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Zonisamide reduces hypoxic-ischemic brain damage in neonatal rats irrespective of its anticonvulsive effect
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European Journal of Pharmacology257 (1994) 131-136
Zonisamide reduces hypoxic-ischemic brain damage in neonatal rats irrespective of its anticonvulsive effect Takahiro Hayakawa *, Yoshihisa Higuchi, Hiroyuki Nigami, Haruo Hattori Department of Pediatrics, Faculty of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan (Received 9 September 1993; revised MS received 23 February 1994; accepted 25 February 1994)
Abstract The neuroprotective effect of a novel anticonvulsant, zonisamide, was investigated in neonatal rats with hypoxic-ischemic brain damage. Rats underwent left carotid ligation followed by hypoxic exposure (8% 0 2) for 2.5 h. When zonisamide (75 mg/kg) was administered i.p. 1 h before hypoxia, it reduced the cortical infarction volume to 6 + 5% (mean + S.E.M.) from 68 + 7% in vehicle-treated controls and the striatal volume to 8 + 4% from 78 + 7%. Zonisamide also reduced neuronal necrosis in 5 hippocampal regions (the dentate gyrus, CA4, CA3, CA1, and the subiculum). The plasma zonisamide concentration before and after hypoxia was 47.9 + 2.0/zg/ml and 42.3 + 3.9/zg/ml, respectively. Epidural electrodes were implanted in 6 pups one day before hypoxia-ischemia. Electroencephalograms were recorded during hypoxia-ischemia in rats given zonisamide or vehicle before the insult. The intensity of seizure activities was similar in the zonisamide-treated pups and the vehicle-treated controls. These findings demonstrate that zonisamide reduces neonatal hypoxic-ischemic brain damage and that this protective effect does not depend on its anticonvulsant action. Key words: Zonisamide; Anticonvulsant; Brain, neonatal, rat; Hypoxia-ischemia, cerebral; Infarction, cerebral; Hypoxic seizure
1. Introduction Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) is a novel anticonvulsant. Its anticonvulsive effect has been demonstrated both in animal seizure models (Masuda et al., 1979; Kamei et al., 1981) and in adults and children with epilepsy, especially those with partial seizures (Sackellares et al., 1985; Wielensky et al., 1985; Kumagai et al., 1991; Seino et al., 1991; Leppik et al., 1993). This article describes the protective effect of zonisamide on neonatal hypoxic-ischemic brain damage and the relationship between its neuroprotection and seizure suppression.
2. Materials and methods 2.1. Hypoxic-ischemic insult Six-day-old Wistar rat pups (the day of birth was defined as day 0) were p r e p a r e d for surgery, and * Corresponding author. Tel. 075-751-3296, fax 075-752-2361. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00130-Y
littermates were divided into 2 groups assigned to zonisamide or vehicle treatment. T h e r e was no difference in sex and body weight ( + 0.5 g) between the two groups. The left common carotid artery was exposed and severed between double ligatures under ether anesthesia. The operation took about 5 min and the pups were allowed to recover for 2.5 h before the hypoxic-ischemic insult. Hypoxic exposure was achieved by placing the pups in an airtight plastic jar submerged in a 37°C water bath that was flushed for 2.5 h with a humidified gas mixture of 8% oxygen and 92% nitrogen. After 1 h of recovery in a 37°C water bath, they were returned to their dams until killing at the age of 9 days. 2.2. Experimental groups The effect of zonisamide was evaluated in two groups that each contained pups from 4 litters. One hour prior to hypoxic exposure, 75 m g / k g of sodium zonisamide (Dainippon Pharmaceutical Co., Japan; n = 21) or the same volume of vehicle (1.5 m l / k g , 40% propylene glycol and 10.5% ethanol, n = 14)was administered i.p.
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T. Hayakawa et al. / European Journal of Pharmacology 257 (1994) 131-136
2.3. Neuropathologic examination
At 9 days of age (72 h after the hypoxic-ischemic insult), the rat pups were anesthetized with pentobarbital and perfusion-fixed with 4% formaldehyde buffered to pH 7. Coronal blocks of brain tissue were processed in a graded ethanol series and xylene. After embedding in paraffin wax, 8-/xm thick coronal sections were cut at every 200-/.~m interval and stained with hematoxylin-eosin. The area of hypoxic-ischemic damage was calculated on 6 coronal levels chosen from a stereotaxic atlas of 9-day-old rat brain (Sherwood and Timiras, 1970). The longitudinal coordinates and anatomical structures of each coronal level were as follows: A7.0 mm, nucleus accumbens; A5.6 mm, caudate-putamen and the anterior commissure; A4.4 m m , globus pallidus; A3.2 ram, anterior tip of the dorsal hippocampus; A2.0 mm, midportion of the dorsal hippocampus; and A0.8 mm, ventral hippocampus. Photographs of all brain sections were enlarged about 10-fold and were input into a personal computer (Macintosh IIcx) using an image scanning system (Color Magician III and DIP station). The magnification was about 2800 pixels per mm 2. All measurements of area were performed at the above-mentioned locations on the 6 coronal levels. The measurements included the cerebral cortex in all 6 coronal planes and the striatum in 2 coronal planes (A5.6 nun and A4.4 mm) as well as the areas showing hypoxic-ischemic damage in the same regions. The percent volume of hypoxic-ischemic necrosis was obtained for the cortex and striatum by dividing the sum of the damaged areas by the sum of the total area of each structure in the ipsilateral hemisphere. The hypoxic-ischemic changes of 5 hippocampal structures (the dentate gyrus, CA4, CA3, CA1, and subiculum) in the dorsal hippocampus in coronal plane A2.0 mm was assessed using a semiquantitative 4-point
scale: 0 = no damage, 1 = occasional eosinophilic neurons ( < 10%), 2 = a moderate number of eosinophilic neurons (10-50%), and 3 = n u m e r o u s eosinophilic neurons ( > 50%). Light microscopic examination was performed by an examiner blinded to the experimental protocol. 2.4. Plasma zonisamide concentration
Following the i.p. injection of zonisamide (75 mg/kg), the pups were decapitated just before hypoxia (1 h after injection; n = 10) and just after the cessation of hypoxia (3.5 h after injection; n = 11), and blood was collected into heparinized plastic tubes. The total plasma concentration of zonisamide was then measured by enzyme immunoassay. 2.5. Core temperature measurement
For measurement of core temperature, the rectal temperature of 6-day-old pups was determined with a probe (Thermo-Finer Termo Co., Japan), at 1 h before 2.5 h of hypoxia, during hypoxia (1.5 h from the start), and 0, 0.5, 1, 2, 3, 4, 24 h after the end of hypoxia. 2.6. E E G examination
Under halothane anesthesia, epidural electrodes for electroencephalography ( E E G ) were implanted in 5day-old rat pups at 1 day before the hypoxic-ischemic insult. Two holes for the electrodes were made on each side of the skull with a dental drill (Minitor, Narishige Scientific Instrument Lab, Japan). The electrodes were placed about 2 mm posterior to the bregma and 1.5 mm lateral to the sagittal suture and were fixed with epoxy resin (Mend-Rex, Nissin Dental Products, Japan). After a 30-min recovery period, the pups were
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Fig. 1. Coronal brain sections showing the middle of the dorsal hippocampus in the A2.0 mm plane accordingto a brain atlas for 9-day-oldrats (Sherwood and Timiras, 1970). (A) is from a vehicle-treated control, and (B) is from a pup given zonisamide 1 h before the hypoxic-ischemic insult. Note the sharply demarcated areas of hypoxic-ischemicinfarction. The neuroprotective effect of zonisamide can be seen in (B). Hematoxylin-eosinstain, original magnification x 10.
