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AR Whitetree, LD Carter-Dawson; Blockage of the GLT-1 Glutamate Transporter Results in Peroxynitrite Formation in the Rat Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4531.
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Purpose: To investigate the hypothesis that increased extracellular glutamate increases peroxynitrite formation in retinal neurons. Methods: Right eyes of male Sprague-Dawley rats were injected intraocularly with 0.25, 0.5, 1.0, or 1.5 mM dihydrokainic acid (DHK) to block GLT-1 glutamate transporters. Left eyes were injected with an equal volume of vehicle. Under deep anesthesia, eyes were enucleated at 2, 4, 7, or 10 days post-injection. Posterior segments were fixed in 4% paraformaldehyde/1% glutaraldehyde in phosphate buffer. Retina, 1mm in diameter, was taken from the temporal inferior region of each eye, 2.5mm from the optic nerve, and vibrotome sections, 40 µM thick, were cut. The sections were immunolabeled with antibody to nitrotyrosine and visualized with Cy3-conjugated secondary antibody. Antibody specificity was tested using dithionite treatment. Images of retina from right and left eyes were taken using identical parameters with a Zeiss confocal microscope. Results: At 0.25 mM DHK, immunolabeling for nitrotyrosine was greater in the inner plexiform layer (IPL) and in neurons of the innermost layer of the inner nuclear layer (INL) of experimental eyes than in control eyes 2 days post-injection. Higher concentrations and longer exposure to DHK produced a graded increase in immunoreactivity in neurons in the innermost layer of the INL, in the IPL, and in neurons in the ganglion cell layer (GCL) of experimental retinas. Conclusion: Blockage of the GLT-1 glutamate transporter by intraocular DHK injection increases nitrotyrosine immunoreactivity in the IPL and cells of the GCL and INL. The increase in nitrotyrosine formation is indicative of peroxynitrite formation and potential compromise of cell function and viability.
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