Anti-Zn2+-Toxicity of 4-Hydroxybenzyl Alcohol in Astrocytes and Neurons Contribute to a Robust Neuroprotective Effects in the Postischemic Brain.

4-Hydroxybenzyl alcohol (4-HBA) is an important phenolic constituent of Gastrodia elata (GE) Blume, which is used as a traditional herbal medicine in East Asia. Many activities have been reported to underlie the beneficial effects of 4-HBA in brain, such as, anti-oxidative, anti-inflammatory, anti-excitotoxic, and anti-apoptotic effects in neurons and microglia. Here, the authors demonstrate the robust neuroprotective effects of 4-HBA in rat middle cerebral artery occlusion (MCAO) model of stroke, and showed anti-Zn2+ toxicity in neurons and astrocytes as a molecular mechanism contributing to these effects. Intraperitoneal administration of 4-HBA (20 mg/kg) in Sprague–Dawley rats 1 h after MCAO reduced infarct volumes to 27.1 ± 9.2% of that of MCAO controls and significantly ameliorated motor impairments and neurological deficits. Significant suppressions of Zn2+-induced cell death, ROS generation, and PARP-1 induction by 4-HBA were observed in primary cortical cultures. 4-HBA also protected astrocytes from Zn2+-induced toxicity and suppressing ROS generation by employing slightly different molecular mechanisms, i.e., suppressing PARP-1 induction and NAD depletion under acute Zn2+-treatment and suppressing p67 NADPH oxidase subunit induction under chronic Zn2+-treatment. Results indicate that the protective effects of 4-HBA against Zn2+-toxicity in neurons and astrocytes contribute to its robust neuroprotective effects in the postischemic brain. Considering the pleiotropic effects of 4-HBA, which have been reported in previous reports and added in the present study, it has therapeutic potential for the amelioration of ischemic brain damage. Electronic supplementary material The online version of this article (doi:10.1007/s10571-017-0508-y) contains supplementary material, which is available to authorized users.

4-Hydroxybenzyl alcohol, MCAO, Anti-Zn2+-toxicity, Astrocyte, Neuroprotection

4-Hydroxybenzyl alcohol (4-HBA) is an important phenolic constituent of Gastrodia elata (GE) Blume, which continues to be used as a traditional herbal medicine in East Asia to treat various pathological conditions, such as headaches, convulsive behavior, dizziness, and vertigo (Hsu et al. 1986). Many functions have been reported to underlie the above-mentioned beneficial effects of 4-HBA, which include anti-oxidative (Yu et al. 2005, 2011), anti-inflammatory (Lim et al. 2007), anti-excitotoxic (Descamps et al. 2009), sedative (Deng and Mo 1979), anti-apoptotic (Yu et al. 2010), and anxiolytic effects (Jung et al. 2006). Several aspects of the molecular mechanism underlying the anti-inflammatory effect of 4-HBA have been reported, such as, down-regulations of the c-Jun NH(2)-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) signaling pathways (Kim et al. 2012), and the up- or down-regulation of glucose-regulated protein 78 (GRP78) or C/EBP homologous protein (CHOP), respectively (Lee et al. 2012). Various molecular mechanisms have also been reported to underlie its anti-oxidative effects, such as, the inductions of various anti-oxidant genes, for example, NAD(P)H dehydrogenase, quinone 1 (NQO1), peroxiredoxin (Prdx6), and protein disulfide isomerase (PDI) (Yu et al. 2011, 2013), and direct radical scavenging (Liu and Mori 1993; Kim et al. 2007; Dhiman et al. 2009). In addition, the suppression of caspase-3 activation has been reported to be responsible for the anti-apoptotic effects of 4-HBA (Yu et al. 2010), and suppression of angiogenesis via decreasing expression of matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF) has also been reported in a mouse model of endometriosis (Laschke et al. 2011) and a dorsal skinfold chamber model (Laschke et al. 2013). 