T. Hayakawa et aL / European Journal of Pharmacology 257 (1994) 131-136 Cerebral c o r t e x
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Fig. 2. Area (mm2) of hypoxic-ischemicnecrosis in different coronal planes in the cerebral cortex (A) and the striatum (B). There are significant differences (P < 0.01; Mann-Whitney U-test) between the vehicle-treated controls (o) and the zonisamide-treated pups (e) in all coronal planes examined in both the cerebral cortex and the striatum. The data represent the means 5: S.E.M.
r e t u r n e d to their dams. T h e surgery lasted for 1 5 - 2 0 min. A t the age of 6 days, the p u p s were subjected to left carotid ligation followed by hypoxic exposure for 2.5 h as m e n t i o n e d above. Z o n i s a m i d e (75 m g / k g ) or the same v o l u m e of vehicle was a d m i n i s t e r e d i.p. 1 h b e f o r e hypoxia. T h e electrodes were c o n n e c t e d to a n E E G r e c o r d e r ( N i h o n K o h d e n , Japan), a n d bilateral m o n o p o l a r a n d b i p o l a r recordings were o b t a i n e d in a shielded r o o m while the p u p s were m o v i n g freely in a plastic jar. E l e c t r o e n c e p h a l o g r a m s were r e c o r d e d during hypoxia in 3 z o n i s a m i d e - t r e a t e d p u p s a n d 3 v e h i c l e - t r e a t e d controls. D u r i n g hypoxia, the p u p s were observed closely for seizure behavior. T h e location of the electrodes was c o n f i r m e d at 9 days of age after perfusion-fixation.
2.7. E E G analysis T h e e l e c t r o e n c e p h a l o g r a m s were analyzed visually. Hypoxia was divided into t h r e e 5 0 - m i n periods, a n d the E E G c h a n g e s in each p e r i o d were analyzed. T h e d u r a -
Table 1 Percent volume of hypoxic-ischemicbrain damage in the neonate Vehicle Zonisamide
n
Cerebral cortex (%)
Striatum (%)
14 16
68 5:7 65:5 a
78 5:7 85:4 a
Pups received 75 mg/kg zonisamide i.p., or 1.5 ml/kg vehicle 1 h before the hypoxic-ischemicinsult. Zonisamide significantly reduced brain damage both in the cortex and striatum. The data are means 5: S.E.M. a p < 0.01 compared with corresponding vehicle-injected controls (Mann-Whitney U-test).
tion of seizure b e h a v i o r (tonic or tonic-clonic convulsions) c o r r e s p o n d i n g to electrical seizure discharges (bursts of polyspikes or spike-and-wave complexes) was m e a s u r e d a n d the total seizure d u r a t i o n s were s u m m e d to give the seizure time.
2.8. Data analysis T h e results are p r e s e n t e d as the m e a n s + S.E.M. T h e d e a t h rate of the a n i m a l s was e v a l u a t e d by the 2-tailed F i s h e r ' s exact probability test, a n d the signific a n c e of differences in b o d y weight was e v a l u a t e d by the 2-tailed u n p a i r e d S t u d e n t ' s t-test. T h e hypoxicischemic v o l u m e a n d area in the cortex a n d s t r i a t u m as well as the n e u r o n a l d a m a g e score in the h i p p o c a m p u s did n o t show a n o r m a l distribution, so differences were assessed by the 2-tailed M a n n - W h i t n e y U-test. Differences in core t e m p e r a t u r e a n d seizure time were as-
Table 2 Semiquantitative ischemic neuronal score for hippocampal structures in a neonatal hypoxic-ischemicrat model n DG
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Vehicle 14 2.3+0.3 2.3+0.3 2.5:1:0.3 2.45:0.3 2.65:0.2 Zonisamide 16 0.35:0.2 a 0.25:0.1 a 0.35:0.2 a 0.35:0.2 a 0.35:0.2 Rat pups received intraperitoneal zonisamide (75 mg/kg) or vehicle (1.5 ml/kg) at 1 h before the hypoxic-ischemicinsult. Zonisamide significantlyreduced neuronal necrosis in all 5 hippocampal regions. DG: dentate gyrus; SUB: subiculum. The number of eosinophilic neurons in each region was assessed semiquantitatively as follows: 0 = no damage, 1 = < 10%, 2 = 10-50%, and 3 = > 50%. The data are means + S.E.M. a p < 0.01 compared with corresponding vehicle-injected controls (Mann-Whitney U-test).
