A doença de Nicmann - Pick tipo C (DNPC) é uma desordem genética lisossomal neurodegenerativa causada pelo acúmulo de lipídios, principalmente colesterol, esfingomielina e glicoesfingolipídios no espaço perinuclear. U18666A é um agente inihidor no transporte de colesterol, que vem sendo utilizado em diversos modelos tanto in vivo quanto in vitro para mimificar DNPC, principalmente em fihrohlastos. Diversas doenças ncurodcgcncrativas estão associadas ao estresse oxidativo c acredita-se que radicais livres, produtos de lipoperoxidação e alterações na fluidez da membrana possam inibir a enzima Na+, K+-A TPase, levando a célula a um "déficit" energético. A deficiência de algumas proteínas como a GF AP parecem tornar os astrócitos menos eficientes em lidar com estados de injúrias no SNC, além de algumas pesquisas mostrarem um decréscimo na fosforilação de vimentina em fihrohlastos humanos com NPC. MK-801 é um receptor não competitivo de NMDA (N-mctil D-aspartato), sendo considerado um ncurotransmissor primário do cérebro. O objetivo desse estudo foi observar os efeitos da droga U 18666A, que causam o acúmulo de colesterol no citoplasma de astrôcitos de ratos jovens e a partir daí verificar a atividade de algumas hidrolascs lisossomais, parâmetros do estresse oxidativo, metabolismo energético e a hipelfosforilação de filamentos intermediários (GFAP e vimcntina) em astrócitos de ratos jovens. Através da coloração de Filippin c microscopia de fluorescência e através da utilização do CellM software, foi feita a visualização e quantificação do colesteroL A melhor dose de U18666A que permitiu o acúmulo de colesterol foi de 0,25 j..tg/mL em um período de incubação de 48 horas. Astrócitos de ratos incubados com a droga (DNPC) mostraram um significativo aumento de colesterol em relação aquelas culturas sem a droga. A medida da atividade da csfingomielinasc c enzimas beta-glicosidase em astrócitos de ratos com NPC foi significativamente menor do que astrócitos controle, o que é consistente com a atividade da DNPC humana. Verificando as alterações sofridas em nível de enzimas antioxidantcs c estresse oxidativo observamos que U 18666A leva a um aumento nos gmpos TBARS e Carbonil, porém a uma diminuição nos grupos tióis medidos por ensaios sulfidril. Além disso, ocorre um decréscimo na atividade de CAT e SOD e aumenta a produção de NO, ao analisarmos a atividade de Na+, K+ A TPase verifica-se um efeito inibitório na atividade dessa enzima. Os presentes resultados mostram que o acúmulo de colesterol nas culturas de células, induz o estresse oxidativo, o que indica que é potencialmente tóxico e confirma os resultados observados em outras doenças neurodegenerativas. Por fim, ao utilizarmos 4 grupos diferentes, fazendo uso ou não da droga c do MK-801 verificou-se que com o acúmulo de colesterol nas células ocorre uma hiperfosforilação dos Filamentos Intermediários, tanto de GFAP quanto de vimentina, porém o MK-801, sendo um antagonista de NMDA e agindo sobre os receptores de glutamato demonstram inibirem a cxcitotoxicidadc provocada pelo Ul8666A. Além dos parâmetros que foram testados em nosso trabalho, como extresse oxidativo, enzimas antioxidantcs c metabolismo energético, fazendo uso da droga para minriticar NPC, outros parâmetros bioquímicas das células, os quais ainda não foram testados em nossos estudos, podem ser testados com este modelo animal, contribuindo assim para uma melhor compreensão da doença. Podemos fazer uso ainda de substâncias que diminuam a excitotocicidade causada pelo aumento do colesterol, observados na NPC, a exemplo do antagonista do receptor NMDA, MK-801, podendo se tomar ferramentas viáveis para auxiliar no tratamento de doenças ncurodcgcncrativas relacionadas a cxcitotocicidadc glutamatérgica. / The Niemann - Pick type C (NPC) is a disorder genetic neurodegenerative caused by lysosomal accumulation of lipids, cspccially cholcstcrol, sphingomyclin and glycosphingolipids in the perinuclear space. U 18666A is an inhibitory agent in choiesteroi transport, which has heen used in several models hoth in vivo and in vitro to mimificar NPC, cspccially in fibroblasts. Various ncurodcgencrativc discases are associatcd with oxidative stress and it is believed that the free radicais, lipid peroxidation products and changes in membrane fluidity may inhibit the enzyme Na+, K + -ATPase, causing the cell suftcring a dcficit of cncrgy. Thc dcficiency of ccrtain protcins as GFAP sccm to makc astrocytes iess efficient in dealing with states of injury in the CNS, and some research shows a decrease in phosphorylation of vimentin in human fibroblasts with NPC. MK-801 is a noncompctitive NMDA receptor, bcing considercd a primary ncurotransmitter. Thc ohjective of this study was to observe the effects of U 18666A drug, eausing the accumulation of cholcstcrol in thc eytoplasm of young rat astrocytcs and thus verify thc activity of some lysosomai hydroiases, oxidative stress parameters, energy metabolism and hyperphosphorylation of intermediate filaments (vimentin and GFAP) in astrocytes of young rat. Through Filippin staining and fluorescence microscopy and by using the software Cel!M was made visualization and quantitation of cholesterol. The hest dose U18666A which allowed thc aceumulation of cholcstcrol was 0.25 mg I mL in a 48 hour period of incubation. Rat astrocytes (NPC) incubated with the drug showed a significant increase in total cholesterol relative to the cultures without drug. The measure of activity of sphingomyclinase and bcta-glucosidasc enzymes in rat astroeytcs NPC was signiticantly lower than contrai astrocytes, which is consistent with the activity of the human NPC. Noting the changcs suftcred in thc levei of antioxidant enzymcs and oxidativc stress U 18666A observed that ieads to an increase in TBARS and carbonyl groups, but a decrease in the thiol groups as measured by sulfl1ydryl tests. Moreover, there is a decrease in SOD and CAT activity and incrcascs the production of NO, by analyzing thc activity ofthe Na+, K + -ATPase there is an inhibitory effect on this enzyme. The present results show that the accumulation of cholesterol in cell cultures induces oxidative stress, which indicates that it is potcntially toxic, confirming the results observed in othcr ncurodcgcnerative discascs. Finally, we used four different groups, with or without the use of the dmg MK-801, and found that the accumulation of cholesterol in cells leads to hyperphosphorylation FI both GFAP and vimcntin, howcver, MK-801, an NMDA antagonist acts on the glutamatc receptor, and inhibit excitotoxicity caused by U 18666A. Besides the parameters that were tcstcd in our study, as oxidative stress, antioxidant cnzymcs and encrgy mctabolism, causing the dmg to mimificar NPC other biochemical parameters of the cells, which were not tested in our study may he tested with this model animal, thus contributing to a better undcrstanding of thc diseasc. Wc can still makc use of substances that rcduec thc excitotocicidade caused by increased choiesteroi, observed in NPC, such as the NMDA receptor antagonist, MK-801, which can hecome viahle tools to aid in the treatment of ncurodcgcnerativc diseascs rclatcd to cxcitotoeicidadc glutamatcrgic.

