published_or_final_version / Anatomy / Master / Master of Philosophy

La compréhension et l'étude du système nerveux est un des grands enjeux du XXIème siècle à la fois pour la recherche fondamentale, mais également pour la mise au point de neuroprothèses implantables pour la réhabilitation fonctionnelle (exemple : implants rétiniens, implants cochléaires). Depuis quelques années, des systèmes basés sur l'utilisation de multi-électrodes (MEA : Multi-Electrode-Array) offrent la possibilité d'enregistrer des milliers de cellules interconnectées entre-elles sur plusieurs jours sur des tranches de tissu nerveux ou des systèmes nerveux complets. Mais une des limites de cette technique est le faible nombre de voies de ces systèmes (64 voies). Les travaux de cette thèse ont consisté à développer une technologie de fabrication permettant la réalisation d'un système multiélectrode s « haute densité 3D ». Cela passe par le développement d'une nouvelle technologie dans la réalisation de micro pointes basée sur la gravure profonde du silicium (DRIE), qui permet d'obtenir des pointes en silicium de 80 µm de haut espacées de 50 µm. Des matrices 60, 256 et 1024 voies ont été fabriquées par cette technique. L'utilisation de la stimulation est aussi un point important dans l'étude de ces grands réseaux, mais il n'est pas possible actuellement de disposer de système permettant une stimulation focale. Pour résoudre ce problème, nous avons développé des matrices spécifiques permettant d'obtenir des stimulations focales du tissu. Nous avons également dans ces travaux de thèse étudié le comportement de l'interface métal/liquide, qui est cruciale pour la réalisation de MEA, en utilisant des techniques d'électrochimie / One challenge of the XXIème century will be to understand dynamics of large neural networks for research and to develop neuroprothesis implant (ex retinal implant, cochlear implant). Today microelectrodes arrays (MEAs) positioned in contact with the neural tissue offer the opportunity to record and simulate neuronal tissue. But the main drawback of his technique is low number of recording sites (typically 64). During this thesis, we have developed a specific process using deep reactive ions etching (DRIE), to achieve high density 3D MEAs containing several hundreds of microelectrodes. We have fabricated microneedles 80 µm of height with spacing of 50 µm and MEAs with 60 – 256 and 1024 microelectrodes have been built with this process. Microstimulation, which makes use of electrodes on the micron scale, is gaining increasing interest in both fundamental and clinical research, opening the possibility to stimulate small groups of neurons instead of large regions. However, controlling the spatial extent of microstimulation to achieve focal activation of neuron networks is a challenge. We have proposed a new configuration of MEA specifically designed to achieve a local stimulation. We have also characterised the interface metal/liquid, that was very important for MEA and we have used electrochemistry techniques

There are approximately 6000 to 8000 rare diseases, each with a prevalence of less than 1 / 10 000, but in aggregate affecting 6 to 8% of the population. It is important to evaluate disease development and progression to know the natural course of any disease. This information can be utilized in diagnostics and in assessing effects of therapeutic interventions as they become available. This thesis describes the natural clinical history and evolution of imaging findings of two rare diseases over approximately two decades. Papers I, II and III present clinical, magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS) and 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) findings in LMNB1-related autosomal dominant leukodystrophy (ADLD). MRI was found to be very sensitive in finding pathology in patients with LMNB1-related ADLD, even before the onset of clinical symptoms. However, even patients with widespread MRI changes can have a relatively mild symptomatology and present only slight disturbances in metabolic examinations such as MRS and FDG-PET. This is compatible with relatively intact axons, even as myelin impairment is widespread. Paper IV presents clinical and MRI findings in the brain and musculature in SIL1-positive Marinesco-Sjögren syndrome (MSS), and describes a new, mild phenotype of the disease with no intellectual disabilities and only slight motor disabilities. With a 19-year-long radiological follow-up, a slow progressive atrophic process in the cerebellum and brainstem could be demonstrated. MRI of the musculature shows early involvement of the quadriceps and gastrocnemii but not the tibialis anterior, progressing to widespread atrophy in the back and upper and lower limbs at the age of 20 years. In the mildest phenotype, the most severely affected muscles were the m gluteus maximus, m sartorius, m peroneus longus, and the lateral head of the m gastrocnemius.

