and 6 days after mercurial poisoning there were higher expressions of p ERK ½ and GFAP, respectively thus characterizing reactive gliosis in retinal tissue. The fish oil supplementation prevents the higher expression of both proteins being similar to control group. There was no change in the expression of COX2 and cleaved caspase-3 proteins between experimental groups. The mercurial poisoning did induce elevation in the total Nrf2 protein expression and altered redox state of the cells, this can be because this heavy metal was able to reduce the activity of catalase and glutathione levels. When exposed to the drug there was an increase in the lipoperoxide concentration in the retina. Our results demonstrate that we succeed in to establish a model in rat for retina lesion by CH3HgCl intoxication characterized by reactive gliosis in retinal tissue and also the poisoning promoted an imbalance of the redox status in this experimental model. Remarkably fish oil supplementation acted as a neuroprotector on the injured retinal tissue by the alterations in GFAP and p ERK ½ protein expressions.

Dissertação de Mestrado Integrado em Medicina Veterinária Este trabalho teve como objectivo caracterizar as alterações retinianas observadas num modelo murino de retinopatia com degenerescência dos fotorreceptores, de forma a poder estabelecer padrões de resposta nas diferentes fases de evolução do processo degenerativo-inflamatório. Material e Métodos: Neste estudo foram utilizadas retinas de murganhos ICR adultos. Foram constituídos grupos de 4 animais. Os murganhos foram injetados intraperitonealmente com uma dose de 100 mg/Kg de iodato de sódio e eutanasiados 24, 48 e 72 horas após a injeção. Um animal não injetado serviu como controlo em cada grupo. As retinas foram analisadas por meio de técnicas de histologia de rotina, imunohistoquímica, Western Blotting e q-RT-PCR. Resultados: A gliose reativa ocorre em resposta a estímulos agressivos. Nos processos de retinopatia, a ativação das células de Müller induz a sobre-expressão da proteína glial fibrilhar acídica (GFAP). As técnicas de imunohistoquímica, Western Blotting e q-RT-PCR utilizadas permitiram caracterizar, no modelo em estudo, níveis de expressão de GFAP específicos de cada uma das fases pré-definidas da resposta inflamatória. Conclusões: No modelo estudado de retinopatia induzida pela injeção de iodato de sódio, as diferenças de expressão de GFAP permitiram estabelecer padrões de resposta passíveis de ser utilizados na identificação da fase do processo inflamatório em curso. ABSTRACT - Identification of Müller cell response patterns during degenerative retinopathy in mice - Introduction The purpose of this study was to characterize the structural retinal alterations in a murine model of retinopathy with photoreceptor degeneration, in order to establish response patterns in the different stages of the degenerative-inflammatory process. Material and Methods: In this work, retinas from adult ICR mice were used. Groups of 4 animals were intraperitoneally injected with 100 mg/Kg of sodium iodate, and euthanized 24, 48 and 72 hours after injection. A non-injected animal was used as a control in each group. The retinas were analyzed by means of conventional histological techniques, immunohistochemistry, Western Blotting and q-RT-PCR. Results: Reactive gliosis occurs in response to aggressive stimuli. During retinopathy, Müller cell activation induces glial fibrillary acidic protein (GFAP) over-expression. Immunohistochemistry, Western Blotting and q-RT-PCR techniques allowed us to characterize the levels of GFAP expression of each predefined inflammatory stage. Conclusions: In the analyzed murine sodium iodate injection-induced model of retinopathy, differences in GFAP expression allowed us to establish expression patterns of this protein which may be used to identify the phase of the inflammatory response in progress.

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.

