*2.1. Global Cerebral Ischemia-Induced Neuronal Death and Zinc Accumulation Is Attenuated by Post-Administration of NAC*

Timeline showing the experimental design for 3 days and 7 days (Figure 1A). Whole GCI conducted experimental animals were monitored for arterial blood pressure and electroencephalography continuously during the before-isoelectric, isoelectric, and after-blood reperfusion condition (Figure 1B–D).

**Figure 1.** (**A**) Timeline representing the experimental design for 3 and 7 days following global cerebral ischemia (GCI). Experimental animals were given vehicle and *N*-acetyl-l-cysteine (NAC) (dosage: 150 mg/kg) once per day for 3 and 7 days. During the 7 days of the experiment, animals were subjected to behavioral outcome assessment by adhesive removal test and modified neurological severity score measurement. Global cerebral ischemia (GCI) induces electroencephalograph (EEG) and blood pressure changes, and timeline of experiment. (**B**) Normal blood pressure, resting EEG pattern. (**C**) Blood pressure decreased up to 40 (diastolic)–50 (systolic) mmHg, and EEG displayed isoelectric pattern. (**D**) Blood pressure and EEG pattern completely normalized after blood reperfusion.

To determine whether GCI-induced neuronal death and zinc accumulation was attenuated by NAC post-administration, we used FJB (Fluoro-Jade B) and TSQ (*N*-(6-methoxy-8-quinolyl) para-toluenesulfonamide staining at 3 days after GCI. The degenerating neurons were strongly present in the GCI–vehicle groups. When compared with vehicle-treated groups, NAC-treated groups showed significantly reduced numbers of degenerating neurons (Figure 2A). Quantified numbers of degenerating neurons are displayed using bar graphs (vehicle: subiculum, 168.48 ± 19.63; CA1, 140.03 ± 16.81; CA2, 132.75 ± 14.96; NAC: subiculum, 52.17 ± 20.09; CA1, 54.97 ± 19.74; CA2, 54.98 ± 19.24; Figure 2B). Consequently, GCI-induced zinc accumulation in post-synaptic neurons was observed in vehicle-treated groups. However, NAC administration after GCI reduced zinc accumulation in hippocampal areas (Figure 2C). Representative bar graph shows number of quantified TSQ-positive cells in both GCI–vehicle and GCI–NAC groups (vehicle: subiculum, 84.6 ± 4.29; CA1, 76.28 ± 5.32; CA2, 76.53 ± 3.18; NAC: subiculum, 37.66 ± 2.18; CA1, 32.63 ± 10.52; CA2, 27.77 ± 4.77; Figure 2D).

**Figure 2.** Number of GCI-induced degenerating neurons and zinc accumulation was attenuated by NAC treatment. (**A**) Brain sections were stained with fluorescence dye for detection of degenerating neurons, Fluoro-Jade B (FJB, green signal) in the hippocampal subiculum, cornus ammonis (CA) 1 and CA2 regions. Brain tissues that suffered GCI condition have numerous degenerating neurons in hippocampal regions and administration of NAC reduces the number of FJB-positive neurons in the same regions. Scale bar = 20 μm. (**B**) Quantification of FJB-positive neurons counted in each hippocampal region. Data are mean ± standard error of mean (SEM), *n* = 6 each group, \* *p* < 0.05 versus vehicle-treated group (Mann–Whitney U test, Subiculum: z = 2.562, *p* = 0.010; CA1: z = 2.402, *p* = 0.016; CA2: z = 2.082, *p* = 0.037). (**C**) Fluorescence microscopic images of free zinc stained by *N*-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) staining. Bright blue fluorescence signal means ischemic condition-induced excessive zinc release and translocation into hippocampal neurons. The NAC-administered group had a remarkably reduced number of TSQ-positive neurons. Scale bar = 20 μm. (**D**) Quantification of TSQ fluorescence signal-positive neurons counted from the hippocampus. Data are mean ± SEM, *n* = 4–5 each group, \* *p* < 0.05 versus vehicle-treated group (Mann–Whitney U test, Subiculum: z = 2.205, *p* = 0.027; CA1: z = 2.449, *p* = 0.014; CA2: z = 2.449, *p* = 0.014).
