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Effect of systemic zinc administration on delayed neuronal death in the gerbil hippocampus
Brain Research 743 Ž1996. 362–365
Short communication
Effect of systemic zinc administration on delayed neuronal death in the gerbil hippocampus Kohji Matsushita a, ) , Kazuo Kitagawa b , Tomohiro Matsuyama a , Toshiho Ohtsuki b , Akihiko Taguchi b , Kenji Mandai b , Takuma Mabuchi b , Yoshiki Yagita b , Takehiko Yanagihara c , Masayasu Matsumoto b,c a
Fifth Department of Internal Medicine, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663 Japan b First Department of Medicine, Osaka UniÕersity Medical School, Osaka 565, Japan c Department of Neurology, Osaka UniÕersity Medical School, Osaka 565, Japan Accepted 24 September 1996
Abstract The divalent cation zinc has been reported to possess several physiological properties such as blocking apoptotic cell death through an inhibitory effect on Ca2q-Mg 2q endonuclease activity, or modulating the neurotoxicity via glutamate receptor subtypes. In the present study, we investigated the effect of peripherally injected zinc on delayed neuronal death seen in the hippocampus after transient global ischemia, in order to elucidate a possible beneficial role on zinc in ischemic neuronal cell death. Forty-five adult Mongolian gerbils of both sexes underwent transient bilateral clipping of the common carotid arteries for 3 min. In the pretreated animals, ZnCl 2 Ž20 mgrkg. was injected subcutaneously once, 1 h before ischemia Žsuperacute group; n s 6. or twice at 24 and 48 h before ischemia Žsubacute group; n s 14.. Histological survey was carried out 3 days later by in situ DNA fragmentation method and 4 days later by hematoxylin-eosin staining by semiquantatively counting dead neurons in the CA1 sector. Subacute zinc pre-administration significantly reduced the nuclear damage and subsequent neuronal death; however, superacutely pre-administered zinc did not protect hippocampal neurons against ischemia but it did not aggravate the effect of ischemia, either. The present study suggested that transfer of exogenous zinc into the intracellular space is required for neuroprotection, presumably via the anti-endonuclease activity. Keywords: Zinc; Delayed neuronal death; Ca2q-Mg 2q endonuclease; Apoptosis
The divalent cation zinc has been a focus of research in central nervous system physiology. Functionally zinc has both inhibitory effect on N-methyl-D-aspartate ŽNMDA. receptor and stimulatory effect on a-amino-3-hydroxy-5methyl-4-isoxasole propionate ŽAMPA. or kainate receptors w14,20x. On the other hand, zinc has been reported to inhibit Ca2q-Mg 2q -dependent endonuclease activity of thymocyte in vitro w3x, suggesting that zinc inhibits DNA fragmentation, an important characteristic of apoptosis. Recently, two distinct types of cell death, necrosis and apoptosis following cerebral ischemia have been well recognized w2x. We previously reported that DNA fragmentation preceded the morphological changes of cell death in the hippocampus of gerbils subjected to transient forebrain ischemia, a model of delayed neuronal death w13x, suggesting that this type of cell death shares the features in
)
Corresponding author. Fax: q81 Ž798. 45-6597.
common with the apoptotic process, in addition to the necrotic pathway through NMDA or AMPA receptors w8,15x. Therefore, it is of great interest to find whether the administration of zinc can influence the outcome of delayed neuronal death caused by the glutamate receptormediated or apoptotic mechanism. The objective of this study was to analyze the effect of systemically injected zinc on histology of the hippocampal lesions, which might have some impact on management of acute stroke. Adult Mongolian gerbils Ž Meriones unguiculatus. of both sexes, aged 16–18 weeks and weighing 60–80 g, were used in the present study. Following experimental procedures were performed according to Osaka University Medical School Guideline for the Care and Use of Laboratory Animals. Under light ether anesthesia, both common carotid arteries were transiently occluded using miniature aneurysmal clips for 3 min. Rectal temperature was monitored and regulated during operation by a thermostatic heating lamp at 37.5 " 0.58C. We prepared the zinc solu-
0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 1 1 2 - 2
K. Matsushita et al.r Brain Research 743 (1996) 362–365
Fig. 1. Histological grading of the hippocampal CA1 lesions. Abscissa indicates the histological grading from 0 to III Žsee text for definition. and ordinate indicates the percentage of animals in the subacute zinc, superacute zinc, or saline-pretreated group. ) P - 0.05.
