Brain Research, 510 (1990) 335-338 Elsevier
335
BRES 23992
Protective effect of a-tocopherol on ischemic neuronal damage in the gerbil hippocampus Hideaki Hara, Hiroyuki Kato and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan)
(Accepted 21 November 1989) Key words: a-Tocopherol; Cerebral ischemia; Free radical scavenger; Gerbil; Hippocampus
The effect of a-tocopherol (vitamin E) on ischemic neuronal damage was studied in the gerbil. The animals were subjected to 5 min of cerebral ichemia by bilateral common carotid artery occlusion. Immediately after ischemia, a-tocopherol at a dose of 50 or 100 mg/kg was administered intravenously. Morphological changes in the CA1 sector of the hippocampus were evaluated after 7 days of survival, a-Tocopherol prevented ischemia-induced neuronal death. The average density of CA1 pyramidal neurons (cells/mm, mean + S.E.M.) was 252 + 8 (n = 8) in the sham-operated group, 50 + 20 (n = 8) in the ischemia group, and 140 + 35 (n = 8) and 182 + 36 (n = 8) in the groups treated with a-tocopherol at the doses of 50 and 100 mg/kg, respectively. The results suggest that free radical scavenging action of a-tocopherol played an important role in preventing the neuronal death. Brief and transient cerebral ischemia produces selective pyramidal cell loss of the hippocampal CA1 subfield in Mongolian gerbils 16'17'29 and rats 24 (delayed neuronal death). Recent experiments have provided definitive evidence supporting a hypothesis that extracellular accumulation of synaptically released neurotransmitters, such as glutamate, is responsible for neuronal death in the hippocampus 4'23'25'32. Some other mechanisms involving protein kinase C ~5, cyclooxygenase22, G A B A 28, and serotonin ~2 have also been thought to participate in the development of delayed neuronal death, Clinical and experimental data have suggested that ischemic neuronal damage is at least partly induced by free radicals 6'9't9'2°'26. Recent studies have unveiled that lipid peroxide levels in the hippocampal CA1 were elevated at 24 h following cerebral ischemia in the rat 2 ' 5 . However, little is known about the action of free radical scavengers on the delayed neuronal death of hippocampal CA1 pyramidal cells. The purpose of this study was, therefore, to examine the effect of a-tocopherol, a free radical scavenger25-27, on the delayed neuronal death of gerbil hippocampal CA1 pyramidal cells, Male adult Mongolian gerbils (Seiwa Experimental Animals Co. Ltd., Japan) weighing 60-80 g were anesthetized with a mixture 0 f 2 % halothane, 70% N20, and 30% 0 2. Bilateral common carotid arteries were exposed and occluded with Sugita No. 51 temporary aneurysm clips for 5 min. a-Tocopherol (50 or 100 mg/kg,
2 ml/kg), pentobarbital (30 mg/kg, 5 ml/kg), or vehicles were injected i.v. for 1 min immediately after ischemia. a-Tocopherol (DL-alpha-tocopherol, vitamin E) and its vehicle were kindly donated by Eisai Co. Ltd., Tokyo, Japan. The a-tocopherol vehicle contained per ml of solution, 100 mg polyoxyethylene hydrogenated castor oil derivatives 60 mole ether (Nikko Chemical, Tokyo, Japan), 100 mg propylene glycol (J.P.), 1 mg citrate and adequate NaOH. Pentobarbital was purchased from Dainippon Co. Ltd., Tokyo, Japan. All surgical procedures were performed on a heating mat kept at 37 °C. Seven days after ischemia, the brains were perfused transcardially with heparinized saline followed by perfusion-fixation with 10% formalin under pentobarbital anesthesia (40 mg/kg, i.p.). The removed brains were postfixed, dehydrated, and embedded in paraffin using standard procedures. Coronal sections, 5 p m thick, were stained with Cresyl violet or Hematoxylin and Eosin. The number of neurons per 1 mm linear length stratum pyramidale of the hippocampal CA1 subfield (neuronal density) was counted using a microscope in each specimen, according to the method of Kirino et al. 18. Statistical comparisons were made with a two-tailed Mann-Whitney U-test. Neuronal densities in each group are presented in Table I and representative photomicrographs of the hippocampal CA1 sector are shown in Fig. 1. In the sham-operated group, the neuronal density of the CA1
Correspondence: Hideaki Hara, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-Machi, Aoba-ku, Sendai 980, Japan.
