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The effects of a free radical scavenger, edaravone, combined with mild hypothermia on ischemic brain damage following transient middle cerebral artery occlusion in rats
International Congress Series 1252 (2003) 109 – 115
The effects of a free radical scavenger, edaravone, combined with mild hypothermia on ischemic brain damage following transient middle cerebral artery occlusion in rats Chikako Nito*, Tatsushi Kamiya, Shimon Amemiya, Kengo Kato, Yasuo Katayama Division of Neurology, Second Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo, Tokyo 113-8603, Japan
Abstract 3-Methyl-1-phenyl-pyrazolin-5-one (edaravone), a novel free radical scavenger, has been reported to reduce ischemic damage in rats subjected to transient focal ischemia. The aim of this study is, therefore, to investigate the effect of a combined therapy with edaravone and mild hypothermia at 35 jC. Sprague – Dawley rats were subjected to middle cerebral artery (MCA) occluding using by an intraluminal suture technique for 2 h. The rats were reperfused for 24 h and decapitated for infarct and edema analysis. Animals were randomly divided into four groups: (I) vehicle + normothermia (control); (II) vehicle + mild hypothermia; (III) edaravone + normothermia; (IV) edaravone + mild hypothermia. Mild hypothermia alone had no reduction of the brain damage. The edaravone alone significantly reduced edema volume. The combined treatment with edaravone and mild hypothermia reduced both infarct and edema volume. In addition, this treatment provided for the best functional outcome. These results demonstrate that a free radical scavenger, edaravone, attenuates brain edema and that the combined therapy with edaravone and mild hypothermia significantly reduces not only edema but also infarct on transient focal cerebral ischemia in rats. The neuroprotective effects seen in this study may be due to the combined interaction of antiedema activity between edaravone and mild hypothermia, suppressing free radical production. D 2003 Published by Elsevier Science B.V. Keywords: Free radical scavenger; Hypothermia; Neuroprotection; Focal ischemia; Rat
* Corresponding author. Tel: +81-3-3822-2131x6496; fax: +81-3-3822-4865. E-mail address: [email protected] (C. Nito). 0531-5131/03 D 2003 Published by Elsevier Science B.V. doi:10.1016/S0531-5131(03)00044-X
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1. Introduction 3-Methyl-1-phenyl-pyrazolin-5-one (edaravone) is a novel antioxidant, and inhibits both hydroxyl radicals and iron-induced peroxidative vascular endothelial cell damage [1]. Furthermore, edaravone has been tested in various different experimental models for evaluation of its protective effects in cerebral ischemia/reperfusion and myocardial ischemia/reperfusion [2,3]. Although any free radical scavengers have been developed for clinical use, edaravone is one of the most expecting of these agents. Therefore, it has been clinically applied for the treatment of acute stroke in Japan since June 2001. Recent experimental studies have demonstrated that hypothermia is a powerful tool in neuronal protection following cerebral ischemia [4]. Studies on the effect of mild or moderate hypothermia on cerebral ischemia were performed in various models of global or focal cerebral ischemia [5,6]. Multiple mechanisms for hypothermia-induced neuroprotection have been identified, such as reduced metabolic rate and energy depletion, decreased excitatory transmitter release, decreased generation of free radicals, improved ion homeostasis, and reduced vascular permeability, blood –brain barrier disruption, and edema [7,8]. Recently, experimental studies suggest that the combination of mild hypothermia with neuroprotective drugs is more effective than each of these alone [9,10]. There are no data available in regard to a combined treatment of free radical scavenger, edaravone, and hypothermia. The present study was designed to evaluate the singular and combined effects of edaravone and/or mild hypothermia of 35 jC on ischemic damage in rats subjected to transient focal ischemia.