tion by dissolving zinc chloride ŽWako Pure Chemical Industries, Ltd., Japan. in distilled water to a final concentration of 214 mmolrl. This solution was injected subcutaneously in a dose of 20 mgrkg w17x both 2 days and 1 day prior to the ischemic insult Žthe subacute group., or at 1 h before ischemia Žthe superacute group.. In choosing this time schedule, we assumed that the subacute group would
363
mainly reflect the biological effect of intracellular zinc and the superacute group would reflect that of extracellular zinc based on the previous studies w16x on the bioavailability of systemically injected zinc. Control animals were treated with injection of an equal volume of physiological saline using the same ischemia protocol. In order to observe in situ DNA fragmentation supposedly preceding nuclear damage, the terminal deoxynucleotidyl transferase ŽTdT. mediated dUTP biotin nick end labeling, the TUNEL method, was carried out after reperfusion for 3 days, based on the method by Gavrieli et al. w5x but was slightly modified to use for frozen sections. For histological examination to count dead neurons, the gerbils were decapitated after reperfusion for four days. Each section, compassing the dorsal hippocampus, was stained with hematoxylineosin ŽHE.. The grade of histological changes in the CA1 sector was semi-quantitatively analyzed according to the previously described scale w6x. In brief, we designated an absence of dead cells as grade 0 Ž0% of CA1 neurons., the presence of only scattered dead cells or a small group of dead cells Ž- 10%. as grade I, the presence of several groups of dead cells comprising less than one half of the total cell count in the CA1 sector Ž10–50%. as grade II, and the presence of dead cells comprising more than one
Fig. 2. Representative histological findings of the hippocampal CA1 lesions. The upper panels show animals with saline pretreatment. The TUNEL method ŽA. shows nuclear DNA fragmentation of dead neurons in the CA1 area after reperfusion for 3 days, while HE staining ŽB. shows dead neurons with nuclear shrinkage in the same area after reperfusion for 4 days. The lower panels show animals with subacute zinc pretreatment. The TUNEL method ŽC. shows no evidence of nuclear DNA fragmentation after reperfusion for 3 days in the CA1 area, while the HE staining ŽD. shows intact neurons after reperfusion for 4 days. Bars s 50 m m. TUNEL, terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling; HE, hematoxylin-eosin.
364
K. Matsushita et al.r Brain Research 743 (1996) 362–365
half of the total cell count Ž) 50%. as grade III. Statistical analysis was performed by using the two-sample Wilcoxon test and regarded significant as P - 0.05. There was no significant difference in rectal temperature between groups and nor did pre-administered zinc affect body temperature before the ischemic insult Ždata not shown.. The histological score is shown in Fig. 1. Subacute injection of zinc resulted in a minor but significant protective potential for hippocampal lesions in the CA1 sector. Superacute injection of zinc, however, did not alter the outcome. Representative histological findings are shown in Fig. 2, where the TUNEL positive nuclei could not be detected in more than 80% of animals subacutely pretreated with zinc. Many in vitro and in vivo studies have shown that zinc is profoundly involved in the underlying mechanism of cell death w4,7,19x. It is generally believed that excessive extracellular zinc concentration is neurotoxic and that the translocation of zinc into intracellular space is the causative phenomenon of ischemic or epileptic cell death. These ideas are mainly based on the observation of the transsynaptic movement in endogeneous zinc. We report here for the first time in vivo effect of systemically administered zinc on ischemic neuronal injury, especially delayed neuronal death. To be emphasized is that the underlying mechanism of delayed neuronal death has been supposed to be apoptotic or similar cell death w12x and that it can raise possibilities that zinc may function there as its antiendonuclease activity. In order to assess the effect of zinc on apoposis more clearly, we