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Levetiracetam enhances endogenous antioxidant in the hippocampus of rats: In vivo evaluation by brain microdialysis combined with ESR spectroscopy
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
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Research Report
Levetiracetam enhances endogenous antioxidant in the hippocampus of rats: In vivo evaluation by brain microdialysis combined with ESR spectroscopy Yuto Ueda a,⁎, Taku Doi a , Mayuko Takaki a , Keiko Nagatomo a , Akira Nakajima b , L. James Willmore c a Section of Psychiatry, Department of Clinical Neuroscience, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan b Section of Chemistry, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan c Department of Neurology and Department of Pharmacology and Physiology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
We have attempted to explore the neuroprotective effectiveness of levetiracetam (LEV) by
Accepted 21 February 2009
measuring its in vivo antioxidant effect in the hippocampus of rats in a freely moving state.
Available online 4 March 2009
Male Wistar rats were used for the estimation of the in vivo antioxidant effect of LEV through microdialysis combined with electron spin resonance spectroscopy. The antioxidant effect
Keywords:
was examined using the principle by which a systemically administered blood–brain
Levetiracetam
barrier-permeable nitroxide radical (PCAM) decreases in an exponential decay manner that
ESR
is correlated with the amount of antioxidant in the brain. The PCAM decay ratio during
Glutamate
perfusion with normal Ringer's solution was compared with that during 32 μM and 100 μM
Antioxidant
LEV co-perfusion. The in vivo antioxidant effect was examined. In addition, the expressions
Hippocampus
of the cystine/glutamate exchanger (xCT) and the inducible nitric oxide synthase (iNOS)
Redox
protein related to redox regulation were measured in the hippocampus of rats after 14 days of administration of LEV at a dose of 54 mg/day i.p. The half-life of PCAM was statistically shortened after LEV perfusion compared with the results of the control experiment. While the expression of the pro-oxidant protein iNOS was decreased, that of the antioxidant protein xCT was statistically increased by the administration of LEV. The role of xCT is to transport cystine, the internal material of glutathione, into the cell. The shortened half-life of the nitroxide radical by co-perfusion of LEV with increased xCT and decreased iNOS expression revealed the enhancement of the endogenous antioxidant effect or free-radical scavenging activity. The results of this study suggest that LEV synergistically enhances the basal endogenous antioxidant effect in the hippocampus with ascorbic acid and αtocopherol. Our findings further suggest that LEV exerts a neuroprotective role by 1)
⁎ Corresponding author. Fax: +81 985 85 5475. E-mail address: [email protected] (Y. Ueda). Abbreviations: ESR, electron spin resonance; NMDA-R, N-methyl-d-aspartate receptor; LEV, levetiracetam; PBT, pentobarbital; PCAM, 3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl; PCA, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl; iNOS, inducible nitric oxide synthase; xCT, cystine/glutamate exchanger; GSH, glutathione; HVSCC, high-voltage activated N-type Ca2+-current; MDA, malondialdehyde; OPA, o-phthalaldehyde 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.02.040
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modifying the expression of xCT and iNOS in connection with lipid peroxidation, 2) synergistically enhancing the increased basal endogenous antioxidant ability in the hippocampus, and 3) decreasing the basal concentration of glutamate followed by upregulation of the intake of cystine, an internal material of GSH. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Levetiracetam (LEV) is an effective anti-epileptic drug (AED) with unique mechanisms of action which appears to inhibit the initiation of kindling in rodents (Loscher et al., 1998; Sasa et al., 2005; Stratton et al., 2003). The molecular effects of LEV remain uncharacterized, although the drug binds specifically to synaptic vesicle protein 2A (SV2A) (Fuks et al., 2003; Gillard et al., 2003; Lynch et al., 2004). LEV also reduces the high-voltage sensitive calcium current (HVSCC) by inhibiting the N-type Ca2+ channel (Lukyanetz et al., 2002; Niespodziany et al., 2001). Some neuroprotective effects of LEV were suggested when the intraperitoneal administration of LEV was associated with the reduction of the product of lipid peroxidation, malondialdehyde (MDA), in the epileptic rat cortex following KA-induced neural toxicity, suggesting that this effect is mediated, at least in part, by the inhibition of lipid peroxidation (Marini et al., 2004). LEV also reduced the infarct volume in an experimental stroke model of ischemia (Hanon and Klitgaard, 2001). Overall, data suggest that LEV may interfere with glutamate neurotoxicity and ischemia. Although further study is needed, the underlying mechanism of neuroprotection may include the stimulation of neurotrophic factors (Cardile et al., 2003) or the stabilization of mitochondrial functions (Gibbs and Cock, 2007) in addition to anti-inflammatory or anti-oxidative actions. In regard to antioxidants, the results of studies on cerebral xCTdependent glutathione production modulating both neuroprotection from oxidative stress and cell proliferation were recently reported (Liu et al., 2007; Shih et al., 2006). Hence, in the current study, we evaluated the following: 1) the effect of LEV on the basal release of amino acids in the hippocampus, 2) the in vivo and in vitro antioxidant effects of LEV, and 3) the alteration of xCT (antioxidant enzyme) and iNOS (pro-oxidant enzyme) expression in rat hippocampus after chronic treatment with LEV.
2.
Results
2.1.
Hippocampal amino acid concentration
successive decrease in amplitude with no change in linewidth compared with the first spectrum in all groups, and the signal intensity of PCAM at the lowest component in the scan field (MI = +1) decayed exponentially in a linear and highly reproducible fashion (Fig. 2A). The half-life of the decay is dependent upon the antioxidant ability of the brain, as explained previously (Ueda et al., 1998). The half-life is an index that allows a comparison of the rate of elimination of the nitroxide radical, which provides an estimate of the brain's antioxidant ability (Ueda et al., 1998). The half-lives of the PCAM signal intensities from the ventral hippocampus with LEV co-perfusion at both 32 μM and 100 μM were significantly briefer than those of the control (Figs. 2A and B).
2.3. In vitro evaluation of the synergistic effect of LEV on the α-tocopherol–ascorbic acid system As shown in Fig. 3, Torox–ascorbic acid decreases the paramagnetism of the nitroxide radical with the addition of LEV. Decay did not occur with Torox or LEV alone. LEV has a synergistic effect on antioxidant agents, such as Torox–ascorbic acid, as demonstrated by the increase in speed when LEV was added to the system of Torox–ascorbic acid compared with the speed of an antioxidant mixture without LEV. When the PCAM solution was dissolved in a mixture of ascorbic acid, Torox, and LEV in vitro, the half-life of the decreasing signal intensity of PCAM was briefer than that in ascorbic acid and Torox. The add-on effect of the antioxidant
The hippocampal basal release of glutamate with a perfusion of 100 μM LEV was significantly lower than that of the control. The other amino acids were within the range of the control (Fig. 1).
