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W Kamphuis, F Dijk; Ischemia-Induced Changes of AMPA-Type Glutamate Receptor Subunit 1 (GluR1) in the Rat Retina: Immunocytochemical and Real-Time QPCR Studies . Invest. Ophthalmol. Vis. Sci. 2002;43(13):781.
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Purpose:To study the underlying mechanisms of the observed changes in immunoreactivity of the AMPA-type subunits GluR1-4 after ischemia-reperfusion in the rat retina. The involvement of calpain-mediated proteolysis and changes at the gene expression level were investigated. Methods:(i) The previously reported reduction of GluR1 immunostaining of GluR1 postischemia was detected using an antibody directed against the GluR1C-terminal domain. This site is also a specific target for calpain activity and ischemia-induced activation of calpain may result in the loss of this epitope. Staining with an N-terminal directed antibody (GluR1N) would reveal the presence of the remainder of the subunit. (ii) Real-time quantitative PCR was used to assess the transcript levels of GluR1-4 with respect to different housekeeping genes, at various postischemic reperfusion times. Results:(i) A profound reduction GluR1C immunostaining in the IPL was observed at 2 hours postischemia and this change precedes the induction of TUNEL-positivity in somata in Inner Plexiform Layer and Ganglion Cell Layer. The staining pattern of the GluR1N antibody in controls was similar to that of the GluR1C antibody. Postischemic changes included a rapid decrease of GluR1N staining, parallel to the GluR1C immunoreactivity decline. (ii) RT-QPCR allowed us to describe the expression profile of the AMPA-type GluR subunits and the different splice variants known. Preliminary data indicate that the initial postischemia stages are not accompanied by altered expression levels of the GluR subunit encoding genes. Conclusion:These results show that: (i) Calpain-mediated proteolysis is not involved in the decrease of GluR1 staining, and (ii) A rapid down-regulation of gene expression is not underlying the decrease of GluR immunostaining.
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