Experiências vivenciadas durante o período pré-natal são determinantes para a saúde do feto. A ocorrência de infecções maternas e a consequente ativação do sistema imune da mãe ocasionam uma série de alterações estruturais e funcionais no cérebro da prole, podendo predispor o indivíduo a transtornos psiquiátricos na vida pós-natal, como esquizofrenia e autismo. No entanto, estudos que investigam as alterações imunes na prole ainda são escassos na literatura. Dessa forma, o objetivo do presente estudo foi avaliar, na prole, o impacto da ativação imune materna sobre a atividade imune periférica, a resposta imune-inflamatória no sistema nervoso central (SNC), e sobre o desenvolvimento da encefalomielite autoimune experimental (EAE), o modelo murino de Esclerose Múltipla. Camundongos fêmeas prenhes receberam uma administração de salina ou lipopolissacarídeo (LPS) ao final da gestação (dia gestacional 17) e, quando adulta, a prole foi submetida a 3 experimentos principais, analisando: (1) produção de citocinas, atividade de células da periferia e desenvolvimento da hipersensibilidade do tipo tardia; (2) produção de mediadores inflamatórios por células residentes do SNC e; (3) desenvolvimento dos sintomas clínicos e da resposta imune no decorrer da EAE. Nossos resultados mostraram que a ativação imune materna provocou na prole alterações imunes periféricas, como aumento da produção de Interleucina(IL)- 12 e exacerbação da resposta de hipersensibilidade do tipo tardia; potencialização da produção das citocinas IL-1β e IL-6 em cultura primária de células residentes do SNC e; piora na severidade dos sintomas clínicos causados pela EAE, que coincide com aumento do infiltrado de linfócitos e macrófagos no SNC e ativação imuneinflamatória das células da glia. Tomados em seu conjunto, os dados do presente trabalho sugerem que condições inflamatórias durante a gestação, particularmente durante o final da gestação, podem predispor o feto a distúrbios autoimunes e neurodegenerativos na vida adulta. / Prenatal period experiences are crucial for the fetal health. The occurrence of maternal infections and subsequent maternal immune system activation cause a number of structural and functional changes in the brain of the offspring that may predispose individuals to psychiatric disorders in post-natal life, such as schizophrenia and autism. However, studies investigating offspring´s immune alterations are still scarce in the literature. The aim of this study was to evaluate, in mice offspring taken from LPS-treated dams, the impact of maternal immune activation on peripheral immune cell activity, central nervous system (CNS) inflammatory response, and development of experimental autoimmune encephalomyelitis (EAE), the murine model of multiple sclerosis. Pregnant female mice received a dose of either saline or lipopolysaccharide (LPS) during late gestation (gestational day 17), and offspring were used in three experiments to analyze: (1) cytokine production and activity by peripheral immune cells and development of delayed type hypersensitivity, (2) production of inflammatory mediators by resident CNS cells and, (3) development of clinical symptoms and immune response during the course of EAE. Our results showed that maternal immune activation resulted in immune alterations in the offspring, such as increased peripheral production of interleukin (IL) -12 and exacerbated response of delayedtype hypersensitivity; enhancement of IL-1β and IL-6 productions in primary CNS resident cells culture and; increased severity of EAE clinical symptoms, which is positively correlated with the increased lymphocytes and macrophages infiltration within the CNS and also with the immune-inflammatory activation of glial cells. Taken together, the data from this study suggest that inflammatory conditions during pregnancy, especially during the late pregnancy, may predispose the fetus to autoimmune and neurodegenerative disorders in adulthood.

Die Infiltration von T Zellen in das Zentrale Nervensystem (ZNS) ist ein Charakteristikum neuroinflammatorischer Erkrankungen wie der Multiplen Sklerose (MS) und ihrem Tiermodell der experimentellen autoimmunen Enzephalomyelitis (EAE), und führt zur Aktivierung intrinsischer Hirnmakrophagen, den Mikrogliazellen, zu axonaler Schädigung sowie zum Zusammenbruch der Blut-Hirnschranke. Die T Zellen, welche als erste im Gehirn erscheinen, sind vom Subtyp Th1, spezifisch für Bestandteile der Myelinscheide, wie das myelinbasische Protein (MBP), produzieren inflammatorische Zytokine und rekrutieren andere unspezifische T Zellen und Makrophagen. Da sich diese Zellen des Immunsystems gegen körpereigene Bestandteile richten, spricht man von autoreaktiven T Zellen und einer autoimmunen Erkrankung. Im ersten Teil meiner Dissertation habe ich den Einfluss dieser autoreaktiven T Zellen auf den Aktivierungszustand von Mikrogliazellen mit Hilfe muriner Schnittkulturpräparate von Hippocampus und entorhinalem Kortex untersucht, welche den myelinisierten Fasertrakt Tractus perforans mit seinen Ursprungsneuronen und Zielzellen enthielten. Gering aktivierte MBP-spezifische T Zellen induzierten die