2.2.
In vivo antioxidant ability
The methods used to evaluate in vivo antioxidant ability have been described in previous reports (Ueda et al., 2005; Ueda et al., 1998; Yokoyama et al., 1999). The ESR spectra of the dialysate samples measured at 2.67-min intervals showed a
Fig. 1 – Comparison of amino acids at the basal concentration in the hippocampus of control rats (black bar) and 100 μM LEV co-perfusion (white bar). The right vertical axis represents the glutamine concentration ((Gln)o). Data represent the means +/− SE. *P < 0.01 LEV versus control (Mann–Whitney U-test). GABA = γ-aminobutyric acid; Gln = glutamine; Glu = glutamate; Gly = glycine; Tau = taurine.
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Fig. 2 – (A) Typical plots of signal intensities of PCAM vs. time after injection of PCAM (indicated by black arrows) in each group (control, black circle; 32 μM LEV, white circle; 100 μM LEV, white square; Fig. 2A). (B) Data represent the means +/− SE value of the half-life for each group (Fig. 2B). Data represent the means +/− SE. *P < 0.01 for control versus 100 μM LEV. †P < 0.05 for control versus 32 μM LEV (Mann–Whitney U-test).
indicates that LEV has a synergistic effect on the endogenous efficacy of antioxidants such as ascorbic and α-tocopherol.
2.4.
Western blot
In rats treated with LEV, xCT increased (129.3± 5.2%), while iNOS (77.5 ± 4.7%) decreased in comparison to the values in rats receiving the vehicle-only treatment (control), as shown in Fig. 4.
Fig. 3 – ESR signal intensity of the nitroxide radical when exposed to an antioxidant agent, such as Torox or ascorbic acid. Elimination of the signal intensity by an antioxidant agent was faster when LEV was added to the Torox–ascorbic acid system. LEV itself did not decay the paramagnetism of the nitroxide radical (closed square).
3.
Discussion
Recent data demonstrated the neuroprotective effects of LEV in clinically relevant models of traumatic brain injury and subarachnoid hemorrhage (Wang et al., 2006a). Although the bulk of the literature supports a neuroprotective effect of LEV in acute brain injury, several studies produced negative results, such as the absence of neuroprotection in a hippocampal slice model (Rekling, 2003). However, Hanon and Klitgaard (2001) reported that the administration of LEV (11 and 44 mg/kg i.p.) reduced the infarct volume in a rat model of focal cerebral ischemia and LEV possessed neuroprotective
Fig. 4 – Changes in the expression of xCT and iNOS in the hippocampus of rats administered i.p. with 54 mg/kg LEV once a day for 14 days. Data represent the means +/− S.E. Representative blots are inserted at the top of each bar. *P < 0.05 LEV versus control (Mann–Whitney U-test).
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properties. Wang et al. (2006a) used the administration of LEV (18 and 54 mg/kg i.v.) in a murine model of closed head injury (CHI) and subarachnoid hemorrhage (SAH) to investigate the neuroprotective properties of LEV, which improved the functional and histological outcomes after CHI and reduced vasospasm following SAH. These results suggest that LEV is neuroprotective in clinically relevant animal models of SAH and CHI. Moreover, in a clinical study, Belcastro et al. (2007) and Kutlu et al. (2008) confirmed the effect of LEV in preventing seizure in patients with Alzheimer's disease and post-stroke disease, respectively. Based on these underlying anti-convulsive and neuroprotective mechanisms, we have pointed out the potent candidature mechanism of the action of LEV (Ueda et al., 2007). After 14 days of LEV treatment (54 mg/kg, i.p.), the ipsilateral hippocampus was removed for Western blot analysis. The expression increased for all of the glutamate and GABA transporters (GLAST, GLT-1, EAAC-1, GAT-1, and GAT-3), while the glutamate transporter-regulating protein (GTRAP318) decreased in comparison to the values in normal rats treated without LEV. The suppression of glutamate excitation and the enhancement of GABA inhibition in the rats by continual LEV administration were demonstrated by our previous result showing the up-regulation of glutamate and GABA transporters with the down-regulation of GTARP3-18. These observations demonstrate the critical molecular mechanism of not only anti-epileptic activity but also potential neuroprotective properties of LEV. The main focus of research on the mechanism of epileptogenesis and neurotoxicity has been the functional significance of excitatory amino acid transporters (Ueda et al., 2001) and Glu receptors (GluRs) (Harrington et al., 2007), and little attention has been given to the possible expression of the cystine/Glu antiporter responsible for the bi-directional transmembrane transport of Glu related to the synthesis of glutathione (GSH) (Wang et al., 2006b), which is important to the exploration of the fundamental mechanism of neuroprotection. In parallel with the in vivo evaluation of antioxidant ability, in the current study, xCT protein expression was increased by LEV chronic treatment, although iNOS decreased in rats after chronic administration of LEV (i.p.) compared with the values of the control. These results show that LEV chronic treatment suppresses the collapse of redox to an oxidized shift. LEV decreased the basal release of the extracellular glutamate level only, which helped the intake of cystine-synthesizing GSH, one of the important endogenous antioxidant agents. The suppressive effect of LEV on glutamate overflow as a neuroprotective action may be derived from the inhibitory modulation of the SV2A receptor. There is very limited information on the function of SV2A; however, knockout mice lacking SV2A are known to exhibit seizures. Without SV2A, presynaptic calcium accumulation during repetitive stimulation causes abnormal increases in the neurotransmitter overflow, inducing epilepsy. Therefore, the binding of the SV2A receptor with LEV may restore the ability of a neuron to reduce excessive glutamate overflow (Lynch et al., 2004; Stahl, 2004). Further study will be required to determine the effect of the inhibition of the SV2A receptor on glutamate overflow. Judging from the results reported here, SV2A-regulating
