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Vitamin E isoforms α-tocotrienol and γ-tocopherol prevent cerebral infarction in mice
Neuroscience Letters 337 (2003) 56–60 www.elsevier.com/locate/neulet
Vitamin E isoforms a-tocotrienol and g-tocopherol prevent cerebral infarction in mice Kenichi Mishima a, Takamitsu Tanaka a, Fengling Pu a, Nobuaki Egashira a,b, Katsunori Iwasaki a,b, Ryoji Hidaka c, Kazuhisa Matsunaga c, Jiro Takata c, Yoshiharu Karube c, Michihiro Fujiwara a,b,* a
Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan b Advanced Materials Institute, Fukuoka University, Fukuoka 814-0180, Japan c Laboratory of Drug Design and Drug Delivery, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan Received 9 July 2002; received in revised form 24 October 2002; accepted 31 October 2002
Abstract a-tocopherol and its derivatives have been shown to be effective in reducing cerebral ischemia-induced brain damage. However, the effects of other vitamin E isoforms have not been characterized. In the present study, we investigated the effects of six different isoforms of vitamin E on the ischemic brain damage in the mice middle cerebral artery (MCA) occlusion model. All vitamin E isoforms were injected i.v., twice, immediately before and 3 h after the occlusion. atocopherol (2 mM), a-tocotrienol (0.2 and 2 mM) and g-tocopherol (0.2 and 2 mM) significantly decreased the size of the cerebral infarcts 1 day after the MCA occlusion, while g-tocotrienol, d-tocopherol and d-tocotrienol showed no effect on the cerebral infarcts. These results suggest that a-tocotrienol and g-tocopherol are potent and effective agents for preventing cerebral infarction induced by MCA occlusion. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vitamin E; a-tocopherol; a-tocotrienol; g-tocopherol; Cerebral ischemia
Vitamin E, a major lipid-soluble chain-breaking antioxidant found in membranes, has been shown to have significant health benefits in preventing a variety of diseases, because it is an important factor in the protection of polyunsaturated fatty acids against peroxidative damage [15,16]. Vitamin E is known to occur in nature in eight different forms; a-, b-, g- and d-tocopherols and a-, b-, g- and dtocotrienols (Fig. 1). Tocopherols have a phytyl side chain attached to their chromanol nucleus, whereas the tail of tocotrienols is unsaturated and forms an isoprenoid chain. These isoforms are determined by the number and position of methyl substituents in the chromanol nucleus. Although there have been many studies about the biological and health effects of a-tocopherol, much attention has recently focused on the effects of other Vitamin E isoforms, g-tocopherol and a-tocotrienol [8,20]. g-Tocopherol has been shown to inhibit platelet aggregation and delay arterial thrombus formation in rats [13], while a-tocotrienols have * Corresponding author. Tel.: 181-92-871-6631 ext. 6634; fax: 181-92-863-0389. E-mail address: [email protected] (M. Fujiwara).
a cholesterol lowering effect [10,12], anticancer and tumor suppressive activities [7], antioxidant properties [4] and anti-aggregation of blood platelets [5]. Importantly, these effects of g-tocopherol and a-tocotrienol are more potent than a-tocopherol. Clinical and experimental findings have suggested that free radical-induced lipid peroxidation is recognized as an important factor in the pathogenesis of ischemic brain damage [3,17,21,22]. Various natural antioxidants are present in cerebral tissue, of which the lipid-soluble nutrient vitamin E scavenges lipid peroxyl radicals and thereby inhibits lipid peroxidation. However, during ischemia and subsequent reperfusion, because of an overwhelming production of radicals, natural antioxidants cannot suppress the radicals. In these circumstances, administration of atocopherol [3,17,22] and a-tocopherol analog [15] as radical scavengers have been shown to reduce ischemic damage. Thus, despite many studies reporting the neuroprotective effects of a-tocopherol, little is known about the neuroprotective effects of tocotrienols and other tocopherols on cerebral brain damage induced by middle cerebral artery (MCA) occlusion. The purpose of this study, therefore, was