T. Hayakawa et aL /European Journal of Pharmacology 257 (1994) 131-136
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Fig. 3. Serial measurements of rectal temperature. Rectal temperature did not differ between the vehicle-treated controls (o) and the zonisamide-treated pups (e) at any of the time points measured (2-tailed unpaired Student's t-test). The data represent the means + S.E.M.
sessed by the 2-tailed unpaired Student's t-test. A probability of less than 0.05 was considered to indicate significance.
The cortical and striatal lesions due to hypoxicischemic damage were sharply demarcated and were easily delineated using the image analyzing system. There were also some scattered ischemic neurons outside the major lesions. Table 1 shows the percent volume of hypoxic-ischemic damage in the cerebral cortex and the striatum. Zonisamide reduced the extent of brain damage (Fig. 1), and a protective effect was seen in all coronal sections of both the cerebral cortex (Fig. 2A) and the striatum (Fig. 2B). Table 2 shows hypoxic-ischemic neuronal damage in 5 areas of the hippocampus. Zonisamide diminished neuronal damage in all 5 hippocampal regions.
3.2. Plasma zonisamide level The plasma concentration of zonisamide was 47.9 + 2.0 ~ g / m l at 1 h after injection and 42.3 + 3.9 ~ g / m l at 3.5 h after injection.
3.3. Core temperature 3. Results
The death rate during hypoxic exposure was higher in the zonisamide-treated pups (23.8%, 5/21) than in the vehicle-treated controls (0%, 0/14), but the difference was not statistically significant. No pup died during the subsequent 3 days before killing. Body weight gain did not differ between the 2 groups, with the weight changing from 10.0 + 0.2 g on day 6 to 10.9 -1-0.2 g on day 9 in zonisamide-treated pups (n = 16) and from 10.1 + 0.2 g on day 6 to 11.6 ___0.4 g on day 9 in vehicle-treated controls (n = 14).
The rectal temperature did not differ significantly between the vehicle-treated controls (n = 4) and the zonisamide-treated pups (n = 4) at any of the time points measured (Fig. 3).
3.4. EEG analysis The pups showed almost isoelectric electroencephalograms during hypoxia. Generalized tonic or
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Fig. 4. Representative electroencephalograms obtained during hypoxic-isehemic damage to a vehicle-treated control and a zonisamide-treated pup. Seizure discharges can be seen in both the pups. Arrowheads indicate the beginning and end of the seizures. L, R: a monopolar recording from each hemisphere. L-R: a bipolar recording.
Fig. 5. The seizure time during hypoxia in the vehicle-treated controis (open bar) and the zonisamide-treated pups (hatched bar). Seizure times observed during the entire hypoxic exposure (2.5 h = 150 min) and the three consecutive periods of the hypoxia (I, II, III; 50 min each) are shown. There was no difference of seizure time between the vehicle-treated controls and the zonisamide-treated pups at any period examined (2-tailed unpaired Student's t-test). Seizure activity decreased with time in both groups.
T. Hayakawa et al. / European Journal of Pharmacology 257 (1994) 131-136
tonic-clonic seizures occurred along with bursts of polyspikes or spike-and-wave complexes. Electrical seizure activity was observed equally in both hemispheres regardless of zonisamide treatment (Fig. 4). Fig. 5 shows the total seizure time and the time in each hypoxic period. Seizure time did not differ significantly between the two groups. Most of the seizures were seen in the first period and seizure activity decreased thereafter.
4. Discussion
Zonisamide reduced hypoxic-ischemic brain damage in neonatal rats when administered within the therapeutic plasma range for anticonvulsant activity in rats (Masuda et al., 1979). The cerebral cortex, striatum, and hippocampus were all protected by the drug. However, the death rate was higher in the zonisamidetreated group than in the vehicle-treated control group. Pups given zonisamide probably died of its sedative effect (Masuda et al., 1979), since respiratory depression was seen after zonisamide treatment. Tonic or tonic-clonic seizures were observed with bursts of polyspikes or spike-and-wave complexes in both the vehicle-treated group and the zonisamidetreated group. In rats, seizure activity and E E G changes (such as spike discharges) are more common during short-term hypoxia ( < 10 min) at a younger age (Jensen et al., 1991). Although the plasma concentration of zonisamide was within the anticonvulsant therapeutic range during hypoxia, the total seizure time was not different in the two groups. The plasma concentrations of zonisamide measured in our experiments were higher than the EDs0 for maximum electric shocks in adult rats (Masuda et al., 1979). However, a higher concentration appears to be needed to suppress hypoxic seizures in neonatal rats. In adult mice, a dose of 200 m g / k g of phenytoin was needed to suppress hypoxic convulsions and 50 m g / k g failed to suppress them (Artru and Michenfelder, 1980). Steen and Michenfelder (1978) reported that the suppression of hypoxic seizures was greater by diazepam than mephobarbital in adult mice, while neuroprotection by mephobarbital was greater. Artru and Michenfelder (1980) also reported that diazepam was more effective than phenytoin to suppress hypoxic seizures, but protection against hypoxic brain damage by phenytoin was greater. Thus the neuroprotective effect of antiepileptic drugs does not seem to depend on their anticonvulsant effect. In our study, far less histological brain damage was seen in the zonisamide-treated pups, although they had seizures as frequently as the control pups. It was reported that hypoxic seizures do not cause histological brain damage in neonatal rats (Jensen et al., 1992). Therefore, the protective effect of zonisamide did not
135
depend on its anticonvulsant effect and seizure activity per se does not necessarily play a significant role in hypoxic-ischemic brain damage, as observed in our experiments. A protective effect during hypoxia a n d / o r ischemia has also been reported for some other anticonvulsants, such as diazepam (Steen and Michenfelder, 1978), mephobarbital (Steen and Michenfelder, 1978), pentobarbital (Imaizumi et al., 1988; Araki et al., 1990; Kato et al., 1990), felbamate (Wasterlain et al., 1992), MK-801 (Hattori et al., 1989; Kato et al., 1990) and phenytoin (Cullen et al., 1979; Artru and Michenfelder, 1980; Imaizumi et al., 1988; Panzenbeck et al., 1989; Taft et al., 1989; Boxer et al., 1990; Kinouchi et al., 1990; Hayakawa et al., 1994). Although these drugs are anticonvulsants, a relationship between brain protection and the suppression of hypoxic seizures has seldom been reported (Steen and Michenfelder, 1978; Artru and Michenfelder, 1980). However, as mentioned above, hypoxic seizures do not seem to be the main factor in brain damage. In previous studies, different mechanisms of neuroprotection by anticonvulsants have been proposed for each model used. Since no common mechanism was found, the protective effect against hypoxic brain damage appears to differ for each antiepileptic drug. The neuroprotective mechanism of zonisamide is unclear, but may partly involve the inhibition of calcium-mediated cell injury by blocking calcium channels (Suzuki et al., 1992), blocking sodium channels (Schauf, 1978; Rock et al., 1989), and longlasting increase of rCBF (Tanaka et al., 1992). Hypothermia can reduce the neonatal hypoxic-ischemic brain damage (McDonald et al., 1991), but this could not be a factor in our study because rectal temperature did not differ significantly between the controls and the zonisamide group.
