Brain Research, 557 (1991) 313-315 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124787Q
313
BRES 24787
Increased activity of superoxide dismutase in kindled brain and suppression of kindled seizure following intra-amygdaloid injection of superoxide dismutase in rats Norio Mori 1, J u h n A. Wada 2, Masaru W a t a n a b e 1 and Hisashi K u m a s h i r o 1 1Department of Neuropsychiatry, Fukushima Medical College, Fukushima (Japan) and 2Divisions of Neurosciences and Neurology, University of British Columbia, Vancouver, B.C. (Canada) (Accepted 30 April 1991) Key words: Superoxide dismutase; Amygdala; Kindling; Oxygen radical; Seizure susceptibility; Elevation of seizure threshold
The possible role played by superoxide dismutase (SOD), a major defense system for counteracting the toxic effects of oxygen free radicals, in amygdaloid (AM) kindling was examined in rats. A significant increase of total SOD activity in the whole brain was observed 30 days after completion of AM kindling. Intra-AM injection of 3 ng of one of the 2 SOD enzymes present in mammalian brain, i.e. cytosolie SOD containing copper and zinc (CuZn-SOD) caused suppression of kindled seizure. These results suggest that SOD participates in the persistence of AM kindled seizure susceptibility and the initiation of kindled AM seizure. Oxygen-derivated free radicals are produced in biological systems by the single-electron reduction of oxygen s'~°. The brain is highly sensitive to the toxic effects of these partially reduced species of oxygen including superoxide anion, hydrogen peroxide, and hydroxyl radical. The superoxide dismutase (SOD) enzymes are a major defense system for aerobic cells combating the toxic effects of oxygen radicals. The enzymes rapidly remove the superoxide anion and so prevent its direct toxic effects as well as its interaction with metal ions to increase products of hydroxyl radical, which is one of the most reactive oxygen radicals 5'1°. Two SOD enzymes are found in mammalian brain, a mitochondrial SOD containing manganese (Mn-SOD) and a cytosolic SOD containing copper and zinc (CuZn-SOD). The CuZn-SOD contributes the most to the total SOD activity5. Recently, Dollwet et al. 2 have demonstrated that in the metrazol and maximum electroshock animal models of epilepsy, copper complexes of antiepileptic drugs exert greater anticonvulsant effect than their parent drugs, presumably by scavenging the superoxide anion. In addition, hyperbaric oxygen-induced convulsion, which is believed to be mediated by membrane lipid peroxidation initiated by the action of oxygen radicals 7, could be suppressed by intravenous injection of CuZn-SODcontaining liposomes as vectors for the blood-brain barrier (BBB)-impermeable enzyme penetrating BBB 19.
A similar anticonvulsant effect with intravenous CuZnSOD-containing liposomes was obtained in amygdaloid (AM) kindled rats in our laboratory 17. We have also shown suppression of limbic status epilepticus induced by intra-AM injection of dibutyryl-cAMP following intravenous CuZn-SOD alone in rats 8, presumably due to the opening of the BBB to the enzyme during sustained seizures 1]. Results of the present study indicate that total SOD activity of the whole brain examined 30 days following completion of AM kindling is significantly increased and that intra-AM injection of CuZn-SOD suppresses kindled AM seizure. For the assay of SOD activity, 10 male Wistar rats (200-240 g) were used. Under pentobarbital anesthesia (50 mg/kg, i.p.), bipolar electrodes made of twisted stainless steel wire 200 g m in diameter were implanted into the left AM. Monopolar dural screw electrodes were placed over the sensorimotor cortex bilaterally. Animals were divided into kindled (K) (n = 5) and control (C) (n = 5) groups. In the K group animals, the afterdischarge (AD) threshold (ADT) was determined at the left AM and daily AM stimulation was delivered bipolarly in a 1 s train of constant current 60 Hz square wave at the ADT. The pattern of clinical seizure development was classified into 5 stages ]3. All the K group animals were kindled at the left AM until a stable Stage 5 seizure was evoked for five successive days. The C group animals were implanted but not stimulated.
