Browsing Graduate Student Dissertations & Theses by Subject "ALTERATIONS"
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ALTERATIONS IN CELLULAR GLUTAMATE TRANSPORT DO NOT CONTRIBUTE TONEURONAL CELL DEATHIN A MIXED CORTICAL CELL CULTURE MODEL OF HYPOGLYCEMIASevere hypoglycemia is associated with neurological deficits thatwhen left untreated can lead to frank neuronal cell death. Despite longstanding evidence in both in vitro andin vivomodels that hypoglycemic neuronalcell death is mediated by glutamateexcitotoxicity, the cellularand molecular mechanisms involved remain incompletelydefined. Toward this end, werecently reported that glutamate efflux from astrocytes via the anionic cystine/glutamate antiporter, system xc-, contributed to glucose-deprivation (GD) induced neuronal cell death in vitro. However,the precise mechanism by which system xc-activity links to glutamate-mediated injury has yet to be determined. Thus, the overall purpose of this thesis was toinvestigate whetherchanges insystem xc-expression in our astrocyte and mixed cortical cell cultures and/or alterations in glutamate handling in a mixed cortical culture modelfollowing glucose deprivationoccur(s). Toward the former, no change in the expression of mRNA (GD up to 4 h) or protein(GD up to 8 h) ofxCT, the functional light chain of system xc-, in either astrocyte or mixed cortical cell cultureswas demonstrated via quantitative RT-PCR or western blot analysis, respectively. Further, aglycemic neuronal injury, induced by 6 or 8 h of glucose deprivation, was not prevented by the addition of either actinomycin D (10 μg/mL) or cycloheximide (1 μg/mL), demonstrating no requirement for transcription or translation, respectively. Toward the latter, alterations in classical glutamate re-uptake transporter function also did not appear to be altered. Media containing added glutamate taken from control astrocytes or astrocytes deprived of glucose (6 h) was equally toxic to pure neuronal cultures, demonstrating no alterations in glutamate removal between control and glucose-deprived cells. However, neurons in mixed cortical cell cultures deprived of glucose showed increased neuronal cell death over those maintained in glucose-containing medium when exposed directly to equimolar concentrations of either glutamate or NMDA.Similarly, this increased neuronal death in glucose deprived mixed cortical cultures was shown across several different time points using constant concentrations of either glutamate or NMDA. Lastly, we show that neurons in our mixed cortical cultures are fully protected from excitotoxic cell death when system xc-and NMDA receptor inhibitors are added up to two hours following the initiation of glucose deprivation. Overall, our data reveal that neither enhanced system xc-expressionnor impaired glutamate uptake could account for the neuronal cell death induced by glucose deprivation, but that energy deprived neurons appear simply more susceptible to excitotoxic insults. Therefore, physiological levels of glutamate releasedfrom astrocyte system xc-maybe sufficient to mediate neuronal cell death under aglycemic conditions.