4-HBA has been reported to have protective effects in various diseases that affect the central nervous system (CNS), in particular, it has been reported to mitigate ischemic injury in rat middle cerebral artery occlusion (MCAO) model (Yu et al. 2010, 2011, 2013) and in a global ischemia animal model (Kim et al. 2007). Among many pathological events known to occur in the postischemic brain, excitotoxicity and Zn2+-toxicity cause acute brain damage and they are known to be interconnected, as Zn2+ aggravates excitotoxicity by inhibiting astrocyte glutamate uptake (Suh et al. 2007; Tang et al. 2010). In fact, Zn2+-toxicity is known to contribute to neuronal death in various diseases affecting brain, including stroke, epilepsy, and traumatic injury. Neuroprotective effect of 4-HBA by suppressing excitotoxicity in CA1 neurons has been reported in transient global ischemia animal model subjecting Mongolian gerbils (Kim et al. 2007); however, no report has been made regarding the protective effect of 4-HBA against Zn2+ toxicity. In the mammalian brain, astrocytes exert pleiotropic functions beneficial to neurons, for example, controlling the energy supply to neurons (Dringen et al. 1993; Pellerin et al. 2007) and modulating the neuronal synapse formation and neurotransmitter turnover (Pfrieger and Barres 1997; Eroglu and Barres 2010). Under pathological conditions, astrocytes become reactive, and display morphological modifications and scar formation, the latter crucially protects adjacent neural tissues by isolating lesions produced by brain injury (Tanaka et al. 1999). In cerebral ischemia, the neuroprotection exerted by reactive astrocytes is also achieved by the regulation of ionic balance and energy metabolism (Nakase et al. 2003). In addition, suppression of Zn2+-toxicity in astrocytes has been known to confer neuroprotection via reducing deteriorating effect on glutamate uptake by astrocytes (Suh et al. 2007; Tang et al. 2010). In the present study, we explored the anti-Zn2+-toxicity effects of 4-HBA in neurons and astrocytes in the postischemic brain and the molecular mechanism responsible for these effects in the belief that this might contribute to its robust neuroprotective effect. We investigated the protective effects of 4-HBA using an established rat model of MCAO, and primary cortical cultures and C6 astrocyte cell line were used to investigate the anti-Zn2+-toxicity effect and the molecular mechanisms responsible.

Neuroprotective Effect of 4-HBA in the Postischemic Brain To examine the neuroprotective effects of 4-HBA in cerebral ischemia, 10, 20, or 40 mg/kg of 4-HBA was administered intraperitoneally at 1 h post-MCAO and infarct volumes were assessed at 1 day post-MCAO. Mean infarct volumes were 72.1 ± 5.7% (n = 5, p < 0.05), 27.1 ± 9.2% (n = 5, p < 0.01), and 36.1 ± 11.6% (n = 5, p < 0.01), respectively, of that of treatment-naïve MCAO controls (Fig. 1a, b). When 20 or 40 mg/kg of 4-HBA was administered at 1 h prior to MCAO, infarct volumes were reduced to 56.9 ± 7.3% (n = 5, p < 0.01) and 60.4 ± 3.4% (n = 5, p < 0.01) of that of treatment-naïve MCAO controls, respectively (Fig. 1c, d). When administered at 6 h post-MCAO, 40 mg/kg of 4-HBA reduced infarct volume to 47.1 ± 6.4% (n = 5, p < 0.01) of that of treatment-naïve MCAO controls (Fig. 1c, d). These results indicate that 4-HBA has a robust neuroprotective effect in the postischemic brain with a wide therapeutic window. Amelioration of Neurological Deficits After MCAO by 4-HBA The mean modified neurological severity score (mNSS) of treatment-naïve MCAO controls at 1 day post-MCAO was 13.2 ± 0.6 (n = 5) (Fig. 2a). When 10, 20, or 40 mg/kg of 4-HBA was administered at 1 h post-MCAO, mNSSs were reduced to 9.4 ± 0.9 (n = 5), 5.0 ± 1.3 (n = 5), and 7.4 ± 0.8 (n = 5), respectively (Fig. 2a). When 20 or 40 mg/kg of 4-HBA was administered at 1 h prior to or 6 h post-MCAO, mNSSs were reduced to 7.6 ± 1.0 (n = 5) and 8.8 ± 0.4 (n = 5), and 9.8 ± 0.5 (n = 5) and 7.0 ± 0.8 (n = 5), respectively (Fig. 2b). These results indicate that the marked infarct suppression by 4-HBA was accompanied by better neurological outcome. We