Kainate-induced hippocampal DNA damage is attenuated in superoxide dismutase transgenic mice

Molecular Brain Research 48 Ž1997. 145–148

Short communication

Kainate-induced hippocampal DNA damage is attenuated in superoxide dismutase transgenic mice Hiroshi Hirata, Jean Lud Cadet

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Molecular Neuropsychiatry Section, NIH r NIDA, DiÕision of Intramural Research, PO Box 5180, Baltimore, MD 21224, USA Accepted 11 March 1997

Abstract Peripheral administration of kainic acid ŽKA. can cause cell death in the hippocampus of rodents. This is thought to involve oxidative stress. In the present study, we tested the possibility that KA-induced neuronal cell death might be attenuated in CuZn superoxide dismutase transgenic ŽSOD-Tg. mice. Acute administration of KA causes animal death in a dose-dependent fashion; this was attenuated in SOD-Tg mice. Similarly, KA caused dose-dependent neuronal cell death in the hippocampus of wild-type mice; this cell death was attenuated in the SOD-Tg mice, in a gene-dosage-dependent fashion, with homozygous mice showing complete protection even at the highest dose Ž45 mgrkg. of KA used in this study. These results provide further support for the involvement of oxygen-based radicals in the toxic effects of KA. Keywords: Kainic acid; Transgenic mice; Superoxide dismutase; Free radical; Hippocampus; Nick translation, in situ

Excessive activation of excitatory amino-acid receptors is thought to be involved in several neurodegenerative diseases w1,5,6x. Receptors for excitatory amino acids are widely distributed in the brain. These include specific receptors for N-methyl-D-asparate ŽNMDA., quisqualate ŽQA., kainic acid ŽKA. and a-amino-3-hydroxy-5methoisoxasole-4-propionate ŽAMPA. and metabotropic receptors. KA binds to both KA and AMPA receptors w14x. KA-induced neuronal damage is thought to be caused by three different types of mechanism. First, KA may reach vulnerable brain areas and may induce direct axonsparing lesions. Second, KA may exert strong excitatory actions upon certain neuronal pathways. Third, secondary seizure-related neuropathological event, such as hypoxia or edema, may play a role in KA-induced brain damage. These possibilities notwithstanding, it has been shown that both intra-amygdala w15x and systemic injection of KA w19x can cause DNA fragmentation in the hippocampus. Stimulation of excitatory amino-acid receptors activates membrane voltage sensitive calcium channels with subsequent Ca2q influx into the cytoplasmic compartments of the cell w11x. This increase in intracellular calcium leads to

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Corresponding author. Fax: q1 Ž410. 550-2745.

the activation of a number of enzymes that generate free radicals. These include phospholipases, nitric oxide synthesis and calcium-dependent proteases w6x. Free radicals generated by these enzymes are probably involved in KA-induced excitotoxicity because that KA-induced neuronal damage is attenuated by superoxide dismutase ŽSOD., catalase, allopurinol and by the free radical trapping agent, a-phenyl-N-tert-butyl-nitrone ŽPBN. in vitro w4,7,13x. These ideas are supported by the further observation that KA can cause free radical production in the gerbil brain w18x and that phenyl-N-tert-butyl-a-Ž2-sulfophenyl.-nitrone ŽS-PBN. can attenuate KA-induced excitotoxic lesions in rats w16,17x. We, thus, wondered if overproduction of SOD in vivo could also protect against the toxic effects of peripheral administration of KA. Towards that end, we have investigated the acute lethal effects and the degenerative actions of KA by using in situ nick translation technique in wild-type mice as well as heterozygous and homozygous SOD-transgenic ŽTg. mice of strain 218r3 that show a respective mean increase of 2.6- and 5.7-fold in SOD enzyme activity compared to wild-type mice. These animals were produced as previously described w9x and have been used in a number of experiments to assess the role of the superoxide radicals in drug-induced neurodegeneration of monoaminergic systems w2,12x. All animal use proce-

0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 1 2 1 - 6

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Fig. 1. Acute lethal effects of KA in wild-type mice and SOD-Tg mice. The mice were administered different doses of KA as described in the text. Key: ) P - 0.05 in comparison to saline-treated mice of the same strain; a P - 0.05 in comparison to the wild-type mice treated with the same dose of KA.

dures were according to the National Institute of Health ŽNIH. guide for the Care and Use of Laboratory Animals and were approved by the local National Institute on Drug Abuse ŽNIDA. Animal Care Committee. All efforts were

made to minimize animal suffering, to reduce the number of animals used. In vitro techniques could not be used as alternative because we wanted to know the effects of KA in vivo in transgenic mice that overproduce CuZn-SOD. 8-week-old mice were housed under conditions of controlled temperature Ž238C. and illumination Ž12-h lightrdark cycle.. Four or five mice were kept in a cage and the cages were kept as close as possible in order to avoid possible environmental influence on the results. 15, 30.0 or 45.0 mgrkg of kainic acid ŽK-0250, monohydrate, Sigma, St. Louis, MO; KA. or saline were administered between 09:00 and 11:00 h via the i.p. route. Seizure onset occurred within 30 min and persisted for 2–3 h. 4 days later, the animals were killed and their brains were rapidly removed, frozen in isopentane on dry ice and stored frozen at y708C. Sections Ž20 m m thick. were cut at y208C and thaw-mounted on silane-coated glass slides. The slides were kept at y708C until used for in situ nick translation or thionin staining as described below. In situ nick translation were performed according to minor modifications of previous protocols w8x. Slide were air-dried and immersed in the following solution: 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min at room temperature ŽRT.; phosphate-buffered saline ŽPBS.