A técnica de mobilização neural (NM) clinicamente é eficaz, porém ainda é pouco fundamentada. Neste trabalho, submetemos ratos Wistar no 14º dia após a lesão constritiva crônica (CCI) do nervo isquiático ao tratamento com NM, em 10 sessões, e avaliamos o comportamento doloroso utilizando testes comportamentais para hiperalgesia e alodinia. Ainda, observamos através de ensaios de Western blotting o envolvimento das células gliais e do fator neurotrófico derivado do cérebro (BDNF). No estudo comportamental, os animais com CCI mostraram diminuição no limiar nociceptivo, tratados com a NM apresentaram melhora no comportamento doloroso. Os ensaios de Western blotting mostraram que após a CCI houve aumento de OX-42, GFAP e BDNF, na medula, tálamo e mesencéfalo, também observado em analise de imuno-histoquímica e após a NM observamos diminuição desses mediadores através da primeira técnica mencionada. Sendo assim, sugerimos que a técnica de NM é eficaz como terapia analgésica, sendo possível observar o envolvimento das células gliais e do BDNF neste modelo experimental. / The technique of neural mobilization (NM) is clinically effective, although it is still poorly reasoned. In this study, Wistar rats on day 14th after chronic constrictive injury (CCI) of the sciatic nerve were submitted to treatment with NM in 10 sessions, and it was evaluated the painful behavior using tests for hyperalgesia and allodynia. Also, we observed through Western blotting assays the involvementof glial cells and brain-derived neurotrophic factor (BDNF). In the behavioral study, animals with CCI showed a decrease in nociceptive threshold, and those treated with NM showed an improvement in pain behavior. Western blotting assays showed an increase after CCI of OX-42, GFAP and BDNF levels in the spinal cord, thalamus and midbrain, also observed in immunohistochemical analysis, and after the NM we observed a decrease of these mediators through the first technique mentioned. Therefore, we suggest that the NM technique is an effective analgesic therapy, and it is possible to observe the involvement of glial cells and BDNF in this experimental model.

O envelhecimento é uma falha progressiva nos processos fisiológicos celulares, produzindo alterações morfológicas nas células e nos tecidos. No sistema nervoso, produz uma redução no número de neurônios, nas fibras nervosas, principalmente nas arborizações dendríticas e nas espinhas sinápticas, e nas células da glia que, de acordo com sua localização e tipo celular, podem diminuir, permanecer constantes ou mesmo aumentar numericamente. Na presente pesquisa, avaliou-se os efeitos do envelhecimento sobre o gânglio trigeminal (GT) de ratos Wistar em animais jovens (2 meses de vida), adultos (12 meses de vida) e idosos (24 meses de vida). Os GT foram submetidos às técnicas histológicas da hematoxilina e eosina e Picro-sírius, onde avaliou-se, respectivamente, a densidade das células satélites glias (CGS) e o componente colágeno ganglionar. Através da técnica histoquímica da NADH-d, avaliou-se a área do perfil do GT, a área do perfil dos corpos celulares dos neurônios ganglionares e a densidade neuronal. Uma avaliação qualitativa foi também realizada relativamente à imunorreatividade dos neurônios ganglionares à substância P (SP) e ao peptídeo intestinal vasoativo (VIP). A densidade das CGS foi maior nos animais jovens do que nos animais adultos e idosos. Verificou-se, qualitativamente, que à medida que o animal envelhece há uma diminuição das fibras colágenas do tipo III, passando a predominar, nos animais idosos, as fibras do tipo I. A área do perfil celular dos corpos neuronais foi maior nos animais adultos sendo que em todos os grupos predominaram neurônios de tamanho médio, com a área do perfil celular entre 490 e 1100 μm2. A densidade neuronal apresentou-se maior nos animais jovens, e sem variações estatísticas entre os animais adultos e idosos. Em todos os grupos estudados, os neurônios pequenos foram os que apresentaram maior imunorreatividade à SP e ao VIP. / Aging is a progressive failure in cellular physiological processes. It determines morphological changes in cells of different tissues. In the nervous system, a reduction in neuron number and in neuron fibers, mainly in dendritic tree and synaptic, are described. With aging the glial cells may increase or decrease in number or also remain constant. In the present work the effects of aging were evaluated on the trigeminal ganglion (TG) comparing young (2 months age), adult (12 months age) and old rats (24 months age). Histological sections of TG were stained with hematoxilin-eosin technique to determine the density of satellite glial cells and Picro-sirius under polarized light to evaluate the Types I and III of collagen fibers. The NADH-diaphorase technique allowed determining the perycarion area. The immunoreactivity of ganglionar neurons to Substance P (SP) and vasoactive intestinal peptide (VIP) were also qualitatively evaluated. The glial cells density was higher in young and adult animals than in old animals. The type I collagen fibers predominates in ganglia of old animals whereas in the young animals is characteristic the presence of the type III collagen fibers. Although the perycarion area was higher in adult animals the medium-sized neurons predominated in all groups. Their areas ranged from 490 to 1100 μm2. It was also observed that the neuron density was higher in young animals. In the adult and old animals the neuron density was similar. In all groups the immunoreactivity both to SP an VIP was detected mainly in neurons of small perycarion.