confirmed that pH, PaO2, PaCO2, and blood glucose levels were similar to those of 4-HBA-treated and treatment-naïve normal and MCAO animals (Table 1), indicating that physiological parameters were not influenced by 4-HBA. Table 1. | Control group | Ischemia group | |||| |---|---|---|---|---|---| | Base | 4-HBA treat | MCAO | MCAO + DMSO | MCAO + 4-HBA | | | Temperature (°C) | 37.08 ± 0.17 | 37.08 ± 0.17 | 37.04 ± 0.14 | 37.06 ± 0.07 | 37.1 ± 0.13 | | pH | 7.64 ± 0.04 | 7.43 ± 0.06 | 7.58 ± 0.03 | 7.56 ± 0.03 | 7.53 ± 0.01 | | pO2 (mmHg) | 145.80 ± 19.2 | 144.00 ± 1.43 | 150.22 ± 6.66 | 145.92 ± 1.78 | 147.36 ± 0.89 | | pCO2 (mmHg) | 32.16 ± 1.95 | 34.30 ± 1.49 | 39.06 ± 1.23 | 32.24 ± 1.31 | 32.86 ± 2.24 | | Glucose (mg/dl) | 109.80 ± 5.63 | 110.00 ± 4.97 | 109.6 ± 5.19 | 105.9 ± 4.81 | 103.50 ± 7.67 | Values are mean ± SD (n = 4). One-way analysis of variance revealed no significant intergroup difference for any variable 4-HBA Suppressed NMDA- or Zn2+-Induced Neuronal Death To investigate underlying mechanism contributing to a robust protective effect of 4-HBA in the postischemic brain, we examined whether 4-HBA confers protective effects against acute damaging processes in neurons, for example, excitotoxicity or Zn2+-toxicity. When primary cortical cultures were treated with NMDA (30 μM) for 10 min, cell death at 24 h after NMDA treatment was induced to 66.1 ± 6.2% (Fig. 3a). However, pre-treatment of cells with 4-HBA (100, 250, or 500 μM) for 6 h significantly suppressed NMDA-induced cell death (Fig. 3a). At 250 μM of 4-HBA, cell death was suppressed to 73.5 ± 4.0% (n = 4) of that of treatment-naïve NMDA control cells (Fig. 3a). Similarly, Zn2+ (200 μM, 15 min)-induced neuronal death decreased to 80.8 ± 1.9% (n = 4) or 69.2 ± 1.6% (n = 4) of that of treatment-naïve Zn2+ control cells by pre-treating 100 or 250 μM of 4-HBA for 6 h, respectively (Fig. 3c). When cells were pre-treated with 4-HBA (50, 100, or 250 μM) for 3 or 9 h, 9 h pre-treatment of 250 μM 4-HBA showed the greatest protective effect both in NMDA- or Zn2+-treated cells, and cell death was reduced to 70.3 ± 2.7% (n = 4) or 65.9 ± 3.0% (n = 4) of that in treatment-naïve NMDA or Zn2+ control cells, respectively (Fig. 3b, d). These results indicate that pre-treatment of 4-HBA (250 μM, 6 or 9 h) has a robust protective effect against NMDA- or Zn2+-toxicity in neurons. 4-HBA Inhibited Zn2+-Induced ROS Generation and PARP-1 Induction in Primary Cortical Cultures It has been reported that Zn2+ overload induces ROS production in neurons (Noh and Koh 2000). When we stained Zn2+ (200 μM, 15 min)-treated primary cortical cultures with CM-H2DCFDA, an intracellular ROS indicator, induction of DCF fluorescence was observed at 15 min after Zn2+ treatment and further increased until 120 min (data not shown). However, pre-treating cells with 100 or 250 μM of 4-HBA for 6 h suppressed the induction of DCF at 60 min after Zn2+ treatment to 69.8 ± 2.9% (n = 4) and 44.7 ± 2.7% (n = 4), respectively, of that in treatment-naïve Zn2+ control cells (Fig. 4a, b), demonstrating that 4-HBA suppressed Zn2+-induced ROS generation. Since poly ADP ribosyl polymerase-1 (PARP-1), a NAD+-catalyzing enzyme, has been known to mediate ATP depletion and neuronal death after excitotoxic insult or exposure to Zn2+ (Kim and Koh 2002), we examined whether 4-HBA affects PARP-1 induction in Zn2+-treated primary cortical cultures. Up-regulation of PARP-1 was detected at 2 h after Zn2+ treatment and peak induction was detected after 4 h (Fig. 4c). PARP-1 up-regulation at 4 h after Zn2+ treatment was significantly suppressed by pre-treating cells with 100 or 250 μM of 4-HBA for 6 h (Fig. 4d) and increase of PAR formation was also suppressed (Fig. 4e). In addition, peak induction of p67 subunit of NADPH oxidase, which was known to mediate Zn2+-mediated ROS induction in neurons and astrocytes (Noh and Koh 2000), was also significantly suppressed by pre-treating cells with 100 or 250 μM of 4-HBA for 6 h (Fig. 4c, d). Furthermore, NAD depletion in Zn2+-treated cells was suppressed by pre-treatment with 100 or 250 μM of 4-HBA for 6 h (Fig. 4f). These results indicate that 4-HBA (250 μM, 6 h pre-treatment) inhibits ROS generation and the inductions of PARP-1 and p67 in neurons exposed to Zn2+. 