Correspondence: J.A. Wada, Divisions of Neurosciences and Neurology, University of British Columbia, Vancouver, B.C. V6T 2A1, Canada.
314 SOD activity assay was by the method of Imanari et al.6 with some modifications as described by McCord and Fridovich 1°. Thirty days after completion of kindling, the animals were decapitated, and the whole brain was removed, weighed, and homogenized in 5 vols. of ice-cold Na 2 CO 3 buffer (pH 10.2). The homogenate was centrifuged at 100,000 g at 4 °C for 1 h. A 0.5 ml of the resulting supernatant was added to 12 ml 0.05 M Na 2 CO 3 buffer (pH 10.2), containing 1.5/~mol xanthine, 1.5 ~mol EDTA, 0.75 mg bovine serum albumin, and 0.375/~mol Nitroblue tetrazolium. The reaction was initiated by adding 0.5 ml xanthine oxidase at 25 °C. After 20-min incubation, the reaction was stopped by the addition of 0.5 ml CuCl2, and the reduction of tetrazolium was measured spectrophotometrically at 540 nm. SOD activity was determined by comparison with the homogenate erythrocyte pure SOD and expressed as units/wet weight. A 50% inhibition of the tetrazolium reduction was defined as one unit of SOD activity. For testing the effect of intra-AM injection of CuZnSOD, ten male Wistar rats (220-240 g) were used. They were implanted with bipolar electrodes into the left AM and monopolar dural screw electrodes in the same manner described above. A 23-gauge guide cannula was attached to the left AM electrode so as to be 1 mm above the electrode tip. The left AM was stimulated at the A D T until a stable Stage 5 seizure was evoked for 5 successive days. Subsequently, the stimulus intensity was gradually reduced and the last intensity to produce Stage 5 seizure was designated as the generalized seizure triggering threshold (GST). Recently, we have found that intravenous administration of 10 mg/kg CuZn-SOD (human SOD; Ube Kousan and Fujisawa Seiyaku) suppresses
6
m
VEHICLE
SOD
status epilepticus induced by intra-AM injection of dibutyryl-cAMP in rats 8. When CuZn-SOD was injected into the AM, the effective doses to suppress the status epilepticus were around 3 ng (unpublished data). Therefore, 3 ng CuZn-SOD was used in this study. CuZn-SOD was dissolved in saline as a vehicle in a concentration of 3 ng//~l. The animals received either 3 ng CuZn-SOD in a volume of 1/tl (n = 5) or an identical volume of vehicle (n = 5) into the kindled AM, through the implanted cannula. Two hours after the injection, the kindled AM was stimulated at the previously established GST. If AD was not induced, the stimulus intensity was increased by 40/~A steps at 10-min intervals until A D was elicited. Subsequently, the animals were stimulated at the GST or increased A D T every 24 h. As shown in Table I, the total SOD activity of the whole brain was 0.37 in the C group and 0.78 in the K group. Therefore, the SOD activity of the K group was significantly higher than that of the C group (P < 0.01). As shown in Fig. 1, intra-AM injection of vehicle alone had no effect. However, SOD injection totally abolished AD generation at the GST in 4/5 animals, although restimulation at 40-80/~A above the GST evoked kindled seizure. This suppression began 2-24 h after the injection and lasted for 2-4 days. The mechanisms underlying the sustained increase of SOD activity in the kindled brain are unknown. Since, however, SOD activity is preferentially stimulated in a response directed toward neutralization of the oxygen radicals 5'14'15, the result observed in this study may reflect the products of oxygen radicals in the course of kindling. The decrease of oxygen tension in the brain during and following epileptic discharges has been observed in different models of epilepsy 4'9'12. This, followed by an increase in blood flow, can in most cases satisfy and even overcompensate for the increased oxygen demand 4' 9,12. These states are similar to those of cerebral ischemia, in which oxygen radicals seem to be augmented 1'3'16"18. Therefore, it is plausible that repeated epileptic activities induced by kindling stimulations cause oxygen radicals, leading to enhanced SOD activity. However, the possibility could also be considered that bio-
2 hrs TABLE I
'-
1
[ ] GST
2
3
4 5 1 2 3 RAT NUMBER
4
5
1 GST+40/~A • GST+80#A Fig. 1. Suppression of kindled seizure following intra-AM injection of 3 ng SOD. When AD was suppressed at the GST, the stimulus intensity was increased by 40/~A steps (40/zA above the GST; 80 /~A above the GST) until Stage 5 seizure was elicited. Electrical stimulation was continued until 3 consecutive Stage 5 seizures were evoked at the GST.