O termo neuroplasticidade se refere às mudanças funcionais ou anatômicas que ocorrem no sistema nervoso decorrentes de experiências. O enriquecimento ambiental (EA) é um dos modelos experimentais utilizados para estudar eventos relacionados à neuroplasticidade, pois aumenta a neurogênese, os níveis de neurotrofinas, a sobrevivência neuronal, a sinaptogênese, além de induzir cascatas de sinalização e uma mudança na arborização dendritica dos neurônios de diversas regiões encefálicas. Nos últimos anos a importância dos astrócitos tem ganho destaque, principalmente no que tange a sua capacidade de modular sinapses e na participação ativa em eventos plásticos no encéfalo, essas mudanças estão relacionadas a alterações funcionais e morfológicas. O EA é um modelo interessante para avaliar performances comportamentais pois é um modelo que ocasiona mudanças na capacidade de armazenar e acessar novas informações. Sendo assim, acreditamos que o EA é capaz de gerar efeitos plásticos nos astrócitos do Stratum Radiatum da região CA1 e alterar as respostas comportamentais a tarefa de reconhecimento de objetos. Após oito semanas de EA iniciado logo após o desmame, camundongos CF-1 albinos não apresentam diferenças significativas no número de astrócitos GFAP-ir e na densidade óptica para GFAP no Stratum Radiatum. Entretanto ocorreu um aumento no número de processos originários do soma, aumento no tamanho destes processos no eixo lateral, paralelo as colaterais de Schaffer e no número de intersecções aos círculos concêntricos de Scholl na mesma região. Os astrócitos adquirem um formato estrelado, este achado pode estar relacionado ao aumento da densidade sináptica nesta região e corroboram com a idéia de que modificações astrocitárias são parte ativa dos processos plásticos que ocorrem no encéfalo. Nossos resultados mostraram também uma mudança comportamental na resposta a tarefa de reconhecimento de objetos. Os animais expostos ao EA despendem menos tempo explorando os objetos, tanto familiar como não familiar e apresentam igual capacidade de discriminar os objetos. Estes resultados demonstram um comportamento mais propicio a sobrevivência da espécie em animais expostos ao EA, o que inclui uma rápida exploração e um possível aumento na capacidade de aprender sobre o ambiente. / Environmental enrichment (EE) induces plastic changes in the brain, including morphological changes in hippocampal neurons, with increases in synaptic and spine densities. In recent years, the evidence for a role of astrocytes in regulating synaptic transmission and plasticity has increased, and it is likely that morphological and functional changes in astrocytes play an important role in brain plasticity. In others hand EE is used to investigate behavioral modifications associated with gene-environmental interaction. Our study was designed to evaluate changes in astrocytes induced by EE in the hippocampus, focusing on astrocytic density and on morphological changes in astrocytic processes and the performance in object recognition task (ORT) for evaluate animals ability to learn about their environment. After 8 weeks of EE starting at weaning, CF-1 mice presented no significant changes in astrocyte number or in the density of glial fibrillary acidic protein immunoreactivity (GFAP-ir) in the Stratum Radiatum. However, in the same region occur significant increase in the ramification of astrocytic processes, as well as by an increase in the number and length of primary processes extending in a parallel orientation to CA1 nerve fibers. This led astrocytes to acquire a more stellate morphology, a fact which could be related to the increase in hippocampal synaptic density observed in previous studies. These findings corroborate the idea that structural changes in astrocytic networks are an integral part of plasticity processes occurring in the brain. In other hand, our results indicate that EE decreased the time the animals spent exploring familiar and unfamiliar objects and total time spent exploring both objects, without affecting the capacity of discrimination of objects. These findings indicate a more propitious behavior for species survival in animals subjected to EE, including rapid exploration and learning about the environment.

