**Pyridoxine Deficiency Exacerbates Neuronal Damage after Ischemia by Increasing Oxidative Stress and Reduces Proliferating Cells and Neuroblasts in the Gerbil Hippocampus**

**Hyo Young Jung 1, Woosuk Kim 2, Kyu Ri Hahn 1, Min Soo Kang 3, Tae Hyeong Kim 3, Hyun Jung Kwon 4, Sung Min Nam 5, Jin Young Chung 6, Jung Hoon Choi 3, Yeo Sung Yoon 1, Dae Won Kim 4, Dae Young Yoo 7,\* and In Koo Hwang 1,\***


Received: 30 June 2020; Accepted: 29 July 2020; Published: 4 August 2020

**Abstract:** We investigated the effects of pyridoxine deficiency on ischemic neuronal death in the hippocampus of gerbil (*n* = 5 per group). Serum pyridoxal 5 -phosphate levels were significantly decreased in Pyridoxine-deficient diet (PDD)-fed gerbils, while homocysteine levels were significantly increased in sham- and ischemia-operated gerbils. PDD-fed gerbil showed a reduction in neuronal nuclei (NeuN)-immunoreactive neurons in the medial part of the hippocampal CA1 region three days after. Reactive astrocytosis and microgliosis were found in PDD-fed gerbils, and transient ischemia caused the aggregation of activated microglia in the stratum pyramidale three days after ischemia. Lipid peroxidation was prominently increased in the hippocampus and was significantly higher in PDD-fed gerbils than in Control diet (CD)-fed gerbils after ischemia. In contrast, pyridoxine deficiency decreased the proliferating cells and neuroblasts in the dentate gyrus in sham- and ischemia-operated gerbils. Nuclear factor erythroid-2-related factor 2 (Nrf2) and brain-derived neurotrophic factor (BDNF) levels also significantly decreased in PDD-fed gerbils sham 24 h after ischemia. These results suggest that pyridoxine deficiency accelerates neuronal death by increasing serum homocysteine levels and lipid peroxidation, and by decreasing Nrf2 levels in the hippocampus. Additionally, it reduces the regenerated potentials in hippocampus by decreasing BDNF levels. Collectively, pyridoxine is an essential element in modulating cell death and hippocampal neurogenesis after ischemia.

**Keywords:** pyridoxine deficiency; ischemia; gerbil; homocysteine; cell death; glia; neurogenesis

#### **1. Introduction**

Vitamin B6 vitamers consist of pyridine derivatives such as naïve and phosphorylated forms of pyridoxine, pyridoxal, and pyridoxamine. Intakes of B6 vitamers are absorbed in the intestine and transformed into its active form, pyridoxal 5 -phosphate (PLP), in the liver. However, among B6 vitamers, naïve forms can cross the blood–brain barrier in the brain [1]. PLP acts as a coenzyme or cofactor in more than 100 reactions associated with energy metabolism and neurotransmitter synthesis. In addition, vitamin B6 has antioxidant properties that quench reactive oxygen [2] and reduce the formation of advanced glycation end-products [3,4].

Brain ischemia is one of the major life-threatening diseases worldwide, and it debases the quality of life in survivors. Mongolian gerbils (*Meriones unguiculatus*) are used in animal models for brain ischemia because they have incomplete posterior communicating arteries that cause brain ischemia only by occlusion of the common carotid artery for 5 min in the neck region [5]. However, more sophisticated models with complete interruption of cerebral blood flow have been implemented in rats [6–8] and mice [9], which are more widely used in research. Several studies demonstrate that PLP has neuroprotective effects against various neurological diseases including ischemia [10,11], vascular dementia [12], Parkinson's disease [13], and cortical damage [14]. In addition, pyridoxine treatment increases proliferating cells and neuroblasts in the dentate gyrus [15]. There is conflicting evidence of dietary vitamin B6 on cardiovascular diseases in human studies [16–19]. Plasma vitamin B6 levels are inversely related to risk and incidence of cardiovascular diseases in the United States, Japan, and Korea [16–18], while a Finnish study demonstrates that there is no relationship between vitamin B6 intake and cardiovascular diseases [19].