Whole brain SOD activity in kindled (K) and control (C) rats
Values are mean + S.E.M. Numbers in parentheses = range. Group
n
SOD activity (units/mg wet weight)
K C
5 5
0.78 + 0.11 (0.53-1.25)* 0.37 + 0.02 (0.32-0.47)
*P < 0.01 compared to controls (Student's t-test).
315 logical systems m e d i a t i n g f o r m a t i o n of oxygen radicals (for e x a m p l e , xithantin/xithantin oxidase system) are activated in kindled brain, although there are no available d a t a in this regard. T h e p r e s e n t result also shows that i n t r a - A M injection of C u Z n - S O D can suppress kindled A M seizure. This is c o m p a r a b l e with our recent study, in which the intravenous C u Z n - S O D containing liposomes m a r k e d l y suppressed kindled A M convulsion in rats, p r e s u m a b l y due to l i p o s o m e - m e d i a t e d brain u p t a k e of the B B B - i m p e r m e a b l e C u Z n - S O D 17. These S O D - i n d u c e d anticonvul-
sant effects do not conflict with o u r assumption that oxygen radicals participate in a certain aspect of kindling phenomenon. Obviously, further w o r k is n e e d e d but at this point we are suggesting that S O D participates in seizure susceptibility induced by A M kindling and initiation of kindled A M seizure in rats.
1 Chan, P.H. and Fishman, R.A., Transient formation of superoxide radicals in polyunsaturated fatty acid-induced brain swelling, J. Neurochem., 35 (1980) 1004-1007. 2 Dollwet, H.H.A., McNicolas, J.B., Pezeshk, A. and Sorenson, J.R.J., Superoxide dismutase-mimetic activity of antiepileptic drug copper complexes, Trace Element Med., 4 (1987) 13-20. 3 Flamm, E.S., Demopoulos, H.B., Seligman, M.L., Poser, R.G. and Ransohoff, J., Free radicals in cerebral ischemia, Stroke, 9 (1978) 445-447. 4 Freund, T.E, Buzaki, G., Prohaska, O.J., Leon A. and Somogyi, P., Simultaneous recording of local electrical activity, partial oxygen tension and temperature in the rat hippocampus with a chamber-type microelectrode. Effects of anesthesia, ischemia and epilepsy, Neuroscience, 28 (1989) 539-549. 5 Halliwell, B. and Gutteridge, J.M.C., Oxygen radicals and the nervous system, Trends Neurosci., 2 (1985) 22-26. 6 Imanari, T., Hirota, M. and Miyazaki, M., Improved assay method for superoxide dismutase, Igaku No Ayumi (in Japanese), 101 (1977) 496-497. 7 Jerret, S.A., Jefferson, D. and Mengel, C.E., Seizure, H202 and lipid peroxidase in brain during exposure to oxygen under high pressure, Aerospace Med., 44 (1973) 40-44. 8 Kurokouchi, A., Moil, N. and Kumashiro, H., Antiepileptic effects of superoxide dismutase on status epilepticus in rats, lgaku No Ayumi (in Japanese), 152 (1990) 465-466. 9 Leninger-Follert, E. and Lubers, D.W., Behavior of microflow and local PO E of the brain cortex during and after direct electrical stimulation. A contribution of the problem of metabolic regulation of microcirculation in the brain, Eur. J. Physiol., 366 (1976) 39-44. 10 McCord, J.M. and Fridovich, I., Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprein), J. Biol. Chem., 244 (1969) 6049-6055. 11 Nitsch, C., Suzuki, R., Fujiwara, K. and Klatzo, I., Incongruence of regional cerebral blood flow increase and blood-brain
barrier opening in rabbits at the onset of seizures induced by bicuculline methoxypyridoxine, and kainic acid, J. Neurol. Sci., 379 (1985) 165-172. 12 Pinard, E., Tremblay, E., Ben-Aft, Y. and Seylaz, J., Blood flow compensates oxygen demand in the vulnerable CA3 region of the hippocampus during kainate-induced seizures, Neuroscience, 13 (1984) 1039-1049. 13 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. 14 Shingh, R. and Pathak, D.N., Lipid peroxidation and glutathion peroxide, glutathione reductase, superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activities in FeC13induced epileptogenic loci in the rat brain, Epilepsia, 31 (1990) 15-26. 