vesicle release by LEV would mainly affect glutamate and not inhibitory amino acids such as glycine, taurine, and GABA. In the normal brain, there is a steady state of redox balance between the production of free radicals and their destruction by endogenous cellular antioxidants such as α-tocopherol– ascorbic acid, GSH–GSSG, and NADP+–NADPH. We believe that the protection of LEV is derived from a synergistic effect with endogenous antioxidants. This hypothesis is strongly supported by the results of the current study, in which the halflife of the decreasing signal intensity of PCAM was shorter than those of ascorbic acid and α-tocopherol when the PCAM solution was dissolved with ascorbic acid and α-tocopherol mixed with LEV in vitro. Although LEV itself did not scavenge PCAM, LEV had a synergistic effect on the endogenous abilities of antioxidants such as ascorbic acid and α-tocopherol. Moreover, repetitive PCAM gradually prolonged the halflife of an injected nitroxide radical (Fig. 2B), which resulted in a decline in the hippocampal antioxidant ability followed by repetitive exposure to the nitroxide radical. Prolongation of the half-life of a free radical in the brain is closely related to neurodegeneration because the prolonged survival of a free radical is related to the enhancement of lipid peroxidation. In other words, LEV prolongs the half-life. In contrast to the prolongation observed in the control group, the half-life of a free radical was shortened by LEV co-perfusion, which indicated that LEV prevented the decline of antioxidant ability in the hippocampus due to repetitive exposure to the nitroxide radical. Thus, LEV may exert neuroprotection against a free radical attack when a seizure occurs. In addition to the increased xCT and decreased iNOS expression, the shortened half-life of the nitroxide radical when co-perfused with LEV revealed the enhancement of the endogenous antioxidant effect or free-radical scavenging activity. Acute LEV exposure certainly increases NO generation through an increase in iNOS expression (Cardile et al., 2003; Dagonnier et al., 2005; Trollmann et al., 2008). This study is the first report suggesting that chronic administration of LEV suppresses NO generation by the depletion of iNOS. The suppression of NO generation could be related to neuroprotection. Although α-tocopherol or ascorbic acid itself has no powerful antioxidant ability, α-tocopherol and ascorbic acid have a powerful ability to eliminate a free radical. The results of this study suggest that LEV synergistically enhances the increased basal endogenous antioxidant effect in the hippocampus with ascorbic acid and α-tocopherol. Our finding that LEV has a synergistic effect on endogenous antioxidants as well as GSH and NADPH is new. Our results, therefore, suggest that LEV exerts a neuroprotective role by 1) modifying the expression regarding oxidative stress, 2) synergistically enhancing the increased basal endogenous antioxidant ability in the hippocampus, and 3) decreasing the basal concentration of glutamate followed by up-regulation of the intake of cystine-synthesizing GSH. Our findings strongly suggest that LEV exerts a neuroprotective effect in the rat hippocampus by selectively decreasing the basal concentration of glutamate and synergistically enhancing the basal endogenous antioxidant activity or free-radical scavenging ability of the αtocopherol and ascorbic acid system via increased elimination of nitroxide radicals in vivo and increased xCT and decreased iNOS expression in vitro after chronic treatment.
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4.
Experimental procedures
All experiments were conducted according to the 1987 guidelines of the Japanese Association for Laboratory Animal Science. The Committee for the Ethics on Animal Experiments of the Faculty of Medicine, University of Miyazaki, reviewed and approved the experimental design (approval number: 1998-158-2). Drugs were purchased from the companies indicated in parentheses: pentobarbital (Dainippon Sumitomo Pharmacology Co., Ltd., Osaka, Japan); blood–brain barrier permeable nitroxide radical (PCAM), synthesized originally from PCA (BBB non-permeable nitroxide radical; Aldrich Chem. Co., Milwaukee); Torox (Aldrich Chem. Co., Milwaukee); ascorbic acid (Wako Pure Chem. Co., Osaka, Japan). The method of PCAM synthesis is described in detail elsewhere (Ueda et al., 2004). 4.1.
Microdialysis operation
Eighteen male Wistar rats weighing 200–250 g at the time of surgery were anesthetized with sodium PBT (37.5 mg/ kg, i.p.). The incisor bar was set on the intraaural line. Each rat was stereotaxically implanted with a guide cannula at coordinates 5.6 mm posterior to and 5.0 mm to the right of the bregma, as determined by the rat brain atlas of Pellegrino et al. (1986). The guide cannula was firmly anchored to the skull with miniature screws and dental cement. After 5 days of recovery, a microdialysis probe (4 mm length, EICOM, Kyoto, Japan) was implanted, and the hippocampus was perfused with Ringer's solution (147 mM NaCl, 4 mM KCl, 2.3 mM CaCl2; pH 6.5) at 2.0 μl/ min (EP-60, EICOM, Kyoto, Japan). For the in vivo microdialysis experiment, we used a dose of 32 μM or 100 μM LEV. This concentration was indicated by Tong and Patsalos (2001), who investigated the extracellular pharmacokinetics of LEV using an SD rat model that allowed serial blood sampling and concurrent brain microdialysis sampling. The mean Coax values for 40 and 80 mg/kg LEV (i. p.) were 58 and 173 μM, respectively. These two conditions were considered to be close to the common clinical doses of LEV (a dose of 1–3 g/day is 17–50 mg/kg in 60 kg body weight). In this experiment, we randomly assigned rats to one of three groups: 1) rats co-perfused with 32 μM LEV, 2) rats co-perfused with 100 μM LEV, and 3) rats infused with Ringer's solution only (control). LEV was diluted with Ringer's solution. 4.2.
Basal release of extracellular amino acids
After a 2-hour stabilization period, sampling from each group was begun. We used HPLC-ECD to measure the basal concentrations of extracellular glutamate, glutamine, glycine, taurine, and γ-aminobutyric acid (GABA) in the ventral hippocampus. Before applying samples to the HPLC-ECD, an OPA solution was made by adding 13.5 mg of OPA and 10 μl of 2-mercaptoethanol to 2.5 ml of a 0.1 M K2CO3 buffer (pH 9.6) with 10% ethanol. The sample (30 μl) was mixed with 10 μl of an OPA solution and incubated for
5
10 min at 4 °C. After mixing was complete, 40 μl of the reactant was applied to the HPLC with an ODS column. Detection was performed by ECD (EICOM, Tokyo, Japan) with + 600 mV/Ag/AgCl. The elution buffer contained 60 mM NaH2PO4U2H2O, 9.6 mM Na2PO4U12H2O, 30% methanol, and 0.5 mM EDTA (pH 6.5). A comparison of the basal release of amino acids was performed between the control and 100 μM LEV groups. 4.3.