References
Araki, T., H. Kato, K. Kogure and T. Inoue, 1990, Regional neuroprotective effects of pentobarbital on ischemia-induced brain damage, Brain Res. Bull. 25, 861. Artru, A.A. and J.D. Michenfelder, 1980, Cerebral protective, metabolic, and vascular effects of phenytoin, Stroke 11, 377. Boxer, P.A., J.J. Cordon, M.E. Mann, L.C. Rodolosi, M.G. Vartanian, D.M. Rock, C.P. Taylor and F.W. Marcoux, 1990, Comparison of phenytoin with noncompetitive N-methyI-D-aspartate antagonists in a model of focal brain ischemia in rat, Stroke 21 (Suppl. 3), 111-47. Cullen, J.P., J.A. Aldrete, L. Jankovsky and F. Romo-Salas, 1979, Protective action of phenytoin in cerebral ischemia, Anesth. Analg. 58, 165. Hattori, H., A.M. Morin, P.H. Schwartz, D.G. Fujikawa and C.G. Wasterlain, 1989, Posthypoxic treatment with MK-801 reduces hypoxic-ischemicdamage in the neonatal rat, Neurology39, 713. Hayakawa, T., Y. Hamada, T. Maihara, H. Hattori and H. Mikawa, 1994, Phenytoin reduces neonatal hypoxic-ischemicbrain damage in rats, Life Sci. 54, 387.
136
T. Hayakawa et al. / European Journal of Pharmacology 257 (1994) 131-136
Imaizumi, S., J. Suzuki, H. Kinouchi and T. Yoshimoto, 1988, Superior protective effects of phenytoin against hypoxia in a pharmacological screening test, Neurol. Res. 10, 18. Jensen, F.E., C.D. Applegate, D. Holtzman, T.R. Belin and J.L. Burchfiel, 1991, Epileptogenic effect of hypoxia in the immature rodent brain, Ann. Neurol. 29, 629. Jensen, F.E., G.L. Holmes, C.T. Lombroso, H.K. Blume and I.R. Firkusny, 1992, Age-dependent changes in long-term seizure susceptibility and behavior after hypoxia in rats, Epilepsia 33, 971. Kamei, C., M. Oka, Y. Masuda, K. Yoshida and M. Shimizu, 1981, Effects of 3-sulfamoylmethyl-l,2-benzisoxazole (AD-810) and some antiepileptics on the kindled seizures in the neocortex, hippocampus and amygdala in rats, Arch. Int. Pharmacodyn. 249, 164. Kato, H., T. Araki and K. Kogure, 1990, Role of the excitotoxic mechanism in the development of neuronal damage following repeated brief cerebral ischemia in the gerbil: protective effects of MK-801 and pentobarbital, Brain Res. 516, 175. Kinouchi, H., S. Imaizumi, T. Yoshimoto and M. Motomiya, 1990, Phenytoin affects metabolism of free fatty acids and nucleotides in rat cerebral ischemia, Stroke 21, 1326. Kumagai, N., T. Seki, H. Yamawaki, N. Suzuki, S. Kimiya, T. Yamada, M. Hara, R. Hashimoto, Y. Takuma and K. Hirai, 1991, Monotherapy for childhood epilepsies with zonisamide, Jpn. J. Psychiatry Neurol. 45, 357. Leppik, I.E., L.J. Willmore, R.W. Homan, G. Fromm, K.J. Oomem, J.K. Penry, J.C. Sackellares, D.B. Smith, R.P. Lesser and J.D. Wallace, 1993, Efficacy and safety of zonisamide: result of a multicenter study, Epilepsy Res. 14, 165. Masuda, Y., Y. Utsui, Y. Shiraishi, T. Karasawa, K. Yoshida and M. Shimizu, 1979, Relationships between plasma concentrations of diphenylhydantoin, phenobarbital, carbamazepine, and 3sulfamoylmethyl-l,2-benzisoxazole (AD-810), a new anticonvulsant agent, and their anticonvulsant or neurotoxic effects in experimental animals, Epilepsia 20, 623. McDonald, J.W., C.K. Chen, W.H. Trescher and M.V. Johnston, 1991, The severity of excitotoxic brain injury is dependent on brain temperature in immature rat, Neurosci. Lett. 126, 83. Panzenbeck, M.J., P. Coyle, R.G. Barton and J.P. Buyniski, 1989,
Phenytoin and MK-801 improve outcome in an occlusion/reperfusion model of cerebral ischemia in Fischer 344 rats, J. Cereb. Blood Flow Metab. 9 (Suppl. 1), S152. Rock, D.M., R.L. Macdonald and C.P. Taylor, 1989, Blockade of sustained repetitive action potentials in cultured spinal cord neurons by zonisamide (AD810, CI912), a novel anticonvulsant, Epilepsy Res. 3, 138. Sackellares, J.C., P.D. Donofrio, J.G. Wagner, B. Abou-Khalil, S. Berent and K. Aasved-Hoyt, 1985, Pilot study of zonisamide (1,2-benzisoxazole-3-methanesulfonamide) in patients with refractory partial seizures, Epilepsia 26, 206. Schauf, C.L., 1987, Zonisamide enhances slow sodium inactivation in Myxicola, Brain Res. 413, 185. Seino, M., H. Miyazaki and T. Ito, 1991, Zonisamide, in: New Antiepileptic Drugs, eds. F. Pisani, E. Perruca, G. Avanzini and A. Richens, Epilepsy Res. Suppl. 3, 169. Sherwood, N.M. and P.S. Timiras, 1970, A Stereotaxic Atlas of the Developing Rat Brain (University of California Press, Berkeley, CA) p. 15. Steen, P.A. and J.D. Michenfelder, 1978, Cerebral protection with barbiturates; relation to anesthetic effect, Stroke 9, 140. Suzuki, S., K. Kawakami, S. Nishimura, Y. Watanabe, K. Yagi, M. Seino and K. Miyamoto, 1992, Zonisamide blocks T-type calcium channel in cultured neurons of rat cerebral cortex, Epilepsy Res. 12, 21. Taft, W.C., G.L. Clifton, R.E. Blair and R.J. DeLorenzo, 1989, Phenytoin protects against ischemia-prodnced neuronal cell death, Brain Res. 483, 143. Tanaka, T., T. Fujita, S. Tanaka, K. Takano and Y. Yonemasu, 1992, Effect of anticonvulsants upon experimental limbic seizure status and regional cerebral blood flow in the hippocampus, No-ToShinkei 44, 234 [in Japanese]. Wasterlain, C.G., L.M. Adams, H. Hattori and P.H. Schwartz, 1992, Felbamate reduces hypoxic-ischemic brain damage in vivo, Eur. J. Pharmacol. 212, 275. Wielensky, A.J., P.N. Friel, L.M. Ojemann, C.B. Dodrill, K.B. McCormick and R.H. Levy, 1985, Zonisamide in epilepsy: a pilot study, Epilepsia 26, 212.