2. Materials and methods Sprague – Dawley rats weighing 250 – 300 g were used. Anesthesia was initially induced with halothane (5% for induction and 1% for maintenance) in a mixture of 70% N2O and 30% O2 under spontaneous breathing. Blood gases and blood glucose levels were measured just before and during cerebral ischemia. Animals were subjected to 2-h middle cerebral artery (MCA) occluding followed by 24-h reperfusion using an intraluminal suture technique [11]. Animals were randomly divided into the following four groups (each, n = 6): (I) vehicle-treated, normothermic group (control); (II) edaravone-treated, normothermic group; (III) vehicle-treated, mild hypothermic (35 jC) group; (IV) edaravone-treated, mild hypothermic(35 jC) group. Temporal muscle and rectal temperatures were maintained during ischemia at 37 F 0.2 jC (normothermic groups) or 35 F 0.2 jC (hypothermic groups). Animals were intravenously administered 3.0 mg/kg of edaravone or vehicle, provided by Mitsubishi Pharma (Tokyo, Japan), just prior to the reperfusion. Twenty four hours after reperfusion, the animals were decapitated and removed. Coronal sections (20 Am) were cut and were stained with hematoxylin and eosin. The cortical or striatal areas (mm2) from each slice were summed and multiplied to obtain an infarct volume (mm3). A volume of edema (mm3) was determined by subtracting the non-ischemic cortical volume from the ischemic cortical volume [12].
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Neurological symptoms of each animal were examined blindly 24 h after reperfusion using an established scoring system [13]. In these tests, the postural reflex test and hemiparesis of animals were observed. Statistical analyses were performed with the use of StatView 5.0. Physiological data for each time point, infarct volumes, edema volumes, and neurological function scores were analyzed with one-way ANOVA. Values are presented as mean F S.D., and differences were considered significant at p < 0.05 level.
3. Results There were no statistically significant differences in the mean arterial blood pressure, pH, PaCO2, PaO2, or blood glucose levels among the groups. 3.1. Infarct volume The cortical infarct volumes in the vehicle-treated normothermic, vehicle-treated hypothermic, edaravone-treated normothermic, and edaravone-treated hypothermic groups were 201 F 37, 181 F 44, 163 F 22, and 90 F 37 mm3, respectively. The cortical infarct volume was significantly reduced in the edaravone-treated hypothermic group compared with the other groups, and the reduction was 55% of those in the vehicletreated normothermic group (Fig. 1). The striatal infarct volumes in the vehicle-treated
Fig. 1. The cortical or striatal infarct volumes after 2-h middle cerebral artery occlusion followed by 24-h reperfusion. Values (mm3) are expressed as mean F S.D. **p < 0.001 vs. vehicle-treated normothermic group by one-way ANOVA.
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normothermic, vehicle-treated hypothermic, edaravone-treated normothermic, and edaravone-treated hypothermic group were 108 F 12, 91 F 10, 96 F 15, and 70 F 15 mm3, respectively. The combination with edaravone and mild hypothermia significantly reduced the striatal infarct volume ( 35%) compared with the vehicle-treated normothermic group (Fig. 1). 3.2. Edema volume The cortical edema volumes in the vehicle-treated normothermic, vehicle-treated hypothermic, edaravone-treated normothermic, and edaravone-treated hypothermic group were 82 F 18, 59 F 16, 42 F 13, and 23 F 17 mm3, respectively. In the edaravone-treated normothermic group, the cortical edema volume was significantly less than that in the vehicle-treated normothermic group ( 49%). Moreover, edaravone with hypothermic group reduced the cortical edema volume significantly compared with that in vehicle-treated normothermic group ( 72%) (Fig. 2). The striatal edema volumes in the vehicle-treated normothermic, vehicle-treated hypothermic, edaravone-treated normothermic, and edaravone-treated hypothermic group were 46 F 10, 34 F 3, 27 F 10, and 21 F 8 mm3, respectively. The striatal edema volume in the edaravone-treated normothermic or the edaravone-treated hypothermic group was significantly smaller than that in the vehicle-treated normothermic group ( 41%, 54%) (Fig. 2).
Fig. 2. The cortical or striatal edema volumes after 2-h middle cerebral artery occlusion followed by 24-h reperfusion. Values (mm3) are expressed as mean F S.D. **p < 0.001, *p < 0.01 vs. vehicle-treated normothermic group by one-way ANOVA.