In vivo evaluation of antioxidant ability
The measurement of antioxidant function in freely moving rats has been reported in detail (Ueda et al., 1998; Yokoyama et al., 1999). Briefly, in the current study, exogenously administered nitroxide radicals were reduced, and their paramagnetism was destroyed by brain antioxidants. When paramagnetism was plotted on a semilogarithmic scale, the signal intensity decayed exponentially. Thus, the half-life of the decay reflected the antioxidant ability in the brain tissue. The PCAM used here as an exogenous nitroxide radical was injected i.p. (0.2 M PCAM, 0.4 mmol/kg). After collection of the dialysis sample, the perfusate was directed into a quartz duct (inner diameter, 4 mm, LST-5HS; Labotec, Tokyo, Japan) using a polyethylene tube (inner diameter, 0.12 mm; BAS). The quartz duct was placed in the resonator of an ESR spectrometer (FR 30; JEOL, Tokyo, Japan) and analyzed with a WIN-RAD ESR data analyzer (Radical Research Inc., Tokyo). The direction of the perfusate from moving animals into the ESR device allowed the detection of numerous time points for the estimation of changes in the paramagnetism of PCAM, reflecting hippocampal redox conditions during LEV perfusion. The antioxidant ability was tested at concentrations of 32 μM and 100 μM LEV. PCAM i.p. injections were repeated 4 times at 40-min intervals. ESR measurements were conducted under the following conditions: microwave power, 4 mW; microwave frequency, 9.42 GHz; static magnetic field, 335.3 mT; modulation width, 0.1 mT; sweep width, 0.86 mT; sweep speed, 0.32 mT/min. Temporal changes in the ESR signal intensity of PCAM were measured as the peak-to-peak height of the lowest field component (MI = + 1; actual sweep range, 333.8 +/− 0.43 mT) of triplet spectra. The analysis was conducted with high-time resolution at 2.67 min. The methods are described in detail elsewhere (Ueda et al., 2005). 4.4. In vitro evaluation of synergistic effect of LEV on antioxidants We used the elimination of the signal intensity of PCAM to evaluate the synergistic effect of LEV on the α-tocopherol– ascorbic acid system. Following the mixture of Torox (a water-soluble chemical compound that mimics α-tocopherol at 1 mM as the working concentration), ascorbic acid (at 10 mM as the working concentration), and LEV (at 1 mM as the working concentration) in a t-tube, PCAM (at 10 mM as the working concentration) was dissolved in the mixture, and the ESR measurement was performed to
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estimate the half-life of PCAM. All chemical compounds were diluted with Ringer's solution. For the control, Ringer's solution without LEV was used.
Science, Sports, and Culture, Japan. LEV was kindly donated by UCB-Belgium.
4.5.
REFERENCES
Western blot
Rats were treated either with daily i.p. injections of 54 mg/ kg LEV (dissolved in physiological saline at a concentration of 50 mg/ml; n = 6) or with the vehicle i.p. (control group; physiological saline 0.1 ml/100 g; n = 6). Systemic administration at 54 mg/kg was determined by Loscher et al. They tested LEV at 13, 27, and 54 mg/kg i.p. on amygdalakindling acquisition in Wistar rats (chronic administration). LEV suppressed the increase in seizure severity and duration induced by repeated amygdala stimulation dosedependently. After termination of daily treatment with 54 mg/kg, the duration of behavioral seizures and discharge recorded from the amygdala were significantly shorter than those in the vehicle controls, although amygdala stimulation continued in the absence of LEV (Loscher et al., 1998). Animals were treated with LEV or saline i.p. injections between 09:00 and 11:00 each morning. The animals were sacrificed on the 14th day of treatment following the last i.p. injection of LEV or saline. In brief, Western blotting was performed as follows. Crude synaptic samples from the bilateral whole hippocampus (30 μg each) were loaded for gel electrophoresis by discontinuous one-dimensional Tris– SDS-PAGE (4% stacking gel and 10% separating gel). Proteins from the gel were transferred by electroblotting to a 0.2-mm nitrocellulose membrane. The nitrocellulose membrane was blocked for 10 min in 10% nonfat milk in a PBS buffer containing 1% Tween 20 (PBS-T). Following this washing, the blot was incubated for 2 h in anti-xCT (purchased from Trans. Genic, Inc.) and anti-iNOS (purchased from Santa Cruz Biotechnology, Inc.) in PBS-T containing a 0.67% blocking agent. The blot was then washed with PBS-T, incubated with horseradish peroxidase-conjugated IgG for 20 min (1:3000), washed again, and processed for immunoreactivity using an enhanced chemiluminescence detection kit (Amersham). For semiquantitative analysis of each protein, each band density in every sample was calculated using NIH Image software. The transferred blotted membrane was stained with Ponceau-S to verify that each lane was loaded with equivalent amounts of protein. The blots were normalized to β-tubulin for densitometry. 4.6.
Statistical analysis
Statistical analysis was carried out using the Mann– Whitney U-test. Data were considered significant at a level of P < 0.05.
Acknowledgments This study was partially supported by a Grant-in-Aid for Scientific Research (C) (2) (16591146, 18591297, and 20591372 to Y.U.) and by a Grant-in-Aid for Encouragement of Young Scientists (18790840 to K.N.) from the Ministry of Education,
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Rekling, J.C., 2003. Neuroprotective effects of anticonvulsants in rat hippocampal slice cultures exposed to oxygen/glucose deprivation. Neurosci. Lett. 335, 167–170. Sasa, M., Yan, H.D., Ishihara, K., 2005. Is levetiracetam distinct from conventional antiepileptic drugs in antiepileptogenic properties in spontaneous epileptic rats? Epilepsia 46, 94–95. Shih, A.Y., Erb, H., Sun, X., Toda, S., Kalivas, P.W., Murphy, T.H., 2006. Cystine/glutamate exchange modulates glutathione supply for neuroprotection from oxidative stress and cell proliferation. J. Neurosci. 26, 10514–10523. Stahl, S.M., 2004. Psychopharmacology of anticonvulsants: levetiracetam as a synaptic vesicle protein modulator. J. Clin. Psychiatry 65, 1162–1163. Stratton, S.C., Large, C.H., Cox, B., Davies, G., Hagan, R.M., 2003. Effects of lamotrigine and levetiracetam on seizure development in a rat amygdala kindling model. Epilepsy Res. 53, 95–106. Tong, X., Patsalos, P.N., 2001. A microdialysis study of the novel antiepileptic drug levetiracetam: extracellular pharmacokinetics and effect on taurine in rat brain. Br. J. Pharmacol. 133, 867–874. Trollmann, R., Schneider, J., Keller, S., Strasser, K., Wenzel, D., Rascher, W., Ogunshola, O.O., Gassmann, M., 2008. HIF-1-regulated vasoactive systems are differentially involved in acute hypoxic stress responses of the developing brain of newborn mice and are not affected by levetiracetam. Brain Res. 1199, 27–36. Ueda, Y., Yokoyama, H., Ohya-Nishiguchi, H., Kamada, H., 1998. ESR spectroscopy for analysis of hippocampal elimination of a nitroxide radical during kainic acid-induced seizure in rats. Magn. Reson. Med. 40, 491–493. Ueda, Y., Doi, T., Tokumaru, J., Yokoyama, H., Nakajima, A., Mitsuyama, Y., Ohya-Nishiguchi, H., Kamada, H., Willmore,
