Neuroprotection by JM-1232(−) against oxygen–glucose deprivation-induced injury in rat hippocampal slice culture

brain research 1594 (2015) 52–60

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Research Report

Neuroprotection by JM-1232(
) against oxygen–glucose deprivation-induced injury in rat hippocampal slice culture Takahiro Oguraa, Tsuyoshi Hamadab, Toshiyasu Matsuib, Shinji Tanakac, Shigeo Okabec, Tomiei Kazamaa, Yasushi Kobayashib,n a

Department of Anesthesiology, National Defense Medical College, Tokorozawa, Japan Department of Anatomy and Neurobiology, National Defense Medical College, Tokorozawa, Japan c Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan b

ar t ic l e in f o

abs tra ct

Article history:

JM-1232(
) (JM) is a novel isoindoline derivative with sedative and hypnotic activities that

Accepted 21 October 2014

are mediated by binding to the benzodiazepine site of the Gamma-aminobutyric acid type

Available online 31 October 2014

A (GABAA) receptor. Although the neuroprotective effects of other GABAA receptor agonists

Keywords:

are well known, there is no published report regarding JM. Thus, we examined the effects

JM-1232(
)

of JM on neurons exposed to oxygen-glucose deprivation (OGD) using rat hippocampal slice

Benzodiazepine

cultures. Hippocampal slices were assigned to either control or JM-administered groups. To

Neuroprotective effects

assess the neuroprotective effects of JM from necrotic changes, we measured the

Oxygen–glucose deprivation

fluorescence of propidium iodide and compared the cell mortality 24 h following OGD

Hippocampal slice culture

between the control and JM-administered groups. We also verified that the effects of JM were mediated by GABAA receptors by adding flumazenil, a benzodiazepine receptor antagonist, in the same experimental settings. JM, at concentrations of 250 and 500 mM, significantly reduced cell mortality in pyramidal neurons after OGD; however, flumazenil did not inhibit this effect. To analyze more immediate effects of JM, we next measured the fluorescence of Oregon Green 488 BAPTA-1 during the OGD and re-oxygenation periods, and evaluated changes in intracellular Ca2þ in single CA1 pyramidal neurons. JM reduced the elevation of intracellular Ca2þ concentration during OGD, and this effect was antagonized by flumazenil. These findings indicate that JM suppressed the elevation of intracellular Ca2þ concentration during OGD through GABAA receptors, but its neuroprotective effects from necrotic changes also involve other unknown mechanisms. & 2014 Elsevier B.V. All rights reserved.

n Corresponding author at: Department of Anatomy and Neurobiology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Fax: þ81 4 2996 5186. E-mail addresses: [email protected] (T. Ogura), [email protected] (T. Hamada), [email protected] (T. Matsui), [email protected] (S. Tanaka), [email protected] (S. Okabe), [email protected] (T. Kazama), [email protected] (Y. Kobayashi).

http://dx.doi.org/10.1016/j.brainres.2014.10.038 0006-8993/& 2014 Elsevier B.V. All rights reserved.

brain research 1594 (2015) 52–60

1.

