Dans le Système Nerveux Central (SNC), les cellules gliales influencent les activités neuronales. Les cellules microgliales, cellules immunitaires résidentes du SNC, contrôlent grandement l’état neuroinflammatoire. Ce contrôle est particulièrement important dans les fonctions physiologiques et s’avère souvent défectueux dans les neuropathologies. Les cellules microgliales sont en relation avec le microenvironnement cérébral et communiquent avec les autres types cellulaires (astrocytes, oligodendrocytes et neurones) afin de contrôler l’état neuroinflammatoire. Parmi les différents modes de communication intercellulaire au sein du SNC, les vésicules extracellulaires (VEs) interviennent largement dans les processus physiologiques (développement, homéostasie…) et pathologiques (maladies neurodégénératives…). C’est pourquoi, ce mode de communication a été étudié dans le dialogue entre la microglie et les neurones chez la sangsue Hirudo medicinalis. Cet annélide est un modèle intéressant de neurobiologie grâce à la structure linéaire de son système nerveux et à l’organisation de ses types cellulaires. Il permet l’étude du dialogue entre les cellules microgliales et les neurones au niveau d’une lésion expérimentale. Dans un premier temps, les résultats ont montré que les cellules microgliales interagissent avec les neurones lors d’une lésion du SNC et que des VEs sont libérées au niveau de cette lésion. De plus, les cellules microgliales produisent des VEs qui interagissent avec les neurones et délivrent un effet neurotrophique in vitro sur des neurones de sangsue et de rat. Dans un deuxième temps, la complexité des composés vésiculaires ainsi que des impératifs d’efficacité liés aux méthodes d’isolement nous ont conduits à développer l’analyse protéomique non ciblée et à grande échelle afin de valider les fractions positives en VEs mais aussi identifier leurs signatures protéiques biologiquement actives. Dans une dernière partie, nous nous sommes intéressés aux microARNs (miARNs) contenus dans les VEs microgliales. Les résultats ont permis l’identification de 6 miARNs dans les VEs microgliales, dont un seul, miR-146a, est décrit à ce jour dans le SNC chez les mammifères. Dans un contexte de dialogue neuroprotecteur entre VEs microgliales et neurones, les analyses neuronales ont prédit des ARNm potentiellement régulés par les miARNs contenus dans les VEs. Ces 6 miARNs ont également été identifiés dans les VEs issues de microglie de souris, de rat et humaine. Dans leur ensemble, les résultats montrent que les cellules microgliales chez la sangsue produisent des VEs, ayant un effet neurotrophique sur les neurones, y compris des neurones de rat. L’identification des molécules présentes dans ces VEs (protéines et miARNs) a permis de soulever des perspectives sur les mécanismes neuroprotecteurs supportant ce dialogue microglie-neurone qu’il sera intéressant d’examiner chez les mammifères dans un contexte de lésion nerveuse. / In the Central Nervous System (CNS), the glial cells influence neuronal activities. The microglial cells, resident immune cells of the CNS, greatly control the neuroinflammatory state. This control is particularly important in physiological functions and is often defective in neuropathologies. The microglial cell activities depend on the brain microenvironment and they communicate with other cell types (astrocytes, oligodendrocytes and neurons) to control the neuroinflammatory state. Among the different mechanisms of intercellular communication within the CNS, extracellular vesicles (EVs) play a major role in physiological processes (development, homeostasis, etc.) and pathological processes (neurodegenerative diseases, etc.). Therefore, this mode of communication was studied in the dialogue between microglia and neurons in the leech Hirudo medicinalis. This annelid is an interesting model of neurobiology thanks to the linear structure of its nervous system and the organization of its cell types. It allows the study of the dialogue between microglial cells and neurons at the level of an experimental lesion. At first, the results showed that microglial cells interact with neurons during CNS injury and that EVs are released at the level of this lesion. In addition, microglial cells produce EVs that interact with neurons and deliver a neurotrophic effect in vitro on leech and rat neurons. In a second step, the complexity of the vesicular compounds as well as efficiency requirements related to the isolation methods led us to develop the non-targeted proteomic analysis on a large scale in order to validate the positive EV fractions but also to identify their biologically active protein signatures. In a last part, we were interested in the microRNAs (miRNAs) contained in microglial EVs. The results allowed the identification of 6 miRNAs in microglial EVs, of which only one, miR-146a, is described to date in the mammalian CNS. In a context of neuroprotective dialogue between microglial EVs and neurons, the analysis of neuronal protein signatures predicted mRNAs potentially regulated by miRNAs contained in EVs. These 6 miRNAs were also identified in EVs derived from mouse, rat and human microglia. Overall, the results show that microglial cells in the leech produce EVs, exerting a neurotrophic effect on neurons, including rat neurons. The identification of the molecules present in these microglial EVs (proteins and miRNAs) made it possible to raise perspectives on the neuroprotective mechanisms supporting this microglia-neuron dialogue that will be interesting to examine in mammals in a context of nerve injury.