Expression der Aktivitätsmarker MHC-II und ICAM-1 auf den Mikroglia und die damit verbundene axonale Schädigung (Phagozytose) im gleichen Maße wie hochaktivierte unspezifische T Zellen. Nur Th1 Zellen konnten Mikroglia aktivieren. MBP-spezifische Th2 Zellen hingegen reduzieren die Th1 induzierte Mikrogliaaktivierung (ICAM-1) auf Kontrollniveau. MBP-spezifische Th1 Zellen konnten die Expression von B7 auf Mikrogliazellen modulieren, während die MBP-spezifischen Th2 Zellen diese Eigenschaft nicht besaßen. Durch diese Befunde kann die prominente Rolle von autoreaktiven Th1 Zellen beim Auslösen neuroinflammatorischer Prozesse auf ihre einmalige Fähigkeit, Mikrogliazellen zu aktivieren und deren kostimulatorische Moleküle zu modulieren, zurückgeführt werden. Gleichzeitig bieten die Daten eine mögliche Erklärung für die protektive Rolle von Th2 Zellen bei MS und EAE. Es ist bekannt, dass autoreaktive T Zellen, wie die MBP-spezifischen Th1 Zellen, auch im gesunden Zustand im humanen und murinen T-Zell-Repertoire vorhanden sind. Die physiologische Funktion dieser Zellen ist unklar. Untersuchungen am Nervus opticus sowie im Rückenmark in vivo belegen, dass autoreaktive T Zellen und Makrophagen die Reorganisationsprozesse im ZNS nach traumatischer Schädigung positiv beeinflussen. Diese bei neuroinflammatorischen Erkrankungen so destruktiv wirkenden autoreaktiven T Zellen verhindern nach einem experimentell gesetzten Primärschaden im ZNS das Fortschreiten der Schädigung und es kommt zu einer fast vollständigen Regeneration des Gewebes. Im zweiten Teil meiner Promotionsarbeit habe ich versucht, die Mechanismen, welche hinter dieser Protektion stecken aufzuspüren. Dazu habe ich ebenfalls das in vitro Hirnschnittmodell benutzt. Für diese Fragestellungen wurden Akutschnitte verwendet, die ein Modell für primäre Schädigung im ZNS darstellen. MBP-spezifische Th2 Zellen hatten ein größeres protektives Potential als MBP-spezifische Th1 Zellen. Die nicht ZNS-spezifischen Th1 und Th2 Zellen benötigten ihr Antigen (OVA-Peptid), um signifikant protektiv zu wirken. Durch eine Superstimulation der OVA- und MBP-spezifischen T Zellen wurde eine Neuroprotektion auf gleichem Niveau erreicht. Die Neuroprotektion nach primärer Schädigung von ZNS Gewebe ist somit antigen- und stimulationsabhängig und wird hauptsächlich von Th2 Zellen unterstützt. / The invasion of T cells into the central nervous system (CNS) is a hallmark of neuro inflammatory diseases like multiple sclerosis (MS) and its rodent model, experimental autoimmune encephalomyelitis (EAE), leading to activation of intrinsic macrophages, the microglia, axonal damage and break down of the blood brain barrier. The initial invading T cells are of the Th1 subtype and specific for parts of the myelin sheet like myelin basic protein (MBP). They produce inflammatory cytokines and recruit peripheral non-specific T cells and macrophages. Because these T cells are directed against a self antigen, they are called auto reactive T cells and the phenomenon an autoimmune disease. In the first part of my study I investigated the influence of auto reactive T cells on microglial cells' utilizing an organotypic slice culture system of hippocampus and entorhinal cortex. The slice culture contains a myelinated fibre tract - the tractus perforans - with its original and target neurons. Low activated MBP-specific T cells induced the expression of the activation markers ICAM-1 and MHC-II on microglia as well as microglial phagocytosis in the same manner as highly activated non-specific T cells. Only Th1 cells were able to activate microglia, while Th2 cells reduced the Th1 induced activation (ICAM-1 expression). MBP-specific Th1 cells could modulate the expression of co-stimulatory molecules B7-1 and B7-2, whereas MBP-specific Th2 cells could not. These findings could show why Th1 cells are responsible for EAE induction while Th2 cells can be protective. Auto reactive T cells like MBP-specific T cells have been found in the normal human and murine T cell repertoire. The physiological function of these cells is still unclear. Studies using the models of optic nerve crush or spinal cord crush have shown that macrophages and auto reactive T cells are involved in reorganisation and regeneration after CNS trauma. These auto reactive T cells, which are usually known to be destructive, could prevent CNS tissue from secondary degeneration. In the second part of my study I tried to identify the mechanisms involved in this phenomenon. I also used the organotypic slice culture system. Immediately after preparation causing the primary injury the slices were cultivated with T cells. Th2 cells were found to be more potent to prevent form secondary damage than Th1 cells. The non-CNS specific OVA Th1 and Th2 cells required their antigen to be fully protective. When over stimulated, MBP- and OVA-specific Th1 and Th2 cells proved to be protective to the same extend. Neuroprotection after primary injury depends on the T cell s state of activation and their antigen specificity. Among the cells examined I found Th2 cells were most effective in preventing CNS tissue from secondary injury.