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L.J., 2001. Collapse of extracellular glutamate regulation during epileptogenesis: down-regulation and functional failure of glutamate transporter function in rats with chronic seizures induced by kainic acid. J. Neurochem. 76, 892–900. Ueda, Y., Yokoyama, H., Tokumaru, J., Doi, T., Nakajima, A., 2004. Kinetics of extracellular nitroxide radical and glutamate levels in the hippocampus of conscious rats: cautionary note to the application of nitroxide radical on clinical arena. Neurochem. Res. 29, 1695–1701. Ueda, Y., Doi, T., Tokumaru, J., Nakajima, A., Nagatomo, K., 2005. In vivo evaluation of the effect of zonisamide on the hippocampal redox state during kainic acid-induced seizure status in rats. Neurochem. Res. 30, 1117–1121. Ueda, Y., Doi, T., Nagatomo, K., Tokumaru, J., Takaki, M., Willmore, L.J., 2007. Effect of levetiracetam on molecular regulation of hippocampal glutamate and GABA transporters in rats with chronic seizures induced by amygdalar FeCl(3) injection. Brain Res. 1151, 55–61. Wang, H., Gao, J., Lassiter, T.F., McDonagh, D.L., Sheng, H., Warner, D.S., Lynch, J.R., Laskowitz, D.T., 2006a. Levetiracetam is neuroprotective in murine models of closed head injury and subarachnoid hemorrhage. Neurocrit. Care 5, 71–78. Wang, L., Hinoi, E., Takemori, A., Nakamichi, N., Yoneda, Y., 2006b. Glutamate inhibits chondral mineralization through apoptotic cell death mediated by retrograde operation of the cystine/glutamate antiporter. J. Biol. Chem. 281, 24553–24565. Yokoyama, H., Lin, Y., Itoh, O., Ueda, Y., Nakajima, A., Ogata, T., Sato, T., Ohya-Nishiguchi, H., Kamada, H., 1999. EPR imaging for in vivo analysis of the half-life of a nitroxide radical in the hippocampus and cerebral cortex of rats after epileptic seizures. Free Radic. Biol. Med. 27, 442–448.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Levetiracetam enhances endogenous antioxidant in the hippocampus of rats: In vivo evaluation by brain microdialysis combined with ESR spectroscopy Yuto Ueda a,⁎, Taku Doi a , Mayuko Takaki a , Keiko Nagatomo a , Akira Nakajima b , L. James Willmore c a Section of Psychiatry, Department of Clinical Neuroscience, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan b Section of Chemistry, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan c Department of Neurology and Department of Pharmacology and Physiology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
We have attempted to explore the neuroprotective effectiveness of levetiracetam (LEV) by
Accepted 21 February 2009
measuring its in vivo antioxidant effect in the hippocampus of rats in a freely moving state.
Available online 4 March 2009
Male Wistar rats were used for the estimation of the in vivo antioxidant effect of LEV through microdialysis combined with electron spin resonance spectroscopy. The antioxidant effect
Keywords:
was examined using the principle by which a systemically administered blood–brain
Levetiracetam
barrier-permeable nitroxide radical (PCAM) decreases in an exponential decay manner that
ESR
is correlated with the amount of antioxidant in the brain. The PCAM decay ratio during
Glutamate
perfusion with normal Ringer's solution was compared with that during 32 μM and 100 μM
Antioxidant
LEV co-perfusion. The in vivo antioxidant effect was examined. In addition, the expressions
Hippocampus
of the cystine/glutamate exchanger (xCT) and the inducible nitric oxide synthase (iNOS)
Redox
protein related to redox regulation were measured in the hippocampus of rats after 14 days of administration of LEV at a dose of 54 mg/day i.p. The half-life of PCAM was statistically shortened after LEV perfusion compared with the results of the control experiment. While the expression of the pro-oxidant protein iNOS was decreased, that of the antioxidant protein xCT was statistically increased by the administration of LEV. The role of xCT is to transport cystine, the internal material of glutathione, into the cell. The shortened half-life of the nitroxide radical by co-perfusion of LEV with increased xCT and decreased iNOS expression revealed the enhancement of the endogenous antioxidant effect or free-radical scavenging activity. The results of this study suggest that LEV synergistically enhances the basal endogenous antioxidant effect in the hippocampus with ascorbic acid and αtocopherol. Our findings further suggest that LEV exerts a neuroprotective role by 1)
⁎ Corresponding author. Fax: +81 985 85 5475. E-mail address: [email protected] (Y. Ueda). Abbreviations: ESR, electron spin resonance; NMDA-R, N-methyl-d-aspartate receptor; LEV, levetiracetam; PBT, pentobarbital; PCAM, 3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl; PCA, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl; iNOS, inducible nitric oxide synthase; xCT, cystine/glutamate exchanger; GSH, glutathione; HVSCC, high-voltage activated N-type Ca2+-current; MDA, malondialdehyde; OPA, o-phthalaldehyde 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.02.040
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modifying the expression of xCT and iNOS in connection with lipid peroxidation, 2) synergistically enhancing the increased basal endogenous antioxidant ability in the hippocampus, and 3) decreasing the basal concentration of glutamate followed by upregulation of the intake of cystine, an internal material of GSH. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Levetiracetam (LEV) is an effective anti-epileptic drug (AED) with unique mechanisms of action which appears to inhibit the initiation of kindling in rodents (Loscher et al., 1998; Sasa et al., 2005; Stratton et al., 2003). The molecular effects of LEV remain uncharacterized, although the drug binds specifically to synaptic vesicle protein 2A (SV2A) (Fuks et al., 2003; Gillard et al., 2003; Lynch et al., 2004). LEV also reduces the high-voltage sensitive calcium current (HVSCC) by inhibiting the N-type Ca2+ channel (Lukyanetz et al., 2002; Niespodziany et al., 2001). Some neuroprotective effects of LEV were suggested when the intraperitoneal administration of LEV was associated with the reduction of the product of lipid peroxidation, malondialdehyde (MDA), in the epileptic rat cortex following KA-induced neural toxicity, suggesting that this effect is mediated, at least in part, by the inhibition of lipid peroxidation (Marini et al., 2004). LEV also reduced the infarct volume in an experimental stroke model of ischemia (Hanon and Klitgaard, 2001). Overall, data suggest that LEV may interfere with glutamate neurotoxicity and ischemia. Although further study is needed, the underlying mechanism of neuroprotection may include the stimulation of neurotrophic factors (Cardile et al., 2003) or the stabilization of mitochondrial functions (Gibbs and Cock, 2007) in addition to anti-inflammatory or anti-oxidative actions. In regard to antioxidants, the results of studies on cerebral xCTdependent glutathione production modulating both neuroprotection from oxidative stress and cell proliferation were recently reported (Liu et al., 2007; Shih et al., 2006). Hence, in the current study, we evaluated the following: 1) the effect of LEV on the basal release of amino acids in the hippocampus, 2) the in vivo and in vitro antioxidant effects of LEV, and 3) the alteration of xCT (antioxidant enzyme) and iNOS (pro-oxidant enzyme) expression in rat hippocampus after chronic treatment with LEV.
