*2.6. Amiloride Prevented Global Cerebral Ischemia-Induced Blood–Brain Barrier Disruption after 24 Hour Post-Insult*

To verify the degree of blood–brain barrier (BBB) disruption, we stained brain sections to evaluate extravasation of serum immunoglobulin G (IgG) by using immunohistochemistry as described before [35,36]. In sham-operated brain sections, leakage of IgG was not detected. However, in ischemia-induced mice, we observed excessive extravascular IgG leakage in the hippocampus (Figure 5A). Figure 5B shows a bar graph of the scale of IgG extravasation from the damaged BBB in the hippocampus. IgG leakage was reduced by 24% in the GCI-amiloride group compared with the GCI-vehicle group (GCI-vehicle, 1.37 ± 0.09; GCI-amiloride, 1.04 ± 0.07) (Figure 5B).

**Figure 5.** GCI-induced blood–brain barrier (BBB) disruption was decreased by amiloride administration. Brain sections were stained with antibodies against IgG to detect BBB disruption. (**A**) Indicates magnification (4×) of a microscopic image of IgG staining in the hippocampus in each group. These images indicate that BBB disruption occurred after GCI. The GCI-amiloride group had decreased leakage of serum IgG in the hippocampus compared with the GCI-vehicle group. Scale bar = 100 μm. (**B**) Bar graph shows the quantification of IgG serum extravasation in the hippocampus (sham-to-GCI ratio, sham-vehicle, *n*=6; sham-amiloride, *n*=5; GCI-vehicle, *n*=7; GCI-amiloride, *n*=8). Data are mean ± SEM. \* Considerably different from the vehicle-treated group, *p* < 0.05; # sham versus vehicle-operated group; *p* < 0.05. (Kruskal–Wallis test (B) Chi square = 11.126, df = 3, *p* = 0.011).