Experiências vivenciadas durante o período pré-natal são determinantes para a saúde do feto. A ocorrência de infecções maternas e a consequente ativação do sistema imune da mãe ocasionam uma série de alterações estruturais e funcionais no cérebro da prole, podendo predispor o indivíduo a transtornos psiquiátricos na vida pós-natal, como esquizofrenia e autismo. No entanto, estudos que investigam as alterações imunes na prole ainda são escassos na literatura. Dessa forma, o objetivo do presente estudo foi avaliar, na prole, o impacto da ativação imune materna sobre a atividade imune periférica, a resposta imune-inflamatória no sistema nervoso central (SNC), e sobre o desenvolvimento da encefalomielite autoimune experimental (EAE), o modelo murino de Esclerose Múltipla. Camundongos fêmeas prenhes receberam uma administração de salina ou lipopolissacarídeo (LPS) ao final da gestação (dia gestacional 17) e, quando adulta, a prole foi submetida a 3 experimentos principais, analisando: (1) produção de citocinas, atividade de células da periferia e desenvolvimento da hipersensibilidade do tipo tardia; (2) produção de mediadores inflamatórios por células residentes do SNC e; (3) desenvolvimento dos sintomas clínicos e da resposta imune no decorrer da EAE. Nossos resultados mostraram que a ativação imune materna provocou na prole alterações imunes periféricas, como aumento da produção de Interleucina(IL)- 12 e exacerbação da resposta de hipersensibilidade do tipo tardia; potencialização da produção das citocinas IL-1β e IL-6 em cultura primária de células residentes do SNC e; piora na severidade dos sintomas clínicos causados pela EAE, que coincide com aumento do infiltrado de linfócitos e macrófagos no SNC e ativação imuneinflamatória das células da glia. Tomados em seu conjunto, os dados do presente trabalho sugerem que condições inflamatórias durante a gestação, particularmente durante o final da gestação, podem predispor o feto a distúrbios autoimunes e neurodegenerativos na vida adulta. / Prenatal period experiences are crucial for the fetal health. The occurrence of maternal infections and subsequent maternal immune system activation cause a number of structural and functional changes in the brain of the offspring that may predispose individuals to psychiatric disorders in post-natal life, such as schizophrenia and autism. However, studies investigating offspring´s immune alterations are still scarce in the literature. The aim of this study was to evaluate, in mice offspring taken from LPS-treated dams, the impact of maternal immune activation on peripheral immune cell activity, central nervous system (CNS) inflammatory response, and development of experimental autoimmune encephalomyelitis (EAE), the murine model of multiple sclerosis. Pregnant female mice received a dose of either saline or lipopolysaccharide (LPS) during late gestation (gestational day 17), and offspring were used in three experiments to analyze: (1) cytokine production and activity by peripheral immune cells and development of delayed type hypersensitivity, (2) production of inflammatory mediators by resident CNS cells and, (3) development of clinical symptoms and immune response during the course of EAE. Our results showed that maternal immune activation resulted in immune alterations in the offspring, such as increased peripheral production of interleukin (IL) -12 and exacerbated response of delayedtype hypersensitivity; enhancement of IL-1β and IL-6 productions in primary CNS resident cells culture and; increased severity of EAE clinical symptoms, which is positively correlated with the increased lymphocytes and macrophages infiltration within the CNS and also with the immune-inflammatory activation of glial cells. Taken together, the data from this study suggest that inflammatory conditions during pregnancy, especially during the late pregnancy, may predispose the fetus to autoimmune and neurodegenerative disorders in adulthood.

La Sclérose en plaques (SEP) est une maladie auto-immune inflammatoire démyélinisante du système nerveux central (SNC), lors de laquelle des cellules inflammatoires du sang périphérique infiltrent le SNC pour y causer des dommages cellulaires. Dans ces réactions neuroinflammatoires, les cellules immunitaires traversent le système vasculaire du SNC, la barrière hémo-encéphalique (BHE), pour avoir accès au SNC et s’y accumuler. La BHE est donc la première entité que rencontrent les cellules inflammatoires du sang lors de leur migration au cerveau. Ceci lui confère un potentiel thérapeutique important pour influencer l’infiltration de cellules du sang vers le cerveau, et ainsi limiter les réactions neuroinflammatoires. En effet, les interactions entre les cellules immunitaires et les parois vasculaires sont encore mal comprises, car elles sont nombreuses et complexes. Différents mécanismes pouvant influencer la perméabilité de la BHE aux cellules immunitaires ont été décrits, et représentent aujourd’hui des cibles potentielles pour le contrôle des réactions neuro-immunes. Cette thèse a pour objectif de décrire de nouveaux mécanismes moléculaires opérant au niveau de la BHE qui interviennent dans les réactions neuroinflammatoires et qui ont un potentiel thérapeutique pour influencer les interactions neuro-immunologiques. Ce travail de doctorat est séparé en trois sections. La première section décrit la caractérisation du rôle de l’angiotensine II dans la régulation de la perméabilité de la BHE. La seconde section identifie et caractérise la fonction d’une nouvelle molécule d’adhérence de la BHE, ALCAM, dans la transmigration de cellules inflammatoires du sang vers le SNC. La troisième section traite des propriétés sécrétoires de la BHE et du rôle de la chimiokine MCP-1 dans les interactions