4-HBA Suppressed Zn2+-Induced Astrocyte Death The protection afforded by 4-HBA in neurons against Zn2+-toxicity prompted us to examined whether 4-HBA exert the same effect on astrocytes. When C6 cells, an astrocyte cell line, were treated with Zn2+ (100, 200, or 300 μM) for 15 min, cell viabilities at 24 h after Zn2+ treatment decreased to 45.7 ± 2.0, 30.1 ± 1.7, or 25.8 ± 0.7%, respectively, of that of non-treated controls (Fig. 5a). However, when cells were pre-treated with 4-HBA (100 or 250 μM) for 6 or 9 h, Zn2+ (200 μM)-induced cell death was significantly suppressed, and 9 h pre-treatment showed greater protective effects at both doses (Fig. 5b). Similarly, when C6 cells were treated with Zn2+ (50 μM) in a chronic manner (for up to 24 h) (Fig. 5c), co-treatment of 4-HBA (50, 100, or 250 μM) also significantly suppressed astrocyte death (Fig. 5d). These results indicate that 4-HBA has a robust protective effect against Zn2+-toxicity in astrocytes. 4-HBA Suppressed ROS Production and PARP-1 Up-regulation Induced by an Acute Zn2+ Treatment in Astrocytes Since, we confirmed Zn2+-induced ROS production in neurons in the present study (Fig. 4), we examined this phenomenon in astrocytes. Staining of C6 cells with CM-H2DCFDA revealed ROS generation in Zn2+ (200 μM, 15 min)-treated C6 cells; DCF fluorescence rapidly increased and maximum fluorescence was observed after 120 min of Zn2+ treatment (Supplementary Fig. 1). However, when C6 cells were pre-treated with 4-HBA (100 μM) for 6 or 9 h, DCF fluorescence after 60 or 120 min of Zn2+ treatment (200 μM) was markedly decreased. It was decreased to 44.5 ± 2.4% (n = 4) and 72.9 ± 6.2% (n = 4) by 9 h of pre-treatment, respectively, which were much lower than those obtained by 6 h of pre-treatment (Fig. 6a, b). Results demonstrated that 4-HBA had a robust suppressive effect on acute Zn2+ treatment-induced ROS generation in astrocytes. Up-regulations of PARP-1 and PAR formation were detected at 30 min after Zn2+ treatment, and peak inductions were detected at 60 or 90 min, respectively (Fig. 6c, d). Up-regulations of the PARP-1 and PAR formation were significantly suppressed by pre-treating cells with 100 or 250 μM of 4-HBA for 9 h (Fig. 6e, f). Furthermore, NAD depletion in Zn2+-treated C6 cells was suppressed by pre-treating 4-HBA (100 or 250 μM, 9 h) and replenished NAD level in 100 μM of 4-HBA-treated cells reached normal level (Fig. 6g). These results indicate that pre-treatment of 4-HBA (250 μM, 9 h) effectively inhibits ROS generation and PARP-1 induction and replenished NAD in astrocytes exposed to an acute Zn2+ treatment. 4-HBA Suppressed ROS Production and p67 Up-regulation in Astrocytes Treated with Zn2+ Chronically When C6 cells were treated with 50 μM of Zn2+ for up to 24 h, i.e., in a chronic manner, DCF fluorescence was detected after 6 h and peaked after 12 h (Supplementary Fig. 2). Suppression of ROS generation by 4-HBA was observed after co-treating C6 cells with 50, 100, or 250 μM of 4-HBA. DCF fluorescence was suppressed to 56.7 ± 8.8, 33.2 ± 4.3, and 49.4 ± 3.7% of that in treatment-naïve Zn2+ control cells, respectively, after 12 h of Zn2+ treatment (Fig. 7a, b), demonstrating that 4-HBA had a robust suppressive effect on Zn2+-induced ROS generation in astrocytes under chronic Zn2+-treated condition. PARP-1 up-regulation was not detected under chronic Zn2+ treatment (data not shown); however, p67 NADPH oxidase subunit was gradually and markedly induced in C6 cells under chronic Zn2+ treatment, and this induction was significantly suppressed by co-treating 4-HBA (100 μM) for 9 h (Fig. 7c, d). Results indicate that 4-HBA inhibits ROS generation and p67 induction in astrocytes exposed to Zn2+ in a chronic manner.

