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NEUROPHARMACOLOGY

Edaravone Protects against Hypoxia/Ischemia-Induced Endoplasmic Reticulum Dysfunction

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

Received for publication March 25, 2004
Accepted June 3, 2004.

	Abstract

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Endoplasmic reticulum (ER) stress-induced cell death plays an important role in cerebral ischemia. In the present study, we investigated whether edaravone (3-methyl-1-phenyl-pyrazolin-5-one), a free radical scavenger, can protect against ER damage induced by cerebral ischemia. In a mouse model of hypoxia/ischemia, treatment with edaravone reduced edema-corrected infarction volume, attenuated hemispheric swelling, and improved neurological status. Moreover, edaravone suppressed ER stress-mediated apoptosis by inhibiting eukaryotic initiation factor phosphorylation, C/EBP homologous protein (CHOP) induction, and caspase-12 activation. In mouse primary cultured glial cells, edaravone attenuated ER stress as evidenced by inhibition of the induction of glucose regulated protein 78 and CHOP and XBP-1 splicing under treatment with tunicamycin (Tm), which induces ER stress. Tm did not induce the production of reactive oxygen species in primary cultured glial cells. In addition, the free radical scavengers N-acetyl-L-cysteine and ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] did not affect ER stress response caused by Tm. These results demonstrated a novel action of edaravone that can protect against ER dysfunction in cerebral ischemia.

Cerebral ischemia is a pathophysiological ER stressor (Paschen and Doutheil, 1999; Kumar et al., 2003). Several studies have shown that ischemic injury causes a severe impairment of ER function, which in turn triggers shutdown of protein translation and apoptosis (Kumar et al., 2003; Tajiri et al., 2004), suggesting that the ER plays an important role in cerebral ischemia. Thus, reducing ER stress may provide a therapeutic way to block the pathological process induced by cerebral ischemia.

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a potent and novel scavenger of free radicals inhibiting not only hydroxyl radicals but also free radical-mediated lipid peroxidative damage (Watanabe et al., 1997). It has been reported that edaravone changes into 2-oxo-3-(phenylbudrazono)-butanoic acid after reacting with peroxy radicals in vitro (Yamamoto et al., 1996). Edaravone has protective effects on cerebral edema and tissue injury after ischemia-reperfusion in rats (Abe et al., 1998) and patients with acute cerebral ischemia (Houkin et al., 1998; Mitsumori et al., 1998). Additionally, edaravone protects against mitochondrial injury in hepatic and myocardial ischemia/reperfusion (Okatani et al., 2003; Rajesh et al., 2003) and inhibits inflammatory reaction (Kono et al., 2003). These effects were predominantly thought to be a result of the protective action of edaravone against lipid peroxidation.

In the present study, we focused on the possible protective effect of edaravone against ER dysfunction. We obtained evidence that edaravone significantly inhibited ER stress-mediated apoptotic signals induced by hypoxia/ischemia and attenuated ER stress in vivo and in vitro.

	Materials and Methods

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Edaravone was a gift from Mitsubishi Chemical Industries (Tokyo, Japan). The drug was dissolved in NaOH (1 M) and adjusted to pH 7.4 according to the manufacturer's instructions. Tunicamycin (Tm) was purchased from Wako Pure Chemicals (Tokyo, Japan). N-Acetyl-L-cysteine (NAC) and ebselen were purchased from Sigma-Aldrich (St. Louis, MO).

Induction of Hypoxia/Ischemia. Male C57BL mice (6 weeks old) were anesthetized with halothane (2% in 70% N₂O, 30% O₂), and the right carotid artery was isolated and double ligated with 4-0 surgical thread. The incision was sutured, and the animals were allowed to recover with access to food and water for 3 h. To induce hypoxia, each animal was placed in a 500-ml glass jar partially submerged in a temperature-controlled water bath and exposed to a humidified gas mixture of 6% O₂, balance N₂ for specific intervals of 30 min. The water bath temperature was maintained at 35.5°C, which in previous experiments was shown to maintain the animals' core body temperature at 37.5–37.7°C throughout the hypoxic interval (Vannucci et al., 2001). Animals were allowed to recover in room air for 30 min and then returned to their cages with free access to food and water. The rectal temperature was measured at several points during the initial hours after hypoxia/ischemia and at longer intervals until they are killed. Animals in which the right common carotid artery was separated but no ligation and hypoxia were performed were used as sham-operated controls. All animal experiments were carried out in accordance with the National Institutes of Health Guide for Care and Use of Laboratory animals and were approved by the animal care and use committee of Hokkaido University.

