*2.2. Activation of Antioxidant Signals by AA Treatment in Aging*

In our aging model of OHSCs with time in culture, we observed age-related changes in the hippocampus. Synapsin-1 and PSD 95, pre- and post-synaptic components implicated in dendritic spine formation and neurotransmission, were observed to confirm the aging statuses of each culture condition. PSD 95 expression was significantly reduced in the 9 w group compared to 3 w group, while the 9 w-daily group showed significantly higher PSD 95 expression than the 9 w group. For synapsin-1, the 9 w group showed markedly lower expression levels than the 3 w and 9 w-daily groups. The expression levels of synapsin-1 and PSD 95 in the 9 w group was markedly reduced compared to the 3 w group, which indicated the age-related phenomena within the hippocampus in our aging model of OHSCs. As for the chronic AA treatment group, the expression levels of both synapsin-1 and PSD 95 proteins showed significant differences between the 9 w and 9 w-daily groups (Figure 3A). These results indicated that chronic AA treatment conserved the decreases in synaptic ability associated with neurodegeneration in aging.

**Figure 2.** Neuroprotective effects of AA against KA-induced oxidative injury in hippocampal neurons. (**A**) Representative PI fluorescence images in OHSCs before (Pre) and 24 h after AA-treatment following oxidative injury. Representative images of PI uptake, as a marker of cell death, showed the effects of AA treatment at different time points. AA-only treatment (500 μM) showed no toxic effect in KA-untreated normal OHSCs (9 w-daily Normal). (**B**) KA treatment induced progressive cell death in the hippocampus compared to the normal group at each time point. After 24 h of AA treatment, PI signals in the CA3 area of the hippocampus were significantly reduced in the treated groups compared to no-treatment (vehicle) groups at each time point. After chronic AA treatment (9 w-daily), there was less cell death following the KA insult compared to the 9 w vehicle group, and the 9 w-daily AA treatment group also showed a significant decrease in the level of PI uptake compared to the 9 w AA treatment group. \*\* *p* < 0.01 compared to normal; † *p* < 0.05, †† *p* < 0.01 compared to vehicle; # *p* < 0.05, ## *p* < 0.01 comparing different time points: two-way ANOVA followed by Tukey's *post hoc* comparison. Scale bar: 500 μm.

To observe the antioxidant role of prolonged AA treatment on cell survival, we examined the expression of superoxide dismutase (SOD) and Nrf2, which are known to be factors in ROS-related cell survival signaling. At 3 w, the expression levels of SOD protein significantly increased in the KA + AA group compared to KA + vehicle group. In the aging model, the 9 w KA + AA group showed significantly higher SOD levels than the KA + vehicle group. Compared to the 3 w KA + AA group, the levels of SOD expression in the 9 w KA + AA group were significantly reduced. Moreover, SOD protein significantly increased in the 9 w-daily KA + AA group compared to the KA + vehicle group. The 9 w-daily KA + vehicle group also showed significantly increased SOD levels compared to the 9 w KA + vehicle group (Figure 3B). Nrf2 expression reflects the susceptibility of the brain to the damaging effects of ROS: the level of Nrf2 expression diminished with age, and consequently, its neuroprotective effect decreased. The expression levels of Nrf2 protein decreased in the vehicle group compared to the normal group at 3 w. With aging, the 9 w group showed no difference in Nrf2 expression between KA + AA and KA + vehicle groups. Compared to 3 w, Nrf2 expression was significantly decreased at 9 w for both KA + AA and KA + vehicle groups. In the 9 w-daily group, Nrf2 expression tended to be higher compared to the 9 w treatment groups (Figure 3C).

