GSH is a major endogenous component of the cellular antioxidant defense. It is capable of scavenging various ROS directly. Research has demonstrated that the order of GSH reactivity towards the radicals is ^•OH > ^•OCH₃ > ^•OOH > ^•OOCCl₃ > ^•OOCHCH₂ > ^•OOCH₃, and that the rate constants range from 2.02 × 10⁴ M⁻¹ s⁻¹ to the diffusion limit (7.68 × 10⁹ M⁻¹ s⁻¹) [16]. These findings indicate that GSH is an excellent free radical scavenger. On the other hand, GSH is also known to act as a substrate for a number of glutathione peroxidases to detoxify ROS [17]. During these reactions, GSH is oxidized and converted into glutathione disulfide (GSSG), which is subsequently reduced to GSH by glutathione reductase [18]. However, cellular GSH levels can be lowered by the export of GSSG when the production of the latter exceeds the capacity for recycling to the former, indicating that this antioxidative defense system cannot offset oxidative stress ^[19][20][19,20]. Indeed, many studies have demonstrated that the brain content of endogenous GSH is depleted during ischemia/reperfusion ^[21][22][23][21,22,23], and that treatment with GSH or glutathione monoethyl ester, a cell-permeable derivative of GSH, ameliorates ischemia-induced oxidative damage, including DNA and lipid oxidation in the hippocampus ^[24][25][24,25]. These observations suggest that maintaining neuronal GSH levels is an important constituent in protecting neurons against oxidative stress in the post-ischemic hippocampus (Figure 1).

Figure 1. Mechanisms of GSH protection against hippocampal neuron damage following brain ischemia. The abbreviations are as follows: cysteine (Cys), glutamate (Glu), glycine (Gly), excitatory amino acid carrier 1 (EAAC1), glutathione (GSH), glutathione disulfide (GSSG), GSH reductase (GR), GSH peroxidase (GPx), reactive oxygen species (ROS).

Interestingly, Won et al. demonstrated that the GSH levels in CA1 pyramidal neurons were decreased during the first few hours of ischemia/reperfusion, accompanied by increased superoxide levels; conversely, the prevention of ischemia/reperfusion-induced increase in superoxide production using an inhibitor of nicotinamide adenine dinucleotide 3-phosphate oxidase, a major source of ROS, was found to suppress the decline in GSH in post-ischemic neurons [26]. On the other hand, they also showed that mice in which neuronal GSH levels were maintained by treatment with N-acetyl cysteine (NAC), a cell-permeable precursor of GSH, exhibited a reduction in neuronal oxidative stress and neuronal cell death in the hippocampus following ischemia/reperfusion [26]. These findings indicate that the depletion of neuronal GSH is both a result and a cause of neuronal oxidative stress following ischemia/reperfusion. In contrast, changes in the levels of GSH in post-ischemic glial cells, such as astrocytes and microglia, remain to be elucidated. Astrocytes play an important role in the synthesis of neuronal GSH by supplying precursor molecules [27]. Therefore, it is necessary to clarify the role of glial GSH in neuronal survival in the post-ischemic hippocampus.

2.2. GSH Protects Neurons from Ischemia-Induced Disruption of Intracellular Zn²⁺ Homeostasis

Zn²⁺ is involved in hippocampal injury following brain ischemia. In the mammalian brain, most of the Zn²⁺ is bound to specialized Zn²⁺ proteins—for example, metallothionein and transcription factors [28]. A smaller part of it is concentrated in the presynaptic vesicles in a subset of glutamatergic neurons throughout the forebrain, especially in the hippocampus ^[29][30][31][29,30,31]. Therefore, the intracellular free Zn²⁺ concentration in the hippocampus is considered to be very low under normal physiological conditions [28]. However, the stored Zn²⁺, along with the protein-bound portion, is massively released during ischemia and the onset of reperfusion, subsequently accumulating within hippocampal neurons ^{[32][33][34][35][36][37][38]}[32,33,34,35,36,37,38]. The prevention of this process using Zn²⁺ chelators, including calcium disodium ethylenediamine tetraacetate and clioquinol, has been shown to protect hippocampal neurons from ischemia-induced neurodegeneration ^[8][39][8,39], suggesting that the ischemia-induced disruption of neuronal Zn²⁺ homeostasis contributes to neurodegeneration in the hippocampus.

The thiol moiety of GSH has a high affinity for Zn²⁺ through non-enzymatic conjugation. Treatment with GSH protects neurons against Zn²⁺ accumulation-induced cell toxicity [40]. The replenishment of GSH using NAC reduces Zn²⁺ accumulation and neurodegeneration in the post-ischemic hippocampus [41]. These findings suggest that GSH acts as an intrinsic factor that buffers intracellular Zn²⁺ concentrations to prevent the ischemia-induced disruption of Zn²⁺ homeostasis; in addition to its antioxidative activity, this mechanism protects neurons (Figure 1).

In contrast, Zn²⁺ binds and inhibits glutathione reductase and peroxidase, the major enzymes of the glutathione redox cycle ^[42][43][42,43]. Chen and Liao demonstrated evidence indicating that Zn²⁺ entry into neurons causes a decrease in intracellular GSH levels and eventually triggers neuronal cell death [40]. GSH can suppress the ischemia-induced disruption of Zn²⁺ homeostasis, but this function may be affected by the accumulation of Zn²⁺ in neurons.