GEB is traditionally used to treat neurological diseases, in particular, convulsive disorder, such as, epilepsy, general paralysis, vertigo, and tetanus, in East Asia. Analytical studies have identified the constituents of GEB and this list continues to grow in line with the introduction of new analytical methods (Zhao et al. 1999; Yang et al. 2007; Guo et al. 2015; Jang et al. 2015). The principal components of GEB are; 4-HBA, gastrodin, parishin-B, -D, and -E, 4-hydroxybenzaldehyde, 4-hydroxybenzyl methyl ether, 4-hydroxybenzyl ethyl ether, and 4-(4′-hydroxybenzyl)phenol (Yang et al. 2007), and of these, 4-HBA has been shown to have many beneficial effects in the animal models of neurological disorders (Kim et al. 2007; Yu et al. 2010, 2011). Protective effects of 4-HBA have been reported in MCAO animal models, wherein 25 or 50 mg/kg of 4-HBA was treated 30 min prior to MCAO (Yu et al. 2010) or 4-HBA (25, 50, or 100 mg/kg) was treated three times before MCAO (Yu et al., 2011). However, in the present study, we showed better protective effect of 4-HBA with a single bolus (20 mg/kg) at 1 h post-MCAO (Fig. 1a, b). In addition, we found that 4-HBA protects neurons and astrocytes from Zn2+ challenges, suggesting that this effect contributes to the robust neuroprotective effect of 4-HBA observed in our rat MCAO model. Although 4-HBA has been reported to have neuroprotective effect in the animal models of transient and global ischemia, this is the first report on its protective effects afforded by astrocyte, and in particular, its protective effects against Zn2+-toxicity. Zn2+ has been implicated in the induction of excitotoxicity and oxidative stress during the acute phase after ischemic insult. In particular, excessive Zn2+ in neurons induces cell death by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), reducing glycolysis and ATP production (Sheline et al. 2000). Zn2+-mediated ROS induction was obtained by activation of NADPH oxidase both in neurons and astrocytes (Noh and Koh 2000) or by the impairment of the anti-oxidative glutathione (GSH) system in astrocyte (Bishop et al. 2007). PKC-dependent generation of ROS has been well-documented in Zn2+-loaded neurons, in which Zn2+-stimulated PKC enhances the expression and translocation of NADPH oxidase subunits to the neuronal membrane (Noh and Koh 2000). In the present study, we showed that 4-HBA significantly suppressed NAD depletion (Fig. 4f, 6f) and markedly suppressed ROS induction in Zn2+-treated neurons and astrocytes (Figs. 4, 6, 7) and significantly and dose-dependently suppressed up-regulations of p67 subunit of NADPH oxidase (Fig. 4d, 7d), demonstrating that it is an important molecular mechanism underlying anti-Zn2+-toxic effect afforded by 4-HBA both in neurons and astrocytes. In terms of the efficacy, while co-treatment has no effect, pre-treatment of 250 μM of 4-HBA for 6 or 9 h showed robust anti-oxidative effects, respectively, in neurons and astrocytes (Figs. 3, 4, 5, 6). Although no enhanced effects were obtained by increasing concentration of 4-HBA up to 500 μM (Fig. 3a, c), 9 h of pre-incubation seemed to show better beneficial effects than that of 6 