O objetivo desta investigação foi avaliar o padrão degenerativo de diversos tratos de substância branca após lesão isquêmica estriatal, correlacionando o processo degenerativo com os padrões de ativação microglial e expressão de Nogo-A. Para isso, foi induzida isquemia focal com injeção estereotáxica de endotelina no estriado de ratos adultos, e nos animais controle apenas injetou-se solução salina estéril. Os animais foram perfundidos 3, 7, 14 e 30 dias após isquemia. O cérebro removido, pós-fixado, crioprotegido, cortado em criostato e os cortes obtidos submetidos à investigação imunoistoquímica com os seguintes anticorpos: Anti-GFAP (1:2000,Dako), Anti-Tau-1 (1:500,Chemicon), Anti-MBP (1:100,Chemicon International), Anti-Nogo A (1:100,Invitrogen), Anti-Iba1 (1:1000, WAKO), Anti-ED1 (1:500, Serotec) e Anti-MHC-II (1:100 Abcam), além da visualização do padrão lesivo com violeta de cresila. As lâminas marcadas pelos diferentes métodos foram avaliadas qualitativamente e algumas também quantitativamente (Anti-Nogo A, Anti-ED1, Anti-MHC-II e Anti-Tau-1), com contagens realizadas no estriado e no corpo caloso. Os dados foram tabulados, submetidos à análise estatística pelo teste de Tukey (p

In the central nervous system, zinc is released along with glutamate during neurotransmission and, in excess, can promote neuronal death. Experimental studies have shown that metallothioneins I/II (MT-I/II), which chelate free zinc, can affect seizures and reduce neuronal death after status epilepticus. Our aim was to evaluate the expression of MT-I/II in the hippocampus of patients with temporal lobe epilepsy (TLE). Hippocampi from patients with pharmacoresistant mesial temporal lobe epilepsy (MTLE) were evaluated for expression of MT-I/II and for neuronal, astroglial, and microglial populations. Compared to control cases, MTLE group displayed widespread increase in MT-I/II expression, astrogliosis and reduced neuronal population. MT-I/II levels did not correlate with any clinical variables, but patients with secondary generalized seizures (SGS) had less MT-I/II than patients without SGS. In conclusion, MT-I/II expression was increased in hippocampi from MTLE patients and our data suggest that it may be associated with different seizure spread patterns. No sistema nervoso central, o zinco é liberado juntamente com o glutamato durante a neurotransmissão e, quando liberado em excesso, pode promover morte neuronal. Estudos indicam que as metalotioneínas I/II (MT-I/II), proteínas quelantes de zinco livre, podem afetar parâmetros relacionados às crises e reduzir a morte neuronal subsequente a um status epilepticus. Nosso objetivo foi avaliar a expressão de MT-I/II no hipocampo de pacientes com epilepsia do lobo temporal (ELT). Hipocampos de pacientes com ELT mesial (ELTM) resistente ao tratamento farmacológico foram avaliados para a expressão de MT-I/II e para as populações neuronal e astroglial. Quando comparadas com o grupo controle, pacientes com ELTM apresentaram aumento na expressão de MT-I/II, astrogliose e redução na densidade neuronal. Não foram observadas correlações entre os níveis de MT-I/II e as características clínicas dos pacientes, mas pacientes com crises secundariamente generalizadas apresentaram um aumento menor nos níveis de MT-I/II que os pacientes sem estas crises. Em resumo, um aumento na expressão de MT-I/II é observado em pacientes com ELTM e nossos dados sugerem que o aumento pode estar associado a diferentes padrões de crises epilépticas.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A doença de Alzheimer é uma doença crônica degenerativa, considerada como a forma mais comum de demência na senilidade. Sua histopatologia é bem definida, com marcas características que incluem a presença de placas senis, novelos neurofibrilares, perda neuronal e gliose reativa em regiões específicas do cérebro, como o hipocampo. A gliose é um evento que ocorre no sistema nervoso central em conseqüência a qualquer dano tecidual, e pode ser definida como um crescimento anormal, por hiperplasia e/ou hipertrofia, de astrócitos, microglia, e, talvez numa extensão qualitativamente