*2.5. Pyridoxine Deficiency Increases Lipid Peroxidation and Decreases Nrf2 and BDNF Expression*

In the PDD-fed sham group, malondialdehyde (MDA) levels in the hippocampus were higher (43.5%) compared to that in the CD-fed sham group. MDA levels in CD- and PDD-fed groups were dramatically increased three hours after ischemia to 368.0% and 395.0% of their respective sham gerbils and thereafter, MDA levels decreased with time after ischemia in both CD- and PDD-fed groups. Twenty-four hours after ischemia, MDA levels in CD- and PDD-fed groups showed 180.4% and 222.5% of their respective sham group. MDA levels were significantly higher in the PDD-fed group compared to that in the CD-fed group after ischemia, and this was not observed in the sham group (Figure 5A).

**Figure 5.** Measurement of malondialdehyde (MDA) levels (**A**) in the hippocampus and Western blot analysis of nuclear Nrf2 (**B**) and total mature brain-derived neurotrophic factor (BDNF) levels (**C**) in the CD- and PDD-fed gerbils of sham- and ischemia-operated groups (*n* = 6–7 per group; \* *p* < 0.05, significant difference between CD- and PDD-fed groups; # *p* < 0.05, significant difference between shamand ischemia-operated groups). All data are expressed as mean with standard deviation.

In the PDD-fed sham group, nuclear factor erythroid-2-related factor 2 (Nrf2) levels in nuclear fraction decreased compared to that in the CD-fed sham group, although statistical significance was not detected between groups. Twenty-four hours after ischemia, nuclear Nrf2 levels were slightly, but not significantly, increased in the CD-fed group, but they were maintained in the PDD-fed group. At this time point, nuclear Nrf2 showed significantly lower levels (71.6% of the CD-fed group) in the PDD-fed group (Figure 5B).

In the sham group, brain-derived neurotrophic factor (BDNF) levels in the PDD-fed gerbils were significantly decreased to 71.1% of CD-fed gerbils. Moreover, BDNF levels were significantly decreased in the hippocampus 24 h after ischemia/reperfusion in CD- and PDD-fed gerbils, and significantly lower levels (67.5% of CD-fed gerbils) of BDNF were found in the PDD-fed gerbils 24 h after ischemia (Figure 5C).

The two-way ANOVA test indicated that ischemia and PDD diets significantly changed MDA levels (df = 3, F = 3.807, *p* = 0.0193) in the hippocampus, while there were no interactive effects of ischemia and PDD diets on Nrf2 and BDNF expression in the hippocampus.

#### **3. Discussion**

Vitamin B6 is one of the essential vitamins that maintain the health of the nervous system in mammals, which should be taken in sufficient quantity from the food because it is not synthesized in mammals [25,26]. Deficiency of vitamin B6 causes cognitive function impairment, hippocampal neurogenesis reduction, cardiovascular disease, and Alzheimer's disease [20,27]. In the present study, we observed the effects of pyridoxine deficiency on cell death in the hippocampal CA1 region and on neuroblasts in the dentate gyrus after transient forebrain ischemia. We observed the changes in serum levels of PLP and homocysteine because vitamins B6, B12, and folic acid have been reported to be associated with the regulation of the methionine–homocysteine cycle [22]. PDD-fed gerbils showed a significant reduction in serum PLP levels in sham-operated animals as well as ischemic animals three and four days after ischemia. This result is consistent with our previous study that PDD-fed mice showed a significant reduction in serum and hippocampal PLP levels [20]. In contrast, pyridoxine deficiency significantly increased serum homocysteine levels in sham- and ischemia-operated animals and showed hyperhomocysteinemia, which is characterized by > 15 μmol/L homocysteine levels in the blood. Vitamin B6, B12, and folic acid are associated with homocysteine metabolism [18], and our previous study showed the presence of hyperhomocysteinemia in folic acid deficient gerbils, and

this condition increased the DNA damage in the hippocampus after ischemia [28]. Moreover, many studies demonstrated that hyperhomocysteinemia caused neuronal damage, cognitive impairments, and psychiatric diseases [29–33].