0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
336 subfield was 252 + 8 cells/mm (n = 8). In the ischemia group treated with the a-tocopherol vehicle, the number of CA1 pyramidal cells was markedly decreased (20% of the sham-operated group; P < 0.01). ct-Tocopherol (50 and 100 mg/kg, i.v.) administered immediately after ischemia, in a dose-dependent manner, prevented neuronal death in the CA1 subfield (56 and 72% of the
sham-operated group; both P < 0.05). These doses ot ct-tocopherol did not cause any side effects. Pentobarbital (30 mg/kg, i.v.) administered immediately after ischemia significantly (P < 0.05) prevented neuronal death (50% of sham-operated group), as compared to its vehicle (saline) group (9% of the sham-operated group). The present study indicates that Ct-tocopherol dose-
Fig. 1. Representative photomicrographs of hippocampal CA 1 subfield, 7 days after 5 min bilateral carotid artery occlusion in gerbils by Cresyl violet staining. A,B: sham-operation. The striatum pyramidale in the CA1 region (between the arrows). CA1 pyramidal cells are well preserved under high magnification, C,D: vehicle-treated animals. Note marked damage to the CA1 pyramidal cells (arrowheads). E,F: a-tocopherol (100 mg/kg, i.v.)-treated animals. Most of the CA1 neurons are preserved. Magnification of A, C and E: xl0; B, D, and F: ×100.
337 TABLE I
edema, promoted resynthesis of ATP, and improved the
Effects of intravenous administration of a-tocopherol and pentobarbital on neuronal death of hippocampal CA1 neurons in the gerbil
severely expressed neurological signs following ischemia in rats and dogs 13'33'34. Superoxide dismutase protects brain
Each value represents mean + S.E.M.
cells against global and focal ischemia 7'27. Further, a lipid peroxidation inhibitor, U74006F, prolongs the survival time and attenuates morphological neuronal necrosis following cerebral ischemia in gerbils TM. Recently, it has been reported that a massive influx of calcium and an abnormal release of neurotransmitters,
Treatment
Sham-operation Vehicle I a-Tocopherol
Dose (mg/ kg, i.v.)
N
Neuronal density % of (no./mm) sham
8 252 _+8** 8 50_+ 20 50 8 140 + 35* 100 8 182 + 36* Vehicle2 9 23 + 7 Pentobarbital 30 11 126 + 29* 1 a-tocopherol vehicle, 2 pentobarbital vehicle (saline), *P **P
335
BRES 23992
Protective effect of a-tocopherol on ischemic neuronal damage in the gerbil hippocampus Hideaki Hara, Hiroyuki Kato and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan)
(Accepted 21 November 1989) Key words: a-Tocopherol; Cerebral ischemia; Free radical scavenger; Gerbil; Hippocampus
The effect of a-tocopherol (vitamin E) on ischemic neuronal damage was studied in the gerbil. The animals were subjected to 5 min of cerebral ichemia by bilateral common carotid artery occlusion. Immediately after ischemia, a-tocopherol at a dose of 50 or 100 mg/kg was administered intravenously. Morphological changes in the CA1 sector of the hippocampus were evaluated after 7 days of survival, a-Tocopherol prevented ischemia-induced neuronal death. The average density of CA1 pyramidal neurons (cells/mm, mean + S.E.M.) was 252 + 8 (n = 8) in the sham-operated group, 50 + 20 (n = 8) in the ischemia group, and 140 + 35 (n = 8) and 182 + 36 (n = 8) in the groups treated with a-tocopherol at the doses of 50 and 100 mg/kg, respectively. The results suggest that free radical scavenging action of a-tocopherol played an important role in preventing the neuronal death. Brief and transient cerebral ischemia produces selective pyramidal cell loss of the hippocampal CA1 subfield in Mongolian gerbils 16'17'29 and rats 24 (delayed neuronal death). Recent experiments have provided definitive evidence supporting a hypothesis that extracellular accumulation of synaptically released neurotransmitters, such as glutamate, is responsible for neuronal death in the hippocampus 4'23'25'32. Some other mechanisms involving protein kinase C ~5, cyclooxygenase22, G A B A 28, and serotonin ~2 have also been thought to participate in the development of delayed neuronal death, Clinical and experimental data have suggested that ischemic neuronal damage is at least partly induced by free radicals 6'9't9'2°'26. Recent studies have unveiled that lipid peroxide levels in the hippocampal CA1 were elevated at 24 h following cerebral ischemia in the rat 2 ' 5 . However, little is known about the action of free radical scavengers on the delayed neuronal death of hippocampal CA1 pyramidal cells. The purpose of this study was, therefore, to examine the effect of a-tocopherol, a free radical scavenger25-27, on the delayed neuronal death of gerbil hippocampal CA1 pyramidal cells, Male adult Mongolian gerbils (Seiwa Experimental Animals Co. Ltd., Japan) weighing 60-80 g were anesthetized with a mixture 0 f 2 % halothane, 70% N20, and 30% 0 2. Bilateral common carotid arteries were exposed and occluded with Sugita No. 51 temporary aneurysm clips for 5 min. a-Tocopherol (50 or 100 mg/kg,