15 Turrens, J.G., Freeman, B.A., Levitt, J.G. and Crapo, J.D., The effect of hyperoxia on superoxide dismutase by lung submitochondrial particles, Arch. Biochem. Biophys., 217 (1982) 401-410. 16 Watson, B.D., Busto, R., Goldberg, W.J., Santio, M., Toshida, S. and Ginsberg, M.D., Lipid peroxidation in vivo induced by reversible global ischemia in rat brain, J. Neurochem., 42 (1984) 268-275. 17 Yokoyama, H., Matsumoto, S., Ishida, S., Mori, N. and Kumashiro, H., Anticonvulsant effect of liposome-entrapped superoxide dismutase on kindled convulsion in rats, Epilepsia, 40 (1989) 647. 18 Yoshida, S., Abe, K., Asano, T., Sano, K., Shimasaki, H. and Ueta, N., Brain free fatty acids, edema and mortality in gerbils subjected to transient, bilateral ischemia, and effect of barbiturate anesthesia, J. Neurochem., 40 (1983) 1278-1286. 19 Yusa, T., Crapo, J.D. and Freeman, B.A., Liposome-mediated augmentation of brain SOD and catalase inhibits CNS 0 2 toxicity, Respir. Environ. Physiol., 57 (1984) 1674-1681.
Supported by grants from the Medical Research Council of Canada to J.A.W.
313
BRES 24787
Increased activity of superoxide dismutase in kindled brain and suppression of kindled seizure following intra-amygdaloid injection of superoxide dismutase in rats Norio Mori 1, J u h n A. Wada 2, Masaru W a t a n a b e 1 and Hisashi K u m a s h i r o 1 1Department of Neuropsychiatry, Fukushima Medical College, Fukushima (Japan) and 2Divisions of Neurosciences and Neurology, University of British Columbia, Vancouver, B.C. (Canada) (Accepted 30 April 1991) Key words: Superoxide dismutase; Amygdala; Kindling; Oxygen radical; Seizure susceptibility; Elevation of seizure threshold
The possible role played by superoxide dismutase (SOD), a major defense system for counteracting the toxic effects of oxygen free radicals, in amygdaloid (AM) kindling was examined in rats. A significant increase of total SOD activity in the whole brain was observed 30 days after completion of AM kindling. Intra-AM injection of 3 ng of one of the 2 SOD enzymes present in mammalian brain, i.e. cytosolie SOD containing copper and zinc (CuZn-SOD) caused suppression of kindled seizure. These results suggest that SOD participates in the persistence of AM kindled seizure susceptibility and the initiation of kindled AM seizure. Oxygen-derivated free radicals are produced in biological systems by the single-electron reduction of oxygen s'~°. The brain is highly sensitive to the toxic effects of these partially reduced species of oxygen including superoxide anion, hydrogen peroxide, and hydroxyl radical. The superoxide dismutase (SOD) enzymes are a major defense system for aerobic cells combating the toxic effects of oxygen radicals. The enzymes rapidly remove the superoxide anion and so prevent its direct toxic effects as well as its interaction with metal ions to increase products of hydroxyl radical, which is one of the most reactive oxygen radicals 5'1°. Two SOD enzymes are found in mammalian brain, a mitochondrial SOD containing manganese (Mn-SOD) and a cytosolic SOD containing copper and zinc (CuZn-SOD). The CuZn-SOD contributes the most to the total SOD activity5. Recently, Dollwet et al. 2 have demonstrated that in the metrazol and maximum electroshock animal models of epilepsy, copper complexes of antiepileptic drugs exert greater anticonvulsant effect than their parent drugs, presumably by scavenging the superoxide anion. In addition, hyperbaric oxygen-induced convulsion, which is believed to be mediated by membrane lipid peroxidation initiated by the action of oxygen radicals 7, could be suppressed by intravenous injection of CuZn-SODcontaining liposomes as vectors for the blood-brain barrier (BBB)-impermeable enzyme penetrating BBB 19.