entre la BHE et les cellules souches. Dans un premier temps, nous démontrons l’importance de l’angiotensinogène (AGT) dans la régulation de la perméabilité de la BHE. L’AGT est sécrété par les astrocytes et métabolisé en angiotensine II pour pouvoir agir au niveau des CE de la BHE à travers le récepteur à l’angiotensine II, AT1 et AT2. Au niveau de la BHE, l’angiotensine II entraîne la phosphorylation et l’enrichissement de l’occludine au sein de radeaux lipidiques, un phénomène associé à l’augmentation de l’étanchéité de la BHE. De plus, dans les lésions de SEP, on retrouve une diminution de l’expression de l’AGT et de l’occludine. Ceci est relié à nos observations in vitro, qui démontrent que des cytokines pro-inflammatoires limitent la sécrétion de l’AGT. Cette étude élucide un nouveau mécanisme par lequel les astrocytes influencent et augmentent l’étanchéité de la BHE, et implique une dysfonction de ce mécanisme dans les lésions de la SEP où s’accumulent les cellules inflammatoires. Dans un deuxième temps, les techniques établies dans la première section ont été utilisées afin d’identifier les protéines de la BHE qui s’accumulent dans les radeaux lipidiques. En utilisant une technique de protéomique nous avons identifié ALCAM (Activated Leukocyte Cell Adhesion Molecule) comme une protéine membranaire exprimée par les CE de la BHE. ALCAM se comporte comme une molécule d’adhérence typique. En effet, ALCAM permet la liaison entre les cellules du sang et la paroi vasculaire, via des interactions homotypiques (ALCAM-ALCAM pour les monocytes) ou hétérotypiques (ALCAM-CD6 pour les lymphocytes). Les cytokines inflammatoires augmentent le niveau d’expression d’ALCAM par la BHE, ce qui permet un recrutement local de cellules inflammatoires. Enfin, l’inhibition des interactions ALCAM-ALCAM et ALCAM-CD6 limite la transmigration des cellules inflammatoires (monocytes et cellules T CD4+) à travers la BHE in vitro et in vivo dans un modèle murin de la SEP. Cette deuxième partie identifie ALCAM comme une cible potentielle pour influencer la transmigration de cellules inflammatoires vers le cerveau. Dans un troisième temps, nous avons pu démontrer l’importance des propriétés sécrétoires spécifiques à la BHE dans les interactions avec les cellules souches neurales (CSN). Les CSN représentent un potentiel thérapeutique unique pour les maladies du SNC dans lesquelles la régénération cellulaire est limitée, comme dans la SEP. Des facteurs qui limitent l’utilisation thérapeutique des CSN sont le mode d’administration et leur maturation en cellules neurales ou gliales. Bien que la route d’administration préférée pour les CSN soit la voie intrathécale, l’injection intraveineuse représente la voie d’administration la plus facile et la moins invasive. Dans ce contexte, il est important de comprendre les interactions possibles entre les cellules souches et la paroi vasculaire du SNC qui sera responsable de leur recrutement dans le parenchyme cérébral. En collaborant avec des chercheurs de la Belgique spécialisés en CSN, nos travaux nous ont permis de confirmer, in vitro, que les cellules souches neurales humaines migrent à travers les CE humaines de la BHE avant d’entamer leur différenciation en cellules du SNC. Suite à la migration à travers les cellules de la BHE les CSN se différencient spontanément en neurones, en astrocytes et en oligodendrocytes. Ces effets sont notés préférentiellement avec les cellules de la BHE par rapport aux CE non cérébrales. Ces propriétés spécifiques aux cellules de la BHE dépendent de la chimiokine MCP-1/CCL2 sécrétée par ces dernières. Ainsi, cette dernière partie suggère que la BHE n’est pas un obstacle à la migration de CSN vers le SNC. De plus, la chimiokine MCP-1 est identifiée comme un facteur sécrété par la BHE qui permet l’accumulation et la différentiation préférentielle de cellules souches neurales dans l’espace sous-endothélial. Ces trois études démontrent l’importance de la BHE dans la migration des cellules inflammatoires et des CSN vers le SNC et indiquent que de multiples mécanismes moléculaires contribuent au dérèglement de l’homéostasie du SNC dans les réactions neuro-immunes. En utilisant des modèles in vitro, in situ et in vivo, nous avons identifié trois nouveaux mécanismes qui permettent d’influencer les interactions entre les cellules du sang et la BHE. L’identification de ces mécanismes permet non seulement une meilleure compréhension de la pathophysiologie des réactions neuroinflammatoires du SNC et des maladies qui y sont associées, mais suggère également des cibles thérapeutiques potentielles pour influencer l’infiltration des cellules du sang vers le cerveau / Multiple Sclerosis is an inflammatory demyelinating disease in which immune cells from the peripheral blood infiltrate the central nervous system (CNS) to cause a pathologic neuroinflammatory reaction. Blood borne leucocytes cross the restrictive cerebral endothelium, the blood brain barrier (BBB), to gain access to the CNS parenchyma and cause cellular damage leading to the characteristic demyelinating lesions. The BBB is the interface between the blood and the CNS and as such is a critical mediator of neuro-immune reactions and an important therapeutic target to modulate neuroinflammation. It is essential to have a better understanding of the molecular mechanisms that regulate the BBB properties to elaborate new therapeutic strategies to modulate the BBB and thus the local neuroinflammation reaction. This Ph.D. thesis describes three distinct molecular mechanisms which regulate key BBB properties. The first section describes a novel role for the renin-angiotensin system (RAS) in the neuro-vascular unit (NVU) as a regulator of paracellular permeability. The second part of this thesis characterises