A doença de Nicmann - Pick tipo C (DNPC) é uma desordem genética lisossomal neurodegenerativa causada pelo acúmulo de lipídios, principalmente colesterol, esfingomielina e glicoesfingolipídios no espaço perinuclear. U18666A é um agente inihidor no transporte de colesterol, que vem sendo utilizado em diversos modelos tanto in vivo quanto in vitro para mimificar DNPC, principalmente em fihrohlastos. Diversas doenças ncurodcgcncrativas estão associadas ao estresse oxidativo c acredita-se que radicais livres, produtos de lipoperoxidação e alterações na fluidez da membrana possam inibir a enzima Na+, K+-A TPase, levando a célula a um "déficit" energético. A deficiência de algumas proteínas como a GF AP parecem tornar os astrócitos menos eficientes em lidar com estados de injúrias no SNC, além de algumas pesquisas mostrarem um decréscimo na fosforilação de vimentina em fihrohlastos humanos com NPC. MK-801 é um receptor não competitivo de NMDA (N-mctil D-aspartato), sendo considerado um ncurotransmissor primário do cérebro. O objetivo desse estudo foi observar os efeitos da droga U 18666A, que causam o acúmulo de colesterol no citoplasma de astrôcitos de ratos jovens e a partir daí verificar a atividade de algumas hidrolascs lisossomais, parâmetros do estresse oxidativo, metabolismo energético e a hipelfosforilação de filamentos intermediários (GFAP e vimcntina) em astrócitos de ratos jovens. Através da coloração de Filippin c microscopia de fluorescência e através da utilização do CellM software, foi feita a visualização e quantificação do colesteroL A melhor dose de U18666A que permitiu o acúmulo de colesterol foi de 0,25 j..tg/mL em um período de incubação de 48 horas. Astrócitos de ratos incubados com a droga (DNPC) mostraram um significativo aumento de colesterol em relação aquelas culturas sem a droga. A medida da atividade da csfingomielinasc c enzimas beta-glicosidase em astrócitos de ratos com NPC foi significativamente menor do que astrócitos controle, o que é consistente com a atividade da DNPC humana. Verificando as alterações sofridas em nível de enzimas antioxidantcs c estresse oxidativo observamos que U 18666A leva a um aumento nos gmpos TBARS e Carbonil, porém a uma diminuição nos grupos tióis medidos por ensaios sulfidril. Além disso, ocorre um decréscimo na atividade de CAT e SOD e aumenta a produção de NO, ao analisarmos a atividade de Na+, K+ A TPase verifica-se um efeito inibitório na atividade dessa enzima. Os presentes resultados mostram que o acúmulo de colesterol nas culturas de células, induz o estresse oxidativo, o que indica que é potencialmente tóxico e confirma os resultados observados em outras doenças neurodegenerativas. Por fim, ao utilizarmos 4 grupos diferentes, fazendo uso ou não da droga c do MK-801 verificou-se que com o acúmulo de colesterol nas células ocorre uma hiperfosforilação dos Filamentos Intermediários, tanto de GFAP quanto de vimentina, porém o MK-801, sendo um antagonista de NMDA e agindo sobre os receptores de glutamato demonstram inibirem a cxcitotoxicidadc provocada pelo Ul8666A. Além dos parâmetros que foram testados em nosso trabalho, como extresse oxidativo, enzimas antioxidantcs c metabolismo energético, fazendo uso da droga para minriticar NPC, outros parâmetros bioquímicas das células, os quais ainda não foram testados em nossos estudos, podem ser testados com este modelo animal, contribuindo assim para uma melhor compreensão da doença. Podemos fazer uso ainda de substâncias que diminuam a excitotocicidade causada pelo aumento do colesterol, observados na NPC, a exemplo do antagonista do receptor NMDA, MK-801, podendo se tomar ferramentas viáveis para auxiliar no tratamento de doenças ncurodcgcncrativas relacionadas a cxcitotocicidadc glutamatérgica. / The Niemann - Pick type C (NPC) is a disorder genetic neurodegenerative caused by lysosomal accumulation of lipids, cspccially cholcstcrol, sphingomyclin and glycosphingolipids in the perinuclear space. U 18666A is an inhibitory agent in choiesteroi transport, which has heen used in several models hoth in vivo and in vitro to mimificar NPC, cspccially in fibroblasts. Various ncurodcgencrativc discases are associatcd with oxidative stress and it is believed that the free radicais, lipid peroxidation products and changes in membrane fluidity may inhibit the enzyme Na+, K + -ATPase, causing the cell suftcring a dcficit of cncrgy. Thc dcficiency of ccrtain protcins as GFAP sccm to makc astrocytes iess efficient in dealing with states of injury in the CNS, and some research shows a decrease in phosphorylation of vimentin in human fibroblasts with NPC. MK-801 is a noncompctitive NMDA receptor, bcing considercd a primary ncurotransmitter. Thc ohjective of this study was to observe the effects of U 18666A drug, eausing the accumulation of cholcstcrol in thc eytoplasm of young rat astrocytcs and thus verify thc activity of some lysosomai hydroiases, oxidative stress parameters, energy metabolism and hyperphosphorylation of intermediate filaments (vimentin and GFAP) in astrocytes of young rat. Through Filippin staining and fluorescence microscopy and by using the software Cel!M was made visualization and quantitation of cholesterol. The hest dose U18666A which allowed thc aceumulation of cholcstcrol was 0.25 mg I mL in a 48 hour period of incubation. Rat astrocytes (NPC) incubated with the drug showed a significant increase in total cholesterol relative to the cultures without drug. The measure of activity of sphingomyclinase and bcta-glucosidasc enzymes in rat astroeytcs NPC was signiticantly lower than contrai astrocytes, which is consistent with the activity of the human NPC. Noting the changcs suftcred in thc levei of antioxidant enzymcs and oxidativc stress U 18666A observed that ieads to an increase in TBARS and carbonyl groups, but a decrease in the thiol groups as measured by sulfl1ydryl tests. Moreover, there is a decrease in SOD and CAT activity and incrcascs the production of NO, by analyzing thc activity ofthe Na+, K + -ATPase there is an inhibitory effect on this enzyme. The present results show that the accumulation of cholesterol in cell cultures induces oxidative stress, which indicates that it is potcntially toxic, confirming the results observed in othcr ncurodcgcnerative discascs. Finally, we used four different groups, with or without the use of the dmg MK-801, and found that the accumulation of cholesterol in cells leads to hyperphosphorylation FI both GFAP and vimcntin, howcver, MK-801, an NMDA antagonist acts on the glutamatc receptor, and inhibit excitotoxicity caused by U 18666A. Besides the parameters that were tcstcd in our study, as oxidative stress, antioxidant cnzymcs and encrgy mctabolism, causing the dmg to mimificar NPC other biochemical parameters of the cells, which were not tested in our study may he tested with this model animal, thus contributing to a better undcrstanding of thc diseasc. Wc can still makc use of substances that rcduec thc excitotocicidade caused by increased choiesteroi, observed in NPC, such as the NMDA receptor antagonist, MK-801, which can hecome viahle tools to aid in the treatment of ncurodcgcnerativc diseascs rclatcd to cxcitotoeicidadc glutamatcrgic.