2.
Results
2.1.
Hippocampal amino acid concentration
successive decrease in amplitude with no change in linewidth compared with the first spectrum in all groups, and the signal intensity of PCAM at the lowest component in the scan field (MI = +1) decayed exponentially in a linear and highly reproducible fashion (Fig. 2A). The half-life of the decay is dependent upon the antioxidant ability of the brain, as explained previously (Ueda et al., 1998). The half-life is an index that allows a comparison of the rate of elimination of the nitroxide radical, which provides an estimate of the brain's antioxidant ability (Ueda et al., 1998). The half-lives of the PCAM signal intensities from the ventral hippocampus with LEV co-perfusion at both 32 μM and 100 μM were significantly briefer than those of the control (Figs. 2A and B).
2.3. In vitro evaluation of the synergistic effect of LEV on the α-tocopherol–ascorbic acid system As shown in Fig. 3, Torox–ascorbic acid decreases the paramagnetism of the nitroxide radical with the addition of LEV. Decay did not occur with Torox or LEV alone. LEV has a synergistic effect on antioxidant agents, such as Torox–ascorbic acid, as demonstrated by the increase in speed when LEV was added to the system of Torox–ascorbic acid compared with the speed of an antioxidant mixture without LEV. When the PCAM solution was dissolved in a mixture of ascorbic acid, Torox, and LEV in vitro, the half-life of the decreasing signal intensity of PCAM was briefer than that in ascorbic acid and Torox. The add-on effect of the antioxidant
The hippocampal basal release of glutamate with a perfusion of 100 μM LEV was significantly lower than that of the control. The other amino acids were within the range of the control (Fig. 1).
2.2.
In vivo antioxidant ability
The methods used to evaluate in vivo antioxidant ability have been described in previous reports (Ueda et al., 2005; Ueda et al., 1998; Yokoyama et al., 1999). The ESR spectra of the dialysate samples measured at 2.67-min intervals showed a
Fig. 1 – Comparison of amino acids at the basal concentration in the hippocampus of control rats (black bar) and 100 μM LEV co-perfusion (white bar). The right vertical axis represents the glutamine concentration ((Gln)o). Data represent the means +/− SE. *P < 0.01 LEV versus control (Mann–Whitney U-test). GABA = γ-aminobutyric acid; Gln = glutamine; Glu = glutamate; Gly = glycine; Tau = taurine.
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Fig. 2 – (A) Typical plots of signal intensities of PCAM vs. time after injection of PCAM (indicated by black arrows) in each group (control, black circle; 32 μM LEV, white circle; 100 μM LEV, white square; Fig. 2A). (B) Data represent the means +/− SE value of the half-life for each group (Fig. 2B). Data represent the means +/− SE. *P < 0.01 for control versus 100 μM LEV. †P < 0.05 for control versus 32 μM LEV (Mann–Whitney U-test).
indicates that LEV has a synergistic effect on the endogenous efficacy of antioxidants such as ascorbic and α-tocopherol.
2.4.
Western blot
In rats treated with LEV, xCT increased (129.3± 5.2%), while iNOS (77.5 ± 4.7%) decreased in comparison to the values in rats receiving the vehicle-only treatment (control), as shown in Fig. 4.
Fig. 3 – ESR signal intensity of the nitroxide radical when exposed to an antioxidant agent, such as Torox or ascorbic acid. Elimination of the signal intensity by an antioxidant agent was faster when LEV was added to the Torox–ascorbic acid system. LEV itself did not decay the paramagnetism of the nitroxide radical (closed square).
3.
Discussion
Recent data demonstrated the neuroprotective effects of LEV in clinically relevant models of traumatic brain injury and subarachnoid hemorrhage (Wang et al., 2006a). Although the bulk of the literature supports a neuroprotective effect of LEV in acute brain injury, several studies produced negative results, such as the absence of neuroprotection in a hippocampal slice model (Rekling, 2003). However, Hanon and Klitgaard (2001) reported that the administration of LEV (11 and 44 mg/kg i.p.) reduced the infarct volume in a rat model of focal cerebral ischemia and LEV possessed neuroprotective
Fig. 4 – Changes in the expression of xCT and iNOS in the hippocampus of rats administered i.p. with 54 mg/kg LEV once a day for 14 days. Data represent the means +/− S.E. Representative blots are inserted at the top of each bar. *P < 0.05 LEV versus control (Mann–Whitney U-test).