h (Figs. 3b, d, 5b). Zn2+-induced activation of NADPH oxidase stimulates the activation of PARP-1, which can trigger NAD/ATP depletion in Zn2+-treated neurons (Kim and Koh 2002). PARP-1 was found to be an important mediator of neuronal death in stroke animal model and the induction of PARP-1 has been reported in neurons in the postischemic brain (Narasimhan et al. 2003). Zn2+-mediated PARP-1 activation has also been reported in astrocyte, which resulted in the inhibition of glutamate uptake, thereby exacerbating excitotoxic damage in neurons (Suh et al. 2007). Recently, Zn2+-mediated PARP-1 activation in astrocytes has also been reported during hypoxia, wherein excess Zn2+ and hypoxia synergistically aggravate cell death by up-regulating PARP-1-induced HIF-1α expression (Pan et al. 2013). In the present study, we demonstrated that 4-HBA significantly suppressed PARP-1 up-regulation and subsequent PAR formation in astrocytes treated with Zn2+ in an acute manner and thus, suppressed Zn2+-induced cell death (Fig. 5). It could be speculated that 4-HBA-mediated PARP-1 suppression might ameliorate neuronal excitotoxicity by suppressing the depletion of bioenergetics and inhibiting glutamate uptake by astrocytes as has been reported by Suh et al. (2007) and Tang et al. (2010). Since astrocytes are required for many functions of neuron and for the maintenance of a stable extracellular environment in the brain, damage or excessive activation of these cells is believed to underlie the pathogeneses of many acute brain diseases and chronic neurodegenerative disorders. Although, astrocytes are known to be more resistant to ischemic damage than neurons, they also lose ATP and ion gradients during prolonged ischemia (Rossi et al. 2007). In particular, as it was mentioned above, release of Zn2+ from neurons during brain insults impairs glutamate uptake of astrocytes by inducing PARP-1 activation in these cells, exacerbating neuronal injury (Suh et al. 2007; Tang et al. 2010). Thus, it appears that the interaction between neurons and astrocytes in terms of Zn2+ homeostasis is critical for aggravating or mitigating excitotoxic neuronal damage in the postischemic brain. The present study shows that 4-HBA protects both neurons and astrocytes against Zn2+ toxicity, therefore, studies need to be performed to identify differential molecular mechanisms conducted by 4-HBA in different cell types. Taken together, our results provide evidence that 4-HBA protects neurons and astrocytes against the toxic effects of Zn2+ by suppressing the inductions of PARP-1 and p67. Although, further studies are needed to confirm that the molecular mechanism proposed in the present study underlies the protective effect of 4-HBA in vivo, the pleiotropic effects of 4-HBA, in terms of its targeting different brain cells and functions, add to its potential as a means of preventing and ameliorating ischemic brain damage. Furthermore, since Zn2+ is known to contribute to neuronal death in different disease states, including stroke, epilepsy, and traumatic injury, it would appear that anti-Zn2+-toxicity effects of 4-HBA might be therapeutically beneficial in those diseases.