In contrast, feeding with pyridoxine deficient diets causes cognitive impairment in normal mice [20] and in animal models for Alzheimer's disease [21]. Additionally, pyridoxine deficiency increases homocysteine levels because pyridoxine is used in homocysteine metabolism [22]. Increased homocysteine is considered as a risk factor for stroke [23], and it facilitates the production of reactive oxygen species by auto-oxidation [24]. However, there are no studies on the effects of pyridoxine deficiency on neuronal cell death after ischemia in the hippocampus. In this study, we examined the effects of pyridoxine deficiency on ischemia-induced cell death based on the oxidative stress in the hippocampus after 5 min of forebrain ischemia.

#### **2. Results**

#### *2.1. Pyridoxine Deficiency Decreases PLP and Increases Homocysteine Levels in Serum*

In the Control diet (CD)-fed sham group, PLP and homocysteine levels were 32.5 ± 12.1 μmol/L and 4.91 ± 2.05 μmol/L in the serum, respectively. In the Pyridoxine-deficient diet (PDD)-fed sham group, PLP levels were dramatically decreased to 0.298 ± 0.205 μmol/L, while homocysteine levels were significantly increased to 57.4 ± 16.9 μmol/L. Transient forebrain ischemia decreased PLP levels in CD- and PDD-fed groups, although the statistical significance was not detected between CD- and PDD-fed groups. PLP levels were significantly lower in the PDD-fed group compared to the CD-fed group three and four days after ischemia. In contrast, homocysteine levels were maintained with significant increases in PDD-the fed group compared to that in the CD-fed group three and four days after ischemia, although homocysteine levels were slightly higher after ischemia compared to that in the sham group. The two-way analysis of variance (ANOVA) test showed that there were no interactions between ischemia and PDD diets in PLP and homocysteine levels in the serum (Figure 1A,B).

**Figure 1.** Pyridoxine deficiency decreases serum pyridoxal 5 -phosphate (PLP) levels (**A**) and increases serum homocysteine levels (**B**) in the control diet (CD)- and pyridoxine-deficient diet (PDD)-fed gerbils of sham- and ischemia-operated groups. Data were analyzed with a two-way ANOVA test followed by Bonferroni post-hoc tests (*n* = 7 per group; \* *p* < 0.05, significant difference between CD- and PDD-fed group). All data are expressed as mean with standard deviation.

#### *2.2. Pyridoxine Deficiency Causes Early Neuronal Death after Ischemia*

In the CD-fed sham group, neuronal nuclei (NeuN)-positive neurons were abundantly observed in all hippocampal regions, including the CA1 region, and the same observation was also obtained in the PDD-fed sham group. In the PDD-fed sham group, the number of NeuN-positive neurons was slightly decreased to 94.8% of the CD-fed sham group. In the CD-fed ischemic group, numerous NeuN-positive neurons were found in the hippocampus three days after ischemia, while NeuN-positive neurons were decreased in the medial side of the CA1 region and not in the lateral region three days after ischemia. The number of NeuN positive neurons was significantly decreased in the PDD-fed group compared to that in the CD-fed sham group to 83.2% of the CD-fed sham group. Four days after ischemia, NeuN-positive neurons were prominently decreased in the hippocampal CA1 region of the CD- and PDD-fed ischemia group to 4.9% and 4.4% of the CD-fed sham group. However, there were no significant differences in the number of NeuN-positive neurons between groups. The two-way ANOVA test demonstrated that there were no interactions between ischemia and PDD diets in neuronal numbers in the hippocampal CA1 region (Figure 2A,B).

**Figure 2.** Immunohistochemical staining for neuronal nuclei (NeuN) in the whole hippocampus (**A**) and its magnified CA1 region (**B**) in the CD- and PDD-fed gerbils of sham- and ischemia-operated groups. Note that fewer NeuN-positive cells (arrows) are found in the medial side of the stratum pyramidale (SP) in the PDD-fed gerbils three days after ischemia. SO, stratum oriens; SR, stratum radiatum. Scale bar = 400 μm (**A**), 50 μm (**B**). The number of NeuN-immunoreactive nuclei in the CA1 region compared to the CD-fed sham group per section, in all the groups, is 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.
