Neurogenesis in the brain continues throughout life and is promoted by brain insults including ischemia. There is no critical conclusion, however, about whether proliferated cells acquire neuronal function after ischemia. Transient global ischemia was produced by a four-vessel occlusion procedure in rats (n = 54). To label proliferative cells, rats were administrated with a single dose of 5-bromo-2'-deoxyuridine (BrdU) at 4, 6, 8, 10, 13, or 15 days after ischemia. Increases in BrdU-positive cells were detected in the hippocampal dentate gyrus at 5, 7, and 9 days after ischemia. To determine the phenotype of BrdU-positive cells, BrdU was administrated twice daily for 3 consecutive days during 6 to 8 days after ischemia. A basic helix-loop-helix transcription factor NeuroD at 7 and 14 days and an immature migrating neuronal marker doublecortin at 14 days after ischemia were expressed transiently in proliferative cells. These proliferative cells after ischemia differentiated to the phenotype of neuron at 28 days after ischemia. Furthermore, BrdU-positive neurons showed phosphorylation of extracellular signal-regulated kinase (ERK) by intracerebroventricular injection of N-methyl-D-aspartate (NMDA) at 28 and 56 days after ischemia as seen in surrounding mature neurons. The number of BrdU-positive neurons, which responded to NMDA stimulation, increased with time after ischemia and was greater than that of sham-operated animals. The present study provides evidence for in vivo ERK phosphorylation in response to NMDA stimulation of BrdU-positive neurons in the adult hippocampus after transient forebrain ischemia.
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