Experimental Animals Male Sprague–Dawley (SD) rats were housed under controlled diurnal lighting conditions and freely accessed food and tap water ad libitum. All animal studies were carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH, USA 2011) and agreed with ARRIVE guidelines (http://www.nc3rs.org/ARRIVE). The protocol for animal experiment was reviewed and approved by the INHA University-Institutional Animal Care and Use Committee (INHA-IACUC) with respect to ethicality (Approval Number INHA-141124-337). Surgical Procedures of Middle Cerebral Artery Occlusion Ischemic brain injury was induced by middle cerebral artery occlusion (MCAO) in SD rats weighing 280–300 g as previously described (Kim et al. 2006). In brief, male SD rats were anesthetized with 5% isoflurane in 70% N2O and 30% O2 mixture and anesthesia was maintained using 0.5% isoflurane in the same gas mixture. Rats were randomly allocated to groups as described below and MCAO was performed for 1 h using a nylon suture (4-0; AILEE, Busan, South Korea) and was followed by reperfusion until 24 h. During surgery, the rectal temperature was maintained at 37.0 ± 0.5 °C using a heating lamp and thermoregulated heating pad. Sham-operated controls were subjected to an identical procedure but the MCA was not occluded. During surgery, the left femoral artery was cannulated for blood sampling to analyze pH, PaO2, PaCO2, and blood glucose concentration (I-STAT; Sensor Devices, Waukesha, WI). Laser Doppler flowmeter (Periflux System 5000; Perimed, Jarfalla, Sweden) was used to monitor regional cerebral blood flow (rCBF) before, during, and after MCAO. Operated rats which show <70% reduction in CBF during MCAO were excluded from the experimental groups. Drug Injection 4-HBA (purity ≥98%) was supplied by Sigma-Aldrich (Sigma, St. Louis, MO) and was dissolved in dimethyl sulfoxide (DMSO; Doctor Protein, Seoul, Korea) and administered intraperitoneally at 1 h prior to or 1, 6 h post-MCAO. Rats were randomly divided into five groups: a MCAO group, treatment-naïve MCAO controls (n = 5); a MCAO + 4-HBA group, 4-HBA-treated MCAO group (n = 35), a 4-HBA group, 4-HBA-treated controls (n = 5), a sham group, animals underwent surgery but not MCAO (n = 5); and a normal group, treatment-naïve control group (n = 5). No animals died during surgery, but the mortality rate after surgery was 7.0% (4/55). Investigators blinded to the experimental groups performed the behavioral analysis and the data analysis. Infarct Volume Assessment To assess infarct volume, rats were decapitated at 24 h post-MCAO and 2 mm brain coronal slices were obtained using a metallic brain matrix (RBM-40000, ASI, Springville, UT). Slices were immediately stained by immersing them in 2% 2,3,5-triphenyltetrazolium chloride (TTC) for 15 min at 37 °C and stored in 4% paraformaldehyde. Volume of infarcted tissue areas was measured using the Scion Image Program (Frederick, MD). To account for edema and shrinkage, volumes of ischemic lesions were calculated as (contralateral hemisphere volume − (ipsilateral hemisphere volume − measured injury volume)). Infarct volumes were quantified (in mm3) by multiplying summed infarct area of each section by section thickness. Evaluation of Neurological Deficits Neurological deficits were evaluated using modified Neurological Severity Scores (mNSSs) at 24 h post-MCAO as previously described (Chen et al. 2001). The mNSS system consists of sensory, motor, reflex and balance tests, and total scores range from 0 to 18 (0 = normal, 18 = maximum deficit). Primary Cortical Neuron Culture and NMDA or Zn2+ Treatment Experiments were carried out in strict accordance with the recommendations in Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH, USA 2011). The animal protocol used in this study was reviewed and approved by INHA-IACUC (Approval Number INHA-141124-337). All efforts were made to reduce the number of animals used and minimize animal suffering. Primary cortical cultures were prepared from embryonic day 15.5 (E15.5) mouse cerebral cortices and cultured as described by Kim et al. (2011). Cortical cells were dissociated using glass Pasteur pipet and plated at a density of six hemispheres per 24-well poly-d-lysine (100 μg/ml)- and laminin (100 μg/ml) (Thermofisher Scientific, Waltham, MA)-coated plate (4 × 105 cells per well). Cultures were maintained in MEM containing fetal bovine serum (FBS, 5%), horse serum (5%), glucose (21 mM), and glutamine (2 mM) without antibiotics. On day 7 in vitro (DIV7), when astrocytes had reached confluence underneath neurons, cytosine arabinofuranoside (ara-C) was treated to reach a final concentration of 10 μM in MEM containing horse serum (10%) and glucose (21 mM), and the