A similar anticonvulsant effect with intravenous CuZnSOD-containing liposomes was obtained in amygdaloid (AM) kindled rats in our laboratory 17. We have also shown suppression of limbic status epilepticus induced by intra-AM injection of dibutyryl-cAMP following intravenous CuZn-SOD alone in rats 8, presumably due to the opening of the BBB to the enzyme during sustained seizures 1]. Results of the present study indicate that total SOD activity of the whole brain examined 30 days following completion of AM kindling is significantly increased and that intra-AM injection of CuZn-SOD suppresses kindled AM seizure. For the assay of SOD activity, 10 male Wistar rats (200-240 g) were used. Under pentobarbital anesthesia (50 mg/kg, i.p.), bipolar electrodes made of twisted stainless steel wire 200 g m in diameter were implanted into the left AM. Monopolar dural screw electrodes were placed over the sensorimotor cortex bilaterally. Animals were divided into kindled (K) (n = 5) and control (C) (n = 5) groups. In the K group animals, the afterdischarge (AD) threshold (ADT) was determined at the left AM and daily AM stimulation was delivered bipolarly in a 1 s train of constant current 60 Hz square wave at the ADT. The pattern of clinical seizure development was classified into 5 stages ]3. All the K group animals were kindled at the left AM until a stable Stage 5 seizure was evoked for five successive days. The C group animals were implanted but not stimulated.
Correspondence: J.A. Wada, Divisions of Neurosciences and Neurology, University of British Columbia, Vancouver, B.C. V6T 2A1, Canada.
314 SOD activity assay was by the method of Imanari et al.6 with some modifications as described by McCord and Fridovich 1°. Thirty days after completion of kindling, the animals were decapitated, and the whole brain was removed, weighed, and homogenized in 5 vols. of ice-cold Na 2 CO 3 buffer (pH 10.2). The homogenate was centrifuged at 100,000 g at 4 °C for 1 h. A 0.5 ml of the resulting supernatant was added to 12 ml 0.05 M Na 2 CO 3 buffer (pH 10.2), containing 1.5/~mol xanthine, 1.5 ~mol EDTA, 0.75 mg bovine serum albumin, and 0.375/~mol Nitroblue tetrazolium. The reaction was initiated by adding 0.5 ml xanthine oxidase at 25 °C. After 20-min incubation, the reaction was stopped by the addition of 0.5 ml CuCl2, and the reduction of tetrazolium was measured spectrophotometrically at 540 nm. SOD activity was determined by comparison with the homogenate erythrocyte pure SOD and expressed as units/wet weight. A 50% inhibition of the tetrazolium reduction was defined as one unit of SOD activity. For testing the effect of intra-AM injection of CuZnSOD, ten male Wistar rats (220-240 g) were used. They were implanted with bipolar electrodes into the left AM and monopolar dural screw electrodes in the same manner described above. A 23-gauge guide cannula was attached to the left AM electrode so as to be 1 mm above the electrode tip. The left AM was stimulated at the A D T until a stable Stage 5 seizure was evoked for 5 successive days. Subsequently, the stimulus intensity was gradually reduced and the last intensity to produce Stage 5 seizure was designated as the generalized seizure triggering threshold (GST). Recently, we have found that intravenous administration of 10 mg/kg CuZn-SOD (human SOD; Ube Kousan and Fujisawa Seiyaku) suppresses
6
m
VEHICLE
SOD
