*2.4. NAC Restores Global Cerebral Ischemia-Induced Sensorimotor Deficit, Neurologic Decline, and Neurodegeneration*

To investigate the effects of NAC post-administration on GCI-induced sensorimotor deficit and loss of neurological function, experimental animals were analyzed using the adhesive removal test and modified neurological severity score (mNSS) methods. First, to evaluate sensorimotor deficit after ischemic damage we used the adhesive removal test method. The GCI–vehicle-treated groups spent longer periods of time recognizing adhesive tapes on palms than the GCI–NAC-treated groups after ischemic insult. Sham–vehicle and NAC groups had no difference in removal time during the test days. These results showed that GCI-induced sensorimotor deficit was restored by NAC treatment (Figure 6A). Next, we also assessed whether NAC administration restored GCI-induced neurological decline. Because the sham groups had no impairment, the mNSS count was zero. Following GCI, neurological impairment lead to high scores in the vehicle group and NAC administration attenuated this neurologic decline (Figure 6B). In addition, we determined whether post-administration of NAC provides neuroprotective effects on GCI-induced neurodegeneration following 7 days of consecutive treatment (Figure 6C). Sham groups show numerous neuronal nuclei (NeuN)-positive cells in hippocampal subiculum, CA1, and CA2 regions. There were no significant differences in the number of NeuN-positive cells between the sham–vehicle and NAC group (sham–vehicle: subiculum, 170.5 ± 5.5; CA1, 180.5 ± 5.5; CA2, 210 ± 11, sham-NAC: subiculum, 177 ± 7; CA1, 179 ± 10; CA2, 205 ± 7; Figure 6D). In contrast, the number of NeuN-positive cells was dramatically decreased in the GCI–vehicle group. However, NAC administration significantly restored the number of NeuN-positive cells in the hippocampal regions (GCI–vehicle: subiculum, 79.95 ± 7.69; CA1, 62.74 ± 11.15; CA2, 66.82 ± 4.91, GCI–NAC: subiculum, 110.47 ± 6.42; CA1, 108.12 ± 9.87; CA2, 110.31 ± 7.81; Figure 6D).

**Figure 6.** *Cont*.

**Figure 6.** Post administration of NAC reduces GCI-induced behavioral outcome deficits and delays neuronal loss. (**A**) The adhesive removal test to assess sensorimotor deficit was conducted at both 3 h and 7 days after sham and GCI surgery. Sham groups showed no difference in time to remove the adhesive tape during the test. GCI–vehicle group took significantly longer to remove the tape. Post treatment of NAC after GCI reduces removal time (time dependently). Data are mean ± SEM, *n* = 5–8 each group, \* *p* < 0.05 versus vehicle-treated group; # *p* < 0.05 versus sham-operated group (repeated measure test followed by ANOVA, time: F = 6.365, *p* < 0.001; group: F = 33.354, *p* < 0.001; time \* group: F = 3.061, *p* < 0.001). (**B**) The mNSS was conducted at 3 h and 7 consecutive days after sham and GCI surgery. Sham groups indicate a 0 score which means all tasks were performed completely without defect. Under the ischemic condition, the vehicle group showed a failure of mNSS sessions. Post administration, NAC improved mNSS procedures and reduced failure of tasks. Data are mean ± SEM, *n* = 6–8 each group, \* *p* < 0.05 versus the vehicle-treated group (repeated measure test followed by ANOVA, time: F = 35.621, *p* < 0.001; group: F = 17.576, *p* = 0.001; time \* group: F = 2.322, *p* = 0.032). (**C**) Immunohistochemistry images show NeuN-positive neurons in the hippocampal subiculum, CA1, and CA2 regions. Scale bar = 50 μm. (**D**) Bar graph shows analysis of NeuN-positive cells in each hippocampal region. Post treatment of NAC restores GCI-induced delayed neuronal loss in the following 7 days. Data are mean ± SEM, *n* = 5–6 each group, \* *p* < 0.05 versus vehicle-treated group; # *p* < 0.05 versus sham-operated group (Kruskal–Wallis test followed by Bonferroni post-hoc test, Subiculum: chi square = 17.792, df = 3, *p* < 0.001; CA1: chi square = 17.958, df = 3, *p* < 0.001; CA2: chi square = 17.735, df = 3, *p* < 0.001).

#### **3. Discussion**

In the present study, we verified whether NAC administration attenuates GCI-induced neuronal death via inhibition of TRPM2 channels. Our findings indicate that NAC reduces accumulation of intracellular free zinc and TRPM2 over-activation by maintaining a sufficient supply of glutathione production. Cysteine is the rate-limiting substrate required for the biosynthesis of glutathione and the cysteine residue present in NAC can serve as an immediate source of cellular cysteine to maintain high levels of intracellular glutathione, which in turn protects against oxidative damage arising from free radicals. In addition, daily administration of NAC for 3 days after GCI also clearly attenuated several GCI-induced cell death cascades involving neurodegeneration, intracellular free zinc accumulation, oxidative damage-induced lipid peroxidation, loss of glutathione, TRPM2 over-activation, and glial activation. Furthermore, post-treatment of NAC for 7 days prevented GCI-induced delayed hippocampal neuron death, sensorimotor impairment, and neurological deficits. Taken together, these results suggest that reduction of intracellular free zinc and restoration of neuronal GSH concentration by NAC administration attenuates GCI-induced neurodegenerative cascades and promotes behavioral impairments. Therefore, NAC may be an outstanding therapeutic agent for preventing GCI-induced neuronal death.