DCLK3 (Doublecortin-like kinase 3) est une protéine kinase neuronale enrichie dans le striatum et dans le gyrus denté. L’expression de DCLK3 est nettement diminuée dans le cerveau des patients atteints de la maladie de Huntington (MH). Cependant, son rôle reste pour l’instant inconnu. Le laboratoire avait précédemment montré que DCLK3 pouvait jouer un rôle neuroprotecteur, en particulier dans différents modèles de la MH. En effet, la surexpression de Dclk3 dans le striatum produit un effet neuroprotecteur contre la toxicité de l’huntingtine mutée (Httm) et améliore les déficits moteurs dans les modèles murins de la MH (knock-in avec 140 répétitions de CAG). Nos résultats récents ont aussi montré que DCLK3 était localisée dans les noyaux des neurones et pourrait réguler la transcription par un mécanisme épigénétique. Le but de la présente étude a été de mieux comprendre le rôle de DCLK3 dans le cerveau. Pour cela, nous avons produit des souris avec des sites LoxP dans le gène Dclk3 (Dclk3flox/flox). Nous avons d’abord croisé ces souris avec les souris CMV-cre pour obtenir des souris constitutivement déficientes en Dclk3 et ceci dans l’ensemble de l’organisme. Nos études comportementales portant sur la mémoire, l’anxiété et les performances motrices, n’ont révélé aucun déficit majeur dans le phénotype. En accord avec cette observation, l’analyse structurale des cerveaux des souris KO Dck3 par histologie et IRM n’a pas révélé d’atteinte notable par rapport aux animaux témoins. Cependant, le profil des métabolites des mâles à 6 mois était légèrement mais significativement perturbé. Nous avons également croisé les souris Dclk3flox/flox avec des souris Rgs9-cre pour avoir une délétion de Dclk3 uniquement dans le striatum. Aucun déficit moteur n’a été observé dans ces souris. De manière intéressante, l’injection d’AAV-cre dans l’hippocampe des souris adultes Dclk3flox/flox tend à créer des déficits de mémoire au test de la piscine de Morris. L’ensemble de nos résultats indique un rôle de DCLK3 dans la plasticité synaptique dans l’hippocampe et suggère que la perte de DCLK3 dans la MH contribuerait aux aspects cognitifs de la maladie. Ainsi, à terme, la signalisation impliquant DCLK3 pourrait constituer une cible thérapeutique intéressante pour améliorer certains processus neurodégénératifs. / DCLK3 (Doublecortin-like kinase 3) is a neuronal kinase enriched in the striatum and in the dentate gyrus of the hippocampus. DCLK3 expression is markedly reduced in the brain of Huntington’s disease (HD) patients. However, its role remains unknown. The laboratory previously showed that DCLK3 could play a neuroprotective role, specifically in different HD models. The overexpression of Dclk3 in the striatum produces neuroprotection against mutant huntingtin (mHtt) toxicity and ameliorates motor deficits in rodent models of HD (knock-in mice with 140 CAG repeats). Our recent results indicate that DCLK3 is localized in the nucleus of neurons and may play a role in transcription through epigenetic mechanisms. Here, we aim at investigating further the role of DCLK3 in the brain. To do so, we generated mice with loxP sites in the Dclk3 gene (DCLK3flox/flox). We first crossed them with CMV-Cre mice to obtain constitutive knockout Dclk3 mice in the whole body. Our behavioral study on memory, anxiety and motor phenotype show no obvious deficit. Likewise, structural analysis in the brain of Dclk3 KO mice in histology and MRI did not highlight any deficit compared to control mice. However the metabolite profile in males at 6 months was slightly but significantly disturbed. Dclk3flox/flox mice were also crossed with Rgs9-cre mice to have Dclk3 deletion in the striatum only. No motor deficits were observed in these mice. Interestingly, the injection of AAV-Cre in the hippocampus of adult Dclk3flox/flox mice tends to produce memory deficits in the water-maze test. These results indicate a potential role of DCLK3 in synaptic plasticity in the hippocampus and suggest that the loss of this protein in HD could contribute to cognitive aspects of the disease. In long term, the signaling involving DCLK3 may constitute an interesting therapeutic target to improve certain neurodegenerative processes.