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0 30 4- 39 40 (0 2) 0 129 3- 4
K. Mishima et al. / Neuroscience Letters 337 (2003) 56–60
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Fig. 1. The chemical structure of vitamin E isoforms
to examine the protective effects of a-, g- and d-tocopherols and a-, g- and d-tocotrienols on cerebral infarction induced by MCA occlusion. Male ddY mice (25–35 g, Kiwa experimental animal laboratory, Wakayama, Japan) were used. The animals were kept under a constant light–dark cycle (light 7:00– 19:00) with a restricted diet (CE-2, Clea Japan, Tokyo, Japan) in an air-conditioned room. All procedures regarding animal care and use were performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University. Anesthesia was induced by 2% halothane and maintained with 1% halothane (Flosen, Takeda, Osaka, Japan). Focal cerebral ischemia was induced by occlusion of the MCA using the intraluminal filament technique. After midline neck incision, the left common and external carotid arteries were isolated and ligated. An 8–0 nylon monofilament (Ethilon, Johinson & Johinson, Tokyo, Japan) coated with silicon resin (Xantopren, Heleus Dental Material, Osaka, Japan) was introduced through a small incision into the common carotid artery and was advanced to a position 9 mm distal from the carotid bifurcation for occlusion of the MCA. Four hours after MCA occlusion, animals were re-anesthetized with halothane, and reperfusion was established by withdrawal of the filament. Twenty-four hours after MCA occlusion, animals were sacrificed by decapitation. The brains were removed and sectioned coronally into four 2 mm slices using a mouse brain matrix. Slices were immediately stained with 2% 2,3,5-triphenyltetrazolium chloride (Sigma, St. Louis, MO, USA). The border between the infarcted and noninfarcted tissue was outlined with an
image analysis system, and the area of infarction was measured and the infarction volume was calculated. All vitamin E isoforms were dissolved in 1% DMSO and were administered immediately before and 3 h after MCA occlusion by bolus intravenous injection. Results are expressed as means ^ SEM. Dunnett’s test after one-way ANOVA was used for infarct volume. A P-value of less than 0.05 was considered to be significant. Saline (n ¼ 9), a-tocopherol (0.2 mM, n ¼ 7; 2 mM, n ¼ 7), a-tocotrienol (0.2 mM, n ¼ 9; 2 mM, n ¼ 5), gtocopherol (0.2 mM, n ¼ 7; 2 mM, n ¼ 8), g-tocotrienol (0.2 mM, n ¼ 9; 2 mM, n ¼ 6), d-tocopherol (0.2 mM, n ¼ 5; 2 mM, n ¼ 7) and d-tocotrienol (0.2 mM, n ¼ 7; 2 mM, n ¼ 6) were administrated immediately before and 3 h after the occlusion by bolus intravenous injection (Fig. 2). Therefore the blood concentration of drugs was maintained [19]. The IV injection of a-tocopherol and a-tocotrienol significantly reduced the infarct volume induced by MCA occlusion in ddY mice [Fð3; 28Þ ¼ 6:580, P , 0:01, Fig. 2A]. Post-hoc analysis revealed that a-tocopherol (2 mM) and a-tocotrienol (0.2 and 2 mM) significantly reduced the infarct volume induced by MCA occlusion in ddY mice (P , 0:01, for Dunnett’s test). g-Tocopherol (0.2 and 2 mM) significantly reduced the infarct volume induced by MCA occlusion in ddY mice [Fð3; 27Þ ¼ 4:469, P , 0:05 for one-way ANOVA, P , 0:01 for Dunnett’s test, Fig. 2B]. On the other hand, d-tocopherol and d-tocotrienol did not reduce the infarct volume induced by MCA occlusion (Fig. 2C). In addition, these effective vitamin E isoforms did not affect both body temperature and blood pressure (data not shown).
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Fig. 2. Effects of a-, g-, d-tocopherols and a-, g-, d-tocotrienols on the infarct volume induced by MCA occlusion in ddY mice. (A) a-forms (n ¼ 5–9), (B) g-forms (n ¼ 6–9), (C) d-forms (n ¼ 5–9). All drugs were injected i.v. immediately before and after the occlusion. Slices were immediately stained with 2% 2,3,5-triphenyltetrazolium chloride 24 h after MCA occlusion. Values are expressed as means ^ SEM. **P , 0:01 vs. vehicle (one-way ANOVA followed by Dunnett’s test).