In general, ionic gradients are actively maintained within finely tuned ranges via several selective and non-selective ion channels throughout the central nervous system, and this process is fundamental to preserving healthy brain function. However, during many neurological injuries that include ischemic stroke [12] and traumatic brain injury [25] damage leads to an ionic imbalance, which in turn allows for the initiation of multiple neurodegenerative cascades. Our previous studies have focused on these mechanisms that follow ionic imbalance in several neurological diseases, such as global ischemia [13,47], hypoglycemia [48], and traumatic brain injury [49]. In particular, we have focused on the abnormal phenomenon of excessive release of vesicular zinc ions following brain injuries and its subsequent cellular pathology. Chelatable zinc that is liberated in this manner moves into post-synaptic neurons through non-selective cation channels, TRPM2 [50], and once chelatable zinc has accumulated within post-synaptic neurons it acts as a neurotoxin [51]. TRPM2 is one of the non-selective cation channels that regulates physiological and pathological processes in the central nervous system via modulation of numerous signaling pathways [38]. In addition, brain disease-induced ROS production triggers TRPM2 activation, leading to calcium influx from the extracellular space, and finally triggers cell death cascades that include PARP-1/PARG and caspase-dependent cell-death pathways [50,52,53]. In this present study, we focused on attenuating the effects of excessively released free zinc ions from synaptic vesicles after GCI that translocate into post-synaptic neurons via the TRPM2 channel. Hence, we hypothesized and verified that by reducing ROS production through supplementation of cysteine by NAC administration to maximize glutathione production, one can reduce hippocampal neurodegeneration through inhibition of zinc accumulation and TRPM2 activation.

*N*-acetyl-l-cysteine (NAC) is a derivative of cysteine and acts as a scavenger for elimination of free radicals [54]. In the present study, we focused on NAC as a cysteine donor to improve glutathione synthesis, and also as a chelator of heavy metal ions. Excessively released and accumulated free zinc ions after ischemic stroke have a deleterious effect on the nervous system. Previous studies determined that NAC can bind heavy metals (nickel, copper, zinc) because NAC has a thiol group (-SH), which allows it to bind cationic metal ions [55]. In addition, NAC can restore neuronal glutathione concentrations and protect against brain injuries. Kho et al. recently demonstrated that hypoglycemic brain disease-induced hippocampal neurodegeneration, lipid peroxidation, and zinc accumulation were restored to pre-insult levels by NAC administration [24]. Furthermore, Choi et al. recently verified that genetic deletion of the cysteine-related transporter, excitatory amino acid carrier 1 (EAAC1), exacerbates traumatic brain injury (TBI)-induced neurological damage, compared with wild type controls. These cell death cascades were attenuated by NAC treatment in both wild type and EAAC1 gene deletion animals [34]. In light of the abovementioned results, we hypothesized that NAC treatment after GCI may chelate zinc ions, and reduce accumulation of zinc-induced neurotoxicity and downregulation of the TRPM2 channel by reducing ROS production.

The post-global ischemic neuro-inflammatory response contributes to formation of the glial scar and exacerbates neuronal damage. Both microglia and astrocytes play an essential role in maintaining ionic gradients within the extracellular space and in supporting neurotransmission in neighboring neurons. In general, inactivated (resting) microglia and astrocytes regulate the maintenance of central nervous system homeostasis [56]. However, neurodegenerative diseases, such as head trauma, multiple sclerosis, epilepsy and stroke, trigger gliosis which is a non-specific alteration of glial cells and this can lead to glial scar formation if unchecked [57]. Lakhan et al. demonstrated that stroke-induced excitotoxicity and oxidative damage triggers activation of microglia and astrocytes, and these cells secrete several cytokines, chemokines, and matrix metalloproteases [58]. In addition, Kauppinen et al. verified that the zinc-overloaded condition alters the morphology of resting microglia to an activated form in cultured microglia, which was attenuated by treatment with the extracellular zinc chelator, CaEDTA, during the ischemic condition [59]. Barreto et al. reported that astrocyte viability was important for the restoration of neurons following ischemic damage. Furthermore, they asserted that although reactive astrocytes trigger the production of pro-inflammatory cytokines, they also perform functions for maintaining neuronal survival [9].