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Fig. 3. The neurological score after 2-h middle cerebral artery occlusion followed by 24-h reperfusion. Each symbol depicts the individual neuro-score of a single animal. #p < 0.05 vs. vehicle-treated normothermic group by one-way ANOVA.
3.3. Neurological symptoms The median neurological score was 2.5 F 0.5 in the control group. The combination treatment of edaravone and hypothermia significantly improved the neurological symptoms (hemiplegia) compared with the control group ( p < 0.05) (Fig. 3).
4. Discussion With use of the suture model in the rat MCA, the present study demonstrated that a low dose of edaravone (3.0 mg/kg) combined with mild intraischemic hypothermia (35 jC) significantly reduces infarct and edema volumes in the cortex and the striatum. However, mild intraischemic hypothermia (35 jC) alone failed to reduce postischemic brain damage. Under normothermic conditions, the protective effects of edaravone were observed in the edema, but not in the infarct. Taken together, these results suggest that the combination of edaravone administration and mild intraischemic hypothermia expands the therapeutic time window for edaravone alone. Moreover, the combination reduced both infarct and edema volumes, suggesting that the combination protects not only ischemic penumbra but also ischemic core. Brain temperature is an important determinant of neuronal injury following cerebral ischemia [4]. Hypothermic treatment has also been used clinically as one of the strategies for reducing ischemic brain injury; however, the usefulness of deep therapeutic hypothermia for cerebral ischemia has been limited by adverse effects such as myocardial
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arrhythmia, hypotension, respiratory insufficiency, blood hypercoagulability, and hemodynamic instability [14]. Severe hypothermia is known to have these side effects; however, mild hypothermia is much safer than the classical deep hypothermia [15]. We chose mild hypothermia of 35 jC because clinical application and continuity can be accomplished both easily and safely, without severe complication. Indeed, mild hypothermia (35 jC) did not affect any physiological variables in the present study. The temperature of 35 jC may improve edaravone’s effect without the adverse effects of more severe hypothermia on physiological functions. Although the mechanisms of ischemic brain damage have not been clearly determined, accumulated experimental evidence suggests that production of free radicals is possibly one of the major factors involved. Several reports have demonstrated that free radicals generated during ischemia play an important role in the development of neuronal damage [16,17]. Although the protective mechanisms of hypothermia are complex, one has been identified as decreased generation of free radicals from microglia [18,19]. Microglial cells produces superoxide anions and nitric oxide, and hypothermia potentially inhibits the production. Accordingly, the neuroprotective effects seen in this study may be due to the combined interaction of antiedema activity between edaravone and mild hypothermia, suppressing free radical production. The present study demonstrates that a free radical scavenger, edaravone, attenuates brain edema and that a combined therapy with edaravone and mild hypothermia significantly reduces not only edema but also infarct on transient focal cerebral ischemia in rats. These data show that this combination is an effective and safe strategy for neuronal protection during ischemia, and may be useful in clinical medical care for acute human ischemic stroke.
Acknowledgements This study was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan (Drs. Kamiya and Katayama). We would like to thank Mr. Toshiki Inaba for his expert technical. Edaravine was generously supplied to us by Mitsubishi Pharma.
References [1] S. Murota, I. Morita, N. Suda, The control of vascular endothelial cell injury, Ann. N.Y. Acad. Sci. 598 (1990) 182 – 187. [2] K. Abe, S. Yuki, K. Kogure, Strong attenuation of ischemic and postischemic brain edema in rats by a novel free radical scavenger, Stroke 19 (1988) 480 – 485. [3] H. Nishi, T. Watanabe, H. Sakurai, S. Yuki, A. Ishibashi, Effect of MCI-186 on brain edema in rats, Stroke 20 (1989) 1236 – 1240. [4] R. Busto, W.D. Dietrich, M.Y. Globus, I. Valdes, P. Scheinberg, M.D. Ginsberg, Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury, J. Cereb. Blood Flow Metab. 7 (1987) 729 – 738. [5] E.A. Bering Jr., Effects of profound hypothermia and circulatory arrest on cerebral oxygen metabolism and cerebrospinal fluid electrolyte composition in dogs, J. Neurosurg. 40 (1974) 199 – 205.
