*2.2. NAC Post-Treatment Regulates Global Cerebral Ischemia-Induced Loss of Neuronal Glutathione and Lipid Peroxidation*

One strategy to solve the problem of ischemic injury is to focus on reducing oxidative damage. We speculated that post-administration of NAC can supply cysteine to produce the powerful anti-oxidant glutathione and influence neuronal oxidative damage. Representative images show glutathione (GS-NEM) levels (Figure 3A). Sham groups show a wide distribution of glutathione within neurons, but the GCI–vehicle group shows a loss of glutathione after injury, particularly oxidative damage. NAC treatment after GCI elevates glutathione concentration in damaged neurons. Based on the above results, we confirmed whether NAC administration directly protects against ROS-induced neuronal oxidative damage by enhancing glutathione levels. ROS triggers lipid peroxidation, and 4-hydroxy-2-nonenal (4-HNE) is a histological marker for lipid peroxidation. GCI led to an increase in ROS, and 4-HNE-positive fluorescence signal was largely increased in

the hippocampal subiculum, CA1, and CA2 regions (Figure 3B). Increased lipid peroxidation by GCI-induced ROS production was significantly attenuated following post-treatment of NAC (mean gray value, GCI–vehicle: subiculum, 25.93 ± 2.08; CA1, 28.89 ± 1.69; CA2, 26.36 ± 2.41; GCI–NAC: subiculum, 17.20 ± 0.97; CA1, 16.34 ± 0.99; CA2, 16.90 ± 0.31; Figure 3C). Sham groups had no differences in 4-HNE signal (sham–vehicle: subiculum, 11.12 ± 0.37; CA1, 9.76 ± 0.64; CA2, 11.12 ± 0.97; sham NAC: subiculum, 10.10 ± 0.92; CA1, 8.65 ± 0.43; CA2, 10.89 ± 1.21; Figure 3C).

**Figure 3.** GCI-induced decreasing concentration of glutathione and increasing oxidative damage was restored by anti-oxidant, NAC. (**A**) Representative histological images show concentration of glutathione marker GS-NEM in the hippocampal CA1. Neuronal glutathione level was remarkably reduced after GCI and was restored to normal levels. Scale bar = 20 μm. (**B**) 4-hydroxy-2-nonenal (4-HNE) immunofluorescence staining was used as a marker for lipid peroxidation. Sham–vehicle and NAC groups show minimized 4-HNE-positive signal (red color) in hippocampal regions. It was remarkably increased in the GCI–vehicle group and NAC administration after GCI attenuates oxidative damage-induced lipid peroxidation. Scale bar = 20 μm. (**C**) Bar graph means intensity of 4-HNE signal in the hippocampal regions. Data are mean ± SEM, *n* = 4–7 each group, \* *p* < 0.05 versus vehicle-treated group; # *p* < 0.05 versus sham-operated group (Kruskal–Wallis test followed by Bonferroni post-hoc test, Subiculum: chi square = 18.688, df = 3, *p* < 0.001; CA1: chi square = 18.783, df = 3, *p* < 0.001; CA2: chi square = 18.229, df = 3, *p* < 0.001).
