*2.3. Amiloride Reduced Global Cerebral Ischemia-Induced Astrocyte and Microglial Activation 24 Hour Post-Insult*

Multiple studies have previously reported that ischemia induces astrocyte and microglial activation in the brain hippocampal region [3,18,33], and it is well known that inflammation contributes to the severity of astrocyte and microglial activation. In addition, activated astrocytes are potentially harmful, because they can produce nitric oxide synthase (NOS) and neurotoxic nitric oxide (NO). To test whether amiloride affects GCI-induced glial activation, we performed immunofluorescence staining using the marker glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba-1). We confirmed an assessment of the microglia number, morphology, and intensity and astrocyte intensity [34]. Global cerebral ischemia triggers astroglia and microglial activation which is considered to have macrophage-like activity. However, activated astrocytes were reduced by approximately 45% in the GCI–amiloride groups compared with the GCI-vehicle groups in the CA1 region (GCI-vehicle, 33.2 ± 3.3; GCI–amiloride, 18.2 ± 1.8). Further, activated microglia were reduced by 32% in the GCI–amiloride groups in the CA1 region (GCI-vehicle, 5.8 ± 0.3; GCI–amiloride, 3.9 ± 0.3) (Figure 2B,D).

**Figure 1.** Amiloride treatment decreased the number of degenerating neurons and zinc accumulation after global cerebral ischemia (GCI). GCI-induced hippocampus neuronal death was confirmed in the subiculum (Sub), cornus ammonis 1 (CA1), CA2, and CA3 regions after ischemic insult. Zinc accumulation was confirmed in the CA1 region after ischemic insult. (**A**) Fluorescent images show degenerated neurons in the Sub, CA1, CA2, and CA3 regions. Intraperitoneal post-treatment with amiloride (10 mg/kg) reduced neuronal death in the Sub, CA1, CA2, and CA3 regions at 24 h after ischemia. Scale bar = 20 μm. (**B**) Bar graph displaying the quantification of degenerating neurons in the hippocampal regions. The number of FJB (+) neurons was decreased in the amiloride-injected (10 mg/kg) group in the Sub, CA1, CA2, and CA3 regions compared with the vehicle-treated group (GCI-vehicle, *n* = 8; GCI-amiloride, *n* = 8). (**C**) Representative images show *N*-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) (+) neurons in the CA1 region. Scale bar = 20 μm. (**D**) The bar graph indicates the TSQ (+) neurons in the hippocampal CA1 region (GCI-vehicle, *n* = 8; GCI-amiloride, *n* = 10). Data are mean ± S.E.M. \* Considerably different from the vehicle-treated group, *p* < 0.05. (Mann–Whitney U test (B) Sub: z = 2.626, *p* = 0.007; CA1: z = 2.838, *p* = 0.003; CA2: z = 2.836, *p* = 0.003; CA3: z = 2.205, *p* = 0.028; (D) CA1: z = 2.134, *p* = 0.034).

**Figure 2.** Amiloride treatment reduced astrocyte and microglial activation. GCI induces an inflammatory response by promoting astrocyte and microglia activation in the damaged brain. This figure shows astrocyte and microglia activation in the CA1 region 24 h after GCI. (**A**,**C**) show glial fibrillary acidic protein (GFAP) and Ionized calcium-binding adaptor molecule-1 (Iba-1) activation and (**B**,**D**) show GFAP and Iba-1 quantification in the CA1 region from sham-operated or GCI-induced mice. It was increased in the GCI-induced group compared with the sham-operated group. However, the amiloride-treated group showed reduced astrocyte and microglia activation after GCI. Scale bar = 20 μm. (Sham-vehicle, *n* = 5; sham-amiloride, *n* = 5; GCI-vehicle, *n* = 6; GCI-amiloride, *n* = 8). Data are mean ± SEM. \* Considerably different from the vehicle-treated group, *p* < 0.05; # sham versus vehicle-operated group; sham versus vehicle-treated group, *p* < 0.05. (Kruskal–Wallis test (B) Chi square = 14.612, df = 3, *p* = 0.002; (D) Chi square = 18.606, df = 3, *p* < 0.001) (SO: stratum oriens; PL: pyramidal cell layer; SR: stratum lacunosum-moleculare).