Recent studies examining cell-cell interactions during CNS development and following disease or trauma have highlighted our limited understanding of the in vivo functions of the myelinating cell of the CNS, the oligodendrocyte. With this in mind, our laboratory has developed techniques by which a profile of proteins derived from or regulated by oligodendrocytes can be elucidated. Specifically, we have demonstrated that oligodendrocytes can be selectively eliminated from one optic nerve of a rat by treating the animal with a unilateral exposure of X-irradiation at the time of birth. Consequently, this approach allowed us to experimentally create, within the same animal, one optic nerve devoid of oligodendrocytes and their progenitors (the X- irradiated side) and one optic nerve containing the normal oligodendrocyte population (the untreated side). Using this experimental animal model we hypothesized that uncharacterized proteins, derived from and regulated by oligodendrocytes, which are crucial for CNS development can be identified. Specifically, by comparing protein profiles found within the normal myelinating optic nerve versus the X-irradiated optic nerve, where oligodendrocytes are absent, potential oligodendrocyte-derived proteins can be quickly identified. Further verification that these proteins are indeed related to oligodendrocytes and/or the processes of myelination can be obtained by their reappearance in the 2-D gel protein profile of P28 X-irradiated nerves that, as we have shown previously, undergo a delayed myelination. We then employed mass spectrometric analysis to determine the identities of oligodendrocyte derivedregulated proteins. In this thesis, I will begin by describing our current knowledge of the proteins expressed by oligodendrocytes and their role(s) in oligodendrocyte function. This will be followed by a detailed description of the experimental model system we utilized in an attempt to elucidate the complete repertoire of oligodendrocyte-regulated proteins. We will then describe the results generated fiom our methodology and discuss the implications of our findings in relation to the functional cooperation between oligodendrocytes and other cells of the developing central nervous system. The results generated fiom this project should lead to a clearer understanding of the role of oligodendrocytes and'of the array of proteins whose expression patterns are associated with these cells during CNS development.

While the classical function of myelin is to facilitate saltatory conduction, this membrane and the myelin-making oligodendrocytes (OLGs) are now recognized as regulators of plasticity and remodeling in the central nervous system (CNS). Thus, OLG maturation and myelination are highly vulnerable processes along CNS development. We previously showed that rat brain myelination is altered by perinatal exposure to buprenorphine, an opioid analogue in clinical trials for the treatment of pregnant opioid addicts. We now found that the in vivo effects on myelination could result from direct alteration in the balance between μ-opioid receptor (MOR) and nociceptin/orphanin FQ receptor (NOPR) activities in the OLGs. Furthermore, we found that myelination could also be affected by the FDA-approved methadone. A delicate balance between MOR and NOPR signaling may play a crucial role timing OLG maturation and myelin formation and exogenous opioids may disrupt this interplay, altering the developmental pattern of brain myelination.