K. Mishima et al. / Neuroscience Letters 337 (2003) 56–60
The results demonstrated that a-tocopherol (2 mM), atocotrienol (0.2 and 2 mM) and g-tocopherol (0.2 and 2 mM) could similarly reduce the infarct volume induced by MCA occlusion, but a-tocotrienol and g-tocopherol were 10-fold more efficient than a-tocopherol. The greater neuroprotective efficacy of a-tocotrienol and g-tocopherol can be interpreted as resulting from the different structure from atocopherol: the side chain of a-tocotrienol and the number of methyl substituents in the chromanol nucleus of g-tocopherol. Thus, we found that vitamin E produced different neuroprotective actions due to small differences between each structure. There have been some studies of a-tocopherol that reported neuroprotective or neuronal rescue effects in animal models of cerebral ischemic brain damage. a-Tocopherol was found to prevent apoptosis in hippocampal neurons caused by cerebral ischemia in stroke-prone spontaneously hypertensive rats [17], delayed neuronal death in the gerbil hippocampus [3], and prevent postischemic lipid peroxidation [22]. In the present study, anti-oxidative effect of a-tocopherol was well demonstrated as in previous studies. In the present study, all isoforms were injected i.v. immediately before and 3 h after the occlusion and a-tocopherol, a-tocotrienol and g-tocopherol could prevent the infarct volume induced by MCA occlusion, suggesting that the period of radical formation is a few hours after the occlusion in this MCA model. Takamatsu et al. [18] reported that EPC-K1, a hydroxyl radical scavenger, when administered 3 h but not immediately or 6 h after the MCA occlusion, significantly reduced brain damage. Moreover, Morimoto et al. [6] demonstrated that the hydroxyl radical levels slowly increased and reached about 2-fold level 2 h after the ischemia, although glutamate release increased immediately after the ischemia. We also found that a delayed evaluation of NO occurred from 2 to 4 h after the first occlusion in a fourvessel repeated occlusion model (unpublished data). These findings suggest that the delayed generation of radicals occurs in cerebral ischemia models and care must be taken with regard to the injection timing of radical scavengers. a-tocotrienol was more potent than a-tocopherol in the present study. Recently, growing interest about the higher antioxidant activity of a-tocotrienol than a-tocopherol has been reported [8,15,16,20]. For example, a-tocotrienol showed 40-fold higher antioxidation against Fe(II) 1 NADPH-induced lipid peroxidation in rat liver microsomes, and was 6.5-fold more effective in the protection of cytochrome P-450 against oxidative damage. The higher antioxidation of a-tocotrienol was suggested to be related to the differences in the side chain structure. In addition, two findings demonstrated their independent antioxidant activity. Firstly, a-tocotrienol acts by inhibiting the enzymic activity of b-hydroxy-b-methylglutaryl coenzymeA (HMG-CoA) reductase [9], while a-tocopherol induces HMG-CoA reductase activity [11]. Moreover, it was reported that
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HMG-CoA reductase inhibitors reduced the infarct volume induced by MCA occlusion [1]. Another findings was that a-tocotrienol counteracted glutamate-induced cell death at much lower concentrations than in a-tocopherol in HT neuronal cells by inhibiting the activation of pp60 c-src kinase [14]. Taken together, these findings suggest that the higher neuroprotective effects of a-tocotrienol may be responsible for both HMG-CoA reductase inhibition and c-src kinase inhibition by adding differences in the side chain structure which depend on antioxidant properties. Another finding demonstrated that g-tocopherol was more potent than a-tocopherol. In recent in vivo and in vitro studies, g-tocopherol was shown to prevent coronary artery disease. For example, g-tocopherol was shown to inhibit lipid peroxidative damage [23] and to trap mutagenic electrophiles [2] more efficiently than a-tocopherol. Further, it was reported that the g-tocopherol but not atocopherol appears to be expressed in low amounts in the plasma of patients with coronary artery disease [10]. These findings suggest that g-tocopherol may act on the vascular system more efficiently than a-tocopherol, and when taken as part of the diet, g-tocopherol may be more important than