Introduction

Transient ischemia in the brain causes irreversible cognitive deficits, including memory disturbances (Levy et al., 1985; Volpe and Petito, 1985), since neurons are extremely susceptible to injury caused by low oxygen and glucose concentrations (Rakic, 1985). Consequently, neuronal protection is an important issue in neurosurgery, cardiac surgery, and intensive care for the management of patients with traumatic brain injuries, where the brain is frequently exposed to ischemia (Arrowsmith et al., 2000; Hoffman et al., 1997; Levine, 1989; Levy et al., 1985; Nussmeier, 1996). Since hippocampal pyramidal neurons are particularly vulnerable to ischemic events, cultured hippocampal slices are often used for the evaluation of neuronal vulnerability, as well as to assess the neuroprotective effects of drugs (Feiner et al., 2005; Gray et al., 2005; Holopainen, 2005; Lee et al., 2006; Son et al., 2004). It is generally accepted that neurons exhaust their intracellular stores of ATP in the low oxygen-glucose environment produced by ischemia. As a result, the membrane potential depolarizes and Ca2þ enters the cell through voltage-gated Ca2þ channels. Moreover, glutamate, which is released from damaged cells, activates glutamate receptors on the cell membrane and induces a further influx of Ca2þ from the extracellular space, as well as Ca2þ release from cellular organelles (Kristian and Siesjo, 1998; Lipton, 1999; Mitani et al., 1993, 1995). These elevated intracellular Ca2þ concentrations activate the intracellular signaling cascades that ultimately lead to cell death. Gamma-aminobutyric acid (GABA) and GABA agonists are considered to be effective in suppressing damage caused by ischemia because they reduce cellular activity and metabolism in the brain (Obradovic et al., 2003; Pellegrini-Giampietro, 2003; Schwartz-Bloom and Sah, 2001). In fact, a number of reports have demonstrated that benzodiazepine derivatives, including diazepam (Corbett et al., 2008; Ricci et al., 2007; Sarnowska et al., 2009) and midazolam (Ito et al., 1999; Lei et al., 2009; Obradovic et al., 2003), as well as anesthetics such as isoflurane (Bickler et al., 2003) and propofol (Hollrigel et al., 1996; Ito et al., 1999), exert their neuroprotective effects via GABAA receptors. Thus, GABAA receptors appear to play an important role in neuroprotection. JM-1232(
) is a novel isoindoline derivative with sedative and hypnotic effects that are mediated through binding to the benzodiazepine site of GABAA receptors (Chiba et al., 2009). JM1232(
) is water soluble, shows less respiratory depression compared to other anesthetics (Kuribayashi et al., 2010), and its metabolites have no sedative effects. Because of these characteristics, JM-1232(
) is considered to be a safe anesthetic with a wide safety margin and limited side effects. Since its safety in clinical use has already been reported (Sneyd et al., 2012), JM-1232 (
) could be particularly useful for anesthesia during surgery and for the management of patients in intensive care units. In spite of these valuable characteristics, there is no published data on the neuroprotective effects of this compound yet. In the present study, we examined whether JM-1232(
) exhibits significant neuroprotective effects during oxygenglucose deprivation (OGD), an experimental model of ischemia. To elucidate the mechanisms underlying the neuroprotective effects of JM-1232(
), we analyzed changes in intracellular Ca2þ concentration during OGD and the effects of JM-1232(
) on these

53

changes. Furthermore, we assessed whether flumazenil, a GABAA receptor antagonist, could block its neuroprotective effects.

2.

Results

2.1.

Cell death

The slices obtained from each hippocampus were divided into control and JM-1232(
) groups. Both groups were exposed to OGD for 1 h, re-oxygenated for 24 h, and subsequently exposed to a high concentration of glutamate for 2 h. Through the OGD and re-oxygenation periods, JM-1232(
) was administered at concentrations of 1, 5, 10, 100, 250, 500, 750, or 1000 mM (n¼ 6 for 5, 100, 250, and 750 mM; n¼7 for 1, 10, 500, and 1000 mM). The fluorescence intensity of propidium iodide (PI) was measured just before OGD, 24 h after OGD, and 24 h after the application of glutamate (Fig. 1A); cell mortality was calculated based on fluorescence intensity (see Section 4). The cell mortality of pyramidal neurons 24 h after OGD was altered by JM-1232(
). JM-1232(
) significantly decreased cell mortality at concentrations of 250 and 500 mM (Fig. 1B), where cell mortalities were 60.178.8% (p¼0.0097) and 42.777.8% (po0.001), respectively. In the experiment determining the best timing of drug administration, 500 mM JM-1232(
) significantly decreased cell mortality when it was administered either through the OGD and re-oxygenation periods, or through the entire periods before, during and after OGD (cell mortalities are 25.474.6% (po0.001) and 62.274.2% (po0.001), respectively; Fig. 1C). Fig. 2 shows PI fluorescence for hippocampal slices both in the control group and in the group that was exposed to 500 mM JM-1232(
). In the JM-1232(
) group, PI uptake in CA1–3 markedly decreased after OGD. These results clearly demonstrated that JM-1232(
) reduced neuronal death induced by OGD. The effects of flumazenil on OGD-induced cell death and on the neuroprotective effects of JM-1232(
) are shown in Fig. 3. Compared to the control group (n¼8), JM-1232(
) had a significant neuroprotective effect and decreased cell death (65.176.9%, n¼7, po0.001); flumazenil did not block this effect (69.875.8%, n¼7, po0.001). The cell death percentage in the flumazenil-only group was not significantly different from that in the control group (92.571.9%, n¼8). When OGD was not performed, 500 mM JM-1232(
) did not cause cell damage even after 48 h. Because of the short survival time before cell death in our experimental settings, the cell death caused by OGD in this study can be regarded as necrosis. To rule out the possibility of apoptosis, we examined the degenerated cells with TUNEL staining. TUNEL positive neurons were rarely observed both in slices before OGD and 24 h after OGD (data not shown), although, the counter staining revealed a marked degeneration of pyramidal cells—darkly stained nuclei with faintly stained perikarya, as well as decreased cell density and broadening of the pyramidal cell layer. Our observations confirmed that the neuronal death caused by OGD in cultured slices was non-apoptotic.

