Summary:The mechanisms of ischemic cell damage are still not fully understood, It has been shown that alpha-amino-3-hy droxy-5 -methy l-isoxazole-4-propionate (AMPA) /kainate receptor antagonists, such as 6-nitro-7-sulphamoyl-benzo-(f)-quinoxaline-2, 3-dione (NBQX) , are neuroprotective in models of transient forebrain isch emia, even when applied during recovery , indicating that nonNMDA receptors may play a pivotal role in ischemic cell damage, In the present series of experiments, we studied whether transient cerebral ischemia causes changes in the extent of mRN A editing of AMP A/kainate receptor subunits, a reaction critical for the control of calcium flux through nonNMDA receptor ion channels. Transient cerebral ischemia was produced in rats using the four-vessel occlusion (4-VO) model. After 30 min of ischemia, brains were recirculated for 4, 8, or 24 h. Total RNA was extracted from the cortex, striatum, and hip pocampus in order to analyze the extent of mRNA editing of the glutamate receptor subunits GluR2, GluR5 , and GluR6. RNA was converted by reverse transcription into cDNA, which was used as a template for subunit-specific polymerase chain reaction (PCR) to amplify a product across the edited base A (A edited to I in the second transmembrane-spanning regions of GluR2, GluR5 , and GluR6). PCR products were analyzed with the restriction enzyme Bbv 1, which recognizes the cDNA sequence GCAGC originating from unedited but not that originating from edited GluR2, GluR5, or GluR6 mRNA (GCGGC, the base I is read as G) . Restriction digests were electro phoresed, and the bands visualized with ethidium bro mide and then photographed. The extent of mRNA edit ing of the different subunits was quantified using image analysis and appropriate standards. In all control brainsThe molecular mechanisms of cell damage in duced by transient cerebral ischemia are still not fully understood (for reviews, see Kogure et aI., 548 studied, GluR2 mRNA was completely edited and re mained so after reversible cerebral ischemia. The extent of GluR5 mRNA editing was significantly upregulated in the striatum (from 39 ± 6% in controls to 57 ± 9 and 56 ± 7 after 4 and 8 h of recovery , respectively , p < 0. 05 ver sus control) but not in the cortex and hippocampus. The extent of GluR6 mRN A editing was significantly reduced after 24 h of recovery : in the cortex, from 92 ± 1 to 78 ± 6% (p < 0. 01); in the striatum, from 91 ± 2 to 79 ± 1% (p < 0. 001) ; and in the hippocampus, from 90 ± 3 to 80 ± 2% (p < 0. 05 ). A significant reduction was already apparent in the striatum after 4 h of recovery (p < 0. 05 ). Results indicate that mRNA editing is regulated differently in each of the glutamate receptor subunits GluR2, GluR5 , and GluR6 after transient cerebral ischemia. The isch emia-induced upregulation of GluR5 mRNA editing ob serve d in the striatum may be indicative of a higher sen sitivity to transient ischemia of neurons that exhibit a large fraction of unedited GluR5 mRNA. This assumption is corroborated by the observation (Mackle...