diferente, oligodendrócitos. Para um melhor entendimento do mecanismo da gliose reativa, realizou-se uma análise imuno-histoquímica no hipocampo, utilizando o marcador de proliferação celular Ki-67, e investigou-se a quantidade proporcional de neurônios e células gliais na mesma área em 16 necropsias de pacientes com a doença de Alzheimer e 5 controles sem demência. Não foi encontrada nenhuma célula Ki-67-positiva no hipocampo do cérebro com doença de Alzheimer. Um decréscimo no número de neurônios e oligodendrócitos e um aumento no número de astrócitos e microglia foram observados no hipocampo com a doença, em comparação com o grupo controle. Os resultados desse estudo sugerem que a gliose reativa que acontece na doença de Alzheimer não ocorre através de hiperplasia, mas de hipertrofia de astrócitos e microglia ou pela migração dessas células para a área de neurodegeneração, como uma resposta compensatória à morte neuronal

In Germany approximately 17.000 people go blind per year. Mainly because of degenarative retinopathias, while current available therapy strategies are limited and mostly failing to effect a cure. For some of this people, there is a new chance to gain their eyelight again within the Projekt RETINA-IMPLANT. In this project researchers are develloping a retina protheses. One part of this work are long time biocompatibility experiments. Therefore, the silicone coat of the protheses is implanted for different periods in one eye of a cat, pig, minipig or rabbit. Afterwards the animal is killed and both eyes are explanted and histologically examinated. The biological attachment of the silicone coat on the retina should be through a gliosis and not through a fibrosis. The histological examination is not always able to clearly distinguish between gliosis and fibrosis. So immunhistological methods are an optimal supplement to standard histological examinations. In routine procedures, material for such examinations is embedded in paraffin. But with this method, it is not possible to investigate the retina-implant-scaffolds. Paraffin is too smooth. We need a much harder embedding material like Technovit®8100 or 9100. Till now, there is no information about the immunhistological staining method with Technovit®8100 or 9100, except for the product information about Technovit®8100. So the goal of this study is to establish the immunhistological staining method with Technovit®8100 and 9100 on normal eyes without any implantations. Therefore, normal eyes from rabbit, swine and minipig were embedded in paraffin and normal eyes from rabbit and swine were embedded in Technovit®8100 and 9100. Afterwards serial sections of 2 µm were made from all embedded eyes succeded by immunhistological labelling. Additionally, sections of 2 µm thickness from a rabbit eye (RI014/02) embedded in paraffin which has a fibrosis were immunhistological labelled. Staining results were compared, depending on the embedding material and the possibility to clearly distinguish between gliosis and fibrosis. Staining results from the eyes embedded in paraffin served as negative control. The outcome of the experiments is, that with immunhistological labelled sections from eyes embedded in Technovit®8100, we could not clearly identify structural changes like we have within a gliosis or fibrosis. Another disadvantage with this embedding material was, that for positive staining results higher antibody concentrations are needed and it is not possible to get any positive results with the labelling method with DAP. Meanwhile staining results from immunhistological labelled sections from eyes embedded in Technovit®9100 show the same qualitity with similar antibody concentrations like labelling results from eyes embedded in paraffin. Therefore the immunhistochemistry with Technovit®9100 is suitable for the exact characterization between gliosis and fibrosis. The disadvantage of this method is the really costly and time-consumingembedding of the material in Technovit®9100, which takes more than four weeks. So at the time, there is no optimal embedding material for exploring tissue-implantat-scaffolds with immunhistological labelling.