2.2.

Intracellular Ca2þ

Neuroprotective effects of JM-1232(
) that were not antagonized by flumazenil prompted us to examine whether JM-1232(
) functions as a GABAA receptor agonist in our

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brain research 1594 (2015) 52–60

experimental conditions. We next evaluated more immediate effects of JM-1232(
) and flumazenil by analyzing changes in the intracellular Ca2þ concentration in single CA1 pyramidal neurons. Each row in Fig. 4A illustrates a cluster of pyramidal neurons loaded by a single application of Oregon Green 488 BAPTA-1 (OGB-1). In the control group, the intensity of OGB-1 fluorescence increased during the OGD period. JM-1232(
) clearly diminished this increase in fluorescence intensity, and the effect was suppressed by flumazenil. Fig. 4B displays the changes in intracellular Ca2þ concentration during the OGD and re-oxygenation periods. Intracellular Ca2þ concentration in pyramidal neurons increased during the 10-min-OGD period in a near-linear manner, and returned to baseline during the re-oxygenation period. The changes in the intracellular Ca2þ concentration, indicated as a ratio of the baseline value, at the end of the OGD period in the control (OGD only; n¼23), JM-1232(
) (n¼ 22), and JM-1232(
)þflumazenil groups

(n¼22) were 113.470.7%, 108.870.9%, and 111.870.6%, respectively (Fig. 4B). JM-1232(
) significantly inhibited the rise in intracellular Ca2þ concentrations in pyramidal neurons during the OGD period (p¼ 0.0002). Flumazenil clearly exhibited an antagonizing effect, and intracellular Ca2þ was elevated to a level close to that of the control group.

3.

Discussion

The results of this study demonstrate for the first time that administration of JM-1232(
) to rat hippocampal slice cultures during OGD and re-oxygenation periods significantly reduces cell death, and that JM ameliorates the elevation of intracellular Ca2þ during OGD. JM-1232(
), a novel isoindoline derivative, has sedative and hypnotic effects that are mediated by binding to the benzodiazepine site of GABAA receptors (Chiba et al., 2009), similar to diazepam and other GABAA receptor agonists. Based on previous studies on the pharmacological mechanisms of these drugs (Bickler et al., 2003; Corbett et al., 2008; Hollrigel et al., 1996; Ito et al., 1999; Lei et al., 2009; Obradovic et al., 2003; Ricci et al., 2007; Sarnowska et al., 2009), we hypothesized that the neuroprotective effects of JM-1232(
) were also mediated by GABAA receptors. In our experiments, however, flumazenil did not exhibit significant antagonistic actions when we evaluated the neuroprotective effects using an assay for cell death. To evaluate the neuroprotective effects of JM-1232(
) in greater detail, it is necessary to examine its immediate influence Fig. 1 – (A) Examples of PI fluorescence images in hippocampal slice cultures at each time point. Panel a shows a dark field image of a hippocampal slice culture. Panels b–d show fluorescent images before OGD, after OGD, and after administration of excessive glutamate, respectively. Bright areas in b–d indicate PI fluorescence (dead neurons). Closed lines show the pyramidal cell layer and its surroundings of CA region. PI fluorescence was measured in these regions using Image J, and cell death percentage was calculated as ([postOGD]
[preOGD])/ ([postGlutamate]
[preOGD])  100. Scale bars¼ 250 lm. B) JM-1232(
) concentration–response relationship on OGDinduced cell death. Cell mortality represents the cell death of the drug-treated group divided by the cell death of each control group at each concentration. JM-1232(
) significantly decreased cell mortality at concentrations of 250 and 500 lM. Data are mean7S.E.M. Statistical analysis was performed using the t-test (**po0.01; ***po0.001). C) Evaluation of the best period for JM-1232(
) administration. JM-1232(
) was administered during 24 h period before OGD (1), during OGD period (2), during 24 h re-oxygenation period (3), through the OGD and re-oxygenation periods (4), and through the entire periods before, during and after OGD (5). Cell mortality represents the cell death of the drug-treated group divided by the cell death of each control group. JM-1232(
) significantly decreased cell mortality when it was administered either through the OGD and re-oxygenation periods (4), or through the entire periods before, during and after OGD (5). Data are mean7S.E.M. Statistical analysis was performed using the t-test (***po0.001).