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properties. Wang et al. (2006a) used the administration of LEV (18 and 54 mg/kg i.v.) in a murine model of closed head injury (CHI) and subarachnoid hemorrhage (SAH) to investigate the neuroprotective properties of LEV, which improved the functional and histological outcomes after CHI and reduced vasospasm following SAH. These results suggest that LEV is neuroprotective in clinically relevant animal models of SAH and CHI. Moreover, in a clinical study, Belcastro et al. (2007) and Kutlu et al. (2008) confirmed the effect of LEV in preventing seizure in patients with Alzheimer's disease and post-stroke disease, respectively. Based on these underlying anti-convulsive and neuroprotective mechanisms, we have pointed out the potent candidature mechanism of the action of LEV (Ueda et al., 2007). After 14 days of LEV treatment (54 mg/kg, i.p.), the ipsilateral hippocampus was removed for Western blot analysis. The expression increased for all of the glutamate and GABA transporters (GLAST, GLT-1, EAAC-1, GAT-1, and GAT-3), while the glutamate transporter-regulating protein (GTRAP318) decreased in comparison to the values in normal rats treated without LEV. The suppression of glutamate excitation and the enhancement of GABA inhibition in the rats by continual LEV administration were demonstrated by our previous result showing the up-regulation of glutamate and GABA transporters with the down-regulation of GTARP3-18. These observations demonstrate the critical molecular mechanism of not only anti-epileptic activity but also potential neuroprotective properties of LEV. The main focus of research on the mechanism of epileptogenesis and neurotoxicity has been the functional significance of excitatory amino acid transporters (Ueda et al., 2001) and Glu receptors (GluRs) (Harrington et al., 2007), and little attention has been given to the possible expression of the cystine/Glu antiporter responsible for the bi-directional transmembrane transport of Glu related to the synthesis of glutathione (GSH) (Wang et al., 2006b), which is important to the exploration of the fundamental mechanism of neuroprotection. In parallel with the in vivo evaluation of antioxidant ability, in the current study, xCT protein expression was increased by LEV chronic treatment, although iNOS decreased in rats after chronic administration of LEV (i.p.) compared with the values of the control. These results show that LEV chronic treatment suppresses the collapse of redox to an oxidized shift. LEV decreased the basal release of the extracellular glutamate level only, which helped the intake of cystine-synthesizing GSH, one of the important endogenous antioxidant agents. The suppressive effect of LEV on glutamate overflow as a neuroprotective action may be derived from the inhibitory modulation of the SV2A receptor. There is very limited information on the function of SV2A; however, knockout mice lacking SV2A are known to exhibit seizures. Without SV2A, presynaptic calcium accumulation during repetitive stimulation causes abnormal increases in the neurotransmitter overflow, inducing epilepsy. Therefore, the binding of the SV2A receptor with LEV may restore the ability of a neuron to reduce excessive glutamate overflow (Lynch et al., 2004; Stahl, 2004). Further study will be required to determine the effect of the inhibition of the SV2A receptor on glutamate overflow. Judging from the results reported here, SV2A-regulating
vesicle release by LEV would mainly affect glutamate and not inhibitory amino acids such as glycine, taurine, and GABA. In the normal brain, there is a steady state of redox balance between the production of free radicals and their destruction by endogenous cellular antioxidants such as α-tocopherol– ascorbic acid, GSH–GSSG, and NADP+–NADPH. We believe that the protection of LEV is derived from a synergistic effect with endogenous antioxidants. This hypothesis is strongly supported by the results of the current study, in which the halflife of the decreasing signal intensity of PCAM was shorter than those of ascorbic acid and α-tocopherol when the PCAM solution was dissolved with ascorbic acid and α-tocopherol mixed with LEV in vitro. Although LEV itself did not scavenge PCAM, LEV had a synergistic effect on the endogenous abilities of antioxidants such as ascorbic acid and α-tocopherol. Moreover, repetitive PCAM gradually prolonged the halflife of an injected nitroxide radical (Fig. 2B), which resulted in a decline in the hippocampal antioxidant ability followed by repetitive exposure to the nitroxide radical. Prolongation of the half-life of a free radical in the brain is closely related to neurodegeneration because the prolonged survival of a free radical is related to the enhancement of lipid peroxidation. In other words, LEV prolongs the half-life. In contrast to the prolongation observed in the control group, the half-life of a free radical was shortened by LEV co-perfusion, which indicated that LEV prevented the decline of antioxidant ability in the hippocampus due to repetitive exposure to the nitroxide radical. Thus, LEV may exert neuroprotection against a free radical attack when a seizure occurs. In addition to the increased xCT and decreased iNOS expression, the shortened half-life of the nitroxide radical when co-perfused with LEV revealed the enhancement of the endogenous antioxidant effect or free-radical scavenging activity. Acute LEV exposure certainly increases NO generation through an increase in iNOS expression (Cardile et al., 2003; Dagonnier et al., 2005; Trollmann et al., 2008). This study is the first report suggesting that chronic administration of LEV suppresses NO generation by the depletion of iNOS. The suppression of NO generation could be related to neuroprotection. Although α-tocopherol or ascorbic acid itself has no powerful antioxidant ability, α-tocopherol and ascorbic acid have a powerful ability to eliminate a free radical. The results of this study suggest that LEV synergistically enhances the increased basal endogenous antioxidant effect in the hippocampus with ascorbic acid and α-tocopherol. Our finding that LEV has a synergistic effect on endogenous antioxidants as well as GSH and NADPH is new. Our results, therefore, suggest that LEV exerts a neuroprotective role by 1) modifying the expression regarding oxidative stress, 2) synergistically enhancing the increased basal endogenous antioxidant ability in the hippocampus, and 3) decreasing the basal concentration of glutamate followed by up-regulation of the intake of cystine-synthesizing GSH. Our findings strongly suggest that LEV exerts a neuroprotective effect in the rat hippocampus by selectively decreasing the basal concentration of glutamate and synergistically enhancing the basal endogenous antioxidant activity or free-radical scavenging ability of the αtocopherol and ascorbic acid system via increased elimination of nitroxide radicals in vivo and increased xCT and decreased iNOS expression in vitro after chronic treatment.
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4.
Experimental procedures
All experiments were conducted according to the 1987 guidelines of the Japanese Association for Laboratory Animal Science. The Committee for the Ethics on Animal Experiments of the Faculty of Medicine, University of Miyazaki, reviewed and approved the experimental design (approval number: 1998-158-2). Drugs were purchased from the companies indicated in parentheses: pentobarbital (Dainippon Sumitomo Pharmacology Co., Ltd., Osaka, Japan); blood–brain barrier permeable nitroxide radical (PCAM), synthesized originally from PCA (BBB non-permeable nitroxide radical; Aldrich Chem. Co., Milwaukee); Torox (Aldrich Chem. Co., Milwaukee); ascorbic acid (Wako Pure Chem. Co., Osaka, Japan). The method of PCAM synthesis is described in detail elsewhere (Ueda et al., 2004). 4.1.
Microdialysis operation
Eighteen male Wistar rats weighing 200–250 g at the time of surgery were anesthetized with sodium PBT (37.5 mg/ kg, i.p.). The incisor bar was set on the intraaural line. Each rat was stereotaxically implanted with a guide cannula at coordinates 5.6 mm posterior to and 5.0 mm to the right of the bregma, as determined by the rat brain atlas of Pellegrino et al. (1986). The guide cannula was firmly anchored to the skull with miniature screws and dental cement. After 5 days of recovery, a microdialysis probe (4 mm length, EICOM, Kyoto, Japan) was implanted, and the hippocampus was perfused with Ringer's solution (147 mM NaCl, 4 mM KCl, 2.3 mM CaCl2; pH 6.5) at 2.0 μl/ min (EP-60, EICOM, Kyoto, Japan). For the in vivo microdialysis experiment, we used a dose of 32 μM or 100 μM LEV. This concentration was indicated by Tong and Patsalos (2001), who investigated the extracellular pharmacokinetics of LEV using an SD rat model that allowed serial blood sampling and concurrent brain microdialysis sampling. The mean Coax values for 40 and 80 mg/kg LEV (i. p.) were 58 and 173 μM, respectively. These two conditions were considered to be close to the common clinical doses of LEV (a dose of 1–3 g/day is 17–50 mg/kg in 60 kg body weight). In this experiment, we randomly assigned rats to one of three groups: 1) rats co-perfused with 32 μM LEV, 2) rats co-perfused with 100 μM LEV, and 3) rats infused with Ringer's solution only (control). LEV was diluted with Ringer's solution. 4.2.