Orientador: Alexandre Leite Rodrigues de Oliveira Tese (doutorado) - Universidade Estadual de Campinas. Instituto de Biologia Resumo: Após uma lesão de um nervo periférico, astrócitos e microglia tornam-se reativos, desencadeando a chamada gliose reativa. Adicionalmente, uma porcentagem significativa dos botões sinápticos em íntima relação com os motoneurônios é eliminada, sendo este mecanismo associado à ação de fagocitose das células gliais. Recentemente, a expressão de MHC I (complexo de histocompatibilidade principal classe I) tem sido relacionada com a plasticidade das sinapses e com o processo de regeneração axonal subseqüente à transecção do nervo isquiático, sendo a manipulação da expressão dessa molécula um possível alvo terapêutico. Por sua vez, a óxido nítrico sintase induzível (iNOS), parece estar envolvida com eventos pró- e anti-apoptóticos e com processos de plasticidade das sinapses após lesão do nervo periférico. Embora alguns trabalhos tenham sugerido a participação do MHC classe I nos processos de plasticidade sináptica após a lesão do Sistema Nervoso (SN), as suas funções bem como os mecanismos moleculares que estão envolvidos nesses processos permanecem obscuros. Portanto, os objetivos principais deste trabalho foram identificar a importância relativa de distintas moléculas de MHC classe I clássico (Ia) e não clássico (Ib) na reatividade astroglial e microglial após a axotomia periférica, bem como avaliar o envolvimento da iNOS em tais processos e no grau de eliminação das sinapses. Para tais fins, foram utilizados camundongos knockout para transportador associado com a apresentação de antígeno - 1 (TAP-1), microglobulina-p2 (mp2) genes K e D do MHC I (H2-Kb / Db), iNOS e camundongos controle C57BL/6J. Os animais que foram submetidos à transecção do nervo isquiático foram processados para imunohistoquímica, histoquímica, western blotting e microscopia eletrônica de transmissão (estudo in vivo). Camundongos neonatos foram utilizados para o cultivo de astrócitos (estudo in vitro). Os resultados indicam que a ausência de complexo principal de histocompatibilidade I não clássico (MHC Ib) pode influenciar o grau de reação astroglial e microglial uma e três semanas após a axotomia periférica. Ainda, animais deficientes em iNOS apresentaram menor capacidade de expressão de complexo principal de histocompatibilidade I clássico (MHC Ia) por células da microglia uma semana após a lesão. A análise quantitativa da microscopia eletrônica indica maior retração das sinapses em animais knockout para iNOS quando comparados com a linhagem selvagem C57BL/6J. O conjunto dos resultados obtidos sugere que a gliose reativa é influenciada pela expressão de MHC I não clássico e que a iNOS pode participar de mecanismos de apresentação da forma clássica na superfície celular microglial, aumentando assim a retração sináptica e contribuindo para a resposta regenerativa neuronal após a axotomia periférica. Abstract: Following a peripheral nerve injury, microglial and astrocytic cells become reactive, triggering the so called 'reactive gliosis'. Also, a significant percentage of the synaptic buttons to the motoneurons is eliminated and such process can be associated to the activation of glial cells. Recently, major histocompatibility complex class I (MHC I) expression has been related to the synaptic plasticity and axonal regeneration process that follows sciatic nerve transection. In this sense, the modulation of MHC I expression can be, in the future, used as a therapeutic approach in several diseases and also after trauma of the nervous system. Similarly to the MHC I, inducible nitric oxide synthase (iNOS) can be involved in the synaptic plasticity process after nerve lesion. The objectives of this work were to investigate the relative importance of MHC I expression (classical / Ia and non-classical / Ib MHC I) on the microglial and astroglial reaction after axotomy and to evaluate iNOS involvement in such process and on the degree of synaptic stripping. For this purpose, knockout mice for transporter associated with antigen processing (TAP-1), P2- microglobulin (mp^) , K and D MHC I genes (H2-Kb / Db), iNOS and wild type C57BL/6J strains were subjected to unilateral sciatic nerve transection and specimens were processed for immunohistochemistry, histochemistry, Western blotting and transmission electron microscopy (in vivo study). Astrocytes from newborn mice were also investigated in primary cell cultures (in vitro study). The results indicate that nonclassical class I major histocompatibility (MHC Ib) absence may influence the microglial and astroglial reaction one and three weeks after axotomy. Also, iNOS deficient mice presented milder classical class I major histocompatibility (MHC Ia) expression by microglia one week after lesion than C57BL/6J. Quantitative transmission electron microscopy (TEM) analysis indicates greater number of synaptic elimination in the iNOS knockout mice as compared to the wild type. Overall, the present results suggest that reactive gliosis is influenced by non-classical MHC Ia expression and iNOS molecules might participate on microglial cell surface presentation of MHC I, therefore contributing to synaptic detachment and the regenerative response after axotomy. Doutorado Anatomia Doutor em Biologia Celular e Estrutural