brain research 1594 (2015) 52–60

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Fig. 2 – Comparison of PI uptake between control and JM-1232(
)-treated groups. PI uptake in CA1 and CA3 clearly decreased after OGD. DG: dentate gyrus. Scale bar¼250 lm.

Fig. 3 – Effects of JM-1232(
) and flumazenil on OGDinduced cell death. JM-1232(
) had a significant neuroprotective effect, thus decreasing cell death; flumazenil did not reduce this effect. The cell death percentage in the flumazenil group was not significantly different from that in the control group. Data are mean7S.E. M. Statistical analysis was performed using a one-way ANOVA followed by Bonferroni’s test for multiple comparisons (***po0.001). JM: JM-1232(
) 500 lM; FLM: flumazenil 500 lM. on neurons. GABAA agonists such as diazepam protect neurons by promoting Cl
influx, which results in cell hyperpolarization, and by suppressing their response to the excitatory stimulation that accompanies OGD. JM-1232(
) indeed enhances GABAergic inhibitory postsynaptic currents (Takamatsu et al., 2011). The suppression of the excitatory response ameliorates the excessive elevation of intracellular Ca2þ concentrations, which is intimately linked to the initiation of the cell death cascade (Kristian and Siesjo, 1996, 1998; Maravall et al., 2000). These effects may

account for the neuroprotective effects of JM-1232(
) at 250 and 500 mM. Thus, we chose to focus on changes in intracellular Ca2þ concentration. We then established that JM-1232(
) inhibits the rise of intracellular Ca2þ concentrations during OGD, and that this effect was antagonized by flumazenil. The results suggest that JM-1232(
), has neuroprotective effects mediated by GABAA receptors during OGD. The suppression of an intracellular Ca2þ increase is likely to prevent, or at least to delay, the initiation of the cell death cascade caused by OGD. Similar effects have also been reported for other GABAA receptor agonists, such as diazepam and midazolam (Abramowicz et al., 1991; SchwartzBloom and Sah, 2001). In our experiments, flumazenil antagonized the effects of JM1232(
) in the Ca2þ imaging but not in the cell death assay. This could possibly be due to a difference in sensitivity between these two methods. In the cell death assay, cell death due to OGD was compared with total cell death after excessive glutamate administration, which included a heterogeneous population of neurons. In contrast, we focused on single pyramidal cells the Ca2þ imaging analysis and were able to detect finer differences in cellular responses. However, given that we were able to detect the decrease in cell death by JM administration, we cannot attribute the lack of antagonistic effects of flumazenil only to the lower sensitivity of the assay. We used hippocampal slices not only in the cell death assay but also in Ca2þ imaging analysis, in which both neurons and glial cells were exposed to OGD. This allowed us to evaluate cellular responses that were closer to in vivo than dispersed cell culture. Accordingly we need to consider the possibility that JM may act on targets other than GABAA receptors. It has been reported that JM-1232(
) prolongs the decay phase of spontaneous inhibitory postsynaptic currents mediated by GABAA receptors, and that this action is not antagonized by flumazenil (Uemura et al., 2012). Furthermore, other benzodiazepine derivatives have effects that are not

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Fig. 4 – (A) OGB-1 fluorescence images of pyramidal neurons during OGD and reperfusion. Bright areas (arrowheads) indicate the pyramidal cells in which intracellular Ca2þ concentrations increased. Area between the dotted line is pyramidal cell layer. Scale bar¼ 20 lm. (B) The time course of intracellular Ca2þ concentration changes during OGD and reperfusion. JM-1232(
) significantly inhibited the rise in intracellular Ca2þ concentration in pyramidal neurons during the OGD period. This effect was antagonized by flumazenil. Data are mean7S.E.M. Error bars are shown in only one direction for clarity. Statistical analysis was performed using a one-way ANOVA followed by Bonferroni’s test for multiple comparison (***po0.001). JM: JM-1232(
); FLM: flumazenil.