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[6] Y. Goto, N.F. Kassell, K. Hiramatsu, S.W. Soleau, K.S. Lee, Effects of intraischemic hypothermia on cerebral damage in a model of reversible focal ischemia, Neurosurgery 32 (1993) 980 – 985. [7] S. Schwab, S. Schwarz, M. Spranger, E. Keller, M. Bertram, W. Hacke, Moderate hypothermia in the treatment of patients with severe middle cerebral artery infarction, Stroke 29 (1998) 2461 – 2466. [8] W.D. Dietrich, R. Busto, M.Y. Globus, M.D. Ginsberg, Brain damage and temperature: cellular and molecular mechanisms, Adv. Neurol. 71 (1996) 177 – 194. [9] C. Nito, T. Kamiya, M. Ueda, T. Arii, A. Terashi, Y. Katayama, Mild hypothermia enhances neuroprotective effects of immunosuppressant FK506 following transient focal ischemia in rats, J. Cereb. Blood Flow Metab. 21 (Suppl. 1) (2001) S405. [10] R. Schmid-Elaesser, E. Hungerhuber, S. Zausinger, A. Baethmann, H.J. Ruelen, Combination drug therapy and mild hypothermia, Stroke 30 (1999) 1891 – 1899. [11] T. Arii, T. Kamiya, K. Arii, M. Ueda, C. Nito, K. Katsura, Y. Katayama, Neuroprotective effect of immunosuppressant FK506 in transient focal ischemia in rat, Neurol. Res. 23 (2001) 755 – 760. [12] M. Jacewicz, J. Tanabe, W.A. Pulsinelli, The CBF threshold and dynamics for focal cerebral infarction in spontaneously hypertensive rats, J. Cereb. Blood Flow Metab. 12 (1992) 359 – 370. [13] J.B. Bederson, L.H. Pitts, M. Tsuji, M.C. Nishimura, R.L. Davis, H. Bartowski, Rat middle cerebral occlusion: evaluation of the model and development of a neurologic examination, Stroke 17 (1986) 472 – 476. [14] A. Schubert, Side effect of mild hypothermia, J. Neurosurg. Anesthesiol. 7 (1995) 139 – 147. [15] A. Kader, M.H. Brisman, N. Maraire, J.T. Huh, R.A. Solomon, The effect of mild hypothermia on permanent focal ischemia in rat, Neurosurgery 31 (1992) 1056 – 1061. [16] H. Kinouchi, C. Epstein, T. Mizui, E. Carlson, S.F. Chen, P.H. Chan, Attenuation of focal cerebral ischemia injury in transgenic mice overexpressing CuZn superoxide dismutase, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 11158 – 11162. [17] K. Kitagawa, M. Matsumoto, T. Oda, M. Niinobe, R. Hata, N. Handa, R. Fukunaga, Y. Isaka, K. Kimura, T. Kamada, Free radical generation during brief period of cerebral ischemia may trigger delayed neuronal death, Neuroscience 35 (1990) 551 – 558. [18] Y.K. Ho, Z. Jing, A.P. Claude, Brain temperature alters hydroxyl radical production during cerebral ischemia/reperfusion in rats, J. Cereb. Blood Flow Metab. 16 (1996) 100 – 106. [19] Q.S. Si, Y. Nakamura, K. Kataoka, Hypothermic suppression of microglial activation in culture: inhibition of cell proliferation and production of nitric oxide and superoxide, Neuroscience 81 (1997) 223 – 229.
