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Myelin is a protective layer wrapped around axons which helps them conduct electrical signals rapidly, and provides them with metabolic support. In the central nervous system (CNS), myelin is produced by specialised glial cells called oligodendrocytes. Loss of myelin (demyelination) is associated with degeneration of axons and many neurodegenerative disorders, including multiple sclerosis (MS). The restoration of myelin sheaths by remyelination may protect axons and help functional recovery of patients, but achieving this requires better understanding of how the process unfolds at the cellular level. To investigate the processes of de- and remyelination in vivo, I have characterised a transgenic zebrafish line in which expression of the bacterial enzyme nitroreductase (NTR) is driven under the myelin basic protein promoter, thus in myelinating glia. I treat larvae with the NTR substrate metronidazole (Mtz). The reaction between NTR and Mtz results in a toxic metabolite which selectively kills NTR-expressing cells. The treatment with Mtz consistently ablates two-thirds of oligodendrocytes while not harming the animals otherwise. Myelin sheaths continue to deteriorate after the end of the treatment, such that seven days later, extensive demyelination is observed by electron microscopy. By 16 days after Mtz-treatment, robust recovery has occurred, with no discernible axon loss and myelin thickness restored to control levels. At this time point, oligodendrocyte numbers have also returned to control levels. During the demyelinated phase, I observe a striking increase in microglia and macrophages in the spinal cord. In order to study the role of the innate immune system in recovery, I used a mutant line, irf8-/- which lacks a transcription factor essential for development of microglia and macrophages. I am in the process of determining the ability of these mutants to regenerate their oligodendrocytes and myelin; preliminary results suggest that they are able to restore their myelin sheaths fully, but seem to have a delay in regenerating their oligodendrocytes compared to wild-types. The model I have established can be used in the future to better understand the consequences of demyelination to axon health, as well as chemical screening to identify compounds that could accelerate the remyelination process or enhance the thickness of myelin generated during remyelination. Insights arising from such studies will be useful in designing strategies to reduce axon loss and improve myelin regeneration in demyelinating diseases.

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国立情報学研究所で電子化したコンテンツを使用している。

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Orientador: Rosemeri de Oliveira Vasconcelos / Banca: Geórgia Modé Magalhães / Banca: Claudia Momo / Banca: Pamela Rodrigues Reina Moreira / Banca: Daniela Bernadete Rozza / Resumo: No sistema nervoso central (SNC) a homeostase é mantida pelo contato célula a célula e por meio da expressão de receptores de membrana em neurônios, que se ligam a microglia para mantê-la em estado quiescente. Os astrócitos também contribuem com este processo, além disso, eles exercem inúmeras funções para a manutenção da imunotolerância local. Em situação de injúria, os astrócitos podem apresentar um efeito deletério, pois ativam a resposta imune e os efeitos da neuroinflamação e podem contribuir para o agravamento dos sinais clínicos neurológicos. Na cinomose canina, enfermidade infecciosa que pode cursar com desmielinização e inflamação no SNC, acredita-se que além do vírus, o processo inflamatório contribua com as lesões na substância branca. Portanto, os objetivos do presente estudo foram avaliar o papel dos astrócitos na encefalite causada pelo vírus da cinomose, por meio da imunodetecção de MHC-II, linfócitos T CD3, MMP9, MIF e GFAP nas áreas de desmielinização do encéfalo, a fim de verificar se estes achados podem estar relacionados à extensão ou gravidade das lesões encefálicas. Para isso realizou-se um estudo retrospectivo do arquivo de blocos de parafina, onde utilizouse 21 blocos de encéfalos (cerebelo) de cães naturalmente infectados com o vírus da cinomose (grupo infectado), bem como, encéfalos de cinco animais livres de doenças sistêmicas ou que afetem o SNC (grupo controle). Na análise imunohistoquímica das amostras verificou-se marcação aumentada de GFAP, MHC... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the central nervous system (CNS) homeostasis is maintained by cellto-cell contact and by the expression of membrane receptors on neurons, which bind to microglia to keep it in a quiescent state. Astrocytes also contribute to this process, in addition, they exert numerous functions for the maintenance of local immunotolerance. In a situation of injury, astrocytes may have a deleterious effect, since they activate the immune response and the effects of neuroinflammation and may contribute to the worsening of clinical neurological signs. In canine distemper, an infectious disease that can occur with demyelination and inflammation in the CNS, it is believed that in addition to the virus, the inflammatory process contributes to lesions in the white matter. Therefore, the objectives of the present study were to evaluate the role of astrocytes in encephalitis caused by the distemper virus, by immunodetection of MHC-II, CD3, MMP9, MIF and GFAP T lymphocytes in the areas of brain demyelination, in order to verify whether these findings may be related to the extent or severity of brain lesions. For this, a retrospective study of the paraffin block file was carried out, using 21 blocks of cerebellum from dogs naturally infected with the distemper virus (infected group), as well as the brains of five disease-free animals systemic or affecting the CNS (control group). In the immunohistochemical analysis of the samples, there was an increased marking of GFAP, MHC-II, MMP9 and MIF in the... (Complete abstract click electronic access below) / Mestre

Stressful life events have been associated with the onset and/or exacerbation of multiple sclerosis (MS). To investigate the effects of stress on the pathogenesis of MS, we employed restraint stress (RST) in the Theiler’s virus-induced demyelination (TVID) model, an animal model for human MS. Intracerebral inoculation of susceptible strain of mice with Theiler’s murine encephalomyelitis virus (TMEV) results in a biphasic disease – an acute encephalomyelitis and chronic demyelination. The establishment of persistent viral infection is critical in inducing immune-mediated demyelination during the chronic disease. The exposure of mice to RST prior to viral infection produced a stress response as evidenced by elevated circulating corticosterone (CORT). To further study the effect of stress on the immune response to TMEV infection and demyelination, we first examined the cytokine and chemokine response during the acute TMEV infection. We demonstrated that RST down-regulated the virus-induced expression of chemokines, Ltn, IP-10, RANTES, and pro-inflammatory cytokines, TNF, IFN and LT in both the brain and spleen during early infection. Histologically, a decreased pattern of inflammation was observed in the brain of restrained mice as compared to non-restrained mice. The increased viral titer was noted in the CNS of restrained mice and was correlated with the decreased production of pro-inflammatory cytokine, suggesting an impaired immune response by RST. Secondly, the duration of stress on the late demyelination was investigated. Repeated and chronically stressed SJL/J mice developed an early onset of clinical signs and a delayed onset was observed in acutely stressed mice. Both acute and chronic RST suppressed the antibody response to TMEV and stressed displayed a higher incidence of demyelination than non-restrained mice. Axonal loss was also noted in chronic stressed mice. Additionally, RST caused an increased systemic viral infection in extraneural organs during the acute infection and cardiotropic TMEV was isolated from the heart of stressed mice. Taken together, stress resulted in profound immunsuppression during acute infection, which may consequently increase the incidence of demyelination. The present study may be generalized in human MS which is potentially triggered by viral infection.

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