Abstract

The objective of this study is to determine the cellular and molecular mechanism of hyperglycemia enhanced neuronal damage induced by transient global ischemia.

Acute hyperglycemia and diabetes were induced in this study. Histopathological examination was used to study ischemic damage. To test whether mitochondria-initiated cell death pathway was involved in diabetes-exaggerated neuronal death, release of cytochrome c, caspase-3 activation, caspase-9 cleavage and caspase-8 activity were measured. Electron microscopy was used to identify whether neuron died of apoptosis or necrosis. To study if inflammatory responses play a role in diabetes-aggravated neuronal death, ICAM-1 expression on microvessels was measured using immunohistochemistry, and further confirmed by Western analysis. Western analysis also was used to measure levels of interlukin-1β (IL-1β) and TNF-α. To study the role of MAPKs in hyperglycemia-aggravated ischemia, Western blot analysis was used to measure phosphorylation of JNK and p38 MAPK in both cytosolic and nuclear fractions in the cortex and dentate gyrus. To study if astrocytes were targets of diabetes-exaggerated ischemia, immunohistochemistry of GFAP was used to measure activation of astrocytes and electron microscopy was used to study astrocytic ultrastructure alteration. To study the role of free radicals in diabetes-aggravated brain damage, iNOS and nitrotyrosine were measured using Western blot analysis. Superoxide production was measured using hydroethidine method, and its colocalization was measured using double immunostaining with mitochondria marker and neuron, macrophages, endothelial cell and astrocytes marker.

The results suggest that (1) diabetic hyperglycemia activates mitochondria-initiated neuronal cell death pathway; (2) inflammatory responses may mediate diabetic hyperglycemia-aggravated brain damage; (3) JNK may play a role in normoglycemic ischemia, but may not contribute to the adverse effect of hyperglycemia; p38 MAPK may not play a major role in ischemia; (4) dysfunction of astrocytes and the increased production of free radicals may play an important role in exaggerating brain damage in diabetic subjects.