No description available.

Primary cilia are essential in brain development, as mediators of sonic hedgehog signaling. However, their role in mature neurons remains elusive. One means to elucidate their function may be to investigate the function of the somatostatin type 3 receptor (SstR3), which is concentrated on the primary cilia of neurons. The inhibitory and anticonvulsant properties of somatostatin suggest that ciliary SstR3 might protect neurons against excitotoxicity, as seen in epileptic seizures. C57BL/6 wild type (wt) and SstR3 knockout mice were administered vehicle or epileptogenic agents kainic acid (KA) or pentylenetetrazole. Seizure behaviors were rated on seizure severity scales. KA-induced seizure behaviors were more severe in SstR3 mutants than in wt. Correspondingly, the mutants showed greater reactive gliosis, as indicated by increased numbers of GFAP immunoreactive (GFAP(+)) astrocyte processes. In addition, seizure severity was associated with a greater percentage of neural stem cells having an ACIII(+) cilium. Following injections of pentylenetetrazole, SstR3 mutants reached maximum seizure levels faster than wt. These results support the hypothesis that ciliary SstR3 are neuroprotective in mature neurons, and may provide a new avenue for the treatment of seizures.

Stroke is a leading cause of disability in Canada. Delayed hypothermia improves outcome in patients following cardiac arrest and reduces lesion volume in rodents after transient focal ischemia, but less is known about the effectiveness of delayed hypothermia following permanent focal ischemia. In Chapter 1, the efficacy of 12, 24 or 48 h of delayed hypothermia was evaluated one week following pMCAO. All treatments attenuated neurological deficits and brain water content, but only the 24 and 48 h treatments reduced stepping error rate and lesion volume. Thus, delayed hypothermia attenuates brain injury and functional deficits following permanent middle cerebral artery occlusion (pMCAO). Longer bouts of cooling provide superior protection; an effect that is not explained by lessened edema. Chapter 3 describes a novel method of focal brain hypothermia in rats. A metal coil was implanted between the Temporalis muscle and adjacent skull and flushed with cold water. Focal, ipsilateral cooling was successfully produced without cooling of the opposite hemisphere or the core. One day of focal hypothermia was maintained in awake rats without significant alterations in blood pressure, heart rate or body temperature. The described simple method allows for safe inductions of focal brain hypothermia in anesthetized or conscious rats, and is ideally suited to trauma or stroke studies. In Chapter 4, long-term efficacy of 12 and 48 h of delayed focal or systemic hypothermia was evaluated following pMCAO. Both systemic treatments equally reduced lesion volume and skilled reaching deficits compared to normothermic controls, but only the 48 h treatment reduced neurological deficits. Conversely, 12 h of focal cooling did not significantly improve outcome, whereas 48 h of focal brain cooling attenuated functional deficits and reduced lesion volume. Thus, both delayed focal and systemic hypothermia attenuate long-term brain injury and functional deficits following pMCAO. Duration of cooling is clearly an important factor that may depend upon the method of cooling. Overall, this data indicates that delayed and prolonged hypothermia provides substantial and persistent protection against pMCAO in the rat. Prolonged hypothermia is a promising neuroprotective therapy for acute stroke and further clinical investigation is warranted.