As teneurinas (TENs) são de proteínas de transmembrana com significante expressão no sistema nervoso central (SNC). Foi verificado que o último éxon dos genes das TENs codifica um peptídeo com elevada identidade ao fator liberador de corticotropina (CRF) sendo este denominado de peptídeo associado ao terminal carboxila da teneurina (TCAP-1 a 4). Estudos mostram que TCAP-1 controla o comportamento emocional possivelmente por modular as ações mediadas pelo (CRF). A distribuição do TCAP-1 no SNC de primatas poderia subsidiar os seus efeitos funcionais e as possíveis aplicações terapêuticas. O presente trabalho analisou a imunorreatividade e a expressão do RNA mensageiro do TCAP-1 no encéfalo de primatas não-humanos. Cortes frontais do SNC de macacos da espécie Cebus apella (n=3) foram selecionados e utilizados nas técnicas de hibridização in situ e de imuno-histoquímica. Neurônios imunorreativos e que expressam o RNAm para TCAP-1 foram encontrados principalmente em áreas que estão direta ou indiretamente envolvidas na modulação da resposta ao estresse e ansiedade. / Teneurins are transmembrane proteins expressed mainly in the central nervous system (CNS). The last exon of the teneurins exhibits a peptide sequence with high homology to corticotrophin-releasing factor (CRF), named teneurin c-terminal associated peptide (TCAP-1 to 4). TCAP-1 effectively modulates stress induced by CRF. Studies have pointed tha TCAP-1 could have important therapeutic applications in stress disorders. The analysis of TCAP-1 distribution in the primate brain could also give anatomical support to the understanding of its functional effects and possible therapeutic use. Thus, the present study focused on the distribution of neurons exhibiting immunoreactivity and mRNA expression to TCAP-1 in the monkey brain (Cebus apella). Frontal brain sections of three young male Cebus apella monkeys were submitted to immunohistochemistry and in situ hybridization. Immunohistochemistry and in situ hybridization results showed that TCAP-1 is preserved in primate brain, mainly in areas directly or indirectly involved in the modulation of stress and anxiety.

A família de Siluriformes Neotropicais Cetopsidae possui cinco gêneros e 43 espécies válidas, com ampla distribuição nas regiões norte e central da América do Sul, tanto cis- como trans-andina. Cetopsidae é proposta como um grupo monofilético, suportado pela maioria dos estudos morfológicos e moleculares. Os estudos evolutivos sobre o sistema nervoso de bagres neotropicais são bastante escassos, apesar desse complexo apresentarimportantes modificações potencialmente informativas para estudos de inferência filogenética. Para testar a significância dos caracteres neuroanatômicos em filogenias, a morfologia externa das principais subdivisões encefálicas de Cetopsidae foi descrita, ilustrada e interpretada. Além disso, análises comparativas do desenvolvimento desse complexo foram realizadas em Helogenes marmoratus e Cetopsis gobioides com o intuito de estabelecer homologias e compreender suas modificações ontogenéticas. Os caracteres foram delimitados e testados separadamente em análises filogenéticas e posteriormente combinados com caracteres morfológicos e moleculares com diferentes métodos de análise. Para determinar o volume de cada região analisada, um modelo elipsoide foi utilizado. Amplas comparações baseadas no formato, posição relativa e volume das principais regiões encefálicas são apresentadas para a maioria das espécies representativas de todos os gêneros de Cetopsidae. As mesmas comparações também foram realizadas ao longo do desenvolvimento de Helogenes marmoratus e Cetopsis gobioides. Além disso, comparações com integrantes de outras famílias filogeneticamente relacionadas em Siluriformes forneceram um amplo contexto para um maior entendimento das principais modificações evolutivas que moldaram o encéfalo doscetopsídeos. Análises filogenéticas de todos os caracteres morfológicos foram primeiramente conduzidas em separado, e posteriormente em matrizes concatenadas sob o critério da Parcimônia. Análises Bayesianas com sequências alinhadas e concatenadas de três genes mitocondriais (COI, 16S e Cytb) foram implementadas. Por fim, análises filogenéticas com todos os dados combinados foram conduzidas sob os critérios da Parcimônia e Bayesiano. Padrões morfológicos distintos foram definidos para cada uma das duas principais subdivisões de Cetopsidae, as subfamílias Helogeninae e Cetopsinae. Poucas variações intraespecíficas na morfologia externa do encéfalo xii existem entre todas as