the role of a novel adhesion molecule of the BBB, ALCAM. The third part of this work studies the interactions between neural stem cells (NSC) and the BBB and identifies MCP-1 as a critical factor involved in NSC recruitment to the CNS. In the first experimental section we provide evidence that angiotensinogen (AGT) produced and secreted by astrocytes, is cleaved into angiotensin II (AngII) and acts on type 1 angiotensin receptors (AT1) expressed by BBB endothelial cells (ECs). Activation of AT1 restricts the passage of molecular tracers across human BBB-derived ECs through threonine-phosphorylation of the tight junction protein occludin and its mobilization to lipid raft membrane microdomains. We also show that AGT knockout animals have disorganized occludin strands at the level of the BBB and a diffuse accumulation of the endogenous serum protein plasminogen in the CNS, as compared to wild type animals. Finally, we demonstrate a reduction in the number of AGT-immunopositive perivascular astrocytes in multiple sclerosis (MS) lesions, which correlates with a reduced expression of occludin similarly seen in the CNS of AGT knockout animals. Such a reduction in astrocyte-expressed AGT and AngII is dependent, in vitro, on the pro-inflammatory cytokines tumor necrosis factor-α and interferon-γ. Our study defines a novel physiological role for AngII in the CNS and suggests that inflammation-induced downregulation of AngII production by astrocytes is involved in BBB dysfunction in MS lesions. In the second experimental part we focus on adhesion molecules of the BBB. Using a lipid raft-based proteomic approach, we identified ALCAM (Activated leukocyte cell adhesion molecule) as an adhesion molecule involved in leukocyte migration across the BBB. ALCAM expressed on BBB endothelium co-localized with CD6 expressed on leukocytes and with BBB endothelium transmigratory cups. ALCAM expression on BBB cells was up-regulated in active multiple sclerosis and experimental auto-immune encephalomyelitis (EAE) lesions. Moreover, ALCAM blockade restricted transmigration of CD4+ lymphocytes and monocytes across BBB endothelium in vitro and in vivo, and reduced the severity and time of onset of EAE. Our findings point to an important role for ALCAM in leukocyte recruitment into the brain and identify ALCAM as a potential therapeutic target to dampen neuroinflammation. The third experimental part of this thesis studies the interactions between NCS and BBB. NCS represent an attractive source for cell transplantation and neural tissue repair. After systemic injection, NCS are confronted with the specialized BBB endothelial cells before they can enter the brain parenchyma. We investigated the interactions of human fetal neural precursor cells with human brain endothelial cells in an in vitro model using primary cultures. We demonstrated that human fetal neural precursor cells efficiently and specifically migrate to sub-endothelial space of human BBB-endothelium, but not pulmonary artery endothelial cells. When migrated across BBB-endothelial cells, fetal neural precursor cells spontaneously differentiate to neurons, astrocytes and oligodendrocytes. Effective migration and subsequent differentiation was found to be dependant on the chemokine CCL2/MCP-1, but not CXCL8/IL-8. Our findings suggest that an intact blood-brain barrier is not an intrinsic obstacle to neural stem cell migration into the brain and that differentiation of neural precursor cells occur in a sub-endothelial niche, under the influence of the chemokine CCL2/MCP-1. These three experimental sections demonstrate the crucial roles that the BBB plays in regulating the CNS homeostasis. Under pathological conditions, such as during neuro-immune reactions, the BBB is altered and becomes an important local player. The three different molecular mechanisms described in this thesis, contribute to our understanding of the BBB and may allow for the development of novel therapeutic strategies to limit neuroinflammation.

Les micelles polyioniques ont émergé comme des systèmes prometteurs de relargage de médicaments hydrophiles ioniques. Le but de cette étude était le développement des micelles polyioniques à base de dextrane pour la relargage de médicaments hydrophiles cationiques utilisant une nouvelle famille de copolymères bloc carboxymethyldextran-poly(éthylène glycol) (CMD-PEG). Quatre copolymères CMD-PEG ont été préparés dont deux copolymères identiques en termes de longueurs des blocs de CMD et de PEG mais différent en termes de densité de charges du bloc CMD; et deux autres copolymères dans lesquels les blocs chargés sont les mêmes mais dont les blocs de PEG sont différents. Les propriétés d’encapsulation des micelles CMD-PEG ont été évaluées avec différentes molécules cationiques: le diminazène (DIM), un médicament cationique modèle, le chlorhydrate de minocycline (MH), un analogue semi-synthétique de la tétracycline avec des propriétés neuro-protectives prometteuses et différents antibiotiques aminoglycosidiques. La cytotoxicité des copolymères CMD-PEG a été évaluée sur différentes lignées cellulaires en utilisant le test MTT et le test du Bleu Alamar. La formation de micelles des copolymères de CMD-PEG a été caractérisée par différentes techniques telles que la spectroscopie RMN 1H, la diffusion de la lumière dynamique (DLS) et la titration calorimétrique isotherme (ITC). Le taux de relargage des médicaments et l’activité pharmacologique des micelles contenant des médicaments ont aussi été évalués. Les copolymères CMD-PEG n'ont induit aucune cytotoxicité dans les hépatocytes humains et dans les cellules microgliales murines (N9) après 24 h incubation pour des concentrations allant jusqu’à 15 mg/mL. Les interactions électrostatiques entre les copolymères