Determination of Infarct Volume and Hemispheric Swelling. Animals were sacrificed 3 days after hypoxia/ischemia, and the brain was removed and cut into four 2-mm sections. The brain slices were immersed in a 2% solution of TTC (Wako Pure Chemicals) in normal saline at room temperature in a dark place for 30 min and then washed in 5% phosphate-buffered saline twice. The slices were fixed in 10% formalin for photography, and the infarction area was measured. An image analysis system (NIH 1.61 software) was used to determine the infracted volume (MV), right hemisphere volume (RV), and left hemisphere volume (LV). To compensate for the effect of brain edema on the measured infarct volume in the ischemic hemisphere, the infarct volume in each mouse was corrected as described (Swanson et al., 1990; Aspey et al., 1998) with the following formula. Corrected infarct volume (%) = [LV - (RV - MV)]/LV x 100. Hemispheric swelling in the ischemic hemisphere was also calculated: swelling (%) = RV - LV/LV x 100.

Evaluation of Neurological Status. Neurological deficits of mice at 1, 2, and 3 days after hypoxia/ischemia were assessed and scored as described previously (Huang et al., 1994) as follows: 0, no observable neurological deficit (normal); 1, failure to extend left forepaw on the lifting of the body by the tail (mild); 2, circling to the contralateral side (moderate); and 3, leaning to the contralateral side at rest or no spontaneous motor activity (severe). Animals not showing neurological deficits were excluded from the study.

Cell Culture. Primary glial cells were prepared from the whole brain of neonatal (<24 h) C57BL/6 mice as described previously (Hosoi et al., 2000). The cells were allowed to grow to confluence in Dulbecco's modified Eagle's medium with 10% (v/v) fetal calf serum, 100 units/ml of penicillin G, and 100 µg/ml of streptomycin (Invitrogen, Carlsbad, CA). All cultured cells were maintained at 37°C in 5% CO₂, 95% air.

RT-PCR. Total RNA was isolated from mouse brain or cultured cells using TRI Reagent (Sigma-Aldrich, St. Louis, MO). RT-PCR was performed as described previously (Hosoi et al., 2002b). The following primers were used: CHOP forward, 5'-ccc tgc ctt tca cct tgg-3'; CHOP reverse, 3'-ccg ctc gtt ctc ctg ctc-5'; XBP-1 forward, 5'-cct tgt ggt tga gaa cca gg-3'; XBP-1 reverse, 3'-cta gag gct tgg tgt ata c-5'; and GAPDH forward, 5'-gat gga gca tca tac tga tcc-3'; GAPDH reverse, 3'-aaa ccc atc acc atc ttc cag-5'.

Western Blotting. Primary antibodies used for Western blotting analysis were as follows: anti-phospho-eIF2 (Ser51) polyclonal antibody (Cell Signaling Technology Inc., Tokyo, Japan), 1:1000; anti-GADD153-polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:1000; anti-caspase-12-polyclonal antibody (ProSci Incorporated, Poway, CA), 1:1000; and anti-KDEL monoclonal antibody (StressGen Biotechnologies, San Diego, CA), 1:1000. Western blotting analysis was performed as described previously (Hosoi et al., 2002a).

Measurement of Intracellular Reactive Oxygen Species (ROS). A cell membrane-permeable and oxidant-sensitive fluorescent dye 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) was used to measure ROS. Tunicamycin (10 µM)-treated or untreated glial cells were washed with prewarmed (37°C) phosphate-buffered saline once and then incubated in culture medium, together with CM-H₂DCFDA (10 µM). After 30 min of incubation, the cells were washed with prewarmed medium twice before being distributed into a 96-well plate in 200 µl. Fluorescence intensity in each well was measured on a fluorescent microplate reader (Tecan, Durham, NC) at excitation and emission wavelengths of 485 and 538 nm, respectively. Finally, the number of cells in each well was counted with Erma cell counting chamber (Erma, Tokyo, Japan). The data were expressed as fluorescent intensity per 2 x 10⁴ cells.