Basal release of extracellular amino acids
After a 2-hour stabilization period, sampling from each group was begun. We used HPLC-ECD to measure the basal concentrations of extracellular glutamate, glutamine, glycine, taurine, and γ-aminobutyric acid (GABA) in the ventral hippocampus. Before applying samples to the HPLC-ECD, an OPA solution was made by adding 13.5 mg of OPA and 10 μl of 2-mercaptoethanol to 2.5 ml of a 0.1 M K2CO3 buffer (pH 9.6) with 10% ethanol. The sample (30 μl) was mixed with 10 μl of an OPA solution and incubated for
5
10 min at 4 °C. After mixing was complete, 40 μl of the reactant was applied to the HPLC with an ODS column. Detection was performed by ECD (EICOM, Tokyo, Japan) with + 600 mV/Ag/AgCl. The elution buffer contained 60 mM NaH2PO4U2H2O, 9.6 mM Na2PO4U12H2O, 30% methanol, and 0.5 mM EDTA (pH 6.5). A comparison of the basal release of amino acids was performed between the control and 100 μM LEV groups. 4.3.
In vivo evaluation of antioxidant ability
The measurement of antioxidant function in freely moving rats has been reported in detail (Ueda et al., 1998; Yokoyama et al., 1999). Briefly, in the current study, exogenously administered nitroxide radicals were reduced, and their paramagnetism was destroyed by brain antioxidants. When paramagnetism was plotted on a semilogarithmic scale, the signal intensity decayed exponentially. Thus, the half-life of the decay reflected the antioxidant ability in the brain tissue. The PCAM used here as an exogenous nitroxide radical was injected i.p. (0.2 M PCAM, 0.4 mmol/kg). After collection of the dialysis sample, the perfusate was directed into a quartz duct (inner diameter, 4 mm, LST-5HS; Labotec, Tokyo, Japan) using a polyethylene tube (inner diameter, 0.12 mm; BAS). The quartz duct was placed in the resonator of an ESR spectrometer (FR 30; JEOL, Tokyo, Japan) and analyzed with a WIN-RAD ESR data analyzer (Radical Research Inc., Tokyo). The direction of the perfusate from moving animals into the ESR device allowed the detection of numerous time points for the estimation of changes in the paramagnetism of PCAM, reflecting hippocampal redox conditions during LEV perfusion. The antioxidant ability was tested at concentrations of 32 μM and 100 μM LEV. PCAM i.p. injections were repeated 4 times at 40-min intervals. ESR measurements were conducted under the following conditions: microwave power, 4 mW; microwave frequency, 9.42 GHz; static magnetic field, 335.3 mT; modulation width, 0.1 mT; sweep width, 0.86 mT; sweep speed, 0.32 mT/min. Temporal changes in the ESR signal intensity of PCAM were measured as the peak-to-peak height of the lowest field component (MI = + 1; actual sweep range, 333.8 +/− 0.43 mT) of triplet spectra. The analysis was conducted with high-time resolution at 2.67 min. The methods are described in detail elsewhere (Ueda et al., 2005). 4.4. In vitro evaluation of synergistic effect of LEV on antioxidants We used the elimination of the signal intensity of PCAM to evaluate the synergistic effect of LEV on the α-tocopherol– ascorbic acid system. Following the mixture of Torox (a water-soluble chemical compound that mimics α-tocopherol at 1 mM as the working concentration), ascorbic acid (at 10 mM as the working concentration), and LEV (at 1 mM as the working concentration) in a t-tube, PCAM (at 10 mM as the working concentration) was dissolved in the mixture, and the ESR measurement was performed to
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estimate the half-life of PCAM. All chemical compounds were diluted with Ringer's solution. For the control, Ringer's solution without LEV was used.
Science, Sports, and Culture, Japan. LEV was kindly donated by UCB-Belgium.
4.5.
REFERENCES
Western blot
Rats were treated either with daily i.p. injections of 54 mg/ kg LEV (dissolved in physiological saline at a concentration of 50 mg/ml; n = 6) or with the vehicle i.p. (control group; physiological saline 0.1 ml/100 g; n = 6). Systemic administration at 54 mg/kg was determined by Loscher et al. They tested LEV at 13, 27, and 54 mg/kg i.p. on amygdalakindling acquisition in Wistar rats (chronic administration). LEV suppressed the increase in seizure severity and duration induced by repeated amygdala stimulation dosedependently. After termination of daily treatment with 54 mg/kg, the duration of behavioral seizures and discharge recorded from the amygdala were significantly shorter than those in the vehicle controls, although amygdala stimulation continued in the absence of LEV (Loscher et al., 1998). Animals were treated with LEV or saline i.p. injections between 09:00 and 11:00 each morning. The animals were sacrificed on the 14th day of treatment following the last i.p. injection of LEV or saline. In brief, Western blotting was performed as follows. Crude synaptic samples from the bilateral whole hippocampus (30 μg each) were loaded for gel electrophoresis by discontinuous one-dimensional Tris– SDS-PAGE (4% stacking gel and 10% separating gel). Proteins from the gel were transferred by electroblotting to a 0.2-mm nitrocellulose membrane. The nitrocellulose membrane was blocked for 10 min in 10% nonfat milk in a PBS buffer containing 1% Tween 20 (PBS-T). Following this washing, the blot was incubated for 2 h in anti-xCT (purchased from Trans. Genic, Inc.) and anti-iNOS (purchased from Santa Cruz Biotechnology, Inc.) in PBS-T containing a 0.67% blocking agent. The blot was then washed with PBS-T, incubated with horseradish peroxidase-conjugated IgG for 20 min (1:3000), washed again, and processed for immunoreactivity using an enhanced chemiluminescence detection kit (Amersham). For semiquantitative analysis of each protein, each band density in every sample was calculated using NIH Image software. The transferred blotted membrane was stained with Ponceau-S to verify that each lane was loaded with equivalent amounts of protein. The blots were normalized to β-tubulin for densitometry. 4.6.
Statistical analysis
Statistical analysis was carried out using the Mann– Whitney U-test. Data were considered significant at a level of P < 0.05.
Acknowledgments This study was partially supported by a Grant-in-Aid for Scientific Research (C) (2) (16591146, 18591297, and 20591372 to Y.U.) and by a Grant-in-Aid for Encouragement of Young Scientists (18790840 to K.N.) from the Ministry of Education,
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