de CMD-PEG et les différentes drogues cationiques ont amorcé la formation de micelles polyioniques avec un coeur composé du complexe CMD-médicaments cationiques et une couronne composée de PEG. Les propriétés des micelles DIM/CMDPEG ont été fortement dépendantes du degré de carboxyméthylation du bloc CMD. Les micelles de CMD-PEG de degré de carboxyméthylation du bloc CMD ≥ 60 %, ont incorporé jusqu'à 64 % en poids de DIM et ont résisté à la désintégration induite par les sels et ceci jusqu'à 400 mM NaCl. Par contre, les micelles de CMD-PEG de degré de carboxyméthylation ~ 30% avaient une plus faible teneur en médicament (~ 40 % en poids de DIM) et se désagrégeaient à des concentrations en sel inférieures (∼ 100 mM NaCl). Le copolymère de CMD-PEG qui a montré les propriétés micellaires les plus satisfaisantes a été sélectionné comme système de livraison potentiel de chlorhydrate de minocycline (MH) et d’antibiotiques aminoglycosidiques. Les micelles CMD-PEG encapsulantes de MH ou d’aminoglycosides ont une petite taille (< 200 nm de diamètre), une forte capacité de chargement (≥ 50% en poids de médicaments) et une plus longue période de relargage de médicament. Ces micelles furent stables en solution aqueuse pendant un mois; après lyophilisation et en présence d'albumine sérique bovine. De plus, les micelles ont protégé MH contre sa dégradation en solutions aqueuses. Les micelles encapsulant les drogues ont maintenu les activités pharmacologiques de ces dernières. En outre, les micelles MH réduisent l’inflammation induite par les lipopolysaccharides dans les cellules microgliales murines (N9). Les micelles aminoglycosides ont été quant à elles capable de tuer une culture bactérienne test. Toutefois les micelles aminoglycosides/CMDPEG furent instables dans les conditions physiologiques. Les propriétés des micelles ont été considérablement améliorées par des modifications hydrophobiques de CMD-PEG. Ainsi, les micelles aminoglycosides/dodecyl-CMD-PEG ont montré une taille plus petite et une meilleure stabilité aux conditions physiologiques. Les résultats obtenus dans le cadre de cette étude montrent que CMD-PEG copolymères sont des systèmes prometteurs de relargage de médicaments cationiques. / Polyion complex (PIC) micelles have emerged as promising delivery systems of ionic hydrophilic drugs. It was the aim of this study to develop dextran-based PIC micelles for the delivery of hydrophilic cationic drugs using a new family of carboxymethyldextranblock- poly(ethylene glycol) (CMD-PEG) copolymers. Four CMD-PEG copolymers were prepared: (i) two copolymers identical in terms of the length of CMD and PEG blocks, but different in terms of the charge density of the CMD block; and (ii) two copolymers in which the charged block is the same, but the PEG block is of different molecular weight. The micellization of CMD-PEG copolymers and drug delivery aspects of the resulting micelles were evaluated using different cationic drugs: diminazene (DIM), a model cationic drug, minocycline hydrochloride (MH), a semisynthetic tetracycline antibiotic with promising neuroprotective properties and different aminoglycoside antibiotics. The cytotoxicity of CMD-PEG copolymers was evaluated in different cell lines using MTT and Alamar blue assays. CMD-PEG micelles encapsulating different drugs were characterized using different techniques, such as 1H NMR spectroscopy, dynamic light scattering (DLS), and isothermal titration calorimetry (ITC). The pattern of drug release and pharmacological activity of micelles-encapsulated drugs were also evaluated. The CMD-PEG copolymers did not induce cytotoxicity in human hepatocytes and murine microglia (N9) in concentrations as high as 15 mg/mL after incubation for 24 h. Electrostatic interactions between CMD-PEG copolymers and different cationic drugs triggered the formation of PIC micelles with a CMD/drug core and a PEG corona. The properties of DIM/CMD-PEG micelles were strongly dependent on the degree of carboxymethylation of the CMD block. Micelles of CMD-PEG copolymers having degree of carboxymethylation ≥ 60%, incorporated up to 64 wt% DIM, resisted salt-induced disintegration in solutions up to 400 mM NaCl and sustained DIM release under physiological conditions (pH 7.4, 150 mM NaCl). In contrast, micelles of CMD-PEG of degree of carboxymethylation ~ 30% had lower drug content (~ 40 wt% DIM) and disintegrated at lower salt concentration (∼ 100 mM NaCl). The CMD-PEG copolymer that showed the most satisfactory micellar properties, in terms of high drug loading capacity, sustained drug release and micelles stability was selected as a potential delivery system of minocycline hydrochloride (MH) and different aminoglycosides. CMD-PEG micelles encapsulating either MH or aminoglycosides had small size (< 200 nm in diameter), high drug loading capacity (≥ 50 wt% drug) and sustained drug release. These micelles were stable in aqueous solution for up to one month, after freeze drying and in the presence of bovine serum albumin. Furthermore, the micelles protected MH against degradation in aqueous solutions. Micelles-encapsulated drugs maintained their pharmacological activity where MH micelles reduced lipopolysaccharides-induced inflammation in murine microglia (N9) cells. And aminoglycosides micelles were able to kill a test micro-organism (E. coli X-1 blue strain) in culture. Aminoglycosides/CMD-PEG micelles were unstable under physiological conditions. Micelle properties were greatly enhanced by hydrophobic modification of CMD-PEG. Thus, aminoglycosides/dodecyl-CMD-PEG micelles showed smaller size and better stability under physiological conditions. The results obtained in this study show that CMD-PEG copolymers are promising delivery systems for cationic hydrophilic drugs.