	Results

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Edaravone Protects against Cerebral Injury Caused by Hypoxia/Ischemia. Treatment with edaravone (1–10 mg/kg i.p., once a day for 3 days) immediately after hypoxia/ischemia dose dependently decreased infarct volume on the 3rd day after hypoxia/ischemia. Remarkable protection was achieved with 3 and 10 mg/kg edaravone by 30 and 60% reduction in edema-corrected infarct volume, respectively (Fig. 1b). However, no significant reduction in infarct volume was observed in mice dosed with 1 mg/kg (Fig. 1b). Hemispheric swelling was also significantly decreased with a dose of 3 (40% reduction) and 10 mg/kg (60% reduction) under the same treatment conditions (Fig. 1c).

View larger version (39K): [in this window] [in a new window]   Fig. 1. Treatment with edaravone attenuated hypoxia/ischemia-induced brain injury. Mice were injected intraperitoneally with saline or edaravone immediately after hypoxia/ischemia and sacrificed 3 days later. Brain coronal sections (2 mm) were stained with 2% TTC. a, representative data from ischemic brain. Treatment with edaravone dose dependently reduced edema-corrected infarction volume (b) and hemispheric swelling (c). Data are means ± S.E. from 10 mice in each group. *, p < 0.05; **, p < 0.01 compared with the saline-injected group, one-way analysis of variance followed by Dunnett's test.

In addition, we examined the effect of edaravone on the rectal temperature of mice in the model of hypoxia/ischemia. No significant change in body temperature was observed in salineand edaravone (10 mg/kg)-treated groups during the experiment (Fig. 2). These results indicate that the protective effect of edaravone on hypoxia/ischemia is not caused by hypothermia.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Edaravone did not affect the body temperature of animals in hypoxia/ischemia. Saline or edaravone (10 mg/kg) was administered (i.p.) immediately after hypoxia/ischemia. The rectal temperature was measured at the indicated time points. Data are means ± S.E. from five mice in each group.

Edaravone Improves Neurological Function. Treatment with edaravone (10 mg/kg) significantly reduced neurological deficits and provided functional recovery from hypoxia/ischemia when examined at 24, 48, and 72 h, compared with the saline group (Fig. 3).

View larger version (26K): [in this window] [in a new window]   Fig. 3. Treatment with edaravone improved neurological function. Saline or edaravone was administered (i.p.) immediately after hypoxia/ischemia. The neurological deficits were assessed at 1, 2, and 3 days of recovery. Data are means ± S.E. from 10 mice in each group, respectively. *, p < 0.05 compared with the neurological score of the saline-treated group on the corresponding day, one-way analysis of variance followed by Dunnett's test.

Edaravone Represses ER Stress-Mediated Apoptotic Signals. A key feature of ER stress induced by cerebral ischemia is the blocking of translation at the initiation step, as indicated by increased phosphorylation of eIF2 (DeGracia et al., 2002; Mengesdorf et al., 2002). Therefore, we first examined whether edaravone affects the phosphorylation of eIF2, which has also been demonstrated to be an inducer of the transcription factor CHOP (Oyadomari and Mori, 2003). The level of phospho-eIF2 in injured cortex was markedly increased after hypoxia/ischemia and detectable until 12 h, whereas the administration of edaravone at 10 mg/kg noticeably reduced the levels from 6 to 12 h (approximately 35 to 50% reduction, compared with the saline-treated group) (Fig. 4a). No change in the level of phosphor-eIF2 was observed in the brains of animals only injected with edaravone (Fig. 4d). In the previous study, we have observed that CHOP was significantly up-regulated in the hypoxia/ischemic cortex, hippocampus, and striatum, and inductions of CHOP in the three regions show similar extent (unpublished observations). Quantification revealed a remarkable increase in the protein level of CHOP in the injured cortex 12 h after hypoxia/ischemia, compared with that in sham-operated animals. However, injection of edaravone remarkably down-regulated CHOP protein level (approximately 90% reduction), compared with that in the saline-treated group (Fig. 4b). The protein level of CHOP was not affected by treatment with edaravone in normal brains (Fig. 4d).