In dieser Arbeit wurde eine auf RNA-Interferenz (RNAi) basierende Methode für die funktionelle Charakterisierung des \(\textit {gap junction}\) Proteins Connexin43 (Cx43) entwickelt. Das Hauptinteresse galt der Frage, ob Cx43 eine Zielstruktur im Prozess der glialen Narbenbildung (\(\textit {glial scarring}\)) darstellt. Wir haben herausgefunden, dass die RNAi vermittelte Herabregulation von Cx43 in kultivierten Astrocyten deren Proliferationsrate verringert. Zudem zeigten mit siRNA behandelte Astrocyten eine verstärkte Migration und geringere Adhäsion \(\textit {in vitro}\). Daneben wurde eine Reihe von Bedingungen getestet, um siRNA gezielt in der Mausretina zu applizieren. Das optimierte Protokoll, in dem die siRNA mit dem Transit-TKO Transfektionsreagenz komplexiert wurde, sorgt für eine Diffusion der Komplexe bis zur Ganglien-Zellschicht der Retina, einschließlich astrocytärer Netzwerke. Das etablierte Protokoll schafft die Grundlage für \(\textit {in vivo}\) Studien an der Mausretina, die ein Modell für \(\textit {glial scarring}\) ist. In this work we explored an RNA interference strategy for functional studies of the gap junction protein Cx43. The major interest of the project was the question if the Cx43 can be used as a target for the prevention of glial scarring. We found that acute knockdown of the Cx43 in cultured astrocytes correlated with the decreased proliferation rate of the cells. In addition, Cx43 impaired astrocytes showed significantly enhanced migration and lower adhesion capacity in vitro. Secondly, we have tested a number of strategies to deliver synthetic siRNA into the mouse retina in vivo. We had shown that under specific protocol siRNA conjugated with Transit-TKO transfection reagent was able to penetrate into the ganglion cell layer of mouse retina. Moreover, siRNA was localized within the retinal astrocytes network. The ultimate goal would be to study the functional effect of the siRNA mediated Cx43 downregulation on glial scar formation in a retina model of neurotrauma.

Retina tissue culture represents an in vitro system to analyze neuronal as well as glial interactions and molecular processes. In the present work different retina tissue culture systems from the adult porcine eyes were examined by immunohistology and results compared to freshly isolated retina as positive control. The control retina displays histological characteristics of in vivo retina, except for an early degeneration of photoreceptor outer segments and a minor reduction of ganglion cell bodies. Astrocytes in control retina could be visualized reproducibly by both GFAP antibodies in immunohistology, whereas variable expression patterns of GFAP and Vim were found in Muller cells depending on variations in antibody specificity. Muller cells in control retina showed GFAP1 labelling however no clear anti-GFAP2 staining could be detected; anti-Vim1 antibody labelled apical Muller cell end feet whereas anti-Vim2 antibody displayed staining of the entire Muller cell. According to the literature S100b-antibodies detect Muller cells and astrocytes in freshly isolated retinal tissue whereas glutamin synthetase antibodies label Muller cells exclusively. Comparison of immunohistological results from freshly isolated, control retina to healthy retina (in vivo situation) verified their identical histological characteristics, except for photoreceptor degeneration. In the present work semiquantitative image analysis of fluorescence labelled, immunhistological sections were used for comparison of different retina cultures to freshly isolated retina. The comparison of this semiquantitative data to the literature proved that anti-GFAP2 and also anti-Vim1-labelled structures of the inner plexiform and the inner nuclear layer correspond to the actual volume of Muller cells. The semiquantitative results for anti-GFAP1, anti-Vim2, anti-S100b and anti-GS staining indicate higher Muller cell volumes than those published previously. The retinas