espécies examinadas aqui. As modificações observadas ao longo do desenvolvimento do encéfalo de Helogenes marmoratus e Cetopsis gobioides foram utilizadas para a formulação correta de caracteres e seus estados. Todos os gêneros foram recuperados como grupos monofiléticos pelos caracteres neuroanatômicos.Mapeamentos filogenéticos revelaram alguns padrões morfológicos sugestivos com especializações alimentares e outras características de história de vida correlacionadas ao sistema nervoso central. Os caracteres morfológicos e moleculares se mostraram bastante congruentes nas hipóteses filogenéticas, comas incongruências restritas à tribo Cetopsini. Todas as análises recuperaram Helogeninae como grupo-irmão dos demais cetopsídeos, seguido por Cetopsidiini, Denticetopsini e Cetopsini como grupos-irmãos sucessivos.Os resultados obtidos no presente estudo suportam a ideia de que análises amplas e concatenadas de dados morfológicos e moleculares resultam em hipóteses de relacionamentos robustas e bem suportadas. Os caracteres neuroanatômicos são altamente informativos para inferências filogenéticas, que podem ser exploradospara uma maior compreensão da evolução de Otophysi. / The Neotropical South American catfish Cetopsidae is a family of Siluriformes that includes five genera and 43valid species distributed over a large portion of northern and central regions of South America, on both sides of the Andean cordilleras. The monophyly of the family is supported by several previous studies, based on both morphological and molecular characters. Despite the scarcity of evolutionary studies on the nervous system of neotropical catfishes, that complex shows great variation potentially informative for phylogenetic inference. In order to test the significance of neurological traits in a phylogenetic framework, the gross morphology of brain subdivisions in the catfish family Cetopsidae is described, illustrated and interpreted. In addition, comparative analyzes of the development of this complex were carried out in Helogenes marmoratus and Cetopsis gobioidesin order to establish homologies and ontogenetic transformations. Characters were delimited and tested separately in phylogenetic analyzes and later combined with other morphological and molecular characters with different methods of analysis. The volume of major brain subdivision was calculated by an ellipsoid model. A comprehensive comparison based on shape, relative position, and volume of the main brain subdivisions is presented for representative species of all genera and most available species in the family. The same comparisons were also made throughout the development of Helogenes marmoratus and Cetopsis gobioides. Comparisons with other phylogenetically related siluriform families provide a broader context for the understanding of the main evolutionary transformations which shaped the cetopsid brain. Phylogenetic analyzes of all morphological characters were first conducted separately, and later in matrices concatenated under the Parsimony criterion. Bayesian analyzes of three aligned and concatenated mitochondrial gene sequences (COI, 16S and Cytb) were implemented. Analyzes of all the data combined were conducted under both Parsimony and Bayesian criteria. Profoundly distinct morphological patterns are identified for each of the two main cetopsid subdivisions, subfamilies Helogeninae and Cetopsinae. Little intraspecific variation on major subdivisions of the brain exists in species examined herein. The modifications observed throughout the development of the brain inHelogenes marmoratus and Cetopsis gobioides were used for the correct delimitation xiv of characters and their states. The monophyly of all genera is supported by putative neuroanatomic characters. Phylogenetic mapping reveals recurrent morphological patterns suggestive of an association with specific feeding specializations and other life-history traits. Morphological and molecular characters were highly congruent in phylogenetic hypotheses, with inconsistencies restricted to the tribe Cetopsini. All analyzes recovered Helogeninae as thesister group toall remaining cetopsids, followed by Cetopsidiini, Denticetopsini and Cetopsini as successive sister groups. Results of the present study support the idea that large and concatenated analyzes of morphological and molecular characters result in robust well-supported hypotheses of relationships. Neuroanatomical characters are highly informative for phylogenetic inferenceand area promising field to be explored in understanding the evolution of Otophysi.

