Diabetes or hyperglycemia exacerbates brain damage induced by ischemia and reperfusion. One factor responsible for this increased damage is enhanced acidosis during ischemia. It has been reported that astrocytes are vulnerable to hypoxia under acidic condition; therefore, astrocytes may also be targets of hyperglycemia. The objectives of the present study are to determine whether hyperglycemia recruits astrocytes during the damage process and whether astrocytes norphological or functional alterations will lead to increased production of inducible NO synthases (iNOS) and NO metabolite, peroxinitrate (ONOO-) Streptozotocin-induced 4 weeks diabetic rats subjected to 5 min of forebrain ischemia and followed by 30 min, 6 hrs, 1-, and 3- days of recovery. Astrocyte morphology was studied using GAFP immunofluorescent staining and electron microscopy. Productions of iNOS and peroxynitrate were measured by immunohistochemistry and western blot analysis. GFAP immunocytochemistry showed that ischemia activated astrocytes in the cortex and hippocampus as reflected by increased numbers of GFAP positive cells, increased numbers of processes and enlarged cell bodies. The astrcoyte activation was observed after 30 min of reperfusion, peaked at 3 days and remained elevated after 7 days reflow. In contrast, with the preexisting diabetic condition, astrocytes were activated after 30 min of reperfusion, however, number of GFAP positive cells significantly reduced after 3 days. Ultrastructural studies revealed that diabetic hyperglycemia caused astrocytic chromatin condensation, nuclear shrinkage and mitochondrial swelling after 1day reperfusion. Western blot analysis demonstrated that while iNOS increased after 6h-3d reperfusion in both diabetic and non-diabetic animals, ONOO- increased only in diabetic rats after 3d reperfusion. Because ONOO- is produced when NO reacts with superoxide, this results suggest that diabetic hyperglycemia may increase ONOO- production by enhancing the formation of superoxide. To test whether was the case, we injected hydroethidine, a marker for in vivo production of superoxide, into the rats before inducing ischemia. After 3 days of recirculation, the animals were sacrificed and brains were sectioned and examined using a confocal laser-scanning microscope at the excitation of 480 nm and emission of 567 nm. The results demonstrated that diabetes enhanced production of superoxide in vivo after ischemia and reperfusion episode. Collectively, these data suggest that astrocytes are targets of hyperglycemic ischemia. Increased production of ONOO- and superoxide my play a major role in mediating the detrimental effects of hyperglycemia on cerebral ischemia