#### *2.3. Effect of AA on Neuronal Activity*

We performed optical imaging to observe cellular activities to visualize the active area based on the overall data and provide information on neuronal activation using a voltage-sensitive dye (VSD). Typical signal transmission and the spatial distribution of the cellular response after electrical stimulation in different age-related groups are shown in Figure 4A. As shown in Figure 4A, KA-untreated hippocampal neurons (normal groups at 3 w, 9 w, and 9 w-daily) demonstrated typical spatiotemporal changes using Schaffer collateral/commissural stimulation. However, limited activation accompanied by synaptic propagation was observed in the KA + vehicle group of OHSCs. In contrast, more activation (indicating more propagation) was observed in the KA + AA group compared to the KA + vehicle group in each age-related group.

**Figure 3.** *Cont.*

**Figure 3.** (**A**) Changes in synaptic protein expression by culture time of OHSCs. PSD 95 and synapsin-1 expression levels significantly reduced at 9 w compared to 3 w. In the chronic AA treatment group, the expression levels of PSD 95 and synapsin-1 proteins showed a significant difference between 9 w and 9 w-daily groups. (**B**) Western blot analysis of SOD and Nrf2 in OHSCs following oxidative injury. The expression of SOD in the vehicle group was significantly reduced compared to the normal group. The expression of SOD significantly increased 24 h after 500 μM AA treatment in the 3 w and 9 w groups, compared to the vehicle group. The chronic AA treatment (9 w-daily) group showed higher SOD expression than the 9 w group. (**C**) The expression level of Nrf2 increased in the AA treatment group compared to the vehicle group at 3 w. The 9 w group showed no difference in Nrf2 expression between the AA treatment and vehicle groups. However, Nrf2 expression was significantly decreased in both AA treatment and vehicle groups at 9 w compared to 3 w. In 9 w-daily groups, Nrf2 expression tended to increase compared to 9 w. The horizontal axis indicates each experimental group (3 w, 9 w, and 9 w-daily AA treatment), and the vertical axis represents the normalized level of each protein expression (ratio of each antibody expression/β-actin expression). \* *p* < 0.05, \*\* *p* < 0.01 compared to normal, † *p* < 0.05, †† *p* < 0.01 compared to vehicle, # *p* < 0.05, ## *p* < 0.01 comparing different time points: two-way ANOVA followed by Tukey's post hoc comparison.

N1 latency, a sign of synaptic activation, increased in both KA + vehicle and KA + AA groups compared to both 3 w and 9 w normal groups by optical signals. The statistical analysis of the neuronal activity of AA is shown in Figure 4B. At 3 w, the AA treatment group showed a significantly reduced latency compared to the KA + vehicle group (normal: 8.84 ± 0.91, KA + vehicle: 30.75 ± 3.66, KA + AA: 16.45 ± 0.97). In 9 w, there was no difference between KA + vehicle and KA + AA groups in N1 latency (9 w normal: 9.05 ± 0.64, KA + vehicle: 26.93 ± 4.27, KA + AA: 23.23 ± 1.56). Meanwhile, N1 latencies in the 9 wdaily groups were significantly shorter than those in the 9 w KA + vehicle and KA + AA groups (9 w-daily normal: 7.66 ± 0.46, KA + vehicle: 12.75 ± 2.52, KA + AA: 8.94 ± 0.81). These results showed that the chronic treatment AA (9 w-daily) in the aged group affected the neuronal activity as an indication of survival neuronal function (Figure 4B). In the activation area in Figure 4C, the KA + AA group showed significantly activated areas compared to the KA + vehicle group at 3 w (normal: 39.70 ± 3.81, KA + vehicle: 15.40 ± 3.58, KA + AA: 27.52 ± 3.00). In 9 w OHSCs, few activated areas were observed around the stimulating electrode in both the KA + vehicle and KA + AA groups (9 w normal: 38.41 ± 4.64, KA + vehicle: 9.39 ± 1.31, KA + AA: 14.38 ± 2.61). There was no

difference between the 9 w-daily groups (9 w-daily normal: 37.81 ± 7.44, KA + vehicle: 26.37 ± 5.44, KA + AA: 30.90 ± 6.02). However, chronic AA treatment in the 9 w-daily KA + AA group elicited significantly more activated areas than the 9 w KA + AA group. In addition, the 9 w-daily KA + vehicle group showed significantly larger activated areas compared to the 9 w KA + vehicle group (Figure 4C). Therefore, synaptic propagation was better preserved and maintained after chronic AA treatment in the 9 w-daily group, and this group showed less vulnerability to oxidative injury compared to other 9 w groups.