selected the model of bilateral carotid occlusion for 3 min in gerbils, based on the idea that a milder ischemic insult might induce more apoptotic cell death w1,2x. Several in vitro studies on apoptosis have shown that DNA fragmentation is inhibited by zinc supplementation and accelerated by zinc deficiency w11x. In vivo study, reported by Thomas et al. w17x, also showed the inhibitory effect of zinc on nuclear damage, in which subcutaneous administration of zinc before 48 h could antagonize lipopolysaccharide-induced DNA fragmentation of thymus cells in a dose-dependent manner Ž1–20 mgrkg.. Our result that ischemic nuclear damage could be rescued by subacute pre-administration of zinc but not by superacute pre-administration is in good agreement with theirs and may suggest that transfer of exogenous zinc into the intracellular space is required for its neuroprotection presumably by inhibiting the endonuclease activity. In addition to our previous report, further evidence of DNA fragmentation has been accumulated in global w10x and focal ischemia w9,18x with and without reperfusion. The present histological result also suggested that intranuclear DNA fragmentation was induced prior to total cell death following transient global ischemia. Although further studies will be necessary to elucidate the precise role of intracellular zinc, the present study has much contributed
to our understanding of the mechanism involved in the type of delayed neuronal death.
Acknowledgements This work was supported by a research grant for cardiovascular diseases from the Japanese Ministry of Health and Welfare and was in part supported by Health Science Research Grants by the Ministry of Health and Welfare of Japan. We also wish to express our thanks to Dr. H. Naritomi ŽNational Cardiovascular Center, Osaka, Japan. who gave us supports for experimental preparations in this study and Ms. M. Shimomura and R. Manabe for their secretarial assistance.
References w1x Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P. and Lipton, S.A., Apoptosis and necrosis: Two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxidersuperoxide in cortical cell cultures, Proc. Natl. Acad. Sci. USA, 92 Ž1995. 7162–7166. w2x Choi, D.W., Calcium: still center-stage in hypoxic-ischemic neuronal death, Trends Neurosci., 18 Ž1995. 58–60. w3x Cohen, J.J. and Duke, R.C., Glucocorticoid activation of a calciumdependent endonuclease in thymocyte nuclei leads to cell death, J. Immunol., 132 Ž1984. 38–42. w4x Frederickson, C.J., Hernandez, M.D. and McGinty, J.F., Translocation of zinc may contribute to seizure-induced death of neurons, Brain Res., 480 Ž1989. 317–321. w5x Gavrieli, Y., Sherman, Y. and Bensasson, S.A., Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation, J. Cell. Biol., 119 Ž1992. 493–501. w6x Kitagawa, K., Matsumoto, M., Oda, T., Niinobe, M., Hata, R., Handa, N., Fukunaga, R., Isaka, Y., Kimura, K., Maeda, H., Mikoshiba, K. and Kamada, T., Free radical generation during brief period of cerebral ischemia may trigger delayed neuronal death, Neuroscience, 35 Ž1990. 551–558. w7x Koh, J., Suh, S.W., Gwag, B.J., He, Y.Y., Hsu, C.Y. and Choi, D.W., The role of zinc in selective neuronal death after transient global cerebral ischemia, Science, 272 Ž1996. 1013–1016. w8x Li, H. and Buchan, A.M., Treatment with an AMPA antagonist 12 hours following severe normothermic forebrain ischemia prevents CA1 neuronal injury, J. Cereb. Blood Flow Metab., 13 Ž1993. 933–939. w9x Linnik, M., Zobrist, R.H. and Hatfield, M.D., Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats, Stroke, 24 Ž1993. 2002–2009. w10x MacManus, J.P., Buchan, A.M., Hill, I.E., Rasquinha, I. and Preston, E., Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain, Neurosci. Lett., 164 Ž1993. 89–92. w11x Martin, S.J., Mazdai, G., Strain, J.J., Cotter, T.G. and Hannigan, B.M., Programmed cell death Žapoptosis. in lymphoid and myeloid cell lines during zinc deficiency, Clin. Exp. Immunol., 83 Ž1991. 338–343. w12x Nitatori T., Sato N., Waguri S., Karasawa Y., Araki H., Shibanai K., Kominami E. and Uchiyama Y., Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis, J. Neurosci., 15 Ž1995. 1001–1011.