The effects of a free radical scavenger, edaravone, combined with mild hypothermia on ischemic brain damage following transient middle cerebral artery occlusion in rats Chikako Nito*, Tatsushi Kamiya, Shimon Amemiya, Kengo Kato, Yasuo Katayama Division of Neurology, Second Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo, Tokyo 113-8603, Japan
Abstract 3-Methyl-1-phenyl-pyrazolin-5-one (edaravone), a novel free radical scavenger, has been reported to reduce ischemic damage in rats subjected to transient focal ischemia. The aim of this study is, therefore, to investigate the effect of a combined therapy with edaravone and mild hypothermia at 35 jC. Sprague – Dawley rats were subjected to middle cerebral artery (MCA) occluding using by an intraluminal suture technique for 2 h. The rats were reperfused for 24 h and decapitated for infarct and edema analysis. Animals were randomly divided into four groups: (I) vehicle + normothermia (control); (II) vehicle + mild hypothermia; (III) edaravone + normothermia; (IV) edaravone + mild hypothermia. Mild hypothermia alone had no reduction of the brain damage. The edaravone alone significantly reduced edema volume. The combined treatment with edaravone and mild hypothermia reduced both infarct and edema volume. In addition, this treatment provided for the best functional outcome. These results demonstrate that a free radical scavenger, edaravone, attenuates brain edema and that the combined therapy with edaravone and mild hypothermia significantly reduces not only edema but also infarct on transient focal cerebral ischemia in rats. The neuroprotective effects seen in this study may be due to the combined interaction of antiedema activity between edaravone and mild hypothermia, suppressing free radical production. D 2003 Published by Elsevier Science B.V. Keywords: Free radical scavenger; Hypothermia; Neuroprotection; Focal ischemia; Rat
* Corresponding author. Tel: +81-3-3822-2131x6496; fax: +81-3-3822-4865. E-mail address: [email protected] (C. Nito). 0531-5131/03 D 2003 Published by Elsevier Science B.V. doi:10.1016/S0531-5131(03)00044-X
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C. Nito et al. / International Congress Series 1252 (2003) 109–115
1. Introduction 3-Methyl-1-phenyl-pyrazolin-5-one (edaravone) is a novel antioxidant, and inhibits both hydroxyl radicals and iron-induced peroxidative vascular endothelial cell damage [1]. Furthermore, edaravone has been tested in various different experimental models for evaluation of its protective effects in cerebral ischemia/reperfusion and myocardial ischemia/reperfusion [2,3]. Although any free radical scavengers have been developed for clinical use, edaravone is one of the most expecting of these agents. Therefore, it has been clinically applied for the treatment of acute stroke in Japan since June 2001. Recent experimental studies have demonstrated that hypothermia is a powerful tool in neuronal protection following cerebral ischemia [4]. Studies on the effect of mild or moderate hypothermia on cerebral ischemia were performed in various models of global or focal cerebral ischemia [5,6]. Multiple mechanisms for hypothermia-induced neuroprotection have been identified, such as reduced metabolic rate and energy depletion, decreased excitatory transmitter release, decreased generation of free radicals, improved ion homeostasis, and reduced vascular permeability, blood –brain barrier disruption, and edema [7,8]. Recently, experimental studies suggest that the combination of mild hypothermia with neuroprotective drugs is more effective than each of these alone [9,10]. There are no data available in regard to a combined treatment of free radical scavenger, edaravone, and hypothermia. The present study was designed to evaluate the singular and combined effects of edaravone and/or mild hypothermia of 35 jC on ischemic damage in rats subjected to transient focal ischemia.