2 ml/kg), pentobarbital (30 mg/kg, 5 ml/kg), or vehicles were injected i.v. for 1 min immediately after ischemia. a-Tocopherol (DL-alpha-tocopherol, vitamin E) and its vehicle were kindly donated by Eisai Co. Ltd., Tokyo, Japan. The a-tocopherol vehicle contained per ml of solution, 100 mg polyoxyethylene hydrogenated castor oil derivatives 60 mole ether (Nikko Chemical, Tokyo, Japan), 100 mg propylene glycol (J.P.), 1 mg citrate and adequate NaOH. Pentobarbital was purchased from Dainippon Co. Ltd., Tokyo, Japan. All surgical procedures were performed on a heating mat kept at 37 °C. Seven days after ischemia, the brains were perfused transcardially with heparinized saline followed by perfusion-fixation with 10% formalin under pentobarbital anesthesia (40 mg/kg, i.p.). The removed brains were postfixed, dehydrated, and embedded in paraffin using standard procedures. Coronal sections, 5 p m thick, were stained with Cresyl violet or Hematoxylin and Eosin. The number of neurons per 1 mm linear length stratum pyramidale of the hippocampal CA1 subfield (neuronal density) was counted using a microscope in each specimen, according to the method of Kirino et al. 18. Statistical comparisons were made with a two-tailed Mann-Whitney U-test. Neuronal densities in each group are presented in Table I and representative photomicrographs of the hippocampal CA1 sector are shown in Fig. 1. In the sham-operated group, the neuronal density of the CA1
Correspondence: Hideaki Hara, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-Machi, Aoba-ku, Sendai 980, Japan.
0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
336 subfield was 252 + 8 cells/mm (n = 8). In the ischemia group treated with the a-tocopherol vehicle, the number of CA1 pyramidal cells was markedly decreased (20% of the sham-operated group; P < 0.01). ct-Tocopherol (50 and 100 mg/kg, i.v.) administered immediately after ischemia, in a dose-dependent manner, prevented neuronal death in the CA1 subfield (56 and 72% of the
sham-operated group; both P < 0.05). These doses ot ct-tocopherol did not cause any side effects. Pentobarbital (30 mg/kg, i.v.) administered immediately after ischemia significantly (P < 0.05) prevented neuronal death (50% of sham-operated group), as compared to its vehicle (saline) group (9% of the sham-operated group). The present study indicates that Ct-tocopherol dose-
Fig. 1. Representative photomicrographs of hippocampal CA 1 subfield, 7 days after 5 min bilateral carotid artery occlusion in gerbils by Cresyl violet staining. A,B: sham-operation. The striatum pyramidale in the CA1 region (between the arrows). CA1 pyramidal cells are well preserved under high magnification, C,D: vehicle-treated animals. Note marked damage to the CA1 pyramidal cells (arrowheads). E,F: a-tocopherol (100 mg/kg, i.v.)-treated animals. Most of the CA1 neurons are preserved. Magnification of A, C and E: xl0; B, D, and F: ×100.
337 TABLE I
edema, promoted resynthesis of ATP, and improved the
Effects of intravenous administration of a-tocopherol and pentobarbital on neuronal death of hippocampal CA1 neurons in the gerbil
severely expressed neurological signs following ischemia in rats and dogs 13'33'34. Superoxide dismutase protects brain
Each value represents mean + S.E.M.
cells against global and focal ischemia 7'27. Further, a lipid peroxidation inhibitor, U74006F, prolongs the survival time and attenuates morphological neuronal necrosis following cerebral ischemia in gerbils TM. Recently, it has been reported that a massive influx of calcium and an abnormal release of neurotransmitters,
Treatment
Sham-operation Vehicle I a-Tocopherol
Dose (mg/ kg, i.v.)
N
Neuronal density % of (no./mm) sham
8 252 _+8** 8 50_+ 20 50 8 140 + 35* 100 8 182 + 36* Vehicle2 9 23 + 7 Pentobarbital 30 11 126 + 29* 1 a-tocopherol vehicle, 2 pentobarbital vehicle (saline), *P **P
