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J. Kogo, Y. Takeba, T. Kumai, K. Kuribayashi, Y. Munemasa, Y. Hayashi, Y. Kitaoka, S. Ueno, S. Kobayashi; TNF– Production in Glutamate–induced Apoptotic Neuronal Cell Death . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5135.
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Purpose:Glutamate–induced apoptotic neuronal cell death has been implicated in several ocular diseases. TNF–α also has shown to be involved in certain ocular disorders such as glaucoma and HIV–related optic neuropathy. We examined the involvement of TNF–α in glutamate–induced apoptotic pathways in neuronal cells. Methods:Pheochromocytoma cells of rat (PC12h) neuronaly differentiated by 100 ng/ml nerve growth factor (NGF) and 1% FCS in three days. Glutamate (1mM) was exposed to the cells for 24 hours, and the cell morphologies were examined using microscopy for confirmation of apoptosis. Apoptotic cells were detected by TDT–dUTP terminal nick–end labeling (TUNEL) assay. Cell proliferation was also assessed using MTS assay. The production of TNFα in the supernatant fluid of cultured medium after exposure of glutamate was measured by Enzyme–linked immunosolvent assay (ELISA) . Results:Glutamate–treated neuronal PC12h cells had morphologically typical alteration of adherent cells with on–going apoptosis as becoming rounded, condensed, detached from the dish. Glutamate significantly increased the number of TUNEL positive cells as compared with non–treated cells. MTS assay showed that the cell proliferation was decreased by glutamate. There was an increase in TNF–α production in glutamate–treated neuronal PC12h cells. Conclusions: Our results suggest the involvement of TNF–α in glutamate–induced apoptotic neuronal cell death.
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