**Figure 4.** *Cont.*

**Figure 4.** Optical recordings were conducted with a voltage-sensitive dye (VSD) in hippocampal slices to assess AA treatment effects. (**A**) Representative pseudo-color optical images show the evoked excitatory neuronal signals and typical spatiotemporal changes that were observed in OHSCs after electrical stimulation. Each frame of the pseudo-color image (left side) is presented by an arrow below the wave form (right side) at each time point (a, before stimulation; b, peak of N1; c, half of the N1; d, post stimulation). The X-axis shows the time (msec), and the y-axis shows the percent change in fluorescent intensity (% ΔF/F). The blue grid line shows the peak of the negative (N1) point, which was used to quantify the N1 latency. A colored scale (color calibration) shows the changes in optical signals (upper-left side). Signal transmission indicating spatiotemporal changes showed an increase in neuronal activity through long-lasting depolarization. The KA + vehicle groups at each time point showed a few signs of activation, only in the focal stimulation area. Spatiotemporal changes, reflecting the distribution of neuronal activity, increased after AA treatment. (**B**) Comparisons of optical signals in the different experimental groups by latency, an indication of basal synaptic transmission. Latencies were significantly delayed in the vehicle groups compared to normal groups at each time point. The latency of the optical signals was decreased in AA-treated hippocampal slices compared to the vehicle group at 3 w. For chronic AA treatment, there was a significant difference between the 9 w and 9 w-daily groups. (**C**) Quantification of pseudo-color activated areas that indicate neuronal activities through neural propagation. The activation area was decreased in the vehicle groups compared to the normal groups at each time point. In particular, the chronic AA-treated 9 w-daily group showed a significantly greater increase in activation area compared to the acute 9 w group. \* *p* < 0.05, \*\* *p* < 0.01 compared to normal, †† *p* < 0.01 compared to vehicle, # *p* < 0.05, ## *p* < 0.01 comparing different time points: two-way ANOVA followed by Tukey's post hoc comparison. Scale bar: 500 μm.

#### **3. Discussion**

Providing cells with AA as an exogenous antioxidant retards their uptake of endogenous antioxidants. In agreement with previous research [21], AA treatment elicited significant reductions in neuronal cell death, compared to vehicle, through a reduction in ROS and oxidative stress by the activation of antioxidant pathways. However, the negative effect of oxidative decay caused by ROS is considered to be an irreversible progression in the biology of aging [22]. Research has shown that the number of neurons and astrocytes remain constant over 21 days in vitro (DIV, 3 w) among OHSCs by cell density analysis and that the density of synaptic contacts within 21 DIV OHSCs is two times higher than those in 7 DIV OHSCs [23]. Moreover, DIV 21 cultures have been found to show similarities to features of the acute slice at P21 [24]. Synapses formed in OHSCs arise from neurons that survive explanation, and synaptic differences in OHSCs according to culture time have been observed, with significant reductions in PSD 95 and evoked synaptic activity at 67-76 DIV [25]. In line with the study by Mielke et al. [25], we also observed significant decreases in PSD 95 and synapsin-1 expressions, which were assessed to reflect age-related phenomena within the hippocampus in our aging model of OHSCs.