K. Matsushita et al.r Brain Research 743 (1996) 362–365 w13x Okamoto, M., Matsumoto, M., Ohtsuki, T., Taguchi, A., Mikoshiba, K., Yanagihara, T. and Kamada T., Internucleosomal DNA cleavage involved in ischemia-induced neuronal death, Biochem. Biophys. Res Commun., 196 Ž1993. 1356–1362. w14x Peters, S., Koh, J. and Choi, D.W., Zinc selectively blocks the action of N-methyl-D-Aspartate on cortical neurons, Science, 236 Ž1987. 589–593. w15x Sheardown, M.J., Nielsen, E.Ø., Hansen, A.J., Jacobsen, P. and Honore´ T., 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzoŽF.quinoxaline. A neuroprotectant for cerebral ischemia, Science, 247 Ž1990. 571– 574. w16x Takeda, A., Akiyama, T., Sawashita, J. and Okada, S., Brain uptake of trace metals, zinc and manganese, in rats, Brain Res., 640 Ž1994. 341–344.
365
w17x Thomas, D.J. and Caffrey, T.C., Lipopolysaccharide induces double-stranded DNA fragmentation in mouse thymus. Protective effect of zinc pretreatment, Toxicology, 68 Ž1991. 327–337. w18x Tominaga, T., Kure, S., Narisawa, K. and Yoshimoto, T., Endonuclease activation following focal ischemic injury in the rat brain, Brain Res., 608 Ž1993. 21–26. w19x Tønder, N., Johansen, F.F., Frederickson, C.J., Zimmer, J. and Diemer, N.H., Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat, Neurosci. Lett., 109 Ž1990. 247–252. w20x Weiss, J.H., Hartley, D.M., Koh, J. and Choi, D.W., AMPA receptor activation potentiates zinc neurotoxicity, Neuron, 10 Ž1993. 43–49.
Short communication
Effect of systemic zinc administration on delayed neuronal death in the gerbil hippocampus Kohji Matsushita a, ) , Kazuo Kitagawa b , Tomohiro Matsuyama a , Toshiho Ohtsuki b , Akihiko Taguchi b , Kenji Mandai b , Takuma Mabuchi b , Yoshiki Yagita b , Takehiko Yanagihara c , Masayasu Matsumoto b,c a
Fifth Department of Internal Medicine, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663 Japan b First Department of Medicine, Osaka UniÕersity Medical School, Osaka 565, Japan c Department of Neurology, Osaka UniÕersity Medical School, Osaka 565, Japan Accepted 24 September 1996
Abstract The divalent cation zinc has been reported to possess several physiological properties such as blocking apoptotic cell death through an inhibitory effect on Ca2q-Mg 2q endonuclease activity, or modulating the neurotoxicity via glutamate receptor subtypes. In the present study, we investigated the effect of peripherally injected zinc on delayed neuronal death seen in the hippocampus after transient global ischemia, in order to elucidate a possible beneficial role on zinc in ischemic neuronal cell death. Forty-five adult Mongolian gerbils of both sexes underwent transient bilateral clipping of the common carotid arteries for 3 min. In the pretreated animals, ZnCl 2 Ž20 mgrkg. was injected subcutaneously once, 1 h before ischemia Žsuperacute group; n s 6. or twice at 24 and 48 h before ischemia Žsubacute group; n s 14.. Histological survey was carried out 3 days later by in situ DNA fragmentation method and 4 days later by hematoxylin-eosin staining by semiquantatively counting dead neurons in the CA1 sector. Subacute zinc pre-administration significantly reduced the nuclear damage and subsequent neuronal death; however, superacutely pre-administered zinc did not protect hippocampal neurons against ischemia but it did not aggravate the effect of ischemia, either. The present study suggested that transfer of exogenous zinc into the intracellular space is required for neuroprotection, presumably via the anti-endonuclease activity. Keywords: Zinc; Delayed neuronal death; Ca2q-Mg 2q endonuclease; Apoptosis
The divalent cation zinc has been a focus of research in central nervous system physiology. Functionally zinc has both inhibitory effect on N-methyl-D-aspartate ŽNMDA. receptor and stimulatory effect on a-amino-3-hydroxy-5methyl-4-isoxasole propionate ŽAMPA. or kainate receptors w14,20x. On the other hand, zinc has been reported to inhibit Ca2q-Mg 2q -dependent endonuclease activity of thymocyte in vitro w3x, suggesting that zinc inhibits DNA fragmentation, an important characteristic of apoptosis. Recently, two distinct types of cell death, necrosis and apoptosis following cerebral ischemia have been well recognized w2x. We previously reported that DNA fragmentation preceded the morphological changes of cell death in the hippocampus of gerbils subjected to transient forebrain ischemia, a model of delayed neuronal death w13x, suggesting that this type of cell death shares the features in
)
Corresponding author. Fax: q81 Ž798. 45-6597.