View larger version (38K): [in this window] [in a new window]   Fig. 4. Treatment with edaravone suppressed the ER stress-mediated apoptosis. Mice were subjected to 30 min hypoxia/ischemia followed by recovery until sacrifice. Saline or edaravone (10 mg/kg) was injected immediately after hypoxia/ischemia. The tissue samples were from the cerebral cortex. a, administration of edaravone decreased the phosphorylation of eIF2. Phospho-eIF2 was examined at the indicated time points by Western blotting. b, change in the CHOP protein level determined at the indicated time points after hypoxia/ischemia by Western blotting. c, treatment with edaravone reduced the activation of caspase-12. Top, representative Western blot of procaspase-12. Bottom, quantification of procaspase-12 levels. The tissue samples were from 24 h after ischemia. d, Western blotting analysis of phospho-eIF2, CHOP, and caspase-12 in extracts from the right hemisphere in normal animals treated with saline or edaravone (10 mg/kg). The data are expressed as the mean ± S.E. from six mice in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the saline-treated group; Fisher's test.

Furthermore, Western blotting showed that hypoxia/ischemia led to the activation of caspase-12 as evidenced by a decrease in the level of the procaspase-12. The activation of caspase-12 was largely restored by edaravone (almost 30% restoration, compared with the saline-treated group) (Fig. 4c). No difference in the activation of caspase-12 was observed between the saline-treated and edaravone-treated groups in normal brains (Fig. 4d).

Edaravone Attenuates ER Stress in Primary Cultured Glial Cells. To elucidate the mechanism underlying the protection against ER dysfunction, first we evaluated the effect of edaravone on ER stress in primary cultured glial cells through treatment with ER stressor Tm (an inhibitor of N-glycoslytion). Western blotting analysis showed that treatment with Tm remarkably up-regulated the expression of GRP78 and CHOP, markers of ER stress response, whereas edaravone (1–10 µM) dose dependently inhibited GRP78 and CHOP induction (Fig. 5, a and b).

View larger version (26K): [in this window] [in a new window]   Fig. 5. Edaravone inhibited ER stress response in primary cultured glial cells. Primary cultured glial cells were preincubated with edaravone for 1 h and then treated with Tm for 24 h, and protein levels were assessed by Western blotting. Edaravone inhibited the induction of GRP78 (a) and CHOP (b). The quantification is expressed as the mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 compared with Tm-treated group; Fisher's test. The spliced form of XBP-1 was analyzed by RT-PCR (c). The data are representative of three independent experiments.

XBP-1 mRNA has been shown to be spliced upon ER stress (Yoshida et al., 2001; Calfon et al., 2002). We observed the splicing of XBP-1 mRNA 6 h after treatment with Tm (1 µg/ml). Edaravone inhibited or even abrogated XBP-1 mRNA splicing (Fig. 5c), as reflected by a decrease in the ratio of spliced to unspliced forms of XBP-1.

In contrast to edaravone, NAC and ebselen, both of which have been demonstrated to be free radical scavengers, did not affect the induction of GRP78 and CHOP under treatment with Tm in primary cultured glial cells (Fig. 6, a and b). Thus, the effect of inhibiting ER stress may be specific to edaravone.

View larger version (36K): [in this window] [in a new window]   Fig. 6. NAC and ebselen provided no protection against ER stress. Primary cultured glial cells were preincubated with NAC or ebselen at the indicated doses for 1 h and then treated with Tm for 24 h. The protein levels were assessed by Western blotting. NAC (a) and ebselen (b) did not affect the protein levels of GRP78 and CHOP under treatment with Tm. The data are representative of two independent experiments. c, Tm did not induce the production of ROS in primary cultured glial cells. Primary cultured glial cells were treated with Tm for the indicated period or H2O2 (1 mM) for 48 h and then incubated with CM-H2DCA (10 µM) for 30 min. The cells were distributed to a 96-well plate in 200 µl. The fluorescent intensity per 2 x 104 cells was measured. The data represent the mean ± S.E. of three independent experiments.

Tm Did Not Induce the Increase in ROS Levels in Primary Cultured Glial Cells. To determine whether the protective effect of edaravone on the ER results from resistance to ROS, which has been suggested to damage the ER and lead to cell death (Hayashi et al., 2003), we assessed the levels of ROS under treatment with Tm in primary cultured glial cells. Tm did not generate ROS in glial cells (Fig. 6c), suggesting that the inhibition of ER dysfunction by edaravone under conditions of Tm treatment may not be based on the radical scavenging action.