a-tocopherol. In conclusion, the findings of the present study showed that a-tocopherol, a-tocotrienol and g-tocopherol significantly prevented the cerebral infarcts induced by the MCA occlusion. Importantly, both a-tocotrienol and gtocopherol were more potent than a-tocopherol. These results suggest that both a-tocotrienol and g-tocopherol may be useful as antioxidants for patients with cerebrovascular disease or cerebrovascular dementia that develops as a sequela of stroke, etc. This study was supported in part by a Grant-in Aid for Scientific Research (No.14572171) from the Ministry of Education, Science and Culture of Japan, and by the Advanced Materials Institute of Fukuoka University and by a grant (No. 991001) from the Central Research Institute of Fukuoka University. We thank Miss Y. Shimoda and Miss R. Matsutani for their excellent technical assistance. [1] Amin-Hanjani, S., Stagliano, N.E., Yamada, M., Huang, P.L., Liano, J.K. and Moskowitz, M.A., Mevastatin, an HMG-CoA reductase inhibitor, reduces stroke damage and upregulates endothelial nitric oxide synthase in mice, Stroke, 32 (2001) 980–986. [2] Christen, S., Woodall, A.A., Shigenaga, M.K., SouthwellKeely Duncan, M.W. and Ames, B.N., g-Tocopherol traps mutagenic electrophiles such as NOx and complements a-tocopherol: physiological implications, Proc. Natl. Acad. Sci. USA, 94 (1997) 3217–3222. [3] Hara, H., Kato, H. and Kogure, K., Protective effect of atocopherol on ischemic neuronal damage in the gerbil hippocampus, Brain Res., 510 (1990) 335–338. [4] Kooyenga, D.K., Gellar, M., Watkins, T.R., Gapor, A., Diakoumakis, E. and Bierenbaum, M.L., Palm oil antioxidants: effects in patients with hyperlipidemia and carotid stenosis – 2 year experience, Asia Pacific J. Clin. Nutr., 6 (1997) 72–75.
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[5] Mahadevappa, V.G., Sicilia, F., and Holub, B.J., Effect of tocotrienol derivatives on collagen and ADP-induced human platelet aggregation, Proc. 1989 Int. Palm Oil Conf. – Nutr. Health Aspect of Palm Oil, PORIM, Kuala Lumpur, Malaysia, (1991) 36-38. [6] Morimoto, T., Globus, M.Y.T., Busto, R., Martinez, E. and Ginsberg, M.D., Simultaneous measurement of salicylate hydroxylation and glutamate release in the penumbral cortex following transient middle cerebral artery occlusion, J. Cereb. Blood Flow Metab., 16 (1996) 92–99. [7] Nesaretnam, K., Stephen, R., Dils, R. and Darbre, P., Tocotrienols inhibit the growth of human breast cancer cells irrespective of estrogen receptor status, Lipids, 33 (1998) 461–469. [8] Packer, L., Weber, S.U. and Rimbach, G., Molecular aspects of a-tocotrienol antioxidant action and cell signalling, J. Nutr., 131 (2001) 369S–373S. [9] Parker, R.A., Pearce, B.C., Clark, R.W., Gordon, D.A. and Wright, J.J.K., Tocotrienols regulat cholesterol production in mammalian cells by post-transcriptional suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, J. Biol. Chem., 268 (1993) 11230–11238. [10] Qureshi, A.A., Sami, S.A., Salser, W.A. and Khan, F.A., Dose-dependent suppression of serum cholesterol by tocotrienol-rich fraction (TRF25) of rice bran in hypercholesterolemic humans, Atherosclerosis, 161 (2002) 199–207. [11] Qureshi, A.A., Mo, H., Pearce, B.C., Nor, R.M., Gapor, A., Peterson, D.M. and Elson, C.E., Dietary a-tocopherol attenuates the impact of g-tocotrienol on hepatic 3-hydroxy-3methylglutaryl coenzyme A reductase activity in chickens, J. Nutr., 126 (1996) 389–394. [12] Qureshi, A.A., Bradlow, B.A., Brace, L., Manganello, J., Peterson, D.M., Pearce, B.C., Wright, J.J.K., Gapor, A. and Elson, C.E., Response of hypercholesterolemic subjects to administration of tocotrienols, Lipids, 30 (1995) 1171–1177. [13] Saldeen, T., Li, D. and Mehta, J.L., Differential effects of aand g-tocopherol on low-density lipoprotein oxidation, superoxide activity, platelet aggregation and arterial thrombogenesis, J. Am. Coll. Cardiol., 34 (1999) 1208–1215. [14] Sen, C.K., Khanna, S., Roy, S. and Packer, L., Molecular