A oclusão permanente e bilateral das artérias carótidas comuns (2VO), resultando em uma significante redução do fluxo sangüíneo cerebral (hipoperfusão) em ratos, é aceita como um modelo experimental bem estabelecido para investigar dano neuronal e déficit cognitivo que ocorrre com o envelhecimento e na doença de Alzheimer. Neste estudo foram avaliadas duas proteínas gliais: S100B e proteína glial fibrilar ácida (GFAP) no tecido cortical cerebral e hipocampo, bem como a S100B no fluído cerebroespinal, e captação de glutamato e a atividade da glutamina sintetase em hipocampo. A cognição foi avaliada através dos protocolos espaciais de memória de referência e de trabalho. O conteúdo de S100B periférico, particularmente no soro e no fluído cerebroespinal (CSF), tem sido usado como parâmetro de ativação glial em diversas condições patológicas. Preliminarmente, investigamos o efeito de anestésicos (tiopental, cetamina e halotano) no conteúdo de S100B no CSF e soro, isto porque alguns estudos têm sugerido um efeito prejudicial desses compostos. Além disso, foi investigado também o possível efeito dependente do sexo sobre o conteúdo de S100B no CSF, já que vários estudos têm sugerido os astrócitos como um dos alvos do estrogênio. Altos níveis de S100B no CSF foram encontrados em ratos anestesiados com tiopental; estes níveis, independente do anestésico, foram sexo-dependentes. Ratos adultos, machos Wistar foram submetidos a 10 semanas de hipoperfusão cerebral crônica após o método 2VO. Um significante aumento da S100B e GFAP em hipocampo foi observado, como também uma significante redução da captação de glutamato. Entretanto, observamos uma diminuição da S100B no CSF. Com relação à função cognitiva, houve um déficit tanto na memória espacial de referência como na de trabalho no labirinto aquático. Nossos dados reforçam a hipótese de que astrócitos têm um papel crucial nos mecanismos das patologias neurodegenerativas, como a doença de Alzheimer, e que a patologia hipocampal resultante da hipoperfusão crônica contribui para os déficits de memória. / The permanent occlusion of common carotid arteries (2VO), resulting in a significant reduction in cerebral blood flow (hypoperfusion) in rats, is accepted as a well established experimental model to investigate neuronal damage and cognitive impairment that occurs in human ageing and Alzheimer's disease. In the present study, we evaluated two astroglial proteins: S100B and glial fibrillary acidic protein (GFAP) in cerebral cortex and hippocampus tissue, as well S100B in cerebrospinal fluid, and glutamate uptake and glutamine synthetase activity in hippocampus tissue. Cognition was assessed by reference and working spatial memory protocols. Peripheral S100B content, particularly in serum and cerebrospinal fluid (CSF), has been used as a parameter of glial activation in several pathological conditions. Preliminary we investigated the effect of anesthetics (thiopental, ketamine and halothane) on CSF S100B levels since some studies have suggested a damage effect of these compounds. Moreover, we investigated a possible gender dependence on CSF S100B, since several studies have suggested astrocytes as putative targets for estrogen. Higher levels of CSF S100B were found when rats were anaesthetized with thiopental; these levels, independently of anesthetic, were gender dependent. Adult male Wistar rats were submitted to 10 weeks of chronic cerebral hypoperfusion after the 2VO method. A significant increase of S100B and GFAP in hippocampus tissue was observed, as well a significant decrease in glutamate uptake. Interestingly, we observed a decrease in S100B in cerebrospinal fluid. As for cognitive function, there was an impairment of both reference and working spatial memory in the water maze. Our data so support the hypothesis that astrocytes play a crucial role in the mechanisms of neurodegenerative disorders, like Alzheimer's disease, and that hippocampal pathology arising after chronic hypoperfusion gives rise to memory deficits.