were cultured without RPE over a period of one, 3 and 5 days. For these cultures a reduction in ganglion cell nuclei, a progressive degeneration of photoreceptor cells and an increase of Muller cell hypertrophy was observed over the cultivation period. Over the culture period of one and 3 days an increase of GFAP, Vim, S100b and glutamin synthetase could be observed whereas after 5 days cultures showed further increase of GFAP, S100b and GS as well as reduction of Vim in Muller cells and astrocytes. After 5 days of culture cellular mechanisms were identified supporting neuronal survival; however at this time an onset glial proliferation was observed. In this study, in contrast previous publications Muller cells did not display co-expression of GFAP and Vim; however this is explained by the fact that these intermediate filaments represented by a group of polypeptides. Various binding options to these polypeptides for different antibodies may result in variable immunohistological results. Furthermore is the relation of GFAP expression to Vim expression object to change in the time course of retina culture. In addition the co-culture of retina/RPE-choroid complexes was established. For this technique retina as well as the RPE-choroid complex were isolated separately and cultured for 3 days after juxtaposing both tissues in their original orientation. It has been shown that RPE co-culture supports survival of retinal cells. It could be observed improved ganglion cell and photoreceptor survival, limited nuclei loss in both nuclear cell layers, and decreased hypertrophy of Muller cells in retina-RPE cultures compared to retinal cultures alone. In addition, retina-RPE cultures were characterized by reduced up-regulation of glia specific proteins such as GFAP1, GFAP2, Vim1 and Vim2. Dynamic retina cultures in Minuth chambers displayed a reduced degeneration and proliferation of retinal cells, which can be explained by improved medium and oxygen supply as well as physiologic pressure conditions within the chamber. It is known from the literature that cultivated Muller cells induce gliotic membranes following in vivo injection and express collagen I abundantly. It has been shown in this study that the addition of Muller cells to the apical surface (ILM) of retina cultures as well as the addition of collagen I to the culture medium results in histological characteristics similar to epiretinal and intraretinal gliosis observed in vivo. Therefore this culture system can be used as in vitro model for retinal gliosis. Immunohistochemistry of Co-Cultures with Muller cells showed an increase in all glial specific proteins such as GFAP, Vim, S100b and GS in apical Muller cell processes up to the INL. The addition of collagen I, known to induce proliferation in pure Muller cell culture, resulted in increased GFAP- and S100b expression in apical Muller cell processes as well as a reduction of Vim, S100b- and GS expression in basal Muller cell processes.

A epilepsia do lobo temporal (ELT), a forma mais freqüente das epilepsias resistentes ao tratamento farmacológico entre a população adulta, se caracteriza por perda neuronal hipocampal e comprometimento cognitivo progressivos. Estudos em pacientes com ELT e em modelos animais têm demonstrado a ocorrência de fenômenos de plasticidade neuronal exuberantes e dinâmicos na formação hipocampal. O estudo desses fenômenos é fundamental para a definição dos mecanismos responsáveis pela gênese e progressão da ELT, e a partir destes, para o desenvolvimento de melhores estratégias terapêuticas. Esta revisão de estudos em humanos e modelos animais trata sobre alterações plásticas que ocorrem como a perda neuronal, neurogênese, mudanças em dendritos, axônios, sinapses e glia, tentando estabelecer o provável papel destas na ELT. Temporal lobe epilepsy (TLE), the most frequent type of focal epilepsy in adults, is characterized by resistance to pharmacological treatment, progressive hipocampal neuronal loss and impairment of cognitive and neuropsychological performances. Studies with human specimens and experimental animal models have demonstrated exuberant and dynamic neuronal plasticity phenomena in the hipocampal formation associated with TLE. The study of these phenomena is fundamental for the definition of the genesis and progression mechanisms of epilepsy and for the development of better therapeutic strategies than those current available. In this article, we review neuronal and glial plastic alterations as neuronal death and birth, dendritic spine formation, axonal sprouting, gliosis and gap junction establishment and their role in the pathophysiology of TLE.