**1. Introduction**

Ischemic stroke is one of the most severe cerebral pathological conditions and can manifest via a number of clinical symptoms such as problems in cognition, dizziness, or loss of vision on one side of the visual field [1]. In particular, the development of ischemic conditions in the brain is very dangerous because only a momentary lack of adequate blood flow to the brain can lead to oxygen deprivation, insufficient nutrient provision, and potentially irreversible neural injury [2–4]. There are two basic types of ischemic injury: global and focal ischemia. Global ischemia impacts wide areas of brain tissue at once due to the blockage of blood flow to an entire region of the brain, while focal ischemia is limited to a specific region of the brain tissue and is due to the more local disruption of cerebral blood flow. Ischemia-induced brain damage can be recovered by early reperfusion, but this reperfusion process can also initiate independent cascades of cell death pathways such as zinc release, microglial activation,

and blood–brain barrier (BBB) disruption [5]. If the interruption of blood circulation happens for an extended period prior to the restoration of circulation, brain damage can be permanent.

Furthermore, after a long period of ischemia, secondary brain damage occurs when blood flow is initiated, which is known as "reperfusion injury". The sudden recovery of blood supply leads to mitochondrial dysfunction, producing excessive reactive oxygen species (ROS) and, finally, cell death [6]. Under healthy conditions, superoxide production via neuronal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is thought to play a part in normal physiological processes such as long-term potentiation and intracellular signaling [7,8]. However, under ischemic conditions, mitochondrial dysfunction and excessive ROS generation predominate and promote pathological responses such as leukocyte invasion and the disruption of the BBB [9,10]. Also, tissue pH is typically reduced to 6.0–6.5 during ischemia, falling even lower during severe ischemic conditions [11–13]. As a result, this study suggests that the increase in NADPH activity after ischemia may also promote cell death by acidifying brain cells.

Zinc is one of the most essential transition metals in our body, especially in the brain. Furthermore, zinc regulates physiological functions that control DNA synthesis, cell division, and signal transduction. Most of the zinc is present in a protein-bound form in neuronal cytoplasm. Free or chelatable zinc is localized within the vesicles of synaptic terminals [14,15]. Taken together, these lines of circumstantial evidence suggest that zinc plays a key role in maintaining cellular homeostasis. However, previous studies have demonstrated that excessive neuronal zinc accumulation occurs after traumatic brain injury, ischemia, hyperglycemia, and epilepsy [16–19]. Under these various pathological conditions, ROS changes the protein-mediated sequestration of zinc and, thus, increases intracellular free zinc levels which increases ROS production. If this condition is maintained for long periods, it can lead to neuronal death [18]. Several neurological injuries, accelerate zinc release from synaptic vesicles, and zinc then moves into neurons via multiple classes of membrane-bound channels [20,21].

Sodium–hydrogen exchangers (NHEs) are membrane transporters that mediate hydrogen efflux into cells. Sodium–hydrogen exchanger-1 (NHE-1) is a ubiquitous and essential membrane ion transporter that mediates the electroneutral exchange of hydrogen and sodium to regulate intracellular pH [22]. Other NHEs, namely, NHE-2–5, indicate more distinct cell-type- and tissue-dependent expressions than NHE-1 and play key roles in regulating transcellular sodium and potassium ion absorption [23]. The NHE-6 expression is localized to early endosomes [24]. The NHE-7 and -8 isoforms have not been localized in the brain, and the NHE-9 isoform is localized to late recycling endosomes [25]. Here, we focused on NHE-1 which is expressed in high abundance in the brain.

Amiloride, also known as thiazide, is related to other loop diuretic agents and blocks sodium hydrogen exchanger-1 [26]. Amiloride, an inhibitor of NHE-1, has demonstrated neuroprotective effects in various neuropathological conditions involving brain injuries such as ischemia, dementia, and epilepsy [27,28]. Previous studies have suggested that injection of amiloride can reduce cerebral-hypoxia-induced neuronal death after seizure and spinal cord injury [29–31]. Under the acidic conditions found in the post-ischemic state, as NHE-1 becomes activated, hydrogen is released into the extracellular space and sodium moves into the intracellular compartment. Thus, intracellular sodium is increased and the sodium–calcium exchanger (NCX) becomes activated, so that sodium is released into the extracellular space driving calcium entry, leading to a calcium overload state and, ultimately, resulting in neuronal death. Considering this, we hypothesized that not only calcium, but also zinc may enter via NCX and that NHE-1 blockade by amiloride decreases zinc accumulation and neuronal death through reduced NCX activity. Therefore, we investigated whether the administration of amiloride (10 mg/kg, intraperitoneally (*i.p.*)) reduces zinc accumulation, neuronal degeneration, oxidative stress, and microtubule damage after global cerebral ischemia. As a result, the present study proved that the administration of amiloride reduces ischemia-induced neuronal degeneration.