2. Materials and methods Sprague – Dawley rats weighing 250 – 300 g were used. Anesthesia was initially induced with halothane (5% for induction and 1% for maintenance) in a mixture of 70% N2O and 30% O2 under spontaneous breathing. Blood gases and blood glucose levels were measured just before and during cerebral ischemia. Animals were subjected to 2-h middle cerebral artery (MCA) occluding followed by 24-h reperfusion using an intraluminal suture technique [11]. Animals were randomly divided into the following four groups (each, n = 6): (I) vehicle-treated, normothermic group (control); (II) edaravone-treated, normothermic group; (III) vehicle-treated, mild hypothermic (35 jC) group; (IV) edaravone-treated, mild hypothermic(35 jC) group. Temporal muscle and rectal temperatures were maintained during ischemia at 37 F 0.2 jC (normothermic groups) or 35 F 0.2 jC (hypothermic groups). Animals were intravenously administered 3.0 mg/kg of edaravone or vehicle, provided by Mitsubishi Pharma (Tokyo, Japan), just prior to the reperfusion. Twenty four hours after reperfusion, the animals were decapitated and removed. Coronal sections (20 Am) were cut and were stained with hematoxylin and eosin. The cortical or striatal areas (mm2) from each slice were summed and multiplied to obtain an infarct volume (mm3). A volume of edema (mm3) was determined by subtracting the non-ischemic cortical volume from the ischemic cortical volume [12].
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Neurological symptoms of each animal were examined blindly 24 h after reperfusion using an established scoring system [13]. In these tests, the postural reflex test and hemiparesis of animals were observed. Statistical analyses were performed with the use of StatView 5.0. Physiological data for each time point, infarct volumes, edema volumes, and neurological function scores were analyzed with one-way ANOVA. Values are presented as mean F S.D., and differences were considered significant at p < 0.05 level.
3. Results There were no statistically significant differences in the mean arterial blood pressure, pH, PaCO2, PaO2, or blood glucose levels among the groups. 3.1. Infarct volume The cortical infarct volumes in the vehicle-treated normothermic, vehicle-treated hypothermic, edaravone-treated normothermic, and edaravone-treated hypothermic groups were 201 F 37, 181 F 44, 163 F 22, and 90 F 37 mm3, respectively. The cortical infarct volume was significantly reduced in the edaravone-treated hypothermic group compared with the other groups, and the reduction was 55% of those in the vehicletreated normothermic group (Fig. 1). The striatal infarct volumes in the vehicle-treated
Fig. 1. The cortical or striatal infarct volumes after 2-h middle cerebral artery occlusion followed by 24-h reperfusion. Values (mm3) are expressed as mean F S.D. **p < 0.001 vs. vehicle-treated normothermic group by one-way ANOVA.
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normothermic, vehicle-treated hypothermic, edaravone-treated normothermic, and edaravone-treated hypothermic group were 108 F 12, 91 F 10, 96 F 15, and 70 F 15 mm3, respectively. The combination with edaravone and mild hypothermia significantly reduced the striatal infarct volume ( 35%) compared with the vehicle-treated normothermic group (Fig. 1). 3.2. Edema volume The cortical edema volumes in the vehicle-treated normothermic, vehicle-treated hypothermic, edaravone-treated normothermic, and edaravone-treated hypothermic group were 82 F 18, 59 F 16, 42 F 13, and 23 F 17 mm3, respectively. In the edaravone-treated normothermic group, the cortical edema volume was significantly less than that in the vehicle-treated normothermic group ( 49%). Moreover, edaravone with hypothermic group reduced the cortical edema volume significantly compared with that in vehicle-treated normothermic group ( 72%) (Fig. 2). The striatal edema volumes in the vehicle-treated normothermic, vehicle-treated hypothermic, edaravone-treated normothermic, and edaravone-treated hypothermic group were 46 F 10, 34 F 3, 27 F 10, and 21 F 8 mm3, respectively. The striatal edema volume in the edaravone-treated normothermic or the edaravone-treated hypothermic group was significantly smaller than that in the vehicle-treated normothermic group ( 41%, 54%) (Fig. 2).
Fig. 2. The cortical or striatal edema volumes after 2-h middle cerebral artery occlusion followed by 24-h reperfusion. Values (mm3) are expressed as mean F S.D. **p < 0.001, *p < 0.01 vs. vehicle-treated normothermic group by one-way ANOVA.
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Fig. 3. The neurological score after 2-h middle cerebral artery occlusion followed by 24-h reperfusion. Each symbol depicts the individual neuro-score of a single animal. #p < 0.05 vs. vehicle-treated normothermic group by one-way ANOVA.