	Discussion

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In the present study, we used a murine model of hypoxia/ischemia. Thirty minutes of hypoxia/ischemia produced markedly damage to the ipsilateral cortex, hippocampus, and striatum, as determined by TTC staining. On the other hand, we showed that peripheral administration of edaravone protected brain from hypoxia/ischemic injury as evidenced by reduction in infarct volume and hemispheric swelling. Improvement of neurological score was also observed in the same animals that showed reduction in infarct volume and hemispheric swelling. Simultaneously, we found that edaravone can effectively suppress ER dysfunction in vivo and in vitro. Moreover, the present findings indicate that the protective effect of edaravone on ischemic injury may be medicated in part by restoration of ER dysfunction.

In cerebral ischemia, excessive generation of oxygen free radicals followed by lipid peroxidation is thought to play an important role in the secondary injury (Seki et al., 1998). Exogenous radical scavengers have been demonstrated to suppress oxidative stress and thus prevent ischemic injury (Zoratti and Szabo, 1995; Crompton, 1999). Edaravone has been shown to exert a protective effect against cerebral ischemic injury by inhibiting OH^- and iron-dependent lipid peroxidations, both of which are essential in mediating ischemic cell damage (Watanabe et al., 1994). On the other hand, ample evidence suggested that ER damage is involved in neuronal cell death induced by cerebral ischemia (Kumar et al., 2003; Tajiri et al., 2004). Thus, in the present study, we investigated the effect of edaravone on ER dysfunction under pathological conditions.

In mice subjected to 30-min hypoxia/ischemia followed by a given period of recovery, we observed an increase in the level of the transcription factor CHOP and activation of caspase-12 in the ischemic cortex. The results indicate that hypoxia/ischemia caused severe ER damage and triggered ER stress-associated apoptosis. On the other hand, treatment with edaravone significantly inhibited caspase-12 activation and CHOP induction. In addition, we observed suppression of phospho-eIF2, upstream of CHOP. Therefore, the protective effects of edaravone in hypoxia/ischemic injury may be partly due to inhibition of ER stress and subsequent apoptotic signaling pathway.

We found that edaravone also inhibited ER stress even if the condition was nonischemic. Treatment of cells with Tm, an inducer of ER stress, caused ER dysfunction as indicated by the induction of representative genes related to ER stress. However, Tm did not cause the production of ROS in primary cultured glial cells. Thus, it was suggested that ER stress does not induce ROS production in glial cells. In addition, other free radical scavengers, such as NAC and ebselen, provided no protection against ER dysfunction. The results indicated that the property of inhibiting ER stress is specific to edaravone. Thus, the protective effect of edaravone against hypoxia/ischemia may involve a novel pathway that directly attenuates ER stress in addition to suppressing ROS production. Considering the findings of the present study, we speculate that edaravone has multiple effects, although the mechanism of its actions needs to be elucidated further.

In summary, the present results suggest that edaravone provided protection against ER dysfunction induced by hypoxia/ischemia, and the protective effect may be mediated by an inhibition of ER stress as well as the radical scavenging action. Therefore, the restoration of ER dysfunction by edaravone may be a novel neuroprotective mechanism. Furthermore, on the basis of its protection against ER damage, edaravone may also be useful for treating other neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.069088.

ABBREVIATIONS: ER, endoplasmic reticulum; CHOP, C/EBP homologous protein; Tm, tunicamycin; NAC, N-acetyl-L-cysteine; TTC, 2,3,5-triphenyltetrazolium chloride; RT-PCR, reverse transcription-polymerase chain reaction; GRP78, glucose regulated protein 78; eIF2, eukaryotic initiation factor ; ROS, reactive oxygen species; CM-H₂DCFDA, 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate.

Address correspondence to: Dr. Yasuyuki Nomura. Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan. E-mail: nomura{at}pharm.hokudai.ac.jp

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				W. Mao, C. Iwai, P. C. Keng, R. Vulapalli, and C.-s. Liang Norepinephrine-induced oxidative stress causes PC-12 cell apoptosis by both endoplasmic reticulum stress and mitochondrial intrinsic pathway: inhibition of phosphatidylinositol 3-kinase survival pathway Am J Physiol Cell Physiol, May 1, 2006; 290(5): C1373 - C1384. [Abstract] [Full Text] [PDF]

				W. Mao, C. Iwai, F. Qin, and C.-s. Liang Norepinephrine induces endoplasmic reticulum stress and downregulation of norepinephrine transporter density in PC12 cells via oxidative stress Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2381 - H2389. [Abstract] [Full Text] [PDF]

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