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basis of vitamin E action: tocotrienol potently inhibits glutamate-induced pp60c-Src kinase activation and death of ht4 neuronal cells, J. Biol. Chem., 275 (2000) 13049–13055. Serbinonva, E.A. and Packer, L., Antioxidant properties of tocopherol and tocotrienol, Methods Enzymol., 234 (1994) 354–367. Serbinova, E., Kangan, V., Han, D. and Packer, L., Free radical recycling and intramembrane mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol, Free Radical Biol. Med., 10 (1991) 263–275. Tagami, M., Ikeda, K., Yamagata, K., Nara, Y., Fujino, H., Kubota, A., Numano, F. and Yamori, Y., Vitamin E prevents apoptosis in hippocampal neurons caused by cerebral ischemia and reperfusion in stroke-prone spontaneously hypertensive rats, Lab. Invest., 79 (1999) 609–615. Takamatsu, H., Kondo, K., Ikeda, Y. and Umemura, K., Hydroxyl radical generation after the third hour following ischemia contributes to brain damage, Eur. J. Pharmacol., 352 (1998) 165–169. Takata, J., Hidaka, R., Yamasaki, A., Hattori, A., Fukushima, T., Tanabe, M., Matsunaga, K., Karube, Y. and Imai, K., Novel d-g-Tocopherol derivative as a prodrug for d-g-Tocopherol and a two-step prodrug for S-g-CEHC, J. Lipid Res., (2002) (in press). Theriault, A., Chao, J-T., Wang, Q., Gapor, A. and Adeli, K., Tocotrienol: a review of its therapeutic potential, Clin. Biochem., 32 (1999) 309–319. van der Worp, H.B., Thomas, C.E., Kappelle, L.J., Hoffman, W.P., de Wildt, D.J. and Ba¨ r, P.R., Inhibition of iron-dependent and ischemia-induced brain damage by the a-tocopherol analogue MDL 74,722, Exp. Neurol., 155 (1999) 103–108. Villalobos, M.A., De La Cruz, J.P., Carrasco, T., SmithAgreda, J.M. and Sanchez de la Cuesta, F., Effects of atocopherol on lipid peroxidation and mitochondrial reduction of tetraphenyl tetrazolium in the rat brain, Brain Res. Bull., 33 (1994) 313–318. Wolf, T., Gamma-tocopherol: an efficient protector of lipids against nitric oxide-initiated peroxidative damage, Nutr. Rev., 55 (1997) 376–378.
Vitamin E isoforms a-tocotrienol and g-tocopherol prevent cerebral infarction in mice Kenichi Mishima a, Takamitsu Tanaka a, Fengling Pu a, Nobuaki Egashira a,b, Katsunori Iwasaki a,b, Ryoji Hidaka c, Kazuhisa Matsunaga c, Jiro Takata c, Yoshiharu Karube c, Michihiro Fujiwara a,b,* a
Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan b Advanced Materials Institute, Fukuoka University, Fukuoka 814-0180, Japan c Laboratory of Drug Design and Drug Delivery, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan Received 9 July 2002; received in revised form 24 October 2002; accepted 31 October 2002
Abstract a-tocopherol and its derivatives have been shown to be effective in reducing cerebral ischemia-induced brain damage. However, the effects of other vitamin E isoforms have not been characterized. In the present study, we investigated the effects of six different isoforms of vitamin E on the ischemic brain damage in the mice middle cerebral artery (MCA) occlusion model. All vitamin E isoforms were injected i.v., twice, immediately before and 3 h after the occlusion. atocopherol (2 mM), a-tocotrienol (0.2 and 2 mM) and g-tocopherol (0.2 and 2 mM) significantly decreased the size of the cerebral infarcts 1 day after the MCA occlusion, while g-tocotrienol, d-tocopherol and d-tocotrienol showed no effect on the cerebral infarcts. These results suggest that a-tocotrienol and g-tocopherol are potent and effective agents for preventing cerebral infarction induced by MCA occlusion. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vitamin E; a-tocopherol; a-tocotrienol; g-tocopherol; Cerebral ischemia
Vitamin E, a major lipid-soluble chain-breaking antioxidant found in membranes, has been shown to have significant health benefits in preventing a variety of diseases, because it is an important factor in the protection of polyunsaturated fatty acids against peroxidative damage [15,16]. Vitamin E is known to occur in nature in eight different forms; a-, b-, g- and d-tocopherols and a-, b-, g- and dtocotrienols (Fig. 1). Tocopherols have a phytyl side chain attached to their chromanol nucleus, whereas the tail of tocotrienols is unsaturated and forms an isoprenoid chain. These isoforms are determined by the number and position of methyl substituents in the chromanol nucleus. Although there have been many studies about the biological and health effects of a-tocopherol, much attention has recently focused on the effects of other Vitamin E isoforms, g-tocopherol and a-tocotrienol [8,20]. g-Tocopherol has been shown to inhibit platelet aggregation and delay arterial thrombus formation in rats [13], while a-tocotrienols have * Corresponding author. Tel.: 181-92-871-6631 ext. 6634; fax: 181-92-863-0389. E-mail address: [email protected] (M. Fujiwara).
