**4. Materials and Methods**

#### *4.1. Experimental Animals*

Male Mongolian gerbils (5 weeks of age) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The animals were housed and cared based on the Guide for the Care and Use of Laboratory Animals (8th edition, 2011). Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-190408-2) on 14 May 2019.

After a week of acclimation, gerbils were fed with pyridoxine-deficient diet (PDD, D10001, Research Diets) and its control diet (CD, D15501R, control, Research Diets, New Brunswick, NJ, USA) for 56 days as described in the previous study [20] since the half-life of the elimination of pyridoxine exceeds 15 to 20 days [56]. All animals used in this study were sacrificed on the 56th day of diet feeding.

#### *4.2. Ischemic Surgery*

Animals were anesthetized with 2.5% isoflurane (Baxter, Deerfield, IL, USA) and then carotid arteries were isolated from adjacent tissue and occluded using aneurysm clips for 5 min as previously reported [57]. Blood flow through carotid arteries was monitored in the central artery of the retinae using an ophthalmoscope (HEINE K180®; HEINE Optotechnik, Herrsching, Germany). Body temperature (37 ± 0.5 ◦C) was regulated by a thermometric blanket under the monitoring using a rectal temperature probe (TR-100; Fine Science Tools, Foster City, CA, USA) until recovered from anesthesia. Two animals with incomplete ischemic induction were excluded. The sham group received the same procedures except for carotid artery occlusion.

#### *4.3. Immunohistochemistry*

To show the morphological evidence of the changes in neurons, astrocytes, and microglia in the hippocampus, immunohistochemical staining was conducted for NeuN, GFAP, and Iba-1, respectively, as previously described [20,57]. In addition, proliferating cells and neuroblasts were visualized with the immunohistochemistry of Ki67 and DCX. Briefly, animals (*n* = 5 per group) were anesthetized with a mixture of 75 mg/kg alfaxalone and 10 mg/kg xylazine on the 56th day of diet feeding, and blood was obtained by cardiac puncture in the right ventricle. Thereafter, animals were perfused transcardially, and the brain was coronally sectioned with a 30 μm thickness between 2.0 and 2.7 mm caudal to the bregma based on gerbil stereotaxic coordinates [58]. Four sections located 150 μm apart were selected and incubated with each antibody; mouse anti-NeuN antibody (1:1000; Merck Millipore, Temecula, CA, USA), rabbit anti-GFAP antibody (1:1000; Merck Millipore), rabbit anti-Iba-1 (1:500; Wako, Osaka, Japan), rabbit anti-Ki67 (1:1000; Abcam, Cambridge, UK), or rabbit anti-DCX (1:2000; Abcam). Immunoreaction was visualized with 3,3 -diaminobenzidine tetrachloride (Sigma, St. Louis, MO, USA) in 0.1 M Tris-HCl buffer (pH 7.2). Sections were dehydrated and mounted on gelatin-coated slides in Canada balsam (Kanto Chemical, Tokyo, Japan).