Thanks to pioneering scientists and clinicians, prosthetic devices that are controlled by intra-cortical electrodes recording one's 'thoughts' are a reality today, and no longer merely in the realm of science fiction. However, widespread clinical use of implanted electrodes is hampered by a lack of reliability in chronic recordings, independent of the type of electrodes used. The dominant hypothesis has been that astroglial scar electrically impedes the electrodes. However, recent studies suggest that the impedance changes associated with the astroglial scar are not high enough to interfere significantly impair neural recordings. Furthermore, there is a time delay between when scar electrically stabilizes and when neural recordings fail (typically >1 month lag), suggesting that scar, per se, does not cause chronic recording unreliability. In this study, an alternative hypothesis was tested in a rat model, namely, that chronic inflammation surrounding microelectrodes causes a local neurodegenerative state. Chronic inflammation was varied in three ways: 1) stab wound control, 2) age-matched control, and 3) inter-shank spacing of a multishank electrode. The results of this study suggest that chronic inflammation, as indicated by activated microglia and reactive astrocytes, is correlated with local neurodegeneration, marked by neuron cell death and dendritic loss. Surprisingly, axonal pathology in the form of hyperphosphorylation of the protein Tau (the hallmark of many tauopathies, including Alzheimer's Disease) was also observed in the immediate vicinity of microelectrodes implanted for 16 weeks. Additionally, work is presented on a fast, non-invasive method to monitor the astrocytic response to intra-cortical electrodes using electrical impedance spectroscopy. This work provides a non-invasive monitoring tool for inflammation, albeit an indirect one, and fills a gap which has slowed the development of strategies to control the inflammatory tissue response surrounding microelectrodes and thereby improve the reliability of chronic neural recordings. The results of these experiments have significance for the field of neuroengineering, because a more accurate understanding of why recordings fail is integral to engineering reliable solutions for integrating brain tissue with microelectrode arrays.

No description available.

No description available.

Mild traumatic brain injury (mTBI), often referred to as concussion, has become increasingly recognized as a serious health issue in the general population. The prevalence of mTBI in athletes, particularly repeated injuries in young athletes, is of great concern as injuries to the developing brain can have long-term detrimental effects. In this study we used a novel awake closed-head injury (ACHI) model in rodents to examine repeated mTBI (rmTBI), to determine if repeated injuries produced the neurological and molecular changes evident with human concussion. Animals were administered 4, 8, and 16 rmTBIs and acute neurological assessments were performed after the injuries. Changes in glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1) levels were assessed using Western blot analysis at one day following rmTBI in the ipsilateral dentate gyrus (DG) and the cornu ammonis (CA) regions of the hippocampus and the cortex (CX) indicative of astrocyte and microglial cell reactivity. Results indicated that the ACHI model produces neurological deficits immediately after the injuries, with the most deficits arising in the rmTBI16 group. Despite deficits in all injury groups, histological staining with cresyl violet revealed no significant morphological tissue damage to the brain. Western blot analysis, however, showed a significant increase in DG and CX GFAP expression in the rmTBI16 group with no changes in Iba-1 levels. This suggests an acute activation of astrocytes in response to injury, with a delay or absence of microglial activation. Our findings show that with repetitive concussions, we are able to detect acute neurological and molecular changes in the juvenile female brain. However, further investigation is necessary to determine if these are transient changes. / Graduate

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Alzheimer s disease (AD) is a progressive neurodegenerative disorder, mainly characterized by memory and intellectual capacity loss. AD is characterized by deposition of amyloid-β peptide, neurofibrillary tangles, neuroinflammation, energy metabolism impairment, oxidative stress and synaptic dysfunction and loss. Its multiple pathological pathways contribute to the difficulty of AD treatment and prevention. Thus, the development of new therapies for AD curing or treatment is a challenge. The purpose of this study was to indicate an organoselenium moiety, p,p -dimethoxyl-diphenyl diselenide [(MeOPhSe)2], as a promising alternative for the treatment and prevention of sporadic dementia of Alzheimer-type (SDAT), using an experimental model of dementia induced by intracerebroventricular (i.c.v.) injection of streptozotocin (STZ) in rodents. Initially, it was investigated the prophylactic action of (MeOPhSe)2. For this, mice were treated with (MeOPhSe)2 (25 mg/kg, by gavage) and STZ (2μl of 2,5mg/ml solution; i.c.v.) or vehicles, and 48h after that, the treatment was repeated. The tasks of step-down-type passive-avoidance (SDPA), Y-maze and Morris water-maze (MWM), that followed this treatment, showed