Glioblastoma (GBM) is the most common and malignant of the glial tumors. These tumors may develop from lower-grade astrocytomas (diffuse astrocytoma; grade II or anaplastic astrocytoma; grade III) through a progressive pathway, but, more frequently, they manifest de novo without any evidence of a pre-malignant lesion. The treatment of GBM includes surgery, radiotherapy, and chemotherapy with temozolomide. Despite improvements in treatment protocols, the median survival of GBM patients remains very low at 12-15 months. The cause of glioma (either development or progression) can be genetic and epigenetic modification driven changes. In contrast to genetic modifications, where DNA sequence is changed, epigenetic modifications are those gene expression regulatory mechanisms which do not involve the change in the DNA sequence. It includes DNA methylation, chromatin modifications and miRNA mediated changes in gene expression. Aberrant DNA methylation is one of the common molecular lesions occurring in the cancer cell. The 5th position of cytosine (CpG) is the most preferred site of DNA methylation in mammalian cells. The methylated cytosines are prone to undergo oxidative deamination, and get mutated to thymine in DNA. Consequently, this led to decrease in CpG abundance in the genome. In normal conditions, promoters of majority of the genes escape methylation, because of which CpG of these regions remain same. This phenomenon led to the restriction of CpGs in the promoter regions of most of the genes. These CpG rich regions of the promoters are known as CpG islands, and the methylation status of these islands have a major role in regulating gene expression. The cancer genome is shown to undergo genome-wide hypomethylation whereas CpG islands undergo hypermethylation compared to normal tissue, resulting in net loss of total methylation, as the CpGs from non-island areas far exceed in number compared to the CpGs from islands. The most studied change of DNA methylation in neoplasms is the silencing of the tumor suppressor genes by CpG island promoter hypermethylation. Apart from few studies, the role of DNA methylation in glioma development and progression is poorly known. With this background, we have focused our study on DNA methylation changes in GBM. To identify GBM specific DNA methylation alterations, we have performed the genome wide methylation profile of 44 GBM and 8 normal samples using Infinium methylation array. Beta value, which is a measure of methylation, was calculated for all the CpG probes. Beta value ranges between 0-1 (from no methylation to complete methylation). We sought to understand the clinical importance, with particular importance to patient survival, of the DNA methylation pattern observed. We also undertook steps to understand the contribution of the differential DNA methylation and the associated gene expression changes in GBM development. This work has been divided into three parts: Part I –Identification of GBM specific methylome and development of a DNA methylation prognostic signature for GBM To identify the differentially methylated genes in GBM, we compared the methylation levels of 27,578 CpGs between GBM and normal control samples using statistical methods. We then compared the list of differentially methylated genes with the expression data generated by The Caner Genome Atlas (TCGA) to find out genes whose expression oppositely correlates with the DNA methylation status. This resulted in the identification of 62 genes hypermethylated and down regulated, while 65 genes hypomethylated and up regulated. We believe that this set of differentially methylated genes may play important role in glioma development. Next, to identify GBM specific DNA methylation survival signature, we correlated the survival data of 44 GBM patients with beta values of all the 27,578 probes. Using Cox regression method, we identified a set of 9 genes, whose methylation predicted the survival in GBM patients. A risk score was then calculated using methylation values and regression co-efficient of each of the genes. The methylation risk score was found to be an independent predictor of survival in a multivariate analysis in TCGA data set and the Bent et al data set (independent validation sets). Using methylation risk score, we were able to divide the patients into low and high risk groups with significant difference in survival. To discover the biology behind the difference in the survival of low and high risk groups, we performed network analysis, using differentially expressed genes between low and high risk patients, which revealed an activated NFkB pathway association with poor prognosis. The inhibition of NFkB pathway sensitized the glioma cells for chemotherapeutic drugs only in NFkB activated cell lines, suggesting a pivotal role for NFkB pathway imparting chemoresistance in poor surviving group. Part II -NPTX2, a methylation silenced gene, inhibits NFkB through a p53-PTEN-PI3K-AKT signaling pathway To understand the mechanism behind the prediction of survival by methylation of 9 genes, we took NPTX2 as a candidate gene for further investigation. NPTX2, a risky methylated gene, is highly methylated in high risk group with poor survival, which suggests that it may have a growth inhibitory activity in GBM. Bisulphite sequencing confirmed the hypermethylation status of NPTX2 promoter in GBM samples and glioma cell lines compared to normal brain tissue. As expected, NPTX2 transcript level was significantly down regulated in GBMs and glioma cell lines compared to normal samples, and could be re-expressed upon methylation inhibitor treatment in glioma cells. Exogenous over expression of NPTX2 inhibited proliferation, colony formation and sensitized glioma cells to chemotherapeutic drugs. Moreover, NPTX2 also inhibited soft agar colony formation in vitro, which confirms its growth inhibitory function in GBM. As NPTX2 was methylated and silenced in the high risk group, which has high activation of NFkB pathway, we then checked if NPTX2 could inhibit NFkB activity. Indeed, we observed that NPTX2 overexpression inhibited expression from NFkB dependent luciferase reporter, sequence-specific DNA-binding of NFkB, nuclear translocation of NFkB sub unit (p65) and it also significantly repressed key NFkB target genes. We also show that NPTX2 mediated inhibition of NFkB could be abrogated by co-expression of constitutively active