Date of Award

2019

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Neuroscience

Abstract

Glaucoma is a progressive optic neuropathy that manifests in a pathological triad of optic nerve head remodeling, damage to optic nerve axons, and retinal ganglion cell death. Optic nerve head astrocytes (ONHAs) are the primary cell type in the optic nerve head. ONHAs undergo reactive astrocytosis, an activated phenotype characterized by altered gene and protein expression and increased cell migration and proliferation, early in the disease pathology. Reactive astrocytosis is thought to underlie optic nerve head remodeling. The first goal of this thesis was to determine if exposure of primary adult rat ONHAs to hyperbaric pressure (25-30 mm Hg above ambient pressure) for 2 - 30 hr induces phenotypes of reactive astrocytosis. ONHAs exposed to hyperbaric pressure exhibited increased expression of glial fibrillary acidic protein and voltage-gated calcium channels, changes to the actin cytoskeleton, and decreased expression of many of the extracellular matrix components involved in normal elastin and collagen crosslinking pathways. Additionally, ONHA cultures exposed to hyperbaric pressure exhibited increased the sensitivity of cells to a subsequent oxidative challenge as determined by cell viability assays. This sensitization was attributed to significantly elevated reactive oxygen species generation as quantified by CellROX® fluorescent staining and dichlorofluorescein fluorescence. The second part of the thesis used the hyperbaric pressure model to induce reactive astrocytosis and investigate whether antioxidants exert glioprotective effects against reactive astrocytosis-induced sensitization to oxidative stress, as clinical evidence also shows that oxidative stress plays a role in the disease. ONHAs were pre-treated for one hour with 100 M Trolox, 50 M resveratrol, 1 M Xanthohumol, 0.005% manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP) or vehicle. ONHAs were then subjected to ambient or to hyperbaric pressure to induce reactive astrocytosis and subsequently exposed to exogenously-applied chemically-induced oxidative stress using tert-butyl hydroperoxide (tBHP; 0 to 1 mM). Oxidative stress was quantified using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) or CellROX®. Assays for cell viability and proliferation were performed to determine glioprotective potential of test compounds. All four compounds were able to provide glioprotection in reactive astrocytes. Protein expression levels of enzymes of the endogenous antioxidant system revealed differential effects by the various antioxidants, elucidating their mechanisms of action. This thesis work provided a novel in vitro model of the diseased optic nerve head, which can be used to test novel therapeutic compounds for the treatment of glaucoma and showed that antioxidants may be used in the treatment of glaucoma.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Available for download on Tuesday, August 13, 2024

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