common with the apoptotic process, in addition to the necrotic pathway through NMDA or AMPA receptors w8,15x. Therefore, it is of great interest to find whether the administration of zinc can influence the outcome of delayed neuronal death caused by the glutamate receptormediated or apoptotic mechanism. The objective of this study was to analyze the effect of systemically injected zinc on histology of the hippocampal lesions, which might have some impact on management of acute stroke. Adult Mongolian gerbils Ž Meriones unguiculatus. of both sexes, aged 16–18 weeks and weighing 60–80 g, were used in the present study. Following experimental procedures were performed according to Osaka University Medical School Guideline for the Care and Use of Laboratory Animals. Under light ether anesthesia, both common carotid arteries were transiently occluded using miniature aneurysmal clips for 3 min. Rectal temperature was monitored and regulated during operation by a thermostatic heating lamp at 37.5 " 0.58C. We prepared the zinc solu-
0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 1 1 2 - 2
K. Matsushita et al.r Brain Research 743 (1996) 362–365
Fig. 1. Histological grading of the hippocampal CA1 lesions. Abscissa indicates the histological grading from 0 to III Žsee text for definition. and ordinate indicates the percentage of animals in the subacute zinc, superacute zinc, or saline-pretreated group. ) P - 0.05.
tion by dissolving zinc chloride ŽWako Pure Chemical Industries, Ltd., Japan. in distilled water to a final concentration of 214 mmolrl. This solution was injected subcutaneously in a dose of 20 mgrkg w17x both 2 days and 1 day prior to the ischemic insult Žthe subacute group., or at 1 h before ischemia Žthe superacute group.. In choosing this time schedule, we assumed that the subacute group would
363
mainly reflect the biological effect of intracellular zinc and the superacute group would reflect that of extracellular zinc based on the previous studies w16x on the bioavailability of systemically injected zinc. Control animals were treated with injection of an equal volume of physiological saline using the same ischemia protocol. In order to observe in situ DNA fragmentation supposedly preceding nuclear damage, the terminal deoxynucleotidyl transferase ŽTdT. mediated dUTP biotin nick end labeling, the TUNEL method, was carried out after reperfusion for 3 days, based on the method by Gavrieli et al. w5x but was slightly modified to use for frozen sections. For histological examination to count dead neurons, the gerbils were decapitated after reperfusion for four days. Each section, compassing the dorsal hippocampus, was stained with hematoxylineosin ŽHE.. The grade of histological changes in the CA1 sector was semi-quantitatively analyzed according to the previously described scale w6x. In brief, we designated an absence of dead cells as grade 0 Ž0% of CA1 neurons., the presence of only scattered dead cells or a small group of dead cells Ž- 10%. as grade I, the presence of several groups of dead cells comprising less than one half of the total cell count in the CA1 sector Ž10–50%. as grade II, and the presence of dead cells comprising more than one
Fig. 2. Representative histological findings of the hippocampal CA1 lesions. The upper panels show animals with saline pretreatment. The TUNEL method ŽA. shows nuclear DNA fragmentation of dead neurons in the CA1 area after reperfusion for 3 days, while HE staining ŽB. shows dead neurons with nuclear shrinkage in the same area after reperfusion for 4 days. The lower panels show animals with subacute zinc pretreatment. The TUNEL method ŽC. shows no evidence of nuclear DNA fragmentation after reperfusion for 3 days in the CA1 area, while the HE staining ŽD. shows intact neurons after reperfusion for 4 days. Bars s 50 m m. TUNEL, terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling; HE, hematoxylin-eosin.
