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UBC Theses and Dissertations

Two-photon imaging of glutathione levels in intact brain indicates sites of enhanced redox buffering Sun, Xiaojian


Oxidative stress, the metabolic imbalance between oxidant creation and destruction is proposed as a final common pathway for neurodegenerative disease and injury. To map sites of antioxidant homeostasis in brain, I used two-photon imaging of monochlorobimane (MCB) fluorescence, a selective enzyme-mediated marker, for reduced glutathione (GSH) in both brain slices and in vivo preparations. I found that cells at the CSF or blood brain interface such as lateral ventricle ependymal cells, meningeal cells, and astroglia contain both high levels of MCB conjugation activity (glutathione S-transferase dependent) and GSH content. In comparison, cortical neurons in Layer II contained approximately 20% of the GSH content of their astrocyte counterparts. Regional mapping of GSH indicated that the highest levels were present in cells lining the lateral ventricles, specifically eperidymal cells and the subventricular zone. The enrichment of GSH content along the lateral ventricle suggested a possible function in oxidant homeostasis for developing neuronal progenitors. Consistent with this, I observed that developing neurons found in the subgranular zone of dentate gyrus, contained 3-fold more GSH than older neurons found in the neighbouring granular layer. Besides imaging GSH distribution with MCB, I also developed an assay to measure different kinetic parameters of GSH metabolism. With this assay, I found that meninges are more active than cortical cells in GSH metabolism. In conclusion, I have developed a powerful approach to map sites of antioxidant homeostasis in brain directly, demonstrating a unique role for GSH in developing neurons and cells at the CSF and blood-brain interface.

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