*2.3. Pyridoxine Deficiency Facilitates the Activation of Astrocytes and Microglia after Ischemia*

In the CD-fed sham group, glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes and ionized calcium-binding adapter molecule 1 (Iba-1)-immunoreactive microglia had small cytoplasm with long and distinct processes. In the PDD-fed sham group, they had enlarged cytoplasm with thickened processes in the hippocampal CA1 region, and GFAP and Iba-1 immunoreactivities were significantly increased in this group, compared to that in the CD-fed sham group. Three days after ischemia/reperfusion, GFAP-immunoreactive astrocytes showed similar morphology in CD- and PDD-fed groups compared to the PDD-fed sham group, and GFAP immunoreactivity was similarly observed. In contrast, Iba-1-immunoreactive microglia showed similar morphology in CD-fed ischemic group three days after ischemia/reperfusion, but in the PDD-fed group, Iba-1-immunoreactive microglia were abundantly found in the medial side of the stratum pyramidale, where the neuronal death occurred. Additionally, Iba-1-immunoreactive microglia that were detected in the stratum pyramidale had round cytoplasm without processes, and Iba-1 immunoreactivity was significantly increased. Four days after ischemia/reperfusion, GFAP-immunoreactive astrocytes had punctuated cytoplasm with thickened processes in CD- and PDD-fed groups. Iba-1-immunoreactive microglia were abundantly observed in the stratum pyramidale of the CA1 region as well as the stratum oriens and radiatum in CD- and PDD-fed groups. There were no significant differences in the GFAP and Iba-1 immunoreactivities between CD- and PDD-fed groups after ischemia. However, the two-way ANOVA test indicated that ischemia and PDD diets significantly increased the GFAP (df = 2, F = 5.341, *p* = 0.0093) and Iba-1 immunoreactivity (df = 2, F = 19.06, *p* < 0.0001) (Figure 3A,B).

**Figure 3.** Immunohistochemical staining for GFAP (**A**) and Iba-1 (**B**) in the hippocampal CA1 region in the CD- and PDD-fed gerbils of sham- and ischemia-operated groups. Note that GFAP-immunoreactive astrocytes (arrows) and Iba-1-immunoreactive microglia (arrows) show morphological changes after PDD-diet and ischemic damage. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 50 μm. Relative optical density (ROD) corresponding to the percentage of GFAP and Iba-1 immunoreactivity value in the hippocampal CA1 region of the CD-fed sham group per section is shown (*n* = 7 per group; \* *p* < 0.05, significant difference between CD- and PDD-fed groups; # *p* < 0.05, significant difference between sham- and ischemia-operated groups). All data are expressed as mean with standard deviation.
