Representative images showing the spatiotemporal profiles of NG2-positive cells in the CA1 region of the hippocampus following transient forebrain ischemia. (A~J) Double-labeling for NeuN and NG2 was performed on free-floating sections. (A, B) Weak NG2 expression is observed in small stellate cells (arrows) in both the pyramidal cell layer (PCL) and the stratum radiatum (SR) in the hippocampal CA1 region of sham-operated rats. The boxed area of the pyramidal cell layer (PCL) and stratum radiatum (SR) in A are enlarged in B. Note that pyramidal neurons are densely distributed in the pyramidal cell layer. (C, D) At day 3 after reperfusion, NG2 immunoreactivity appears to be increased in the CA1 hippocampus with prominent enrichment in the pyramidal cell layer, in which NeuN expression declines. (E~H) NG2 immunoreactivity tends to increase in both the pyramidal cell layer and the stratum radiatum at days 7 (E, F) and 14 (G, H) after reperfusion. Note that NeuN immunoreactivity has disappeared in the pyramidal cell layer by day 7. (I, J) At day 28 after reperfusion, NG2 immunoreactivity is still prominent in the pyramidal cell layer but appears to decrease in the stratum radiatum. Cell nuclei are stained with DAPI. Scale bars represent 50 μm in A, C, E, G and I, and 20 μm in B, D, F, H, and J. (K, L) Quantitative analysis of the intensity of NG2 expression in the pyramidal cell layer (K) and the stratum radiatum (L) in sham-operated control and ischemic rats. Note that the NG2 staining intensities in both the pyramidal cell layer and stratum radiatum significantly increase and peak at 7 days after reperfusion, and this enhanced expression is maintained for up to 28 days. n=3 rats per group. In each animal, five randomly selected areas in both strata of each section were analyzed in each of three sections. The data are expressed in arbitrary units (A.U.) as mean±SEM. ***p<0.001 vs sham-operated controls based on oneway ANOVA followed by Bonferroni’s multiple comparisons test.