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K. Matsushita et al.r Brain Research 743 (1996) 362–365
half of the total cell count Ž) 50%. as grade III. Statistical analysis was performed by using the two-sample Wilcoxon test and regarded significant as P - 0.05. There was no significant difference in rectal temperature between groups and nor did pre-administered zinc affect body temperature before the ischemic insult Ždata not shown.. The histological score is shown in Fig. 1. Subacute injection of zinc resulted in a minor but significant protective potential for hippocampal lesions in the CA1 sector. Superacute injection of zinc, however, did not alter the outcome. Representative histological findings are shown in Fig. 2, where the TUNEL positive nuclei could not be detected in more than 80% of animals subacutely pretreated with zinc. Many in vitro and in vivo studies have shown that zinc is profoundly involved in the underlying mechanism of cell death w4,7,19x. It is generally believed that excessive extracellular zinc concentration is neurotoxic and that the translocation of zinc into intracellular space is the causative phenomenon of ischemic or epileptic cell death. These ideas are mainly based on the observation of the transsynaptic movement in endogeneous zinc. We report here for the first time in vivo effect of systemically administered zinc on ischemic neuronal injury, especially delayed neuronal death. To be emphasized is that the underlying mechanism of delayed neuronal death has been supposed to be apoptotic or similar cell death w12x and that it can raise possibilities that zinc may function there as its antiendonuclease activity. In order to assess the effect of zinc on apoposis more clearly, we selected the model of bilateral carotid occlusion for 3 min in gerbils, based on the idea that a milder ischemic insult might induce more apoptotic cell death w1,2x. Several in vitro studies on apoptosis have shown that DNA fragmentation is inhibited by zinc supplementation and accelerated by zinc deficiency w11x. In vivo study, reported by Thomas et al. w17x, also showed the inhibitory effect of zinc on nuclear damage, in which subcutaneous administration of zinc before 48 h could antagonize lipopolysaccharide-induced DNA fragmentation of thymus cells in a dose-dependent manner Ž1–20 mgrkg.. Our result that ischemic nuclear damage could be rescued by subacute pre-administration of zinc but not by superacute pre-administration is in good agreement with theirs and may suggest that transfer of exogenous zinc into the intracellular space is required for its neuroprotection presumably by inhibiting the endonuclease activity. In addition to our previous report, further evidence of DNA fragmentation has been accumulated in global w10x and focal ischemia w9,18x with and without reperfusion. The present histological result also suggested that intranuclear DNA fragmentation was induced prior to total cell death following transient global ischemia. Although further studies will be necessary to elucidate the precise role of intracellular zinc, the present study has much contributed
to our understanding of the mechanism involved in the type of delayed neuronal death.
Acknowledgements This work was supported by a research grant for cardiovascular diseases from the Japanese Ministry of Health and Welfare and was in part supported by Health Science Research Grants by the Ministry of Health and Welfare of Japan. We also wish to express our thanks to Dr. H. Naritomi ŽNational Cardiovascular Center, Osaka, Japan. who gave us supports for experimental preparations in this study and Ms. M. Shimomura and R. Manabe for their secretarial assistance.