Age-dependent changes in brain homeostasis and function occur gradually, particularly by the combination of ROS and the aging brain's impaired ability to repair increased oxidative stress damage [26]. Although we observed decreased neuronal cell death with acute AA treatment after 3 w and 9 w, compared to vehicle, PI uptake quantitation, as an indicator of cell death, was significantly different (based on age) between the 3 w and 9 w AA treatment groups. The 9 w group showed more vulnerability to oxidative injury and cell death compared to the 3 w group. Aging of the brain was also associated with an increase in seizure susceptibility, as well as with seizure-induced neuronal damage, after KA-induced oxidative injury [27,28]. While Siqueira et al. [19] reported that 2 weeks of chronic supplementation with ascorbate was unable to protect the hippocampus from age-related oxidative damage (H2O2), we observed that the 9 w-daily vehicle and 9 w-daily AA treatment groups showed significantly greater reductions in cell death than did the 9 w AA treatment group. As a consequence of aging, decreased ascorbate synthesis or altered ascorbate transport characteristics [19] may explain this reduced neuroprotective effect of acute AA treatment in the 9 w group compared to chronic AA treatment in the 9 w-daily group. We hypothesized that chronic AA treatment may lead to persistent regulation of reactions against increased reactive oxygen and nitrogen species with various target molecules during aging, a period during which the antioxidant system appears to be less functional in the brain. Mild prooxidant activity can enhance antioxidant defense systems through Nrf2 signaling [29–31]. In this regard, chronic treatment of AA, which is not only able to scavenge free radicals but also maintain an optimal ROS flow, may upregulate cytoprotective enzymatic antioxidants, thus improving neuronal survival. Chronic treatment of AA might act as a parahormetic phytochemical, which can be attributed to both antioxidant and mild prooxidant activities that affect the intracellular antioxidant defense system. As part of the antioxidant defense system, any decrease in SOD and Nrf2 levels would result in increased ROS [32]. In our Western blot analysis, the expressions of SOD and Nrf2 significantly decreased in the 9 w groups compared to the 3 w groups. Imbalances in ROS production due to impaired expression of SOD and decreased levels of antioxidant molecules occurred with aging, as well as in neurodegenerative diseases [33]. Similar to the PI results, chronic AA treatment (9 w-daily AA treatment group) significantly increased SOD levels. The neurons in the 9 w group suffered more severe attacks by free radicals compared to the neurons with chronic AA treatment, and AA supplementation might have scavenged free radicals from plasma directly. The present study indicates that the aging brain is highly susceptible to hippocampal cell loss by KA-induced oxidative stress, and that chronic AA treatment can aid the cellular antioxidant system against oxidative injury.

To assess neuronal survival and functional property, optical imaging of VSD was used to examine synaptic changes and strength in OHSCs. Optical imaging data represent the visualization of electrical signals from the population activity of postsynaptic

neurons in the activated area as a distribution map of stimulus-induced activities [34]. Here, the differences in activities of surviving neurons were examined between 3 w and 9 w slice cultures that underwent AA treatment after oxidative injury. The 9 w group showed increased latencies of optical signals and less activated areas compared to the 3 w group, indicating reduced functionality of surviving neurons. This result may be related to decreased uptake of radiolabeled ascorbate in aged rats [19], as well as to a decline in sodium-dependent ascorbate transport during the aging process [20]. Siqueria et al. [19] reported a 40% decrease in radiolabeled 14C-ascorbate uptake in 11-month-old rats compared to 4-month-old rats, and Michels et al. [20] described an age-related reduction in the plasma transport of dietary ascorbate. Neurons have a 10-fold-higher level of ascorbate than the glia, making them more sensitive to ascorbate deficiency and any reduction in total antioxidant capacity in an older hippocampus [19]. The VSD activation area is dependent on the spatial distribution of cellular responses to electrical stimulation. In this study, we demonstrated enhanced synaptic transmission resulting from chronic AA treatment in older 9 w OHSCs after oxidative injury. To our knowledge, there are no reports that chronic AA supplementation protects hippocampal cells in the brain from the age-related increases in susceptibility to oxidative damage. Therefore, with the optical recording results demonstrating better survival of hippocampal neurons and their connections, chronic AA supplementation may not only be neuroprotective, but also help restore the endogenous antioxidant system with advancing age.