Fig. 2. Photomicrographs of nick-translation processed cells in the hippocampus of wild-type mice ŽA,C. and homozygous SOD-Tg mice ŽB,D.. Note the appearance of apoptotic cells in CA1 and CA3 in wild-type mice ŽA,C. killed 4 days after administration of KA Ž45 mgrkg ip.. No apoptotic cells are seen in homozygous SOD-Tg mice ŽB,D.. Magnification levels are =50 ŽA,B. and =400 ŽC,D.. The quantitative data are given in Fig. 3.

H. Hirata, J.L. Cadetr Molecular Brain Research 48 (1997) 145–148

Fig. 3. KA causes apoptosis in the hippocampus of wild-type mice but SOD-Tg mice. The mice were administered different doses of KA as described in the text. Key: ) P - 0.05 and ) ) P - 0.005 in comparison to saline-treated mice of the same strain; a P - 0.05 and aa P - 0.005 in comparison to the wild-type mice treated with the same dose of KA.

pH 7.4 for 2 = 5 min at RT; 2 = SSC for 20 min at 808C; 10 m grml proteinase K in 0.01 M Tris pH 7.8, 0.005 M ethylenediaminotetraacetate ŽEDTA., 0.5% sodium dodecyl sulfate ŽSDS. for 10 min at RT; 0.2% glycine in PBS for 1 min at RT. Subsequently, the slides were equilibrated in 50 mM Tris, 5 mM MgCl 2 , 10 mM b-mercaptoethanol,

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0.005% bovine serum albumin ŽBSA. pH 7.5 for 5 min at RT, then incubated in the presence of 50 Urml DNA Polymerase I ŽPromega, Madison, WI., 10 m M each of dCTP, dATP, dGTP ŽPromega. and biotin-21-dUTP ŽClontech Laboratories, Palo Alto, CA. for 90 min at RT. Sections were rinsed in PBS for 5 min, immersed 0.1% H 2 O 2 in PBS for 15 min at RT and washed in PBS for 2 = 5 min. To detect biotinylated dUTP incorporation, slides were incubated in 0.1 M PBS containing 1% horseradish peroxidase avidin D ŽVector Laboratories, Burlingame, CA., 1% BSA and 0.5% Tween-20 for 30 min at RT. After washing in PBS for 3 = 5 min at RT, sections were then placed in 3,3X-diaminobenzidine ŽDAB. containing solution for f 10 min at RT, washed in ddH 2 O for 2 = 5 min and dried under a stream of cool air. As a negative control, sections were incubated in the absence of DNA polymerase I. Thionin staining was performed in a buffer containing 0.25% thionin, 0.5 M NaOH and 10% acetic acid for 30 s at RT. Statistical analyses were done using x 2 test. Criteria for significance was set at the 0.05 level. Fig. 1 shows that administration of KA can cause death of a significant number of mice. At doses of - 30.0 mgrkg KA, there was no significant difference in the

Fig. 4. Photomicrographs of thionin-stained cell in the hippocampus of wild-type mice ŽA,C. and homozygous SOD-Tg mice ŽB,D.. Positive staining was observed in the wild-type mice but not in CA1 and CA3 in the homozygous SOD-Tg mice. Note also the enlargement of the hippocampus in the KA-treated wild-type mice ŽC.. No such changes were seen in the SOD-Tg mice. Magnification levels are =50 ŽA,B. and =400 ŽC,D..

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percentage survival between the three strains of mice. However, the highest dose of KA Ž45 mgrkg. used in this study killed 40% of wild-type mice but only 14% of heterozygous SOD-Tg mice. None of homozygous SOD-Tg mice died after that dose of KA. KA injections resulted in the appearance of neuronal cell death in a dose-dependent fashion ŽFigs. 2 and 3.. There was no cell death in the hippocampus of any of the strains of mice injected with saline nor with 15 mgrkg KA. In the animals injected with 30 mgrkg KA, 25% of wild-type mice and 9% of heterozygous SOD-Tg mice showed cell death in their hippocampus; however, no cell death was seen in the homozygous SOD-Tg mice. In the groups injected with 45.0 mgrkg KA, 73% wild-type mice and 20% heterozygous SOD-Tg mice showed cell death whereas none of the homozygous SOD-Tg mice showed similar changes. Fig. 4 showed thionin-stained sections from KA-treated mice. The hippocampi from KA-treated wild-type mice were edematous; this is probably due to cell swelling secondary to changes cellular calcium homeostasis and secondary osmotic alterations. This is the first demonstration that KA-induced neuronal cell death in the hippocampus is attenuated in mice which have high levels of CuZn-SOD enzyme activity in their brains. Because CuZn-SOD dismutates the superox. and forms the first line of defense against ide anion ŽOy 2 oxygen-based radicals w10x, the present data indicate that superoxide radicals must play an important role in KA-induced neuronal cell death. This view is supported by the observations that homozygous SOD-Tg mice that have higher SOD activity than the heterozygous mice are also more protected against KA-induced neuronal cell death. A number of investigators had shown that KA-induced cell damage involves free radical production in vitro w4,7,13x and in vivo w16,18x. I.p. melatonin administration can also attenuate KA-induced cell death in the hippocampus w19x. Melatonin-mediated neuroprotection is probably related to its antioxidative activity w3x. Thus, when taken together with these previous studies, the present results indicate that KA-induced cell death may in part be mediated by free radicals. In summary, the present study documents, for the first time, that KA-induced neuronal cell death is attenuated in SOD-Tg mice. The study also supports the view that oxygen-based radicals generated by KA must be the main culprits in the manifestation of KA-induced neuronal cell death in the brain.

Acknowledgements We thank the staff of our Animal Care Facility at the Division of Intramural Research Center of NIHrNIDA for the impeccable care of the animals.

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