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Frederike Dijk, Elza Kraal-Muller, Willem Kamphuis; Ischemia-Induced Changes of AMPA-Type Glutamate Receptor Subunit Expression Pattern in the Rat Retina: A Real-Time Quantitative PCR Study. Invest. Ophthalmol. Vis. Sci. 2004;45(1):330-341. doi: https://doi.org/10.1167/iovs.03-0285.
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purpose. To investigate whether the previously observed decrease in immunoreactivity of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits GluR1, -2, -3, and -4 after ischemia–reperfusion in the rat retina is associated with changes at the mRNA expression level. Furthermore, to study possible changes in the ratios of alternative splice variants of GluR2 and -4 and possible changes in the subunit composition of the receptor complex after ischemia–reperfusion. The ischemia-induced changes were related to expression levels of immediate early genes, c-fos and c-jun, and to expression levels of different cell-type–specific transcripts.
methods. A 60-minute ischemic event was induced unilaterally in the rat eye by cannulating the anterior chamber and raising the intraocular pressure. Reperfusion was allowed to occur for 2 hours up to 28 days. Total RNA was isolated from the retinas and transcript levels were assessed by real-time quantitative PCR (qPCR).
results. A differential decrease was observed in the expression levels of all AMPA-type GluR subunits 2 hours after ischemia–reperfusion, with a significant downregulation of GluR2 and -3 transcript levels. At the long-term (72 hours–4 weeks), expression levels for all four subunits were decreased by approximately 64%. No changes were observed, either in the expression ratio of GluR2 and -4 splice variants, or in the relative expression of the different subunits. Immediate early genes c-fos and c-jun were transiently upregulated. Expression levels of the ganglion-cell–specific transcripts Thy-1 and neurofilament and of the AII-amacrine–specific transcript parvalbumin decreased after ischemia–reperfusion, whereas the ON bipolar cell transcripts mGluR6 and PKCα did not show ischemia-induced changes.
conclusions. Shortly after ischemia–reperfusion immunolabeling of GluR1, -2/3, and -4 is strongly decreased, whereas the corresponding mRNA levels are not affected, indicating degradation at the protein level. In contrast, the GluR2 mRNA level is reduced, whereas immunostaining is not yet affected, suggesting that the GluR2 protein is relatively stable under postischemia conditions. The long-term decrease in mRNA levels of all AMPA-type GluR subunits suggests that ischemia affects a main component of the excitatory retinal neurotransmission. It remains to be investigated whether these changes contribute to the subsequent neurodegeneration.
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