#### *2.4. Amiloride Reduces Global Cerebral Ischemia-Induced Oxidative Damage after 24 Hour Post-Insult*

We estimated oxidative stress by using 4-hydroxynonenal (4HNE) staining. The brain was immunohistochemically stained with a 4HNE antibody 24 h after global cerebral ischemia induction to discover whether hippocampal neurons had experienced oxidative stress. The sham-vehicle and amiloride-injected groups showed no difference in 4HNE fluorescence signals. However, the 4HNE fluorescence signal of the GCI-vehicle groups increased in the hippocampal Sub, CA1, CA2, and CA3 regions. The amiloride-treated group showed a significant reduction of 4HNE intensity compared with the vehicle-treated groups (Figure 3A). Oxidative damage was reduced in the GCI-amiloride groups compared with the GCI-vehicle groups by approximately 42% in the Sub (GCI-vehicle, 21.8 ± 2.4; GCI-amiloride, 12.6 ± 0.5), 48% in the CA1 (GCI-vehicle, 23.5 ± 2.3; GCI-amiloride, 12.2 ± 1.5), 36% in the CA2 (GCI-vehicle, 21.5 ± 2.4; GCI-amiloride, 13.7 ± 1.4), and 48% in the CA3 (GCI-vehicle, 31.2 ± 3.3; GCI-amiloride, 16.1 ± 2.2) regions (Figure 3B).

**Figure 3.** Amiloride reduced oxidative injury after GCI. Oxidative injury was detected by 4-hydroxynonenal (4HNE, red color) staining from the hippocampal Sub, CA1, CA2, and CA3 regions 24 h after GCI. (**A**) Sham-operated groups showed minimal 4HNE fluorescence signals in the hippocampus. Amiloride-treated groups showed reduced immunoreactive fluorescence intensity for 4HNE in the hippocampus compared with the vehicle-treated group after GCI. Scale bar = 20 μm. (**B**) The bar graph presents the 4HNE fluorescence intensity in the Sub, CA1, CA2, and CA3 regions. The fluorescence intensity showed a significant difference among groups (sham-vehicle, *n* = 6; sham-amiloride, *n* = 5; GCI-vehicle, *n* = 8; GCI-amiloride, *n* = 8). Data are mean ± S.E.M. \* Considerably different from the vehicle-treated group, *p* < 0.05; # sham versus vehicle-operated group, sham versus vehicle-treated group, *p* < 0.05. (Kruskal–Wallis test (B) Sub: Chi square = 22.444, df = 3, *p* < 0.001; CA1: Chi square = 17.896, df = 3, *p* < 0.001; CA2: Chi square = 20.967, df = 3, *p* < 0.001; CA3: Chi square = 20.986, df = 3, *p* < 0.001).

#### *2.5. Amiloride Reduced Global Cerebral Ischemia-Induced Microtubule Damage after 24 Hour Post-Insult*

To evaluate whether GCI-induced microtubule damage occurred, brain sections were histologically processed with antibodies against microtubule-associated protein 2 (MAP2) 24 h after GCI. The GCI-vehicle groups showed a significant reduction in MAP2 immunoreactivity (IR) in the hippocampus and cortex compared with the GCI-amiloride group, indicating a loss of microtubules. Amiloride injection reduced GCI-induced microtubule damage compared with the vehicle-treated group (Figure 4A). Microtubule intensity was increased in the GCI-amiloride groups compared with the GCI-vehicle groups by approximately 42% in the Sub (GCI-vehicle, 41.9 ± 6.3; GCI-amiloride, 72.5 ± 7.2), 60% in the CA1 (GCI-vehicle, 43.3 ± 8.4; GCI-amiloride, 109.7 ± 12.5), 43% in the CA2

(GCI-vehicle, 56.2 ± 11; GCI-amiloride, 99.6 ± 10.1), and 59% in the CA3 (GCI-vehicle, 23.2 ± 5.9; GCI-amiloride, 57.2 ± 10.6) regions (Figure 4B).

**Figure 4.** Microtubule damage was detected by microtubule-associated protein 2 (MAP2, green color) staining at the hippocampal Sub, CA1, CA2, and CA3 regions 24 h after GCI. (**A**) Sham-operated groups showed MAP2 fluorescence signals in the hippocampus. Amiloride-administered groups showed a reduced microtubule loss in the hippocampal regions compared with the vehicle-treated group. Scale bar = 20 μm. (**B**) The bar graph indicates the MAP2 fluorescence intensity in the hippocampus. (**C**) The bar graph indicates the MAP2 percent area in the hippocampus (sham-vehicle, *n* = 6; sham-amiloride, *n* = 5; GCI-vehicle, *n* = 8; GCI-amiloride, *n* = 8). Data are mean ± S.E.M. \* Considerably different from the vehicle-treated group, *p* < 0.05; # sham versus vehicle-operated group, sham versus vehicle-treated group, *p* < 0.05. (Kruskal–Wallis test (B) Sub: Chi square = 18.901, df = 3, *p* < 0.001; CA1: Chi square = 15.166, df = 3, *p* < 0.002; CA2: Chi square = 15.054, df = 3, *p* < 0.002; CA3: Chi square = 17.137, df = 3, *p* < 0.001).