Spinal cord injuries can cause the catastrophic loss of motor and sensory function. The neurological deficits that result are the consequence of not only the primary injury to the spinal cord, but also a complex milieu of secondary pathological processes that are now beginning to be understood. The major mechanisms that underlie this secondary pathology include vascular disruption, ischemia, oxidative stress, excitotoxicity, and inflammation. In light of this, the fact that this secondary pathology occurs after the initial impact makes it potentially amenable to therapeutic intervention. Pharmacotherapies may attenuate some of these processes and minimize secondary damage. Some of the promising treatments that are emerging for acute spinal cord injury are drugs that are already used by physicians for the treatment of unrelated diseases. These drugs, which have already been established to be safe for humans, offer the unique advantage over other novel therapeutic interventions that have yet to be tested in humans. This would save a tremendous amount of time and money needed for human safety studies, if considered as a treatment for spinal cord injury. Examples of such drugs include minocycline (an antibiotic), erythropoietin (a recombinant hormone used to treat anemia), and statins (a popular class of blood cholesterol reducers), all of which have demonstrated the ability to attenuate the various pathophysiological processes initiated after trauma to the central nervous system. In a series of studies, erythropoietin, darbepoetin, atorvastatin, simvastatin, and minocycline were all evaluated for their ability to improve neurologic recovery in a clinically relevant model of spinal cord injury. My experiments revealed that erythropoietin, darbepoetin, atorvastatin and minocycline did not significantly improve neurological recovery. These negative results were in stark contrast to the positive findings which had been published in the literature suggesting that differences in experimental models and methodology influence the neuroprotective efficacy of these drugs. Simvastatin, on the other hand, demonstrated significant improvements in locomotor and histological outcomes. Although this is indeed exciting, the results were modest at best. My results highlight the need for further preclinical work on the above treatments to refine and optimize them prior to proposing them for human testing.

Spinal cord injuries can cause the catastrophic loss of motor and sensory function. The neurological deficits that result are the consequence of not only the primary injury to the spinal cord, but also a complex milieu of secondary pathological processes that are now beginning to be understood. The major mechanisms that underlie this secondary pathology include vascular disruption, ischemia, oxidative stress, excitotoxicity, and inflammation. In light of this, the fact that this secondary pathology occurs after the initial impact makes it potentially amenable to therapeutic intervention. Pharmacotherapies may attenuate some of these processes and minimize secondary damage. Some of the promising treatments that are emerging for acute spinal cord injury are drugs that are already used by physicians for the treatment of unrelated diseases. These drugs, which have already been established to be safe for humans, offer the unique advantage over other novel therapeutic interventions that have yet to be tested in humans. This would save a tremendous amount of time and money needed for human safety studies, if considered as a treatment for spinal cord injury. Examples of such drugs include minocycline (an antibiotic), erythropoietin (a recombinant hormone used to treat anemia), and statins (a popular class of blood cholesterol reducers), all of which have demonstrated the ability to attenuate the various pathophysiological processes initiated after trauma to the central nervous system. In a series of studies, erythropoietin, darbepoetin, atorvastatin, simvastatin, and minocycline were all evaluated for their ability to improve neurologic recovery in a clinically relevant model of spinal cord injury. My experiments revealed that erythropoietin, darbepoetin, atorvastatin and minocycline did not significantly improve neurological recovery. These negative results were in stark contrast to the positive findings which had been published in the literature suggesting that differences in experimental models and methodology influence the neuroprotective efficacy of these drugs. Simvastatin, on the other hand, demonstrated significant improvements in locomotor and histological outcomes. Although this is indeed exciting, the results were modest at best. My results highlight the need for further preclinical work on the above treatments to refine and optimize them prior to proposing them for human testing.

No description available.

No description available.