European Journal of Pharmacology257 (1994) 131-136
Zonisamide reduces hypoxic-ischemic brain damage in neonatal rats irrespective of its anticonvulsive effect Takahiro Hayakawa *, Yoshihisa Higuchi, Hiroyuki Nigami, Haruo Hattori Department of Pediatrics, Faculty of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan (Received 9 September 1993; revised MS received 23 February 1994; accepted 25 February 1994)
Abstract The neuroprotective effect of a novel anticonvulsant, zonisamide, was investigated in neonatal rats with hypoxic-ischemic brain damage. Rats underwent left carotid ligation followed by hypoxic exposure (8% 0 2) for 2.5 h. When zonisamide (75 mg/kg) was administered i.p. 1 h before hypoxia, it reduced the cortical infarction volume to 6 + 5% (mean + S.E.M.) from 68 + 7% in vehicle-treated controls and the striatal volume to 8 + 4% from 78 + 7%. Zonisamide also reduced neuronal necrosis in 5 hippocampal regions (the dentate gyrus, CA4, CA3, CA1, and the subiculum). The plasma zonisamide concentration before and after hypoxia was 47.9 + 2.0/zg/ml and 42.3 + 3.9/zg/ml, respectively. Epidural electrodes were implanted in 6 pups one day before hypoxia-ischemia. Electroencephalograms were recorded during hypoxia-ischemia in rats given zonisamide or vehicle before the insult. The intensity of seizure activities was similar in the zonisamide-treated pups and the vehicle-treated controls. These findings demonstrate that zonisamide reduces neonatal hypoxic-ischemic brain damage and that this protective effect does not depend on its anticonvulsant action. Key words: Zonisamide; Anticonvulsant; Brain, neonatal, rat; Hypoxia-ischemia, cerebral; Infarction, cerebral; Hypoxic seizure
1. Introduction Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) is a novel anticonvulsant. Its anticonvulsive effect has been demonstrated both in animal seizure models (Masuda et al., 1979; Kamei et al., 1981) and in adults and children with epilepsy, especially those with partial seizures (Sackellares et al., 1985; Wielensky et al., 1985; Kumagai et al., 1991; Seino et al., 1991; Leppik et al., 1993). This article describes the protective effect of zonisamide on neonatal hypoxic-ischemic brain damage and the relationship between its neuroprotection and seizure suppression.
2. Materials and methods 2.1. Hypoxic-ischemic insult Six-day-old Wistar rat pups (the day of birth was defined as day 0) were p r e p a r e d for surgery, and * Corresponding author. Tel. 075-751-3296, fax 075-752-2361. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00130-Y
littermates were divided into 2 groups assigned to zonisamide or vehicle treatment. T h e r e was no difference in sex and body weight ( + 0.5 g) between the two groups. The left common carotid artery was exposed and severed between double ligatures under ether anesthesia. The operation took about 5 min and the pups were allowed to recover for 2.5 h before the hypoxic-ischemic insult. Hypoxic exposure was achieved by placing the pups in an airtight plastic jar submerged in a 37°C water bath that was flushed for 2.5 h with a humidified gas mixture of 8% oxygen and 92% nitrogen. After 1 h of recovery in a 37°C water bath, they were returned to their dams until killing at the age of 9 days. 2.2. Experimental groups The effect of zonisamide was evaluated in two groups that each contained pups from 4 litters. One hour prior to hypoxic exposure, 75 m g / k g of sodium zonisamide (Dainippon Pharmaceutical Co., Japan; n = 21) or the same volume of vehicle (1.5 m l / k g , 40% propylene glycol and 10.5% ethanol, n = 14)was administered i.p.
