Impact Of Culture Conditions on Primary Astrocyte Phenotype




Prah, Jude K.


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Although 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.