a cholesterol lowering effect [10,12], anticancer and tumor suppressive activities [7], antioxidant properties [4] and anti-aggregation of blood platelets [5]. Importantly, these effects of g-tocopherol and a-tocotrienol are more potent than a-tocopherol. Clinical and experimental findings have suggested that free radical-induced lipid peroxidation is recognized as an important factor in the pathogenesis of ischemic brain damage [3,17,21,22]. Various natural antioxidants are present in cerebral tissue, of which the lipid-soluble nutrient vitamin E scavenges lipid peroxyl radicals and thereby inhibits lipid peroxidation. However, during ischemia and subsequent reperfusion, because of an overwhelming production of radicals, natural antioxidants cannot suppress the radicals. In these circumstances, administration of atocopherol [3,17,22] and a-tocopherol analog [15] as radical scavengers have been shown to reduce ischemic damage. Thus, despite many studies reporting the neuroprotective effects of a-tocopherol, little is known about the neuroprotective effects of tocotrienols and other tocopherols on cerebral brain damage induced by middle cerebral artery (MCA) occlusion. The purpose of this study, therefore, was
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0 30 4- 39 40 (0 2) 0 129 3- 4
K. Mishima et al. / Neuroscience Letters 337 (2003) 56–60
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Fig. 1. The chemical structure of vitamin E isoforms
to examine the protective effects of a-, g- and d-tocopherols and a-, g- and d-tocotrienols on cerebral infarction induced by MCA occlusion. Male ddY mice (25–35 g, Kiwa experimental animal laboratory, Wakayama, Japan) were used. The animals were kept under a constant light–dark cycle (light 7:00– 19:00) with a restricted diet (CE-2, Clea Japan, Tokyo, Japan) in an air-conditioned room. All procedures regarding animal care and use were performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University. Anesthesia was induced by 2% halothane and maintained with 1% halothane (Flosen, Takeda, Osaka, Japan). Focal cerebral ischemia was induced by occlusion of the MCA using the intraluminal filament technique. After midline neck incision, the left common and external carotid arteries were isolated and ligated. An 8–0 nylon monofilament (Ethilon, Johinson & Johinson, Tokyo, Japan) coated with silicon resin (Xantopren, Heleus Dental Material, Osaka, Japan) was introduced through a small incision into the common carotid artery and was advanced to a position 9 mm distal from the carotid bifurcation for occlusion of the MCA. Four hours after MCA occlusion, animals were re-anesthetized with halothane, and reperfusion was established by withdrawal of the filament. Twenty-four hours after MCA occlusion, animals were sacrificed by decapitation. The brains were removed and sectioned coronally into four 2 mm slices using a mouse brain matrix. Slices were immediately stained with 2% 2,3,5-triphenyltetrazolium chloride (Sigma, St. Louis, MO, USA). The border between the infarcted and noninfarcted tissue was outlined with an
image analysis system, and the area of infarction was measured and the infarction volume was calculated. All vitamin E isoforms were dissolved in 1% DMSO and were administered immediately before and 3 h after MCA occlusion by bolus intravenous injection. Results are expressed as means ^ SEM. Dunnett’s test after one-way ANOVA was used for infarct volume. A P-value of less than 0.05 was considered to be significant. Saline (n ¼ 9), a-tocopherol (0.2 mM, n ¼ 7; 2 mM, n ¼ 7), a-tocotrienol (0.2 mM, n ¼ 9; 2 mM, n ¼ 5), gtocopherol (0.2 mM, n ¼ 7; 2 mM, n ¼ 8), g-tocotrienol (0.2 mM, n ¼ 9; 2 mM, n ¼ 6), d-tocopherol (0.2 mM, n ¼ 5; 2 mM, n ¼ 7) and d-tocotrienol (0.2 mM, n ¼ 7; 2 mM, n ¼ 6) were administrated immediately before and 3 h after the occlusion