Browsing by Subject "astrogliosis"
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Item Endothelin-1-Induced Proliferation of Human Optic Nerve Head Astrocytes Under Hypoxia(2003-11-01) Desai, Devashish; Thomas Yorio; Ganesh Prasanna; Clark, Abbot F.Desai, Devashish, Endothelin-1-Induced Proliferation of Cultured Human Optic Nerve Head Astrocytes under Hypoxia. Master of Science (Biomedical Sciences). Purpose: Optic nerve head astrocytes (ONAs) normally support and protect the axons of retinal ganglion cells exiting the eye. Along with effects related to elevated intraocular pressure (IOP), proliferation and activation of ONAs, known as ‘astrogliosis’, is also thought to contribute to the pathophysiology of glaucoma by distributing axonal transport and preventing axon regeneration. Concentrations of endothelin-1 (ET-1) are elevated in glaucomatous eyes and in animal models for glaucoma. ET-1 injection into the eye causes reduction of ocular blood flow. ET-1 causes a time-dependent proliferation of human ONAs. Tumor necrosis factor-α (TNF-α), a cytokine, which is also elevated in glaucomatous optic nerve head, promotes ET-1 release from ocular cells and could potentially stimulate ET-1 secretion from the ONAs. Hypoxia resulting from ischemia, which is produced by the elevation of IOP or vasospasm in the retinal vasculature, is considered a significant factor contributing to the stress as the glaucomatous optic nerve head. Methods: Concentrations of ET-1 secreted by hONAs into cell culture media after hypoxia and TNF-α treatment was measured using an enzyme-linked immunosorbent assay (ELISA). Proliferation of hONAs was measured using a proliferation assay (formazan assay), performed at the end of various time periods of incubation with TNPα and ET-1 under normoxia or hypoxia. The involvement of mitogen activated protein kinase (MAPK) in hONA proliferation was examined using MAPK inhibitors and Western blot analyses. Results: Cell culture media collected from hONAs after 24-hour hypoxia with concurrent TNF-α treatment showed a 500% increase in the irET-1. Under normoxia, both TNF-α and ET-1 caused moderate proliferation of hONAs. Under hypoxia, TNF-α-induced proliferation was greatly increased. Conclusion: Hypoxia augments TNF-a and ET-1 growth of optic nerve head astrocytes, by way of increasing ET-1 synthesis and release as well as mitogenesis. Therefore reactive ONAs could be the common denominator underlying optic nerve damage in glaucoma since their localization makes them susceptible to mechanistic and ischemic influences in addition to influences of ET-1 and TNF-α. Keywords: astrocyte; endothelin-1; tumor-necrosis factor-α; hypoxia; proliferation; astrogliosis; glaucoma; optic nerveItem Impact Of Culture Conditions on Primary Astrocyte Phenotype(2019-05) Prah, Jude K.; Yang, Shaohua; Singh, Meharvan; Forster, Michael J.; Yan, Liang-Jun; Fudala, RafalAlthough previously thought to be passive support cells in the central nervous system (CNS), recent findings introduced critical contributions of astrocytes to numerous CNS functions like energy metabolism, ion and water homeostasis, blood brain barrier formation and neurotransmission. Their dysfunction has been implicated in the initiation and progression of specific CNS pathologies with astrocyte now given serious attention as cellular target for neuroprotection and treatment of neurological disorders. In spite of the aforementioned advances, our understanding of the mechanisms and pathways regulating astrocytic function, dysfunction and astrogliosis is still rudimentary. This is as a result of the complex interwoven nature of different cells in the CNS. Because of the complexities of the brain structure and function in vivo, methods of in vitro primary culture that overcome the influence of complex brain environment provide critical tools for understanding brain cell function at the cellular and molecular levels. The current primary astrocytes cultures are mostly maintained in serum-containing hyperglycemic medium which is non-physiological and produces astrocyte with a reactive, morphological and functional phenotype different from in vivo quiescent astrocytes. The first study presented in the dissertation delineates a serum free astrocyte culture condition that maintains primary astrocytes in a quiescent state. Results showed that primary astrocytes isolated from the cerebral cortex of postnatal day 1 C57BL6 mice and cultured in an astrocyte base medium supplemented with fibroblast growth factor (FGF2) and epidermal growth factor (EGF) (ABM- FGF2-EGF) have higher process bearing morphologies similar to in vivo astrocytes and different from the flat polygonal fibroblast like morphologies exhibited by astrocytes cultured under the traditional FBS condition developed by McCarthy and de Vellis (1980) (MD-10% FBS). Additionally astrocytes cultured in ABM-FGF2-EGF had enhanced glycolytic metabolism, higher glycogen content, lower GFAP and vimentin, increased glutamine synthase and glutamate transporter mRNA levels compared to astrocytes in the MD-10% FBS condition. These findings strongly indicates that astrocytes cultured in ABM-FGF2-EGF medium compared to the usual FBS medium promote quiescent and biosynthetic phenotype similar to in vivo astrocytes. This media provides a novel method for studying astrocytes function in vitro under physiological and pathological condition. Hyperglycemia could increase neuronal glucose level which leads to neuronal damage in a phenomenal referred to as glucose neurotoxicity. On the other hand the impact of hyperglycemia on astrocytes has been less explored although astrocytes are critical for glucose uptake and metabolism and many primary astrocyte cultures are maintained in high glucose conditions. In the second part of this dissertation we investigated the impact of hyperglycemia on astrocyte phenotype and function. Our studies demonstrated that hyperglycemic levels (25 mM) induce cell cycle arrest, ROS production, cytokine expression and inhibited astrocyte proliferation. High glucose enhanced glycolysis and increased metabolic potentials of astrocyte. In addition high glucose activated AMP-activated kinase (AMPK) signaling pathways and induces reactive astrocyte phenotype. In conclusion both studies presented a unique perspective of how culture conditions influence astrocyte phenotype and experimental outcome. Our study also provided a mechanism which may underline the role of astrocytes in hyperglycemia induced neurological complications.