that (MeOPhSe)2 protected against the impairment in learning and memory caused by i.c.v. injection of STZ in mice. (MeOPhSe)2 protected against the increase in reactive species (RS) and the reduction of glutathione (GSH) levels, as well as modulated the antioxidant enzymes. (MeOPhSe)2 inhibited the acetylcholinesterase (AChE) activity, which was increased by STZ. Subsequently, it was investigated the effectiveness of (MeOPhSe)2 in reversing the cognitive impairment and neuronal damage induced by STZ. Therefore, rats were injected with STZ (1.0 mg/8μl; 4μl/ventricle) twice, 48h apart. After 21 days of STZ injection, regular diet fed rats were supplemented with 10ppm of (MeOPhSe)2 during 30 days. At the end of this period, it was observed that (MeOPhSe)2 dietary supplementation reversed STZ-induced memory impairment in MWM, SDPA and object recognition tasks. The results on SDPA and object recognition tasks demonstrated that the (MeOPhSe)2 improved memory in rats per se. STZ enhanced the RS and protein nitration levels in cortex and decreased GSH levels on hippocampus of rats, (MeOPhSe)2 reversed these alterations. (MeOPhSe)2 normalized AChE activity (which was enhanced by STZ) in both cortex and hippocampus, but did not reverse the deficit in cerebral glucose metabolism (ATP turnover was decrease by STZ). (MeOPhSe)2 was effective in reducing STZ-induced neuronal (apoptosis) loss. Moreover, (MeOPhSe)2 suppressed neuroinflammation induced by STZ in the rats hippocampus. The organoselenium inhibited activation of microglia and astrogliosis. Based on these results, it was concluded that: 1) (MeOPhSe)2 protected and reversed the cognitive abilities decline; 2) the mechanisms involved in the neuroprotective effect of (MeOPhSe)2 are: antioxidant, AChE inhibitor; inflammation suppressor; 3) (MeOPhSe)2 did not alter the energy metabolism; and 4) (MeOPhSe)2 reduced the neuronal death. Therefore, the present study demonstrated that (MeOPhSe)2 is a promising alternative for the drug studies for treatment of cognitive disorders such as SDAT. / A doença de Alzheimer (DA) é uma síndrome neurodegenerativa progressiva caracterizada principalmente por uma perda da memória e da capacidade intelectual. Ela é caracterizada pelo depósito de fragmentos β-amilóides; emaranhados neurofibrilares; neuroinflamação; déficit do metabolismo energético; estresse oxidativo e deficiência da neurotransmissão. As múltiplas vias patológicas da DA dificultam sua prevenção e tratamento. Logo, o desenvolvimento de novas terapias para a DA é um desafio. Por esta razão, este trabalho procurou apontar uma nova molécula orgânica contendo selênio, o disseleneto de p-metoxi fenila [(MeOPhSe)2], como uma alternativa promissora para o tratamento e prevenção da demência decorrente da DA (DEDA), usando um modelo experimental de demência induzida pela injeção intracerebroventricular (i.c.v.) de estreptozotocina (ETZ) em roedores. Inicialmente, avaliou-se o efeito profilático do (MeOPhSe)2. Para tal, camundongos receberam uma dose oral do organoselênio (25mg/kg, gavage) 30 minutos antes da ETZ (2μl de uma solução 2,5mg/ml), esse procedimento foi repetido 48horas depois. Os testes da esquiva passiva, do labirinto em Y e aquático de Morris, que sucederam esse tratamento, revelaram que o (MeOPhSe)2 protegeu os camundongos do prejuízo cognitivo induzido pela ETZ. O (MeOPhSe)2 protegeu o tecido cerebral do aumento das espécies reativas (ER) e da diminuição dos níveis de glutationa (GSH) induzidos pela ETZ, assim como modulou a atividade de enzimas antioxidantes. O (MeOPhSe)2 inibiu a atividade da acetilcolinesterase (AChE), a qual foi estimulada pela ETZ. Posteriormente, investigou-se a efetividade do (MeOPhSe)2 em reverter o prejuízo cognitivo e os danos neuronais induzidos pela ETZ. Para isso, a ETZ foi injetada nos ratos (1μg/8μl, 4μl/ventrículo) em 0 e 48horas. Passados 21dias, iniciou-se uma suplementação dietética com 10ppm de (MeOPhSe)2 durante 30dias. Ao final deste período, observou-se que o (MeOPhSe)2 restaurou as habilidades cognitivas prejudicadas pela ETZ nos ratos, nos testes do labirinto aquático de Morris, esquiva passiva e reconhecimento do objeto. Os resultados referentes aos testes do reconhecimento do objeto e da esquiva passiva apontaram que o (MeOPhSe)2 melhorou per se a memória dos ratos. A ETZ aumentou os níveis de ER e de nitração de proteínas no córtex e diminuiu os níveis de GSH no hipocampo dos ratos, o (MeOPhSe)2 reverteu estas alterações. O organoselênio inibiu a atividade da AChE (aumentada pela ETZ) tanto no córtex como no hipocampo dos ratos, mas não modulou o metabolismo da glicose (ETZ diminuiu ATP-turnover). O (MeOPhSe)2 evitou a perda neuronal (apoptose) e inibiu os eventos neurodegenerativos (ativação da caspase-3) induzidos pela ETZ. O (MeOPhSe)2 suprimiu a neuroinflamação induzida pela ETZ no hipocampo dos ratos. O organoselênio inibiu a ativação das células gliais e astrócitárias. Baseado nestes resultados, conclui-se que: 1) O (MeOPhSe)2 protegeu e reverteu o declínio das habilidades cognitivas; 2) Os mecanismos envolvidos no efeito neuroprotetor do (MeOPhSe)2 são: antioxidante; inibidor da AChE; supressor da neuroinflamação; 3) O (MeOPhSe)2 não altera o metabolismo energético; e 4) O (MeOPhSe)2 reduziu a morte neuronal. Assim sendo, este trabalho demonstrou que o (MeOPhSe)2 é uma alternativa promissora para o estudo de drogas para o tratamento de desordens cognitivas como a DEDA.