*4.1. Ethics Statement and Experimental Animals*

All animal care protocols and experimental procedures were approved by the Committee on Animal Use for Research at Hallym University (protocol # Hallym 2019-70, 21 February 2020). To test our hypothesis, experimental animal groups were divided as follows: sham (Vehicle, NAC) and GCI (Vehicle, NAC). All rats were housed in a consistently controlled environment (temperature: 22 ± 2 ◦C, humidity: 55 ± 5%, light regulation: 12-h light/dark cycle) and given water and standard feed by Purina (Gyeonggi-do, Korea) ad libitum. To minimize any suffering of experimental animals, we used 2~3% isoflurane anesthesia during all procedures. Additionally, this manuscript was written in accordance with the standards put forth in ARRIVE (Animals in Research: Reporting In Vivo Experiments) [69].

### *4.2. Global Cerebral Ischemia Surgery*

The experimental disease model of global cerebral ischemia was performed as previously described and reported [70]. To explain again in detail, rats were deeply anesthetized with 2~3% isoflurane, which was ventilated using mixed 70% nitrous oxide and 30% oxygen and kept at a body temperature of 37 ± 1 ◦C using a homeothermic monitoring system (Harvard Apparatus, Holliston, MA, USA). To consistently monitor systemic arterial blood pressure and to remove blood as needed, a catheter filled with 10 units of heparin was inserted into the femoral artery. Then, common carotid arteries located beside the tracheal muscle were carefully isolated and transiently occluded. In this dissecting process, we used a surgical microscope (SZ61, Olympus, Shinjuku, Japan) to enhance surgical accuracy and avoid vagus nerve impairment. At the same time, to monitor the electroencephalograph (EEG), electrodes were placed in two bilateral burr holes. To induce the ischemic condition, we drained blood (8~10 cc) from the femoral artery and set systemic arterial blood pressure within the range of 40 ± 10 mmHg. The isolated bilateral common carotid arteries were occluded with a surgical clamp (Fine Science Tools, Foster city, CA, USA). When the systemic arterial blood pressure range was within 40 ± 10 mmHg and the electroencephalograph reached a sustained isoelectric point, we maintained these conditions for 7 min to induce the global cerebral ischemic condition. After occlusion, blood circulation to the brain was restored by unclamping the device and reperfusing the removed blood. During this period, the systemic arterial blood pressure and EEG signal were consistently monitored and vital signs of the experimental animal were periodically monitored and adjusted as required. Experimental animal groups were immediately administered NAC to the intraperitoneal space following restoration of blood perfusion and normal EEG activity; vehicle groups were given the same volume of 0.9% normal saline.

### *4.3. Experimental Procedures and NAC Administration*

Planned experimental procedures were 3 days and 7 days. These time points mean the termination of GCI surgery. In the acute phase (3 days), GCI-induced neurodegenerative cascades were assessed using several immunostaining processes for histological evaluation. In the chronic phase (7 days), GCI-induced cognitive decline and neurological deficits were verified by behavior tests. Vehicle and NAC was administered daily during the acute and chronic phases. To investigate the effects of NAC post-administration to reduce GCI-induced hippocampal neurodegenerations, we used NAC (Sigma-Aldrich, St. Louis, MO, USA). NAC was dissolved with 0.9% normal saline and injected into the intraperitoneal space once per day for 3 days and 7 days at a dose of 150 mg/kg in the present study. Vehicle groups (sham and GCI) were administered the same volumes of 0.9% normal saline only.