O estudo dos efeitos do estresse em modelos animais contribui para investigar os mecanismos de sinalização envolvidos nas enfermidades humanas. O projeto foi dividido em duas partes. Primeiramente investigou-se o efeito do estresse crônico imprevisível (ECI) na evolução clínica da encefalomielite autoimune experimental (EAE) e as concentrações circulantes de corticosterona (CORT). O ECI agravou os sinais clínicos da EAE em ratos, mas as concentrações plasmáticas de CORT durante o desenvolvimento da doença pareceram depender unicamente do desafio imunológico. Posteriormente investigaram-se os mediadores inflamatórios e o estresse oxidativo em diversas áreas do SNC de ratos submetidos à isquemia e reperfusão mesentérica. Foram observadas alterações distintas, conforme o tecido analisado, na expressão gênica de NOS e COX, atividade da NOS Ca2+-dep e da arginase e no conteúdo de TBARS. Conclui-se que, nas primeiras horas de reperfusão intestinal, as estruturas analisadas reagem diferentemente ao estresse oxidativo. / The study of stress effects in different animal models help to clarify signalization mechanisms involved in human diseases. The present project was divided in two parts. Firstly, we investigated the effects of chronic unpredictable stress (CUS) on the course of experimental autoimmune encephalomyelitis (EAE) and circulating corticosterone (CORT) concentrations. CUS aggravated the clinical signs of the disease in Lewis rats. However, plasma CORT during the development of clinical signs seemed to be solely dependent on the immunological challenge. Secondly, we investigated the oxidative and defense mechanisms in several CNS regions of rats submitted to an intestinal ischemia-reperfusion model. There were distinct results, as tissue analysis, in genic expression of NOS and COX, calcium-dependent nitric oxide synthase and arginase activities and TBARs content. Based on these results, we concluded that, at least during the first two hours of intestinal reperfusion, CNS lesions may be efficiently prevented by defense mechanisms able to modulate the oxidative injury.

A avaliação histopatológica de uma lesão se baseia nas alterações microscópicas predominantes no tecido, e descreve a gravidade, cronicidade, distribuição, localização, e natureza da lesão. Entretanto, no campo da Neurologia, a obtenção de amostras para avaliação histopatológica de tecidos pode causar danos irreversíveis ao paciente, o que torna extremamente importante a utilização de exames menos invasivos, como os de imagem. A Ressonância Magnética (RM) é uma ferramenta diagnóstica bastante sensível para lesões neurológicas de uma forma geral, devido ao contraste que suas imagens fornecem aos tecidos moles. Todavia, os achados histopatológicos, que são a base para um diagnóstico definitivo, não podem ser observados diretamente pelas técnicas imaginológicas. Por se tratar de uma técnica de aplicação relativamente recente, ainda é escassa a literatura que correlaciona os achados imaginológicos com os histopatológicos de lesões neurológicas. Foram incluídos neste estudo 17 animais, entre cães e gatos, que foram a óbito com sinais neurológicos e que foram submetidos a um exame por RM antemortem. O sistema nervoso central (SNC) de todos os animais foi avaliado histologicamente, e de um deles, imunoistoquimicamente. Foram diagnosticadas doenças de múltiplas naturezas: vasculares, inflamatórias, traumáticas, neoplásicas e degenerativas; e seus achados imaginológicos foram correlacionados com a histopatologia. O presente estudo demonstrou que a técnica de RM pode ser utilizada como ferramenta diagnóstica antemortem de lesões no SNC de pequenos animais em ampla diversidade de acometimentos, demonstrando o potencial da RM de antecipar o diagnóstico de lesões no SNC, permitindo intervenções mais precoces e melhorando, dessa forma, o prognóstico desses animais. / The histopathological evaluation of a lesion is based on the predominant microscopic changes in the tissue, and describes the gravity, chronicity, distribution location and nature of the lesion. Nevertheless, in the field of Neurology, obtaining samples for the histopathological evaluation of tissues can cause permanent damage to the patient, making the usage of less invasive techniques, like imaging exams, extremely important. Magnetic Resonance Imaging (MRI) is a very sensitive diagnostic tool for neurological lesions in general, given the contrast between soft tissues it provides. Despite that, histopathological findings, which are the basis for a definitive diagnosis, cannot be directly seen in imaging exams. Because of its relatively recent application, literature on the correlation between imaging and histopathological findings is still scarce. In the present study, 17 animals, among dogs and cats, which died with neurological signals and were submitted to MRI examination antemortem, were included. The central nervous system (CNS) of all animals was evaluated histologically, and one of them, immunohistochemically. Diseases of multiple natures were diagnosed: vascular, inflammatory, traumatic, neoplastic and degenerative; and their imaging features were correlated to their histopathological findings. The present study demonstrated that MRI can be used as an antemortem diagnostic tool for CNS lesions in small animals, allowing prompt interventions and improving, therefore, the prognosis for these animals.

