*2.4. Pyridoxine Deficiency Decreases Ischemia-Induced Proliferating Cells and Neuroblasts*

In all groups, Ki67-positive nuclei and cell bodies of doublecortin (DCX)-immunoreactive neuroblasts were mainly found in the subgranular zone of the dentate gyrus, and processes of neuroblasts branched out to the molecular layer of the dentate gyrus. However, there were significant changes in the number of proliferating Ki67-positive cells and the immunoreactivity of DCX-immunoreactive neuroblasts among groups. In the sham group, the DCX immunoreactivity was significantly decreased in the PDD-fed group compared to that in the CD-fed group, while the number of Ki67-positive cells was slightly decreased. Three and four days after ischemia/reperfusion, the number of Ki67-positive cells and the DCX immunoreactivity were significantly increased compared to the sham group, and there were fewer Ki67-positive cells and lower DCX immunoreactivity in the

PDD-fed group compared to that in the CD-fed group. However, the two-way ANOVA test indicated that ischemia and PDD diets significantly changes the number of proliferating Ki67-positive cells (df = 2, F = 8.816, *p* = 0.0008), and there were no interaction effects of ischemia and PDD diets in the density of DCX-immunoreactive neuroblasts (Figure 4A,B).

**Figure 4.** Immunohistochemical staining for Ki67 (**A**) and doublecortin (DCX) (**B**) in the dentate gyrus in the CD- and PDD-fed gerbils of sham- and ischemia-operated groups. Note that fewer proliferating Ki67-positive cells (arrows) and lower DCX immunoreactivity (arrows) are found in the PDD-fed gerbils than in the CD-fed ones. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 50 μm (**A**) and 100 μm (**B**). The number of Ki67-positive nuclei in the dentate gyrus and the ROD of DCX compared to the CD-fed sham group per section, in all the groups, are shown (*n* = 7 per group; \* *p* < 0.05, significant difference between CD- and PDD-fed group; # *p* < 0.05, significant difference between sham- and ischemia-operated groups). All data are expressed as mean with standard deviation.