To confirm whether pyridoxine deficiency facilitates neuronal death in the hippocampal CA1 region, we conducted immunohistochemical staining of NeuN, a marker for mature neurons, in the hippocampus three and four days after ischemia because neuronal death was detected in the CA1 region four days after ischemia. In the sham group, NeuN-positive neurons slightly decreased in the CA1 region, although there is no statistical significance between the CD- and PDD-fed gerbils. A study showed that hyperhomocysteinemia significantly increased neuronal death in the rat hippocampus [34]. In the present study, we observed that pyridoxine deficiency significantly reduced the number of NeuN-positive neurons in the medial side of the CA1 region, and this suggests that pyridoxine deficiency facilitates neuronal death in the CA1 region after ischemia. However, we did not observe any significant changes in the number of NeuN-positive nuclei four days after ischemia in CD- and PDD-fed gerbils because most of the neurons showed degeneration in the CA1 region. Pyridoxine deficiency also showed activation of astrocytes and microglia in the hippocampus of sham-operated gerbils, and microglial aggregation was found in the medial side of the stratum pyramidale three days after ischemia/reperfusion because of neuronal death in this region. These results suggest that pyridoxine deficiency facilitates glial activation in the hippocampal CA1 region of sham- and ischemia-operated gerbils three days after ischemia. Our study provides new insight into the relationship between pyridoxine deficiency and glial activation in the hippocampus. We could not elucidate the mechanisms of glial activation in the hippocampus of PDD-fed gerbils. One hypothesis is hyperhomocysteinemia induced by pyridoxine deficiency because treatment with homocysteine significantly increased reactive gliosis in astrocytes and microglia in rats [35], and excess homocysteine causes toxicity in the brain [36,37].

We examined the possible mechanisms of pyridoxine deficiency in the hippocampal neuronal death after ischemia based on oxidative stress due to the presence of antioxidant potentials in vitamin B6, which can quench hydroxyl radicals similar with vitamin C. In contrast, transient forebrain ischemia increases oxidative stress, including DNA damage and lipid peroxidation in the hippocampus [24,38–41]. In the present study, lipid peroxidation measured by MDA levels was significantly increased 3 h after ischemia in the CD-fed gerbils and thereafter decreased with time after ischemia. This result is consistent with previous studies that MDA products increased as an accumulation of aldehyde in various neurological disorders such as Alzheimer's disease, ischemia, and Parkinson's disease [24,40–45]. Additionally, MDA levels in this study were significantly higher in PDD-fed gerbils than in CD-fed ones 3, 12, and 24 h after ischemia, and not in the sham-operated group. This result suggests that PDD increases reactive aldehyde in the hippocampus, which aggravates neuronal death in the hippocampal CA1 region after transient forebrain ischemia. Changes in Nrf2 expression were also observed in the hippocampus because it is believed to be a key transcription factor in decreasing reactive oxygen species [46]. There have been reports that Nrf2 decreased ischemic damage by reducing oxidative stress [47], while Nrf2 deficient mice showed vulnerability to oxidative stress [48]. In the present study, lower Nrf2 expression levels were found in the PDD-fed gerbils than in the CD-fed gerbils 24 h after ischemia, and this result suggests that the reduction of Nrf2 may be associated with early neuronal damage after transient forebrain ischemia.

In this study, we also observed the effects of pyridoxine deficiency on regenerative potentials based on proliferating cells and neuroblasts in the dentate gyrus because we observed the reduction of neuroblasts in the mice hippocampus [20]. In gerbil brain, the proliferating cells and neuroblasts were reduced in the dentate gyrus of sham- and ischemia-operated animals, and this result suggests that pyridoxine deficiency reduces the regeneration potentials in ischemic brain. Additionally, we observed BDNF levels in the hippocampus because it modulates the hippocampal neurogenesis [49,50]. Pyridoxine deficiency and/or brain ischemia decreased BDNF levels in the hippocampus, and BDNF levels were lowest in PDD-fed ischemic gerbils. This result supports the immunohistochemical finding

for proliferating cells and neuroblasts in the dentate gyrus. Interestingly, Nrf2 is one of the essential elements to regulate the hippocampal neurogenesis [51] and homocysteine reduced cell differentiation in chicken embryonic brain [52]. However, neurogenesis in the dentate gyrus is unlikely to be related to regeneration; rather, migrating neuroblasts from SVZ may contribute to regeneration [53,54]. Moreover, the initial increase in neurogenesis in the dentate gyrus may be accompanied by its significant decrease at a later stage after ischemia [7] due to the limited pool of stem cells in this region [55].

In conclusion, pyridoxine deficiency facilitates neuronal death in the hippocampal CA1 region after transient forebrain ischemia by increasing homocysteine levels in the serum and lipid peroxidation in the brain, as well as reducing the Nrf2 levels in the hippocampus. Moreover, pyridoxine deficiency reduces the proliferating cells and neuroblasts, probably by reducing BDNF levels. This result suggests that pyridoxine is an essential element in fighting neuronal death and in increasing the regenerative potentials after ischemia.