Orientador: Alexandre Leite Rodrigues de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T10:28:31Z (GMT). No. of bitstreams: 1 Chiarotto_GabrielaBortolanca_M.pdf: 2946434 bytes, checksum: 5981e0da5b0dde27b2c22c03e945752a (MD5) Previous issue date: 2013 / Resumo: A lesão do nervo periférico em animais neonatos resulta em extensa morte neuronal na medula espinal e nos gânglios das raízes dorsais. Como a maior parte da perda neuronal é devido ao estresse oxidativo e mecanismos apoptóticos, vários estudos têm sido realizados a fim de investigar o efeito de substâncias neuroprotetoras. Dentre esses fármacos, o antioxidante Tempol tem mostrado resultados promissores, uma vez que é capaz de quelar espécies reativas de oxigênio (ROS) e minimizar, ou mesmo prevenir, danos teciduais. Neste sentido, o presente trabalho teve como objetivo avaliar os efeitos neuroprotetores do Tempol sobre a morte neuronal induzida pela secção do nervo isquiático em ratos recém-nascidos. Para as análises morfológicas, ratos Wistar com dois dias de idade (P2), foram submetidos à secção do nervo isquiático esquerdo e foram divididos em dois grupos: (1) grupo tratado com Tempol (24mg/kg), 10 minutos, 6 horas e a cada 24 horas após a lesão por até 1 semana; (2) grupo veículo - tratado com o veículo de diluição do Tempol nos mesmos períodos de tratamento. Os animais de ambos os grupos foram sacrificados em tempos de sobrevida de 8, 12, 24, 72 horas e uma semana após a lesão. O lado contralateral da medula foi utilizado como controle interno para análise dos resultados. Após os respectivos dias de sobrevida, os animais foram anestesiados e submetidos à toracotomia para perfusão com solução salina e fixadora. Em seguida, o conjunto contendo a intumescência lombar e as raízes nervosas foram processadas para posterior confecção dos cortes histológicos. Secções transversais de 12 ?m da intumescência lombar foram utilizadas nas técnicas de Microscopia de luz para avaliação da sobrevivência neuronal e TUNEL para detecção e quantificação de células apoptóticas. Os resultados demonstraram que o tratamento com Tempol aumentou a sobrevivência de motoneurônios da medula espinal em 15% após 8 horas; 19% após 12 horas; 13% após 24 horas; 15% após 72 horas e 21% após uma semana pós-lesão (p<0,0001). Da mesma forma, os animais tratados com Tempol apresentaram uma diminuição do número de células TUNEL positivas, indicando uma redução dos eventos apoptóticos. Para a realização da qRT-PCR, a intumescência lombar foi dissecada 12 e 24 após lesão. Os resultados mostraram um aumento na expressão gênica de 13, 3 e 28 vezes para bax, caspase3 e bcl2, respectivamente, após 12 horas da axotomia e tratamento com Tempol. Após 24 horas houve redução na expressão dos genes estudados, porém, esta não foi estatisticamente significativa. Como um todo, os presentes resultados mostraram que o tratamento com Tempol é neuroprotetor, levando à maior sobrevivência de motoneurônios após a lesão de nervo periférico, minimizando a ocorrência de eventos apoptóticos / Abstract: The peripheral nerve injury in newborn animals results in extensive neuronal death in the spinal cord and dorsal root ganglia. Since most neuronal loss is due to oxidative stress and apoptotic mechanisms, several studies have been conducted to investigate the effect of neuroprotective substances. Among these drugs, the antioxidant Tempol has shown promising results as it is capable of chelating reactive oxygen species (ROS) and to minimize or even prevent, tissue damage. In this sense, the present study aimed to evaluate the neuroprotective effects of Tempol on neuronal death induced by sciatic nerve section in newborn rats. For this purpose, two days old (P2) Wistar rats underwent left sciatic nerve section and were divided into two groups: (1) Tempol-treated group (24mg/kg) - 10 minutes and 6 hours, and every 24 hours after injury for up to 1 week; (2) Group Vehicle - Vehicle treated with the same dilution of Tempol. The animals of both groups were sacrificed at survival times of 8, 12, 24, 72 hours and one week after the lesion. The contralateral side of the spinal cord was used as internal control. After the respective days of survival, the animals were anesthetized and underwent thoracotomy for transcardial perfusion with saline and fixative. The assembly containing the lumbar intumescence and nerve roots were processed for subsequent preparation of histological sections. Cross sections of 12 ?m were used for evaluation of neuronal survival and TUNEL for the detection and quantification of apoptotic cells. The results showed that Tempol treatment increased the survival of motoneurons in the spinal cord 15% after 8 hours, 19% after 12 hours, 13% after 24 hours, 15% after 72 hours and 21% after one week post-lesion ( p <0.0001). Likewise, Tempol treated animals showed a decrease in the number of TUNEL positive cells, indicating reduction in apoptotic events. To further investigate the apoptotic events observed herein, we have performed qRT-PCR evaluation of bax, caspase3 and bcl2 transcript levels, 12 and 24 after injury. The results showed an increase in gene expression of 13, 3 and 28 times for bax, caspase 3 and bcl2, respectively, after 12 hours of axotomy and treatment with Tempol. After 24 hours no differences were observed relative to the control. As a whole, these results showed that Tempol is neuroprotective, leading to improved survival of motor neurons after peripheral nerve injury, minimizing the occurrence of apoptotic events / Mestrado / Anatomia / Mestra em Biologia Celular e Estrutural