*2.1. Amiloride Reduced Global Cerebral Ischemia-Induced Hippocampus Neuronal Death after 24 Hour Post-Insult*

To investigate whether amiloride has neuroprotective effects after global cerebral ischemia (GCI)-induced hippocampus neuronal death, experimental mice were immediately intraperitoneally injected with amiloride (10 mg/kg) after GCI. The mice were sacrificed 24 h after GCI with or without amiloride treatment. After insult, a histological evaluation to detect degenerating neurons was conducted in the hippocampal subiculum (Sub), cornus ammonis 1 (CA1), CA2, and CA3 regions. Fluoro-Jade B (FJB) staining showed widespread neuronal degeneration in the Sub, CA1, CA2, and CA3 regions of the hippocampus (*p* < 0.05) (Figure 1A). This staining protocol is a sensitive and selective marker of degenerating neurons. The number of degenerating neurons was increased in the GCI-induced group compared with the sham-operated group. When compared with the GCI-vehicle-treated groups, the amiloride-injected groups showed a dramatically reduced number of degenerating hippocampal neurons. Figure 1B shows the quantified FJB (+) neurons in the Sub, CA1, CA2, and CA3 regions. Amiloride-administered groups displayed a reduction of FJB (+) neurons of approximately 64% in the Sub (GCI-vehicle, 76.9 ± 8.4; GCI-amiloride, 27.2 ± 10.5), 84% in the CA1 (GCI-vehicle, 126.2 ± 18.5; GCI-amiloride, 19.5 ± 17.6), 83% in the CA2 (GCI-vehicle, 125.3 ± 22; GCI-amiloride, 20.7 ± 17.9), and 50% in the CA3 (GCI-vehicle, 220.6 ± 30.3; GCI-amiloride, 109.5 ± 29) regions compared with the vehicle-treated groups.