References w1x Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P. and Lipton, S.A., Apoptosis and necrosis: Two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxidersuperoxide in cortical cell cultures, Proc. Natl. Acad. Sci. USA, 92 Ž1995. 7162–7166. w2x Choi, D.W., Calcium: still center-stage in hypoxic-ischemic neuronal death, Trends Neurosci., 18 Ž1995. 58–60. w3x Cohen, J.J. and Duke, R.C., Glucocorticoid activation of a calciumdependent endonuclease in thymocyte nuclei leads to cell death, J. Immunol., 132 Ž1984. 38–42. w4x Frederickson, C.J., Hernandez, M.D. and McGinty, J.F., Translocation of zinc may contribute to seizure-induced death of neurons, Brain Res., 480 Ž1989. 317–321. w5x Gavrieli, Y., Sherman, Y. and Bensasson, S.A., Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation, J. Cell. Biol., 119 Ž1992. 493–501. w6x Kitagawa, K., Matsumoto, M., Oda, T., Niinobe, M., Hata, R., Handa, N., Fukunaga, R., Isaka, Y., Kimura, K., Maeda, H., Mikoshiba, K. and Kamada, T., Free radical generation during brief period of cerebral ischemia may trigger delayed neuronal death, Neuroscience, 35 Ž1990. 551–558. w7x Koh, J., Suh, S.W., Gwag, B.J., He, Y.Y., Hsu, C.Y. and Choi, D.W., The role of zinc in selective neuronal death after transient global cerebral ischemia, Science, 272 Ž1996. 1013–1016. w8x Li, H. and Buchan, A.M., Treatment with an AMPA antagonist 12 hours following severe normothermic forebrain ischemia prevents CA1 neuronal injury, J. Cereb. Blood Flow Metab., 13 Ž1993. 933–939. w9x Linnik, M., Zobrist, R.H. and Hatfield, M.D., Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats, Stroke, 24 Ž1993. 2002–2009. w10x MacManus, J.P., Buchan, A.M., Hill, I.E., Rasquinha, I. and Preston, E., Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain, Neurosci. Lett., 164 Ž1993. 89–92. w11x Martin, S.J., Mazdai, G., Strain, J.J., Cotter, T.G. and Hannigan, B.M., Programmed cell death Žapoptosis. in lymphoid and myeloid cell lines during zinc deficiency, Clin. Exp. Immunol., 83 Ž1991. 338–343. w12x Nitatori T., Sato N., Waguri S., Karasawa Y., Araki H., Shibanai K., Kominami E. and Uchiyama Y., Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis, J. Neurosci., 15 Ž1995. 1001–1011.
K. Matsushita et al.r Brain Research 743 (1996) 362–365 w13x Okamoto, M., Matsumoto, M., Ohtsuki, T., Taguchi, A., Mikoshiba, K., Yanagihara, T. and Kamada T., Internucleosomal DNA cleavage involved in ischemia-induced neuronal death, Biochem. Biophys. Res Commun., 196 Ž1993. 1356–1362. w14x Peters, S., Koh, J. and Choi, D.W., Zinc selectively blocks the action of N-methyl-D-Aspartate on cortical neurons, Science, 236 Ž1987. 589–593. w15x Sheardown, M.J., Nielsen, E.Ø., Hansen, A.J., Jacobsen, P. and Honore´ T., 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzoŽF.quinoxaline. A neuroprotectant for cerebral ischemia, Science, 247 Ž1990. 571– 574. w16x Takeda, A., Akiyama, T., Sawashita, J. and Okada, S., Brain uptake of trace metals, zinc and manganese, in rats, Brain Res., 640 Ž1994. 341–344.
365
w17x Thomas, D.J. and Caffrey, T.C., Lipopolysaccharide induces double-stranded DNA fragmentation in mouse thymus. Protective effect of zinc pretreatment, Toxicology, 68 Ž1991. 327–337. w18x Tominaga, T., Kure, S., Narisawa, K. and Yoshimoto, T., Endonuclease activation following focal ischemic injury in the rat brain, Brain Res., 608 Ž1993. 21–26. w19x Tønder, N., Johansen, F.F., Frederickson, C.J., Zimmer, J. and Diemer, N.H., Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat, Neurosci. Lett., 109 Ž1990. 247–252. w20x Weiss, J.H., Hartley, D.M., Koh, J. and Choi, D.W., AMPA receptor activation potentiates zinc neurotoxicity, Neuron, 10 Ž1993. 43–49.