status epilepticus induced by intra-AM injection of dibutyryl-cAMP in rats 8. When CuZn-SOD was injected into the AM, the effective doses to suppress the status epilepticus were around 3 ng (unpublished data). Therefore, 3 ng CuZn-SOD was used in this study. CuZn-SOD was dissolved in saline as a vehicle in a concentration of 3 ng//~l. The animals received either 3 ng CuZn-SOD in a volume of 1/tl (n = 5) or an identical volume of vehicle (n = 5) into the kindled AM, through the implanted cannula. Two hours after the injection, the kindled AM was stimulated at the previously established GST. If AD was not induced, the stimulus intensity was increased by 40/~A steps at 10-min intervals until A D was elicited. Subsequently, the animals were stimulated at the GST or increased A D T every 24 h. As shown in Table I, the total SOD activity of the whole brain was 0.37 in the C group and 0.78 in the K group. Therefore, the SOD activity of the K group was significantly higher than that of the C group (P < 0.01). As shown in Fig. 1, intra-AM injection of vehicle alone had no effect. However, SOD injection totally abolished AD generation at the GST in 4/5 animals, although restimulation at 40-80/~A above the GST evoked kindled seizure. This suppression began 2-24 h after the injection and lasted for 2-4 days. The mechanisms underlying the sustained increase of SOD activity in the kindled brain are unknown. Since, however, SOD activity is preferentially stimulated in a response directed toward neutralization of the oxygen radicals 5'14'15, the result observed in this study may reflect the products of oxygen radicals in the course of kindling. The decrease of oxygen tension in the brain during and following epileptic discharges has been observed in different models of epilepsy 4'9'12. This, followed by an increase in blood flow, can in most cases satisfy and even overcompensate for the increased oxygen demand 4' 9,12. These states are similar to those of cerebral ischemia, in which oxygen radicals seem to be augmented 1'3'16"18. Therefore, it is plausible that repeated epileptic activities induced by kindling stimulations cause oxygen radicals, leading to enhanced SOD activity. However, the possibility could also be considered that bio-
2 hrs TABLE I
'-
1
[ ] GST
2
3
4 5 1 2 3 RAT NUMBER
4
5
1 GST+40/~A • GST+80#A Fig. 1. Suppression of kindled seizure following intra-AM injection of 3 ng SOD. When AD was suppressed at the GST, the stimulus intensity was increased by 40/~A steps (40/zA above the GST; 80 /~A above the GST) until Stage 5 seizure was elicited. Electrical stimulation was continued until 3 consecutive Stage 5 seizures were evoked at the GST.
Whole brain SOD activity in kindled (K) and control (C) rats
Values are mean + S.E.M. Numbers in parentheses = range. Group
n
SOD activity (units/mg wet weight)
K C
5 5
0.78 + 0.11 (0.53-1.25)* 0.37 + 0.02 (0.32-0.47)
*P < 0.01 compared to controls (Student's t-test).
315 logical systems m e d i a t i n g f o r m a t i o n of oxygen radicals (for e x a m p l e , xithantin/xithantin oxidase system) are activated in kindled brain, although there are no available d a t a in this regard. T h e p r e s e n t result also shows that i n t r a - A M injection of C u Z n - S O D can suppress kindled A M seizure. This is c o m p a r a b l e with our recent study, in which the intravenous C u Z n - S O D containing liposomes m a r k e d l y suppressed kindled A M convulsion in rats, p r e s u m a b l y due to l i p o s o m e - m e d i a t e d brain u p t a k e of the B B B - i m p e r m e a b l e C u Z n - S O D 17. These S O D - i n d u c e d anticonvul-
sant effects do not conflict with o u r assumption that oxygen radicals participate in a certain aspect of kindling phenomenon. Obviously, further w o r k is n e e d e d but at this point we are suggesting that S O D participates in seizure susceptibility induced by A M kindling and initiation of kindled A M seizure in rats.
