*2.2. Amiloride Reduced Global Cerebral Ischemia-Induced Hippocampal Zinc Accumulation after 24 Hour Post-Insult*

To estimate GCI-induced zinc accumulation, brain sections were histologically evaluated by *N*-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) staining 24 h after GCI. Zinc accumulation is known to advance the neuronal NADPH oxidase activity and ROS responsible for neuronal death. Under normal conditions, zinc levels are controlled by zinc transporters and zinc-binding proteins [32]. However, under conditions such as ischemia, traumatic brain injury, and seizure, neuronal death occurs in part by the destruction of zinc homeostasis. Thus, we performed TSQ fluorescence staining to confirm whether amiloride can reduce zinc accumulation in the brain hippocampal CA1 region. The intensity of TSQ staining in the CA1 region was reduced in the amiloride-treated group compared with the GCI-induced group (*p* < 0.05) (Figure 1C,D). Amiloride-administered groups displayed an approximately 44% reduction of TSQ (+) neurons in the CA1 region (GCI-vehicle, 36.9 ± 5.9; GCI-amiloride, 20.6 ± 4.1) compared with the vehicle-treated groups.