mediated by GABAA receptor activation. These drugs activate Ca2þ-dependent Kþ channels (Polc, 1988), inhibit voltage-gated Ca2þ channels in guinea pigs (Cherubini and North, 1985), inhibit voltage-dependent Naþ channels in mice (Macdonald and McLean, 1986), and activate opioid receptors (Cox and Collins, 2001). Several reports have demonstrated neuroprotective effects mediated by the K-ATP channel (Ballanyi, 2004; Soundarapandian et al., 2007; Yamada and Inagaki, 2005), as well as opioid receptors (Zhao et al., 2006). It is possible that a decrease in excitability accompanying Naþ channel inhibition and a decrease in an influx of intracellular Ca2þ caused by the

inhibition of Ca2þ channels are neuroprotective in combination. Therefore, our findings strongly support the possibility that JM1232(
) also has neuroprotective effects that are not mediated by GABAA receptors. An important issue is whether the effects of JM-1232(
) observed in this study are clinically relevant or not. For clinical purposes, the final effects of cell death prevention are more important than other short-term effects on neurons. Thus, we determined the most effective concentration of JM-1232(
) using a cell death assay. The long-term neuroprotective effects were significant at concentrations of 250 and 500 mM and weaker

brain research 1594 (2015) 52–60

at lower and higher concentrations. Certainly, a concentration of 500 mM is much higher than the concentration of other benzodiazepine derivatives, such as diazepam and midazolam, used clinically in humans for sedation. Several reports showed that other benzodiazepine derivatives have neuroprotective effects at lower concentrations than JM-1232(
) in our study (Galeffi et al., 2000; Sarnowska et al., 2009; Xue et al., 2004). There are a number of possible reasons for this observation. First, differences in the sensitivity to drugs between rats and humans may be a critical factor. Second, we used slice cultures positioned on a membrane, in which drugs diffuse only from the bottom of the slices. Therefore, the drug concentration in the extracellular space of the slices was lower than that in the medium. This is particularly different from cell culture in which neurons are fully immersed in medium. Finally, we focused on the neuroprotection against necrotic changes, and adopted severer OGD conditions and earlier assessments of cell death than other reports using slice culture (Bickler et al., 2003; Choi et al., 2007; Sullivan et al., 2002). These factors may explain why a higher dose of JM-1232(
) was necessary for the neuroprotection in our experiments. Previous studies have indeed shown that the neuroprotective effects of anesthetics are observed at concentrations several times higher than those useful for sedation (Berns et al., 2009; Lei et al., 2009). Since JM-1232(
) has a very wide safety margin for a benzodiazepine derivative, it is reasonable to expect that the neuroprotective effects of JM-1232(
) will be evaluated clinically in the near future. It is imperative that future studies explore the neuroprotective mechanisms of JM-1232(
) that are not mediated by GABAA receptors. Moreover, since it has been reported that neuroprotective effects are modulated by development and aging (Zhan et al., 2006; Zhao et al., 2005), further research is also needed on the long-term effects of JM-1232(
) in vivo at different ages.

4.

Experimental procedures

All the experimental procedures were approved by the Animal Care and Ethics Committee of the National Defense Medical College.

4.1.

Organotypic hippocampal slice culture

Hippocampal slices were prepared and cultured according to the modified interface culture method (Stoppini et al., 1991). Wistar rats were decapitated at postnatal day 7–8. Brains were aseptically removed and immersed in ice-cold dissection buffer containing 2.5 mM KCl, 10 mM MgSO4, 0.5 mM CaCl2, 1.6 mM NaH2PO4, 11 mM glucose, 20 mM HEPES, 8 mM NaOH, 18 mM NaCl, and 0.23 M sucrose. The hippocampus was dissected from each hemisphere and cut vertically along its long axis into 400mm slices using a McIlwain Tissue Chopper (The Mickle Laboratory Engineering Co., Surrey, UK). The hippocampal slices were carefully separated from each other in the dissection buffer and placed on a semi-porous membrane (Millicell-CM 0.4-mm Culture Plate Insert; Millipore, Billerica, MA, USA) in a 35 mm cell culture dish. Each dish contained 1 mL of culture medium composed of 50% Eagle’s Basal Medium, 25% normal horse serum, 25% Earle’s Balanced Salt Solution, 0.5% D-glucose, 10 mM HEPES, 0.146 mg/mL L-glutamine, 50 units/mL Penicillin G sodium, and

57

50 mg/mL streptomycin sulfate. Six to eight hippocampal slices were placed on each membrane in culture medium, and slices were maintained at 37 1C in a 5% CO2/100% humidity incubator. Every 3 days, 70% of the culture medium was replenished with fresh medium. Slices were kept in culture for 14 days before use.