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T. Hayakawa et al. / European Journal of Pharmacology 257 (1994) 131-136
2.3. Neuropathologic examination
At 9 days of age (72 h after the hypoxic-ischemic insult), the rat pups were anesthetized with pentobarbital and perfusion-fixed with 4% formaldehyde buffered to pH 7. Coronal blocks of brain tissue were processed in a graded ethanol series and xylene. After embedding in paraffin wax, 8-/xm thick coronal sections were cut at every 200-/.~m interval and stained with hematoxylin-eosin. The area of hypoxic-ischemic damage was calculated on 6 coronal levels chosen from a stereotaxic atlas of 9-day-old rat brain (Sherwood and Timiras, 1970). The longitudinal coordinates and anatomical structures of each coronal level were as follows: A7.0 mm, nucleus accumbens; A5.6 mm, caudate-putamen and the anterior commissure; A4.4 m m , globus pallidus; A3.2 ram, anterior tip of the dorsal hippocampus; A2.0 mm, midportion of the dorsal hippocampus; and A0.8 mm, ventral hippocampus. Photographs of all brain sections were enlarged about 10-fold and were input into a personal computer (Macintosh IIcx) using an image scanning system (Color Magician III and DIP station). The magnification was about 2800 pixels per mm 2. All measurements of area were performed at the above-mentioned locations on the 6 coronal levels. The measurements included the cerebral cortex in all 6 coronal planes and the striatum in 2 coronal planes (A5.6 nun and A4.4 mm) as well as the areas showing hypoxic-ischemic damage in the same regions. The percent volume of hypoxic-ischemic necrosis was obtained for the cortex and striatum by dividing the sum of the damaged areas by the sum of the total area of each structure in the ipsilateral hemisphere. The hypoxic-ischemic changes of 5 hippocampal structures (the dentate gyrus, CA4, CA3, CA1, and subiculum) in the dorsal hippocampus in coronal plane A2.0 mm was assessed using a semiquantitative 4-point
scale: 0 = no damage, 1 = occasional eosinophilic neurons ( < 10%), 2 = a moderate number of eosinophilic neurons (10-50%), and 3 = n u m e r o u s eosinophilic neurons ( > 50%). Light microscopic examination was performed by an examiner blinded to the experimental protocol. 2.4. Plasma zonisamide concentration
Following the i.p. injection of zonisamide (75 mg/kg), the pups were decapitated just before hypoxia (1 h after injection; n = 10) and just after the cessation of hypoxia (3.5 h after injection; n = 11), and blood was collected into heparinized plastic tubes. The total plasma concentration of zonisamide was then measured by enzyme immunoassay. 2.5. Core temperature measurement
For measurement of core temperature, the rectal temperature of 6-day-old pups was determined with a probe (Thermo-Finer Termo Co., Japan), at 1 h before 2.5 h of hypoxia, during hypoxia (1.5 h from the start), and 0, 0.5, 1, 2, 3, 4, 24 h after the end of hypoxia. 2.6. E E G examination
Under halothane anesthesia, epidural electrodes for electroencephalography ( E E G ) were implanted in 5day-old rat pups at 1 day before the hypoxic-ischemic insult. Two holes for the electrodes were made on each side of the skull with a dental drill (Minitor, Narishige Scientific Instrument Lab, Japan). The electrodes were placed about 2 mm posterior to the bregma and 1.5 mm lateral to the sagittal suture and were fixed with epoxy resin (Mend-Rex, Nissin Dental Products, Japan). After a 30-min recovery period, the pups were
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Fig. 1. Coronal brain sections showing the middle of the dorsal hippocampus in the A2.0 mm plane accordingto a brain atlas for 9-day-oldrats (Sherwood and Timiras, 1970). (A) is from a vehicle-treated control, and (B) is from a pup given zonisamide 1 h before the hypoxic-ischemic insult. Note the sharply demarcated areas of hypoxic-ischemicinfarction. The neuroprotective effect of zonisamide can be seen in (B). Hematoxylin-eosinstain, original magnification x 10.
T. Hayakawa et aL / European Journal of Pharmacology 257 (1994) 131-136 Cerebral c o r t e x
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Fig. 2. Area (mm2) of hypoxic-ischemicnecrosis in different coronal planes in the cerebral cortex (A) and the striatum (B). There are significant differences (P < 0.01; Mann-Whitney U-test) between the vehicle-treated controls (o) and the zonisamide-treated pups (e) in all coronal planes examined in both the cerebral cortex and the striatum. The data represent the means 5: S.E.M.
r e t u r n e d to their dams. T h e surgery lasted for 1 5 - 2 0 min. A t the age of 6 days, the p u p s were subjected to left carotid ligation followed by hypoxic exposure for 2.5 h as m e n t i o n e d above. Z o n i s a m i d e (75 m g / k g ) or the same v o l u m e of vehicle was a d m i n i s t e r e d i.p. 1 h b e f o r e hypoxia. T h e electrodes were c o n n e c t e d to a n E E G r e c o r d e r ( N i h o n K o h d e n , Japan), a n d bilateral m o n o p o l a r a n d b i p o l a r recordings were o b t a i n e d in a shielded r o o m while the p u p s were m o v i n g freely in a plastic jar. E l e c t r o e n c e p h a l o g r a m s were r e c o r d e d during hypoxia in 3 z o n i s a m i d e - t r e a t e d p u p s a n d 3 v e h i c l e - t r e a t e d controls. D u r i n g hypoxia, the p u p s were observed closely for seizure behavior. T h e location of the electrodes was c o n f i r m e d at 9 days of age after perfusion-fixation.
2.7. E E G analysis T h e e l e c t r o e n c e p h a l o g r a m s were analyzed visually. Hypoxia was divided into t h r e e 5 0 - m i n periods, a n d the E E G c h a n g e s in each p e r i o d were analyzed. T h e d u r a -
Table 1 Percent volume of hypoxic-ischemicbrain damage in the neonate Vehicle Zonisamide
n
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Striatum (%)
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Pups received 75 mg/kg zonisamide i.p., or 1.5 ml/kg vehicle 1 h before the hypoxic-ischemicinsult. Zonisamide significantly reduced brain damage both in the cortex and striatum. The data are means 5: S.E.M. a p < 0.01 compared with corresponding vehicle-injected controls (Mann-Whitney U-test).
tion of seizure b e h a v i o r (tonic or tonic-clonic convulsions) c o r r e s p o n d i n g to electrical seizure discharges (bursts of polyspikes or spike-and-wave complexes) was m e a s u r e d a n d the total seizure d u r a t i o n s were s u m m e d to give the seizure time.