Este estudo teve como objetivo avaliar a influência do tratamento com estrógeno em ratos submetidos à lesão medular aguda experimental. A lesão foi produzida por meio de um equipamento computadorizado para impacto medular, o NYU Impactor, que promoveu a queda de um peso de altura de 12,5 mm sobre a medula do animal. Utilizamos 20 ratos Wistar que foram separados em dois grupos de 10 animais cada: Grupo 1, com ratos submetidos a lesão medular e depois a terapia com 17-beta estradiol ainda sob anestesia, o grupo experimental; Grupo 2, com ratos submetidos apenas à lesão medular, o grupo controle. Os animais foram observados por 42 dias. A recuperação funcional motora foi avaliada pela escala de Basso, Beattie e Bresnahan (BBB) nos dias 2, 7, 14, 21, 28, 35 e 42 após a lesão, e pelo exame de potencial evocado motor no dia 42. Foi também realizada avaliação histopatológica da área da lesão medular após eutanásia, no dia 42. Os resultados das avaliações da escala BBB evidenciaram que o grupo experimental apresentou melhora significativamente superior em relação ao outro grupo desde o 28o dia até o 42o dia de observação. Os resultados das avaliações por exame de potencial evocado revelaram que o grupo experimental apresentou melhora estatisticamente significante em relação ao grupo controle. Os resultados das avaliações anatomopatológicas pela histomorfometria mostraram melhor recuperação neurológica do grupo experimental com relação à proporção numérica e ao diâmetro das fibras nervosas contadas. A conclusão é que a administração de estrógeno em ratos submetidos à lesão medular mostrou benefícios na recuperação neurológica e funcional motora dos animais tratados / This study aimed to evaluate the influence of estrogen treatment in rats with experimental acute spinal cord injury. The injury was produced using a computerized device for spinal cord impact, the NYU Impactor, which promoted the injury by the falling of a weight on the animal\'s spine from a 12.5 mm-height. Twenty male Wistar rats were divided into two groups of 10 animals each: Group 1, rats with spinal cord injury and undergoing estrogen therapy with 17-beta estradiol, while still anesthetized, the experimental group; Group 2, rats that underwent spinal cord injury only, the control group. Animals were observed for 42 days. The neurological recovery was verified by assessing functional motor recovery by the scale of Basso, Beattie and Bresnahan (BBB) on the 2nd, 7th, 14th, 21st, 28th, 35th and 42nd days after injury, and by quantifying motor evoked potential in the 42nd day. Histopathological evaluation of the area of spinal cord injury was performed after euthanasia in the 42nd day. Results of the BBB scale evaluation showed that the experimental group had significantly greater improvement compared to the other group since the 28th day until the 42nd day of observation. The results of evaluations by the evoked potential test revealed that the experimental group showed statistically significant improvement compared to the control group. The results of the histomorphometry evaluations showed better neurological recovery in the experimental group with respect to the numerical proportion and diameter of nerve fibers counted. The conclusion is that the administration of estrogen in rats with spinal cord injury showed benefits in neurological and functional motor recovery of the treated animals