White matter injury often results in clinical neurodevelopmental deficits; however, molecular mechanisms remain partially understood and there is currently no clinically effective treatment. The purpose of this study was to elucidate the molecular and cellular mechanisms underlying developmental white matter ischaemia to aid the search for therapeutic interventions. In this thesis, a particular priority was to develop an ischaemia paradigm enabling study of the extended effects of ischaemia in a controlled environment. To achieve this, I sought to use organotypic slice cultures (OSC), whereby cerebellar slices could be generated from animals where the white matter is developing and maintain it whilst exposing it to a short oxygen-glucose deprivation (OGD) insult at some defined point in the culture cycle. Using this approach, I show that culture treatment at 7 days in vitro (DIV) with OGD for twenty-minutes triggered significant injury as judged by a 58.6% reduction in cell viability 3 days post-injury. TUNEL labelling showed about 60% of cell death was apoptotic in nature. Gene expression studies using Q-RT -PCR confirmed caspase-dependent cell death. OGD also produced marked oligodendrocyte loss and myelination disturbances as seen by immunocytochemistry. Post-OGD, astrocytes and microglia became activated, and cytokine and iNOS mRNA expression was upregulated. After a transient demyelinating insult with OGD, GluR-antagonists were effective against cellular damage and myelination disturbances. Myelin gene, oligodendroglial transcription factor, and BCL-2 mRNA expression were also maintained following administration of GluR-antagonists. In addition, there seemed to be some NG2+ OPCs differentiated into MBP+ oligodendrocytes during the recovery phase as seen by proliferation- marker BrdU. Western blotting showed that a mechanism by which GluR-antagonists confer protection was independent of the CREB transcriptional activity. The results also suggest ischaemia-induced NF-kB and p38MAPK signalling pathway activation may be attributable to ionotropic GluR stimulation. The cellular damage and myelination disturbances in this ex vivo model of white matter ischaemia provides a system to study remyelination following CNS injury- induced demyelination, and may serve as a valid pre-animal test-bed for examining potential pharmacotherapies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:589408 |
| Date | January 2012 |
| Creators | Al-Griw, Mohamed A. |
| Publisher | University of Leeds |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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