4.2.

Assessment of cell death

The amount of cell death was evaluated using PI (Molecular Probes, Eugene, OR, USA), a highly polar fluorescent dye, which penetrates damaged plasma membranes and binds to DNA. We used PI for quantitative evaluation of cell death and measured its fluorescence in the pyramidal layer of the hippocampus. Our preliminary experiments showed that the intensity of PI fluorescence after OGD increased to the peak level in 24 h and remained high for the following 30 h. We thus analyzed the fluorescence intensity of PI at 3 different stages of the experiment—immediately before OGD (preOGD), 24 h after OGD (postOGD), and 24 h after glutamate exposure (postGlutamate). JM1232(
) and other drugs were added to the medium from the start of OGD until 24 h after OGD. Before OGD, the slices were washed 3 times with glucose-free Hank’s balanced salt solution. 1 mL of glucose-free Hank’s balanced salt solution was placed below a semi-porous membrane. The cultures were then inserted into an air-tight anoxic chamber (gas exchange desiccator; Eiko Kagaku, Tokyo, JAPAN) and perfused with 95% N2/5% CO2. The flow rate of the gas was 6 L/min for the initial 5 min and was decreased to 1 L/min for the following 55 min. The temperature of the chamber was kept at 37 1C by placing it in an incubator. After exposure to OGD, the slices were transferred to the standard slice culture medium and incubated for re-oxygenation under normoxic conditions. After the assessment of cell death induced by OGD, all of the neurons in the slice were killed with the administration of 20 mM glutamate for 2 h and then transferred to the standard slice culture medium and maintained in the incubator. PI fluorescence was imaged using a Lumar V12 microscope (excitation light wave length of 538 nm, 465009 filter) and AxioVisionLE.4.6.3.sp1 software (Carl Zeiss, Jena, Germany). The intensity of the fluorescence was measured in the CA1–3 regions of the hippocampal slices using Image J software (National Institutes of Health, Bethesda, ML, USA) at each time point (shown in Fig. 1Ab–d). The net percentage of cell death due to OGD was calculated based on the fluorescence intensity values at the different time points as follows: cell death percentage¼ ([postOGD]
[preOGD])/([postGlutamate]
[preOGD])  100 because a linear relation exists between cell death and PI fluorescence intensity (Laake et al., 1999; Newell et al., 1995). We determined the most effective dose of JM-1232(
) by comparing 8 different concentrations of JM-1232(
): 1, 5, 10, 100, 250, 500, 750, and 1000 mM. To normalize individual differences in the response to OGD and the neuroprotective effects of JM-1232(
), slice cultures obtained from the same hippocampus were divided into control and JM-1232(
) groups. Cell mortality was calculated for each pair of slices from the same hippocampus using the following equation: cell mortality ¼cell death percentage of JM-1232(
)-treated slice/cell death percentage of control slice.

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brain research 1594 (2015) 52–60

We also determined the most effective period of JM-1232 (
) administration by comparing 5 different conditions. We administered JM-1232(
) during 24 h period before OGD (1), during OGD period (2), during 24 h re-oxygenation period (3), through the OGD and re-oxygenation periods (4), and through the entire periods before, during and after OGD (5). Cell mortality was calculated in the same manner as above. Since JM-1232(
) was most effective when administered at 500 mM during OGD and re-oxygenation periods, the following experiments were all performed in this condition. To determine whether the JM-1232(
) effects were mediated by GABAA receptors, we added 500 mM of flumazenil during OGD and reoxygenation periods. This concentration is consistent with usage in previous reports in which flumazenil was applied to antagonize diazepam (Ricci et al., 2007; Takamatsu et al., 2011). The slices made from each rat were divided into 4 groups: control, administration of JM-1232(
) only, administration of both JM1232(
) and flumazenil, and administration of flumazenil only. The cell death percentages were compared among the 4 groups.

4.3.