forms of PI3 kinase, AKT and IKKα, suggesting an involvement of PI3K-AKT-IKKα axis in NPTX2 mediated NFkB inhibition. Further, we found that NPTX2 repressed NFkB activity by inhibiting AKT through an ATM-p53-PTEN-PI3K dependent pathway. Thus, these results explain the need for hypermethylation and down regulation of NPTX2 in high risk GBM wherein the NFkB pathway is activated. Part III -Methylation silencing of ULK2, an autophagy gene, is important for astrocyte transformation and cell growth Among the differentially methylated genes (see part I), ULK2 was one of the most hypermethylated and down regulated genes. ULK2 is a known initiator protein in autophagy pathway, which is a type II cell death mechanism. There are many contradictory reports with respect to the role of autophagy in GBM development. For example, it has been shown that autophagy has a tumor suppressor activity and is essential for temozolomide mediated cell toxicity in GBM cells, whereas others studies implicate its involvement in tumor growth and progression. Hence, we carried out experiments to understand the role of ULK2 in GBM development. Using bisulphite sequencing, we validated ULK2 promoter hypermethylation status in GBM and glioma cell lines. In good correlation, ULK2 was found to be down regulated in GBMs and glioma cell lines, which was reexpressed by methylase inhibitor treatment in glioma cell lines. The over expression of ULK2 was found to inhibit colony formation, proliferation and soft agar colony formation of glioma cells. As expected, ULK2 overexpressing cells showed higher autophagy, compared to control cells. Interestingly, we also found increased apoptosis in ULK2 overexpressing cells. The cell death caused by ULK2 overexpression was compromised when cells were treated with 3-MA (an autophagy inhibitor) or Z-VAD-FMK (a pan caspase inhibitor). However, ULK2 failed to inhibit cell growth in autophagy deficient cells (ATG5-/-), thereby suggesting the importance of autophagy in ULK2 induced cell death. Further, ULK2 overexpression, increased catalase degradation and Reactive Oxygen Species (ROS) generation, which suggests that increase in ROS may play a role in ULK2 dependent cell death. In good correlation, N-Acetyl Cysteine, a ROS inhibitor, treatment rescued the cells from ULK2 mediated cell death, confirming the role of ROS in ULK2 induced cell death. Kinase deficient ULK2 overexpression failed to induce cell growth inhibition, autophagy and apoptosis, suggesting kinase activity of ULK2 is important for ULK2 function. Co-transfection of ULK2 inhibited Ras mediated transformation of immortalized normal human astrocytes. Taken together, we have identified and validated ULK2 as one of the DNA methylation silenced genes in GBM. ULK2 was found to be growth inhibitory in GBM cells by increasing autophagy dependent apoptosis. ULK2 inhibited Ras mediated transformation, suggesting essentiality of DNA methylation mediated ULK2 down regulation in GBM. In conclusion, the present work sheds light on the importance of methylation of genes in GBM progression. As observed, two of the genes, NPTX2 and ULK2 play as critical growth inhibitors in GBM. Also, we have identified a robust, independent and a highly sensitive 9 gene methylation signature, for GBM patient’s survival prediction.

Notre approche biomimétique permet de régénérer une dent entière. Un protocole en deux étapes à partir de réassociations de cellules dentaires embryonnaires permet le développement de la couronne in vitro et, après implantation, la différenciation fonctionnelle des cellules, l’initiation du développement radiculaire et la vascularisation dentaire. Cependant, l’absence d’innervation a nécessité des expériences complémentaires :- La co-implantation de réassociations cellulaires avec un ganglion trigéminal permet la croissance d’axones autour de la dent formée, mais pas dans le mésenchyme dentaire.- Pour tenter de résoudre ce problème, la régénération axonale a été testée dans un contexte immunodéprimé en utilisant la cyclosporine A (CsA). Dans ces conditions, des fibres nerveuses entrent dans la pulpe dentaire, jusqu’aux odontoblastes. Cependant, la CsA a aussi un effet direct sur la croissance axonale.- Des co-implantations chez des souris immunodéprimées (Nude) montrent que l’immunomodulation seule suffit pour l’innervation de la dent.- Dans la dent, les axones assurent différentes fonctions en interagissant avec les cellules voisines. Les relations entre axones et autres cellules (odontoblastes, cellules endothéliales, péricytes et cellules gliales) ont été analysées dans les mésenchymes dentaire et péri-dentaire de réassociations implantées et comparées à ce que l’on observe pour une molaire physiologique à un stade similaire.Ce travail décrit les conditions permettant l’innervation des dents régénérées. Des expériences préliminaires encourageantes ont été réalisées avec des cellules souches pour remplacer la CsA. / Our biomimetic approach allowed the regeneration of a whole tooth. Using embryonic dental cells, a two-steps protocol allowed crown formation in vitro and, after implantation, functional cells differentiation, initiation of root formation and tooth vascularization. However, the teeth were not innervated, which led to complementary experiments:- The co-implantation of cell re-associations with a trigeminal ganglion allowed axonal growth around the forming teeth, but not in the dental mesenchyme. - To try to solve this point, axonal regeneration was tested in immunodepressed conditions, using cyclosporin A (CsA). In these conditions, nerve fibers entered the dental pulp and reached odontoblasts. However, CsA shows multiple effects, including direct ones on nerve growth. - Co-implantations were performed in immunocompromised Nude mice allowed axons to reach the odontoblast layer, thus showing that immunomodulation is sufficient.- Axons in the dental mesenchyme interfere with several functions by interacting with neighbor cells. Relationships between axons and other cells (odontoblasts, endothelial cells, pericytes and glial cells) were analyzed in the peridental and dental mesenchymes of implanted reassociations and compared to the physiological situation in developing molars at similar stage. This work describes conditions allowing the innervation of engineered teeth. Preliminary encouraging attempts have been made to replace CsA by using stem cells.