by bolus intravenous injection (Fig. 2). Therefore the blood concentration of drugs was maintained [19]. The IV injection of a-tocopherol and a-tocotrienol significantly reduced the infarct volume induced by MCA occlusion in ddY mice [Fð3; 28Þ ¼ 6:580, P , 0:01, Fig. 2A]. Post-hoc analysis revealed that a-tocopherol (2 mM) and a-tocotrienol (0.2 and 2 mM) significantly reduced the infarct volume induced by MCA occlusion in ddY mice (P , 0:01, for Dunnett’s test). g-Tocopherol (0.2 and 2 mM) significantly reduced the infarct volume induced by MCA occlusion in ddY mice [Fð3; 27Þ ¼ 4:469, P , 0:05 for one-way ANOVA, P , 0:01 for Dunnett’s test, Fig. 2B]. On the other hand, d-tocopherol and d-tocotrienol did not reduce the infarct volume induced by MCA occlusion (Fig. 2C). In addition, these effective vitamin E isoforms did not affect both body temperature and blood pressure (data not shown).
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K. Mishima et al. / Neuroscience Letters 337 (2003) 56–60
Fig. 2. Effects of a-, g-, d-tocopherols and a-, g-, d-tocotrienols on the infarct volume induced by MCA occlusion in ddY mice. (A) a-forms (n ¼ 5–9), (B) g-forms (n ¼ 6–9), (C) d-forms (n ¼ 5–9). All drugs were injected i.v. immediately before and after the occlusion. Slices were immediately stained with 2% 2,3,5-triphenyltetrazolium chloride 24 h after MCA occlusion. Values are expressed as means ^ SEM. **P , 0:01 vs. vehicle (one-way ANOVA followed by Dunnett’s test).
K. Mishima et al. / Neuroscience Letters 337 (2003) 56–60
The results demonstrated that a-tocopherol (2 mM), atocotrienol (0.2 and 2 mM) and g-tocopherol (0.2 and 2 mM) could similarly reduce the infarct volume induced by MCA occlusion, but a-tocotrienol and g-tocopherol were 10-fold more efficient than a-tocopherol. The greater neuroprotective efficacy of a-tocotrienol and g-tocopherol can be interpreted as resulting from the different structure from atocopherol: the side chain of a-tocotrienol and the number of methyl substituents in the chromanol nucleus of g-tocopherol. Thus, we found that vitamin E produced different neuroprotective actions due to small differences between each structure. There have been some studies of a-tocopherol that reported neuroprotective or neuronal rescue effects in animal models of cerebral ischemic brain damage. a-Tocopherol was found to prevent apoptosis in hippocampal neurons caused by cerebral ischemia in stroke-prone spontaneously hypertensive rats [17], delayed neuronal death in the gerbil hippocampus [3], and prevent postischemic lipid peroxidation [22]. In the present study, anti-oxidative effect of a-tocopherol was well demonstrated as in previous studies. In the present study, all isoforms were injected i.v. immediately before and 3 h after the occlusion and a-tocopherol, a-tocotrienol and g-tocopherol could prevent the infarct volume induced by MCA occlusion, suggesting that the period of radical formation is a few hours after the occlusion in this MCA model. Takamatsu et al. [18] reported that EPC-K1, a hydroxyl radical scavenger, when administered 3 h but not immediately or 6 h after the MCA occlusion, significantly reduced brain damage. Moreover, Morimoto et al. [6] demonstrated that the hydroxyl radical levels slowly increased and reached about 2-fold level 2 h after the ischemia, although glutamate release increased immediately after the ischemia. We also found that a delayed evaluation of NO occurred from 2 to 4 h after the first occlusion in a fourvessel repeated occlusion model (unpublished data). These findings suggest that the delayed generation of radicals occurs in cerebral ischemia models and care must be taken with regard to the injection timing of radical scavengers. a-tocotrienol was more potent than a-tocopherol in the present study. Recently, growing interest about the higher