TUNEL staining

To determine that the observed cell death was not due to apoptosis, hippocampal slice cultures were fixed for 3 h in 4% paraformaldehyde 24 h after exposure to OGD. After immersion in 0.1 M phosphate buffer containing 30% sucrose, frozen sections (10 mm in thickness) were prepared using a cryostat (HM 560 MV; Microm, Walldorf, Germany) and stained with the TUNEL method using an in situ apoptosis detection kit (Takara Bio, Ohtsu, Japan). Briefly, frozen sections were washed in phosphate buffered saline (pH 7.4) and treated with protease K (10 mg/mL) for 10 min. Sections were then incubated with FITClabeled TdT enzyme and reacted with anti-FITC HRP conjugates followed by DAB development.

4.4.

Assessment of intracellular Ca2þ

Slice cultures were divided into control, JM-1232(
), and JM-1232(
)þflumazenil groups to assess the effects of JM1232(
) and flumazenil on intracellular Ca2þ concentration. Both the JM-1232(
) and flumazenil concentrations were 500 mM. Hippocampal slices were observed in a superfusion chamber (DTC-FB, Eiko Kagaku, Tokyo, Japan) mounted to the imaging setup, which was placed on the stage of an inverted microscope. The chamber was superfused at a rate of approximately 3 mL/min with oxygenated Artificial cerebrospinal fluid (ACSF) containing 2.5 mM KCl, 2.1 mM CaCl2, 1.25 mM NaH2PO4, 10 mM Glucose, 126 mM NaCl, 2.0 mM MgCl2, and 26 mM NaHCO3. Slices were exposed to OGD for 10 min with glucose-free ACSF perfused with 95% N2/5% CO2. After OGD exposure, slices were recovered in normal ACSF equilibrated with 95% O2/5% CO2 for 5 min. The temperature of the ACSF was kept at 37 1C. JM-1232(
) and flumazenil were added to the ACSF during the OGD and re-oxygenation periods. Single-cell intracellular Ca2þ concentrations were evaluated using OGB-1 (Molecular Probes, Eugene, OR, USA). The dye application protocol was modified slightly from one previously described (Delaney et al., 2001). Briefly, 0.5 mg crystals of dye were dissolved in 2 mL of 15% bovine serum albumin and then

air-dried. A droplet of distilled water was then placed on the desiccated dye. Fine glass micropipettes were coated with a film of dye by dipping them in the dye solution and then allowing the dye to dry onto the pipette. The dye was applied locally to the stratum radiatum of the hippocampal slice cultures under a stereoscopic microscope. The dye was applied to 3–4 locations in 1 slice. Typically, 4–8 pyramidal cells were loaded with the dye at each location. The slices were then incubated in oxygenated ACSF for at least 1 h at room temperature. Slices were imaged using a Nipkow disk confocal system (CSU21; Yokogawa, Tokyo, Japan) with a 40  objective (PlanNeofluar, n.a. 1.3, oil immersion lens; Carl Zeiss), as previously described (Ness et al., 2008). OGB-1 was excited at 488 nm by an Argon ion laser. The images were conveyed by a relay lens to an intensified ICCD camera (XR/MiniICCD CAMERA; Solamere Technology Group, UT, USA). Images were captured and recorded on a computer every 15 s. OGB-1 fluorescence was measured in single hippocampal pyramidal neurons using Image J, and the relative changes in fluorescence were calculated based on the pre-OGD measurement. We compared the changes in fluorescence among the 3 groups of slices (control, JM-1232(
), and JM-1232(
)þflumazenil) that were derived from the same rat.

4.5.

Statistical analysis

In the experiment determining the optimal dose of JM-1232(
), we used Student’s t-test to compare the cell death percentages between JM-1232(
)-treated and control slices. In the other experiments, in which data were compared among 3 or 4 groups, we used a one-way ANOVA followed by Bonferroni’s test for multiple comparisons. Statistical analysis was performed using Graph Pad Prism 4.0c software (GraphPad Software; CA, USA). Differences were considered significant if po0.01.

Disclosure None of the authors have any conflicts of interest to disclose.

Acknowledgments The authors thank Dr. Norio Ishizuka (Tokyo Metropolitan Institute of Medical Science) for invaluable advice in slice experiments, and Ms. Mayumi Watanabe (Department of Anatomy and Neurobiology, National Defense Medical College) for assistance in histological processing.

r e f e r e n c e s

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