Dans la rétine, la protéine dystrophine Dp71 est principalement exprimée dans les cellules gliales de Müller (CGM), qui contribuent à la stabilisation de la barrière hémato-rétinienne (BHR). Les CGM sont aussi les principales sources de facteurs inflammatoires. Ainsi, nous avons étudié les effets de l’absence de Dp71 sur l’homéostasie potassique et aqueuse, ainsi que sur l’expression de médiateurs de l’inflammation et la perméabilité vasculaire rétinienne.L'absence de Dp71 diminue l'expression de la protéine AQP4 et induit la redistribution de Kir4.1 tout le long des CGM. Par ailleurs, nous avons également constaté que le décollement expérimental de la rétine chez les souris WT induit une diminution de Dp71 associée à une délocalisation de Kir4.1, une régulation à la baisse de la protéine AQP4 dans les CGM.Nos données montrent clairement que l'absence de la Dp71 entraîne une augmentation de l'expression du VEGF, d’ICAM-1, une augmentation du nombre de leucocytes adhérents rétiniens, une dégénérescence accrue des capillaires associée à une forte perméabilité vasculaire chez les souris Dp71-null.L’ensemble de nos résultats a mis en évidence le rôle de la Dp71 dans les mécanismes visant à réguler l'homéostasie rétinienne et à assurer la stabilisation de la BHR. Nous apportons la preuve que la perte de Dp71 favorise l'inflammation vasculaire rétinienne et la dégénérescence des capillaires associée à une perméabilité vasculaire. Ensemble, ces observations suggèrent que la souris Dp71-null serait un modèle approprié pour étudier les pathologies vasculaires rétiniennes telles que la rétinopathie diabétique, l’uvéite rétinienne et l’occlusion veineuse rétinienne. / In the retina, the Dp71 dystrophin protein is mainly expressed in Müller glial cells (MGC), which contribute to the stabilization of the blood-retinal barrier (BRB). MGC are also the main sources of inflammatory factors. Thus, in our thesis project we studied the effects of the absence of the Dp71 protein on potassium and water homeostasis, as well as the expression of inflammatory mediators and retinal vascular permeability.The absence of the Dp71 protein decreased the expression of AQP4 protein and induces the redistribution of Kir4.1, initially restricted to the end-feet of MGC and around vessels, all along the cell membrane. Moreover, we have also shown that the experimental retinal detachment in WT mice induces a reduction of Dp71 which is associated with Kir4.1 mislocation, a down regulation of AQP4 protein in MGC.Our data clearly demonstrate that the absence of the Dp71 leads to increased retinal VEGF and ICAM-1 expression in Dp71-null mouse compared to WT mouse strain. There is also an increase of the number of retinal adherent leukocytes, capillary degeneration associated with high BRB permeability observed in Dp71-null mice.Our findings highlight Dp71 as an important component in the mechanisms leading to the regulation of retinal homeostasis; and to the maintaining of the BRB stabilization. We provide evidence that deficiency of Dp71 promotes retinal vascular inflammation and significantly exacerbated degeneration of capillaries and BRB breakdown. Together these results suggest that the Dp71-null mouse could be a good model to study retinal vascular diseases such as diabetic retinopathy, retinal uveitis and retinal vein occlusion.

Nos últimos anos, os estudos de nosso grupo foram focados na área de dor, avaliando diferentes modelos experimentais de dor aguda, neuropática e muscular. A busca por mecanismos moduladores destes tipos de dores são alvo de grande estudo, uma vez que o fenômeno da dor é peculiar e torna-se de difícil tratamento em muitos casos. Distúrbios (VER) musculoesqueléticos são as principais causas de incapacidade nas pessoas durante seus anos de trabalho. Diversos estudos tem sido realizados lesionando o músculo gastrocnêmio como modelo experimental em diferentes animais para melhor entendimento deste tipo de dor. O nosso objetivo foi observar possíveis alterações histológicas no tecido muscular, avaliar a alteração na sensibilidade nociceptiva e a atividade locomotora dos animais após a indução de dor muscular crônica, bem como observar o envolvimento das células gliais na medula espinal destes animais. Em adição, avaliamos a participação de determinadas citocinas com o intuito de obter um perfil inflamatório em nosso modelo experimental. Nossos resultados demonstraram um quadro de inflamação instalada no tecido muscular de animais com miosite crônica através das analises histológicas realizadas. Os testes comportamentais tanto para hiperalgesia mecânica como térmica e alodinia confirmaram a instalação do quadro álgico uma vez que os animais com miosite apresentaram uma queda em seus limiares nociceptivos em relação aos grupos controle. A atividade locomotora dos animais também se demonstrou comprometida após a indução de miosite. Em relação à participação das células gliais neste modelo, demonstramos que houve um aumento na expressão de GFAP e OX-42, correspondentes à marcação astrócitos e células da microglia na porção lombar da medula espinal dos animais com miosite, quando comparados ao grupo controle. Quanto à participação dos mediadores sistêmicos, observamos um aumento nos níveis de IL-1β e fractalquina (FKN) no sangue dos animais, enquanto o nível de IL-10 permaneceu baixo em relação ao grupo controle. Com nossos achados esperamos colaborar com o aprimoramento de estratégias terapêuticas para tratamento de dores musculares. / In the last years, the studies of our group were focused on the area of pain, evaluating different experimental models of acute, neuropathic and muscular pain. The search for mechanisms modulating types of pain are the subject of great study, since the phenomenon of pain is peculiar and becomes difficult in many cases. Musculoskeletal (VER) disorders are the leading causes of disability in people during their working years. Several studies have been carried out with the model of experimental model in different animals to better understand this type of pain. Our objective was to observe the non-muscular histological changes, to evaluate the nociceptive sensitivity and a locomotor activity of the animals after an induction of chronic muscular pain, as well as to observe the involvement of the glial cells in the spinal cord of the animals. In addition, we evaluated the participation of certain cytokines in order to obtain an inflammatory profile in our experimental model. Our results demonstrated a picture of inflammation installed without muscle tissue of animals with chronic myositis through histological analysis. Behavioral tests for both mechanical and thermal hyperalgesia and allodynia confirmed the onset of pain since animals with myositis showed a decrease in their nociceptive thresholds in relation to the control groups. The locomotor activity of the animals was also shown to be impaired after an induction of myositis. Regarding the participation of glial cells in this model, we demonstrated that there was an increase in the expression of GFAP and OX-42, corresponding to marking astrocytes and microglia cells in the portion of the spinal cord of animals with myositis, when compared to the control group. Regarding the participation of systemic mediators, we observed an increase in the levels of IL-1β and fractalkin (FKN) in the blood of the animals, while the level of IL-10 remained low in relation to the control group. With the findings we hope to collaborate with the improvement of therapeutic strategies for the treatment of muscular pain.