2.8. Data analysis T h e results are p r e s e n t e d as the m e a n s + S.E.M. T h e d e a t h rate of the a n i m a l s was e v a l u a t e d by the 2-tailed F i s h e r ' s exact probability test, a n d the signific a n c e of differences in b o d y weight was e v a l u a t e d by the 2-tailed u n p a i r e d S t u d e n t ' s t-test. T h e hypoxicischemic v o l u m e a n d area in the cortex a n d s t r i a t u m as well as the n e u r o n a l d a m a g e score in the h i p p o c a m p u s did n o t show a n o r m a l distribution, so differences were assessed by the 2-tailed M a n n - W h i t n e y U-test. Differences in core t e m p e r a t u r e a n d seizure time were as-
Table 2 Semiquantitative ischemic neuronal score for hippocampal structures in a neonatal hypoxic-ischemicrat model n DG
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Vehicle 14 2.3+0.3 2.3+0.3 2.5:1:0.3 2.45:0.3 2.65:0.2 Zonisamide 16 0.35:0.2 a 0.25:0.1 a 0.35:0.2 a 0.35:0.2 a 0.35:0.2 Rat pups received intraperitoneal zonisamide (75 mg/kg) or vehicle (1.5 ml/kg) at 1 h before the hypoxic-ischemicinsult. Zonisamide significantlyreduced neuronal necrosis in all 5 hippocampal regions. DG: dentate gyrus; SUB: subiculum. The number of eosinophilic neurons in each region was assessed semiquantitatively as follows: 0 = no damage, 1 = < 10%, 2 = 10-50%, and 3 = > 50%. The data are means + S.E.M. a p < 0.01 compared with corresponding vehicle-injected controls (Mann-Whitney U-test).
T. Hayakawa et aL /European Journal of Pharmacology 257 (1994) 131-136
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Fig. 3. Serial measurements of rectal temperature. Rectal temperature did not differ between the vehicle-treated controls (o) and the zonisamide-treated pups (e) at any of the time points measured (2-tailed unpaired Student's t-test). The data represent the means + S.E.M.
sessed by the 2-tailed unpaired Student's t-test. A probability of less than 0.05 was considered to indicate significance.
The cortical and striatal lesions due to hypoxicischemic damage were sharply demarcated and were easily delineated using the image analyzing system. There were also some scattered ischemic neurons outside the major lesions. Table 1 shows the percent volume of hypoxic-ischemic damage in the cerebral cortex and the striatum. Zonisamide reduced the extent of brain damage (Fig. 1), and a protective effect was seen in all coronal sections of both the cerebral cortex (Fig. 2A) and the striatum (Fig. 2B). Table 2 shows hypoxic-ischemic neuronal damage in 5 areas of the hippocampus. Zonisamide diminished neuronal damage in all 5 hippocampal regions.
3.2. Plasma zonisamide level The plasma concentration of zonisamide was 47.9 + 2.0 ~ g / m l at 1 h after injection and 42.3 + 3.9 ~ g / m l at 3.5 h after injection.
3.3. Core temperature 3. Results
The death rate during hypoxic exposure was higher in the zonisamide-treated pups (23.8%, 5/21) than in the vehicle-treated controls (0%, 0/14), but the difference was not statistically significant. No pup died during the subsequent 3 days before killing. Body weight gain did not differ between the 2 groups, with the weight changing from 10.0 + 0.2 g on day 6 to 10.9 -1-0.2 g on day 9 in zonisamide-treated pups (n = 16) and from 10.1 + 0.2 g on day 6 to 11.6 ___0.4 g on day 9 in vehicle-treated controls (n = 14).
The rectal temperature did not differ significantly between the vehicle-treated controls (n = 4) and the zonisamide-treated pups (n = 4) at any of the time points measured (Fig. 3).
3.4. EEG analysis The pups showed almost isoelectric electroencephalograms during hypoxia. Generalized tonic or
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Fig. 4. Representative electroencephalograms obtained during hypoxic-isehemic damage to a vehicle-treated control and a zonisamide-treated pup. Seizure discharges can be seen in both the pups. Arrowheads indicate the beginning and end of the seizures. L, R: a monopolar recording from each hemisphere. L-R: a bipolar recording.
Fig. 5. The seizure time during hypoxia in the vehicle-treated controis (open bar) and the zonisamide-treated pups (hatched bar). Seizure times observed during the entire hypoxic exposure (2.5 h = 150 min) and the three consecutive periods of the hypoxia (I, II, III; 50 min each) are shown. There was no difference of seizure time between the vehicle-treated controls and the zonisamide-treated pups at any period examined (2-tailed unpaired Student's t-test). Seizure activity decreased with time in both groups.
T. Hayakawa et al. / European Journal of Pharmacology 257 (1994) 131-136
tonic-clonic seizures occurred along with bursts of polyspikes or spike-and-wave complexes. Electrical seizure activity was observed equally in both hemispheres regardless of zonisamide treatment (Fig. 4). Fig. 5 shows the total seizure time and the time in each hypoxic period. Seizure time did not differ significantly between the two groups. Most of the seizures were seen in the first period and seizure activity decreased thereafter.