antioxidant activity of a-tocotrienol than a-tocopherol has been reported [8,15,16,20]. For example, a-tocotrienol showed 40-fold higher antioxidation against Fe(II) 1 NADPH-induced lipid peroxidation in rat liver microsomes, and was 6.5-fold more effective in the protection of cytochrome P-450 against oxidative damage. The higher antioxidation of a-tocotrienol was suggested to be related to the differences in the side chain structure. In addition, two findings demonstrated their independent antioxidant activity. Firstly, a-tocotrienol acts by inhibiting the enzymic activity of b-hydroxy-b-methylglutaryl coenzymeA (HMG-CoA) reductase [9], while a-tocopherol induces HMG-CoA reductase activity [11]. Moreover, it was reported that
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HMG-CoA reductase inhibitors reduced the infarct volume induced by MCA occlusion [1]. Another findings was that a-tocotrienol counteracted glutamate-induced cell death at much lower concentrations than in a-tocopherol in HT neuronal cells by inhibiting the activation of pp60 c-src kinase [14]. Taken together, these findings suggest that the higher neuroprotective effects of a-tocotrienol may be responsible for both HMG-CoA reductase inhibition and c-src kinase inhibition by adding differences in the side chain structure which depend on antioxidant properties. Another finding demonstrated that g-tocopherol was more potent than a-tocopherol. In recent in vivo and in vitro studies, g-tocopherol was shown to prevent coronary artery disease. For example, g-tocopherol was shown to inhibit lipid peroxidative damage [23] and to trap mutagenic electrophiles [2] more efficiently than a-tocopherol. Further, it was reported that the g-tocopherol but not atocopherol appears to be expressed in low amounts in the plasma of patients with coronary artery disease [10]. These findings suggest that g-tocopherol may act on the vascular system more efficiently than a-tocopherol, and when taken as part of the diet, g-tocopherol may be more important than a-tocopherol. In conclusion, the findings of the present study showed that a-tocopherol, a-tocotrienol and g-tocopherol significantly prevented the cerebral infarcts induced by the MCA occlusion. Importantly, both a-tocotrienol and gtocopherol were more potent than a-tocopherol. These results suggest that both a-tocotrienol and g-tocopherol may be useful as antioxidants for patients with cerebrovascular disease or cerebrovascular dementia that develops as a sequela of stroke, etc. This study was supported in part by a Grant-in Aid for Scientific Research (No.14572171) from the Ministry of Education, Science and Culture of Japan, and by the Advanced Materials Institute of Fukuoka University and by a grant (No. 991001) from the Central Research Institute of Fukuoka University. We thank Miss Y. Shimoda and Miss R. Matsutani for their excellent technical assistance. [1] Amin-Hanjani, S., Stagliano, N.E., Yamada, M., Huang, P.L., Liano, J.K. and Moskowitz, M.A., Mevastatin, an HMG-CoA reductase inhibitor, reduces stroke damage and upregulates endothelial nitric oxide synthase in mice, Stroke, 32 (2001) 980–986. [2] Christen, S., Woodall, A.A., Shigenaga, M.K., SouthwellKeely Duncan, M.W. and Ames, B.N., g-Tocopherol traps mutagenic electrophiles such as NOx and complements a-tocopherol: physiological implications, Proc. Natl. Acad. Sci. USA, 94 (1997) 3217–3222. [3] Hara, H., Kato, H. and Kogure, K., Protective effect of atocopherol on ischemic neuronal damage in the gerbil hippocampus, Brain Res., 510 (1990) 335–338. [4] Kooyenga, D.K., Gellar, M., Watkins, T.R., Gapor, A., Diakoumakis, E. and Bierenbaum, M.L., Palm oil antioxidants: effects in patients with hyperlipidemia and carotid stenosis – 2 year experience, Asia Pacific J. Clin. Nutr., 6 (1997) 72–75.
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