Characterization of NG2-positive microglia/macrophages in the CA1 region of the hippocampus following transient forebrain ischemia using double-labeling for NG2 and Iba1. (A) Resting microglia in both the pyramidal cell layer (PCL) and the stratum radiatum (SR) are devoid of NG2 in the hippocampal CA1 region of sham-operated rats. (B) The three-dimensional renderings of the above images indicate that the resting microglial cells of both strata exhibit a ramified morphology. (C) At 3 days after reperfusion, NG2 expression is induced in amoeboid-like macrophages with retracted processes (arrows) in the pyramidal cell layer, as well as in activated stellate microglial cells with evident processes (arrowheads) in the stratum radiatum. (D) The three-dimensional renderings of the above images indicate that NG2 expression is more prominent in activated microglia/macrophages of the pyramidal cell layer compared those of the stratum radiatum. (E) At 14 days after reperfusion, the labeling patterns of NG2 expression in activated microglia/macrophages in the pyramidal cell layer (arrows) and the stratum radiatum (arrowheads) remain unchanged in comparison to the 3-day time point. (F) Three-dimensional renderings of the above images, indicating that NG2 expression is more prominent in amoeboid-like macrophages in the pyramidal cell layer than in activated stellate microglia in the stratum radiatum. Cell nuclei are stained with DAPI. (G) Quantitative temporal analysis of the intensity of NG2 immunoreactivity in activated microglia/macrophages in the CA1 hippocampus during the post-ischemic period. Note that the relative intensity of microglial NG2 immunoreactivity in the pyramidal cell layer was significantly higher than that in the stratum radiatum by 7 days after reperfusion. 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. ns, not significant; ***p<0.001 vs sham-operated controls; ###p<0.001 vs pyramidal cell layer based on two-way ANOVA followed by Bonferroni’s multiple comparisons test. Scale bars represent 10 μm in A, C, and E; and 4 μm in B, D, and F.