*4.3. Induction of Severe IR Injury*

Severe IR injury in the hippocampus was induced as follows. As described in a previous study [6], in brief, the gerbils were anesthetized with 2.5% isoflurane (4 L/min; JW Pharmaceutical Corporation, Seoul, Korea). Midline cervical incision was executed in the neck, and bilateral common carotid arteries, which supply arterial blood to the brain, were isolated from the carotid sheath and ligated with clips (Fine Science Tools, Foster City, CA, USA) for 15 min to develop severe IR injury in the forebrain, which contains the hippocampus. The complete stop of the blood supply was confirmed by the right and left central arteries located in the retinae using HEINE K180 ophthalmoscope (Heine Optotechnik, Herrsching, Germany). The clips were removed to restore the blood supply and the incision region was sutured. Body temperatures were controlled at normothermia (37 ± 0.5 ◦C) using a heating lamp (Harvard Apparatus, Holliston, MA, USA) and TR-100 rectal temperature probe (Fine Science Tools, Foster City, CA, USA). The gerbils of all sham groups received identical surgery without the ligation of the arteries. After the sham and IR operation, the gerbils were housed in adequate rooms (about 24 ◦C of temperature and about 55% of relative humidity).

## *4.4. Preparation of Histological Sections*

Brain sections containing the hippocampus were prepared in the usual way. In brief, the gerbils were deeply anesthetized with urethane (1.5 g/kg, intraperitoneal injection). Under the deep anesthesia, the gerbils were rinsed by transcardial perfusion of 50 mM phosphate-buffered saline (pH 7.4) and fixed with 4% paraformaldehyde. Thereafter, the fixed brains were obtained from the skulls and more fixed in the 4% paraformaldehyde overnight and soaked in 25% sucrose for cryoprotection. Thereafter, coronal sections of 30-μm thickness were made using SM2010 R sliding microtome (Leica Biosystems, Wetzlar, Germany).
