**1. Introduction**

Recent decades have seen a dramatic increase in the attention and focus on the study of brain disease. In particular, cerebral ischemia is a global problem and is the leading cause of brain disease and morbidity [1,2]. There are many reasons why cerebral ischemia is so widespread, in part due to the diverse number of individual pathologies that converge on the emergence of the ischemic state; the largest risk factor for development of cerebral ischemia is occlusion of blood vessels to the brain. Upon the occlusion of these blood vessels, cerebral blood flow is considerably reduced to regions of the brain and essential supplies of oxygen, nutrients, glucose, and many other cellular substrates are reduced [3]. Cerebral ischemia is divided into two sub-categories. The first, focal cerebral ischemia, involves transiently or permanently reduced blood flow and nutrient supply to particular brain areas that are directly fed by the occluded vasculature. Damage to the brain after focal ischemia is thus focused on the affected areas and injury occurs locally [4]. Global cerebral ischemia (GCI) is similar to focal cerebral ischemia, although the mechanisms of induction and patterns of damage to specific brain regions are different. This brain injury occurs due to several reasons, but is best described by a sudden and near-complete stop in cerebral blood-flow, such as is seen under cardiac arrest-induced cessation of blood circulation [5].

Once the ischemic insult has occurred, abnormal physiological changes to brain function have already began to occur. Under the ischemic condition, disruption of brain homeostasis is disturbed and cell death mechanisms are initiated. This cerebral injury leads to an alteration in ionic balance within the intra-extracellular space [6], reactive oxygen species (ROS) production [7], microglia [8] and astrocyte [9] activation, zinc accumulation [10], and neuronal cell degeneration [11–13] in the brain. Blockage of blood flow to the brain by ischemic insult causes tissue infarction, dysfunction of ionic balance, and intracellular calcium overload [14]. In a previous study, excessive release of calcium ions from synaptic vesicles after brain ischemia was shown to have deleterious effects on neurons and glia [15]. Overloaded intracellular calcium ions trigger glutamate release, proteolysis, mitochondrial dysfunction, and other deleterious cascades [16]. As discussed above, calcium overload contributes to ischemic injury-induced cell death mechanisms. According to a recent report, the cation zinc has an essential role in brain ischemia, in addition to the calcium ion [17].

Zinc is the most widely distributed metal ion in the central nervous system, and is especially abundant in the hippocampus. Zinc has many important roles in enzymes and transcription factors, and its concentration in neurons and glia is controlled by binding proteins, known as metallothionein, as well as several transporters and channels [17–19]. In addition, zinc regulates neural synaptic transmission, innate and adaptive immunity, signal transduction, and cell proliferation, particularly for DNA synthesis [20,21]. In the pre-synaptic vesicles, chelatable zinc is abundant. It is released into the synaptic cleft, moved into pre-synaptic neurons via several transporters, and subsequently undergoes reuptake from the synaptic cleft under normal physiological conditions [22,23]. Under abnormal physiological conditions, such as neurological disorders, zinc homeostasis is disturbed and dysregulated. In the case of neurological disorders, vesicular zinc is excessively released and largely accumulates within post-synaptic neurons. In addition to releasing zinc from synaptic vesicles, zinc that is loosely bound by proteins can be divided into the categories of chelatable free zinc and proteins [12,24,25]. Pathological zinc accumulation can influence cellular dysfunction in several ways. In a previous study, it was demonstrated that zinc-treated cultured neurons produce ROS from their mitochondria [18] and trigger microglial activation [26]. In addition, previous studies by our group have observed that excessive zinc accumulation within brain hippocampal regions can lead to neurodegeneration after cerebral insult, such as ischemia [10,27], head trauma [25], hypoglycemia [28], and cultured neurons [29].

*N*-acetyl-l-cysteine (NAC), which contains an acetylated cysteine residue, acts as a powerful antioxidant by supplementation of cysteine and promotion of glutathione biosynthesis [30,31]. Injury-induced oxidative damage plays a primary role in central nervous system dysfunction and contributes to neurodegenerative cascades [32,33]. To protect against brain injury-induced oxidative damage, our group has previously used NAC to treat hypoglycemia and traumatic brain injury. NAC stabilized glutathione concentrations, reduced degenerating neurons, and decreased oxidative damage within hippocampal regions [24,34]. Hippocampal neurodegeneration can be prevented or reduced when oxidative damage and other deleterious cascades are prevented by regulating physiologic concentrations of glutathione via providing ample cysteine, which is supplied from NAC [35]. Moreover, NAC has binding sites for trace metals that include copper, magnesium, and zinc. These studies demonstrate that NAC can neutralize toxically accumulated trace metals occurring in neuropathies [24,36].

Transient receptor potential melastatin 2 (TRPM2) is one of the non-selective cationic channels, and the activity of this channel is triggered by ROS production promoted by deleterious cell death mechanisms in several neurological disorders. Recently, the TRPM2 channel has been reported to be included in both physiological and pathological conditions [37,38]. Several previous studies have shown that the TRPM2 channel is expressed in the central nervous system (CNS), which is largely expressed within the hippocampus, stratum, and cortex [39]. These studies found that several divalent cations, for example, calcium and magnesium, moved into the intracellular space via TRPM2, and that its activity was regulated by external stimuli such as ROS, hydrogen peroxide, and tumor necrosis factor-α (TNF-α) [40–42]. In addition, TRPM2 is distributed in both neurons and glia, and is involved in pathophysiological conditions of nitric oxide (NO) and ROS production signaling [38,43]. Following several neurological injuries, excessive calcium influx via TRPM2 can trigger release of diverse cytokines and thus contributes to the inflammatory response [44]. Several brain diseases, such as stroke or head trauma, lead to excitotoxicity, which is caused by the inappropriate activation of voltage-dependent calcium channels and excitatory amino acid release from the dendritic and presynaptic space into the extracellular cleft. Previous studies verified that in a rodent disease model of ischemia, TRPM2 mRNA levels were significantly increased. Interestingly, a recent study demonstrated that TRPM2 deficiency in mice decreased cytosolic zinc concentrations and reduced zinc influx from the extracellular space [45]. These findings suggest that, during several brain diseases, TRPM2 is activated and this contributes to cell death mechanisms [44,46].

In the present study, we tested the hypothesis that if ischemia-induced oxidative damage leads to TRPM2 activation, inhibition of this channel may have therapeutic effects through blockade of zinc influx-induced cell damage, which is supported by evidence that NAC administration decreased brain damage in several other neurological diseases [24,34]. Following this logic, we hypothesized that if the TRPM2 level was increased by global cerebral ischemia-induced cell death cascades, regulation of TRPM2 as a potential target for preventing ischemia-induced injury could ameliorate a diverse number of cerebral pathologies associated with ischemia. In addition, we speculated that the zinc ion is closely related to TRPM2 and is excessively moved into the intracellular space via this channel. To test our hypothesis, we used a GCI brain disease model in adult rats and conducted several types of histological evaluations and behavioral assessments.