In the present study, our findings indicate that TRPM2 activation in neurons and glial cells were found after GCI, but were significantly correlated with astrocytes. Astrocytes have a dual nature when activated that can both alleviate or exacerbate the effects of brain damage depending on a sensitive mix of cellular factors. Accordingly, Barreto et al. assert that any solution that adequately addresses the problem of stroke-induced neurodegenerative must involve astrocytes [9]. No details about the interaction of TRPM2 and astrocytes has been reported, but we assumed that there must be some ischemia-related cascades between TRPM2 channels and astrocytes, and began to test this hypothesis. In the case of our research, TRPM2 activation in microglia was only mildly observed compared with astrocytes. However, microglia have a strong influence on CNS diseases, and it is also necessary to consider them for understanding injury-induced neuroinflammation and neurodegeneration in several neurological conditions. Malko et al. reported that calcium influx via TRPM2 after external stimuli triggers microglial activation and generates pro-inflammatory mediators from activated microglial cells, which further exacerbate damage. Furthermore, the TRPM2 activation process was closely related with NADPH oxidase-mediated ROS production, and PARP-1 (poly ADP-ribose polymerase-1) and ADPR (ADP-ribose) activation [60]. As mentioned in the introduction, oxidative damage following ischemic stroke is an essential factor for understanding brain damage, and is closely related with regulation of cation channel activity.

Based on our study and other previous studies, we speculated that a possible neuronal death cascade could be triggered by zinc influx via TRPM2 (Figure 7). In Figure 7A, schematic drawings describe the process of GCI-induced zinc accumulation, ROS production, and TRPM2 activation. Predicted steps of zinc-related neuronal death are as follows. (1) GCI induces vesicular zinc release from the pre-synaptic terminal to the synaptic cleft [10,13], where it translocated into post-synaptic neurons via the non-selective cation channel, TRPM2 [50,61]. In the post-synaptic neurons, translocated zinc contributes several deleterious cascades such as apoptosis [62,63], necrosis [63], as well as ROS-producing signaling. (2) Following ischemia zinc aggravates ROS production via neuronal NADPH oxidase activation [64]. Synaptically released zinc after GCI stimulates protein kinase C (PKC), and PKC contributes NADPH oxidase activation [65]. (3) A major activator of TRPM2 is ADP-ribose (ADPR), which is released upon oxidative stress including ROS [66,67]. (4) ROS-induced TRPM2 activation triggers increased secondary zinc influx [61]. (5) Secondary translocation of zinc promotes increased ROS production [59,68]. (6) Zinc and ROS induce neuronal death. In Figure 7B, the predicted mechanism of NAC on TRPM2 is shown, as follows. (7) GCI induces zinc translocation from the synaptic terminal into neurons through TRPM. Zinc increases ROS production via NADPH oxidase activation. (8) NAC-supplied cysteine enters into post-synaptic neurons through EAAC1. EAAC1 acts as a cysteine carrier in the brain. (9) Cysteine is converted to glutathione, which has an anti-oxidative effect. ROS production is attenuated by glutathione. (10) Reduction of ROS production decreases TRPM2 activation. (11) Reduced TRPM2 induces diminished secondary zinc influx. Zinc influx reduction contributes neuroprotection. (12) NAC administration induced reduced ROS production and diminished zinc influx, which finally attenuates neuronal death. Thus, as discussed above, steps (1)–(6) shown in the Figure 7A have been demonstrated by our and other group's previous studies. However, steps (7)–(12) in the Figure 7B have been verified in this study; it is shown in Figures 2–6 that NAC treatment reduced GCI-induced zinc accumulation, oxidative stress, TRPM2 activation and neuronal death.

Taken together, the present study demonstrates the efficacy of NAC in preventing GCI-induced neuronal death, sensorimotor deficits, and neurological pathology via several signaling cascades. NAC inhibits GCI-induced zinc accumulation and influx via TRPM2 into postsynaptic neurons. Therefore, we speculate that inhibition of zinc influx and TRPM2 activation by NAC may have a potential for the treatment of ischemic stroke.

**Figure 7.** Predicted reactions of NAC post treatment on GCI-induced neuronal death. Representative schematic illustration indicates NAC action via reduction of reactive oxygen species (ROS) production and subsequent decrease in TRPM2 activation. (**A**) (1) Released zinc after GCI moves into the post-synaptic neurons through TRPM2. (2) Translocated zinc contributes to ROS production. (3) Oxidative damage including ROS activated TRPM2. (4) ROS-induced TRPM2 activation increased secondary zinc influx. (5) Increased translocated of zinc contributes to greater ROS production. (6) Zinc and ROS induces neuronal death. (1)–(6) Image shows the hypothesized cascade produced upon GCI-induced zinc influx and TRPM2 activation. (**B**) (7) Zinc translocated into the post-synaptic neurons via TRPM2 after GCI. (8) Cysteine supplied by NAC administration moves into post-synaptic neurons through excitatory amino acid carrier 1 (EAAC1). (9) Cysteine is converted to glutathione which has an anti-oxidant effect and inhibits ROS production. (10) Inhibition of ROS production leads to reduced TRPM2 activation. (11) Reduced TRPM2 activation leads to less secondary zinc influx. (12) Reduction of ROS production and secondary zinc influx reduces neuronal death. (7)–(12) Process shows possible neuroprotective cascade of NAC after GCI.
