*2.3. NAC Attenuates TRPM2 Activation in Hippocampal Neurons and Glial Cells*

GCI activates TRPM2 channels in the brain, which is localized to glial cells in addition to hippocampal neurons. To demonstrate whether TRPM2 is located within neurons or glial cells in our experimental setting, we conducted immuno-fluorescence staining. Figure 4A shows distribution of TRPM2 channels in the hippocampal pyramidal layer CA1 region, where it was co-localized with neuronal nuclei (NeuN). There was no difference in TRPM2-positive fluorescence signals in sham groups (mean gray value, sham–vehicle: 11.08 ± 0.58; sham NAC: 11.09 ± 0.62; Figure 4B). However, the ischemic condition contributes to activation of TRPM2 in the brain and this is highly localized to the hippocampal pyramidal CA1 layer, which is also known to be especially vulnerable to ischemic injury. The TRPM2-positive signal was dramatically increased in the GCI–vehicle group and significantly decreased in the NAC post-treatment group (GCI–vehicle: 29.55 ± 1.15; GCI–NAC: 18.25 ± 1.76, Figure 4B).

**Figure 4.** GCI-induced transient receptor potential melastatin 2 (TRPM2) activation was attenuated by NAC treatment. (**A**) Representative histological images of TRPM2-positive signal in the hippocampal CA1. The neuronal TRPM2 level was remarkably increased in the GCI–vehicle group and NAC administration after GCI reduced TRPM2 activation. Scale bar = 20 μm. (**B**) Bar graph shows intensity of TRPM2-positive signal (red color) within the hippocampal pyramidal layer. Data are mean ± SEM, *n* = 5 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: chi square = 16.097, df = 3, *p* = 0.001).

To explore the localization of TRPM2 in non-neuronal cells, we conducted glial cell immunostaining. Sham groups showed that Iba-1-positive microglia were distributed throughout the hippocampus in

an inactivated state. However, GCI triggers microglial activation, which was significantly decreased by NAC post-treatment (Figure 5A). The bar graph shows quantified microglia intensity (mean gray value, sham–vehicle, 7.59 ± 0.70; sham NAC, 7.82 ± 0.54; GCI–vehicle, 48.35 ± 4.60; GCI–NAC, 23.59 ± 1.01; Figure 5B). Figure 5C shows co-localized TRPM2 with microglia in GCI–vehicle groups. Similarly, astrocytes were distributed throughout the hippocampus, and there were no differences in sham groups. GCI triggers astrocyte activation, which was significantly reduced by NAC post-treatment (Figure 5D). The bar graph shows quantified astrocyte activation (mean gray value, sham–vehicle, 12.52 ± 1.29; sham NAC, 11.88 ± 1.96; GCI–vehicle, 61.46 ± 5.21; GCI–NAC, 36.75 ± 4.58; Figure 5E). Figure 5F shows co-localized TRPM2 with astrocyte in GCI–vehicle groups.

**Figure 5.** GCI-induced glial cell activation was attenuated by NAC treatment. (**A**) Microglial activation was estimated by Iba-1 immuno-staining. Sham groups (vehicle and NAC) had no difference in Iba-1-positive signal (green color). GCI triggers excessive microglial activation in the hippocampal pyramidal layer, and this was attenuated by NAC administration for 3 days after onset of GCI. Scale bar = 10 μm. (**B**) Bar graph representing intensity of Iba-1-positive signals from hippocampal pyramidal layer administered with vehicle and NAC for 3 days after sham and GCI surgery. Data are mean ± SEM, *n* = 5–7 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: chi square = 20.248, df = 3, *p* < 0.001). (**C**) Distribution of TRPM2 in microglial cells (merged image). (**D**) Astrocyte activation was evaluated by GFAP immuno-staining. Sham groups had no difference in GFAP-positive signal (red color). Astrocyte activation was stimulated by GCI, and reduced by NAC administration. Scale bar = 10 μm. (**E**) Bar graph displaying intensity of GFAP-positive signals from the hippocampal pyramidal layer in vehicle and NAC-treated groups after sham and GCI surgery. Data are mean ± SEM, *n* = 5–6 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: chi square = 18.747, df = 3, *p* < 0.001). (**F**) Distribution of TRPM2 in astrocyte following GCI (merged image).