3.3. Neurological symptoms The median neurological score was 2.5 F 0.5 in the control group. The combination treatment of edaravone and hypothermia significantly improved the neurological symptoms (hemiplegia) compared with the control group ( p < 0.05) (Fig. 3).
4. Discussion With use of the suture model in the rat MCA, the present study demonstrated that a low dose of edaravone (3.0 mg/kg) combined with mild intraischemic hypothermia (35 jC) significantly reduces infarct and edema volumes in the cortex and the striatum. However, mild intraischemic hypothermia (35 jC) alone failed to reduce postischemic brain damage. Under normothermic conditions, the protective effects of edaravone were observed in the edema, but not in the infarct. Taken together, these results suggest that the combination of edaravone administration and mild intraischemic hypothermia expands the therapeutic time window for edaravone alone. Moreover, the combination reduced both infarct and edema volumes, suggesting that the combination protects not only ischemic penumbra but also ischemic core. Brain temperature is an important determinant of neuronal injury following cerebral ischemia [4]. Hypothermic treatment has also been used clinically as one of the strategies for reducing ischemic brain injury; however, the usefulness of deep therapeutic hypothermia for cerebral ischemia has been limited by adverse effects such as myocardial
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arrhythmia, hypotension, respiratory insufficiency, blood hypercoagulability, and hemodynamic instability [14]. Severe hypothermia is known to have these side effects; however, mild hypothermia is much safer than the classical deep hypothermia [15]. We chose mild hypothermia of 35 jC because clinical application and continuity can be accomplished both easily and safely, without severe complication. Indeed, mild hypothermia (35 jC) did not affect any physiological variables in the present study. The temperature of 35 jC may improve edaravone’s effect without the adverse effects of more severe hypothermia on physiological functions. Although the mechanisms of ischemic brain damage have not been clearly determined, accumulated experimental evidence suggests that production of free radicals is possibly one of the major factors involved. Several reports have demonstrated that free radicals generated during ischemia play an important role in the development of neuronal damage [16,17]. Although the protective mechanisms of hypothermia are complex, one has been identified as decreased generation of free radicals from microglia [18,19]. Microglial cells produces superoxide anions and nitric oxide, and hypothermia potentially inhibits the production. Accordingly, the neuroprotective effects seen in this study may be due to the combined interaction of antiedema activity between edaravone and mild hypothermia, suppressing free radical production. The present study demonstrates that a free radical scavenger, edaravone, attenuates brain edema and that a combined therapy with edaravone and mild hypothermia significantly reduces not only edema but also infarct on transient focal cerebral ischemia in rats. These data show that this combination is an effective and safe strategy for neuronal protection during ischemia, and may be useful in clinical medical care for acute human ischemic stroke.
Acknowledgements This study was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan (Drs. Kamiya and Katayama). We would like to thank Mr. Toshiki Inaba for his expert technical. Edaravine was generously supplied to us by Mitsubishi Pharma.
References [1] S. Murota, I. Morita, N. Suda, The control of vascular endothelial cell injury, Ann. N.Y. Acad. Sci. 598 (1990) 182 – 187. [2] K. Abe, S. Yuki, K. Kogure, Strong attenuation of ischemic and postischemic brain edema in rats by a novel free radical scavenger, Stroke 19 (1988) 480 – 485. [3] H. Nishi, T. Watanabe, H. Sakurai, S. Yuki, A. Ishibashi, Effect of MCI-186 on brain edema in rats, Stroke 20 (1989) 1236 – 1240. [4] R. Busto, W.D. Dietrich, M.Y. Globus, I. Valdes, P. Scheinberg, M.D. Ginsberg, Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury, J. Cereb. Blood Flow Metab. 7 (1987) 729 – 738. [5] E.A. Bering Jr., Effects of profound hypothermia and circulatory arrest on cerebral oxygen metabolism and cerebrospinal fluid electrolyte composition in dogs, J. Neurosurg. 40 (1974) 199 – 205.
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