Quantitative analysis of the number and the proliferation status of NG2 glia in the CA1 region of the hippocampus following transient forebrain ischemia. (A, B) The number of NG2 glia (namely, NG2⁺/Iba1⁻ cells) in the pyramidal cell layer (PCL) was counted in five areas from the middle third of the CA1 subfield along 160 μm of the pyramidal band in each hemisphere. The number of labeled cells in the stratum radiatum (SR) was counted in five areas (160×160 μm per field) in each hemisphere. Three sections from three animals per group were included in this analysis. The numbers of NG2 glia in the pyramidal cell layer significantly increase, reach a maximum 3 days after reperfusion, and remain elevated until day 28, while those in the stratum radiatum progressively increase until day 14 and then slightly decrease on day 28. (C, D) Representative images of triple-labeling for NG2, Iba1, and Ki67, showing that NG2 glia (arrowheads in C and D) and NG2-positive microglia/macrophages (arrows in C and D) have Ki67-labeled nuclei in both the pyramidal cell layer (C) and the stratum radiatum (D) at 3 days after reperfusion. Cell nuclei are stained with DAPI. (E, F) Temporal profile of proliferating NG2 glia in the pyramidal cell layer (E) and the stratum radiatum (F) in the hippocampal CA1 region after reperfusion. The number of NG2⁺/ Iba1⁻/Ki67⁺ cells is divided by the total number of NG2⁺/Iba1⁻ cells for each area described above to indicate the percentage of proliferating NG2 glia. The proportion of Ki67-labeled NG2 glia is more than 20% of all NG2 glia in both layers on day 3 and decreases abruptly on day 7. Note that in the stratum radiatum, this proportion remains at about 5% up to day 28. The data are expressed as the mean ± SEM. *p<0.05, **p<0.01, and ***p<0.001 vs shamoperated controls based on one-way ANOVA followed by Bonferroni’s multiple comparisons test. Scale bars represent 10 μm in C and D.