1 Chan, P.H. and Fishman, R.A., Transient formation of superoxide radicals in polyunsaturated fatty acid-induced brain swelling, J. Neurochem., 35 (1980) 1004-1007. 2 Dollwet, H.H.A., McNicolas, J.B., Pezeshk, A. and Sorenson, J.R.J., Superoxide dismutase-mimetic activity of antiepileptic drug copper complexes, Trace Element Med., 4 (1987) 13-20. 3 Flamm, E.S., Demopoulos, H.B., Seligman, M.L., Poser, R.G. and Ransohoff, J., Free radicals in cerebral ischemia, Stroke, 9 (1978) 445-447. 4 Freund, T.E, Buzaki, G., Prohaska, O.J., Leon A. and Somogyi, P., Simultaneous recording of local electrical activity, partial oxygen tension and temperature in the rat hippocampus with a chamber-type microelectrode. Effects of anesthesia, ischemia and epilepsy, Neuroscience, 28 (1989) 539-549. 5 Halliwell, B. and Gutteridge, J.M.C., Oxygen radicals and the nervous system, Trends Neurosci., 2 (1985) 22-26. 6 Imanari, T., Hirota, M. and Miyazaki, M., Improved assay method for superoxide dismutase, Igaku No Ayumi (in Japanese), 101 (1977) 496-497. 7 Jerret, S.A., Jefferson, D. and Mengel, C.E., Seizure, H202 and lipid peroxidase in brain during exposure to oxygen under high pressure, Aerospace Med., 44 (1973) 40-44. 8 Kurokouchi, A., Moil, N. and Kumashiro, H., Antiepileptic effects of superoxide dismutase on status epilepticus in rats, lgaku No Ayumi (in Japanese), 152 (1990) 465-466. 9 Leninger-Follert, E. and Lubers, D.W., Behavior of microflow and local PO E of the brain cortex during and after direct electrical stimulation. A contribution of the problem of metabolic regulation of microcirculation in the brain, Eur. J. Physiol., 366 (1976) 39-44. 10 McCord, J.M. and Fridovich, I., Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprein), J. Biol. Chem., 244 (1969) 6049-6055. 11 Nitsch, C., Suzuki, R., Fujiwara, K. and Klatzo, I., Incongruence of regional cerebral blood flow increase and blood-brain
barrier opening in rabbits at the onset of seizures induced by bicuculline methoxypyridoxine, and kainic acid, J. Neurol. Sci., 379 (1985) 165-172. 12 Pinard, E., Tremblay, E., Ben-Aft, Y. and Seylaz, J., Blood flow compensates oxygen demand in the vulnerable CA3 region of the hippocampus during kainate-induced seizures, Neuroscience, 13 (1984) 1039-1049. 13 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. 14 Shingh, R. and Pathak, D.N., Lipid peroxidation and glutathion peroxide, glutathione reductase, superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activities in FeC13induced epileptogenic loci in the rat brain, Epilepsia, 31 (1990) 15-26. 15 Turrens, J.G., Freeman, B.A., Levitt, J.G. and Crapo, J.D., The effect of hyperoxia on superoxide dismutase by lung submitochondrial particles, Arch. Biochem. Biophys., 217 (1982) 401-410. 16 Watson, B.D., Busto, R., Goldberg, W.J., Santio, M., Toshida, S. and Ginsberg, M.D., Lipid peroxidation in vivo induced by reversible global ischemia in rat brain, J. Neurochem., 42 (1984) 268-275. 17 Yokoyama, H., Matsumoto, S., Ishida, S., Mori, N. and Kumashiro, H., Anticonvulsant effect of liposome-entrapped superoxide dismutase on kindled convulsion in rats, Epilepsia, 40 (1989) 647. 18 Yoshida, S., Abe, K., Asano, T., Sano, K., Shimasaki, H. and Ueta, N., Brain free fatty acids, edema and mortality in gerbils subjected to transient, bilateral ischemia, and effect of barbiturate anesthesia, J. Neurochem., 40 (1983) 1278-1286. 19 Yusa, T., Crapo, J.D. and Freeman, B.A., Liposome-mediated augmentation of brain SOD and catalase inhibits CNS 0 2 toxicity, Respir. Environ. Physiol., 57 (1984) 1674-1681.
Supported by grants from the Medical Research Council of Canada to J.A.W.
