Figure 2.

Reduced ischemia-induced Zn²⁺ accumulation and neuronal injury in response to increased GSH levels via elevated EAAC1 protein expression at 23:00. (A) Representative images showing TSQ fluorescence (indicative of Zn²⁺ accumulation) in the hippocampus 72 h after transient brain ischemia. Zn²⁺ accumulation was observed in the CA3 and hilar but not the CA1 region in ischemic mice. Notably, the hilar Zn²⁺ accumulation was remarkably reduced in mice subjected to transient brain ischemia at 23:00 compared to that at 09:00. (B) Fluorescent images showing neurodegeneration by Fluoro-Jade B staining in the hippocampus 72 h after transient brain ischemia in the CA3 and hilar but not in the CA1 region in ischemic mice. In the hilus, Fluoro-Jade B fluorescent signals were remarkably reduced in mice subjected to transient brain ischemia at 23:00 compared to those at 09:00. (C) Representative images depicting double immunofluorescence staining for glutathione adduct with N-ethylmaleimide (GS-NEM, represents GSH levels; green) and DAPI (blue) in the hilar region. GSH levels were significantly higher in the hilus at 23:00 than at 09:00. (D) EAAC1 protein expression was analyzed by Western blotting. EAAC1 protein levels in the hippocampus were significantly increased at 23:00 compared to at 09:00. * p < 0.05, relative to the hippocampus harvested at 09:00 (Mann-Whitney U test). (E) Representative images highlighting TSQ fluorescence in the hilar region 72 h after ischemia in mice pretreated with L-aspartic acid β-hydroxamate (LAβHA), a selective EAAC1 inhibitor, at 23:00. Ischemia-induced Zn²⁺ accumulation was significantly increased in LAβHA-pretreated mice.