culture was maintained for 2 days to halt microglial growth. Medium was changed every 2 days after DIV7 and FBS and glutamine was not supplemented from DIV7. Cultures were used at DIV12-14. For N-methyl-D-aspartate (NMDA, Sigma, St. Louis) treatment, cells were treated with 30 μM NMDA in MEM (serum free) for 10 min and replaced with MEM containing 21 mM glucose. For Zn2+ treatment, cells were treated with 200 μM ZnSO4 in a HEPES-controlled salt solution (HCSS) containing 120 mM NaCl, 5 mM KCl, 1.6 mM MgCl2, 2.3 mM CaCl2, 15 mM glucose, 20 mM HEPES, and 10 mM NaOH for 15 min and then medium was replaced with MEM containing 21 mM glucose. C6 Cell Culture and Zn2+ Treatment C6 astroglioma cells were seeded in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO) supplemented with 5% fetal bovine serum (FBS; Thermo, Waltham, MA) at 37 °C in a humidified 95% air/5% CO2 atmosphere. Approximately 4 × 104 cells were treated with 50 μM of Zn2+ (Sigma, St. Louis, MO) for 3, 6, 9, 12, or 24 h in serum-free DMEM or with 100, 200, or 300 μM of Zn2+ for 15 min in serum-free HCSS. Reactive Oxygen Species Quantification C6 cells (4 × 104) and cortical neurons (4 × 105) were seeded into 24-well plates and cultured for 24 h. After treatment with Zn2+ and/or 4-HBA for the indicated times, cells were then incubated in DMEM containing 5 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermo Fisher Scientific, Waltham, MA) for 30 min. After washing cells with PBS, fluorescence and differential interference contrast images were obtained using a Zeiss (Oberkochen, Germany) microscope. For primary cortical cultures, similar procedures were carried out. We quantified fluorescence changes using ImageJ (http://rsbweb.nih.gov/ij/) (Kim et al. 2011). Cell Viability Assays Cell viabilities after Zn2+ treatment were determined using an MTT (3-[4,5-dimethylthiazol-3-yl] 2,5-diphenyltetrazolium bromide) assay. At 24 h after Zn2+-treatment, C6 cells were incubated with 500 μg/ml of MTT (Sigma, St. Louis, MO) for 30 min, and formazan produced was solubilized using 200 μl of DMSO. Optical densities were read at 550 nm. Neuronal cell death after Zn2+ treatment was determined using a LDH (lactate dehydrogenase) assay according to the manufacturer’s instructions by incubating primary cortical culture medium with 50 μl LDH assay reagent (Roche, Mannheim, Germany) for 15 min and reading optical densities at 490 nm. Immunoblot Analysis Cells were washed using cold PBS and lysed with RIPA buffer (50 mM Tris–HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 complete Mini protease inhibitor cocktail tablet (Roche Diagnostics, Basel, Switzerland). Cell lysates were centrifuged at 17,500×g for 10 min at 4 °C and total protein content was measured by BCA protein assay kit (Thermofisher Scientific, Waltham, MA). Thirty μg of protein was loaded onto 8–12% SDS-PAGE gels. The primary antibodies used are as follows: anti-PARP-1 (1: 3000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-p67 (1:3000; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-α-Tubulin (1:2000; Cell Signaling, Danvers, MA). Primary antibodies were detected using anti-rabbit or anti-mouse secondary antibody (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA) and a chemiluminescence kit (Roche, Basel, Switzerland). Measurement of NAD Levels Cyclic enzymatic assay (Szabó et al. 1996) was used to measure NAD concentrations in primary cortical neurons. Cells were extracted in 0.25 ml of 0.5 N HClO4 and neutralized with 3 M KOH/125 mM Gly-Gly buffer (pH 7.4). Cells were centrifuged at 10,000×g for 5 min and supernatants were mixed with 100 μl of reaction solution containing 0.9 mM phenazine methosulfate, 0.1 mM MTT, 5.7% ethanol, and 13 U of alcohol dehydrogenase (Sigma, St Louis, MO) in 61 mM Gly-Gly buffer (pH 7.4) for 30 min in the dark at 37 °C. Absorbance was measured at 550 nm. NAD levels were normalized to protein concentration as determined by the bicinchoninic acid (BCA) method. Statistical Analysis Statistical analysis was performed using analysis of variance (ANOVA) followed by the Newman–Keuls test. All results are presented as means ± SEMs and statistical significance was accepted at the 5% level. Electronic supplementary material Below is the link to the electronic supplementary material.

This work was supported by a Translational Research Grant (HI13C1417) funded by Korea Health Industry Development Institute (KHIDI) (to J.-K.L.) and Mid-carrier Research Grant (2015R1A2A2A01003395) funded by the National Research Foundation (NRF) of Korea (to J.-K.L.). Compliance with Ethical Standards Conflict of interest All authors have no conflicts of interest.

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