4. Discussion
Zonisamide reduced hypoxic-ischemic brain damage in neonatal rats when administered within the therapeutic plasma range for anticonvulsant activity in rats (Masuda et al., 1979). The cerebral cortex, striatum, and hippocampus were all protected by the drug. However, the death rate was higher in the zonisamidetreated group than in the vehicle-treated control group. Pups given zonisamide probably died of its sedative effect (Masuda et al., 1979), since respiratory depression was seen after zonisamide treatment. Tonic or tonic-clonic seizures were observed with bursts of polyspikes or spike-and-wave complexes in both the vehicle-treated group and the zonisamidetreated group. In rats, seizure activity and E E G changes (such as spike discharges) are more common during short-term hypoxia ( < 10 min) at a younger age (Jensen et al., 1991). Although the plasma concentration of zonisamide was within the anticonvulsant therapeutic range during hypoxia, the total seizure time was not different in the two groups. The plasma concentrations of zonisamide measured in our experiments were higher than the EDs0 for maximum electric shocks in adult rats (Masuda et al., 1979). However, a higher concentration appears to be needed to suppress hypoxic seizures in neonatal rats. In adult mice, a dose of 200 m g / k g of phenytoin was needed to suppress hypoxic convulsions and 50 m g / k g failed to suppress them (Artru and Michenfelder, 1980). Steen and Michenfelder (1978) reported that the suppression of hypoxic seizures was greater by diazepam than mephobarbital in adult mice, while neuroprotection by mephobarbital was greater. Artru and Michenfelder (1980) also reported that diazepam was more effective than phenytoin to suppress hypoxic seizures, but protection against hypoxic brain damage by phenytoin was greater. Thus the neuroprotective effect of antiepileptic drugs does not seem to depend on their anticonvulsant effect. In our study, far less histological brain damage was seen in the zonisamide-treated pups, although they had seizures as frequently as the control pups. It was reported that hypoxic seizures do not cause histological brain damage in neonatal rats (Jensen et al., 1992). Therefore, the protective effect of zonisamide did not
135
depend on its anticonvulsant effect and seizure activity per se does not necessarily play a significant role in hypoxic-ischemic brain damage, as observed in our experiments. A protective effect during hypoxia a n d / o r ischemia has also been reported for some other anticonvulsants, such as diazepam (Steen and Michenfelder, 1978), mephobarbital (Steen and Michenfelder, 1978), pentobarbital (Imaizumi et al., 1988; Araki et al., 1990; Kato et al., 1990), felbamate (Wasterlain et al., 1992), MK-801 (Hattori et al., 1989; Kato et al., 1990) and phenytoin (Cullen et al., 1979; Artru and Michenfelder, 1980; Imaizumi et al., 1988; Panzenbeck et al., 1989; Taft et al., 1989; Boxer et al., 1990; Kinouchi et al., 1990; Hayakawa et al., 1994). Although these drugs are anticonvulsants, a relationship between brain protection and the suppression of hypoxic seizures has seldom been reported (Steen and Michenfelder, 1978; Artru and Michenfelder, 1980). However, as mentioned above, hypoxic seizures do not seem to be the main factor in brain damage. In previous studies, different mechanisms of neuroprotection by anticonvulsants have been proposed for each model used. Since no common mechanism was found, the protective effect against hypoxic brain damage appears to differ for each antiepileptic drug. The neuroprotective mechanism of zonisamide is unclear, but may partly involve the inhibition of calcium-mediated cell injury by blocking calcium channels (Suzuki et al., 1992), blocking sodium channels (Schauf, 1978; Rock et al., 1989), and longlasting increase of rCBF (Tanaka et al., 1992). Hypothermia can reduce the neonatal hypoxic-ischemic brain damage (McDonald et al., 1991), but this could not be a factor in our study because rectal temperature did not differ significantly between the controls and the zonisamide group.
References
Araki, T., H. Kato, K. Kogure and T. Inoue, 1990, Regional neuroprotective effects of pentobarbital on ischemia-induced brain damage, Brain Res. Bull. 25, 861. Artru, A.A. and J.D. Michenfelder, 1980, Cerebral protective, metabolic, and vascular effects of phenytoin, Stroke 11, 377. Boxer, P.A., J.J. Cordon, M.E. Mann, L.C. Rodolosi, M.G. Vartanian, D.M. Rock, C.P. Taylor and F.W. Marcoux, 1990, Comparison of phenytoin with noncompetitive N-methyI-D-aspartate antagonists in a model of focal brain ischemia in rat, Stroke 21 (Suppl. 3), 111-47. Cullen, J.P., J.A. Aldrete, L. Jankovsky and F. Romo-Salas, 1979, Protective action of phenytoin in cerebral ischemia, Anesth. Analg. 58, 165. Hattori, H., A.M. Morin, P.H. Schwartz, D.G. Fujikawa and C.G. Wasterlain, 1989, Posthypoxic treatment with MK-801 reduces hypoxic-ischemicdamage in the neonatal rat, Neurology39, 713. Hayakawa, T., Y. Hamada, T. Maihara, H. Hattori and H. Mikawa, 1994, Phenytoin reduces neonatal hypoxic-ischemicbrain damage in rats, Life Sci. 54, 387.
136
T. Hayakawa et al. / European Journal of Pharmacology 257 (1994) 131-136
Imaizumi, S., J. Suzuki, H. Kinouchi and T. Yoshimoto, 1988, Superior protective effects of phenytoin against hypoxia in a pharmacological screening test, Neurol. Res. 10, 18. Jensen, F.E., C.D. Applegate, D. Holtzman, T.R. Belin and J.L. Burchfiel, 1991, Epileptogenic effect of hypoxia in the immature rodent brain, Ann. Neurol. 29, 629. Jensen, F.E., G.L. Holmes, C.T. Lombroso, H.K. Blume and I.R. Firkusny, 1992, Age-dependent changes in long-term seizure susceptibility and behavior after hypoxia in rats, Epilepsia 33, 971. Kamei, C., M. Oka, Y. Masuda, K. Yoshida and M. Shimizu, 1981, Effects of 3-sulfamoylmethyl-l,2-benzisoxazole (AD-810) and some antiepileptics on the kindled seizures in the neocortex, hippocampus and amygdala in rats, Arch. Int. Pharmacodyn. 249, 164. Kato, H., T. Araki and K. Kogure, 1990, Role of the excitotoxic mechanism in the development of neuronal damage following repeated brief cerebral ischemia in the gerbil: protective effects of MK-801 and pentobarbital, Brain Res. 516, 175. Kinouchi, H., S. Imaizumi, T. Yoshimoto and M. Motomiya, 1990, Phenytoin affects metabolism of free fatty acids and nucleotides in rat cerebral ischemia, Stroke 21, 1326. Kumagai, N., T. Seki, H. Yamawaki, N. Suzuki, S. Kimiya, T. Yamada, M. Hara, R. Hashimoto, Y. Takuma and K. Hirai, 1991, Monotherapy for childhood epilepsies with zonisamide, Jpn. J. Psychiatry Neurol. 45, 357. Leppik, I.E., L.J. Willmore, R.W. Homan, G. Fromm, K.J. Oomem, J.K. Penry, J.C. Sackellares, D.B. Smith, R.P. Lesser and J.D. Wallace, 1993, Efficacy and safety of zonisamide: result of a multicenter study, Epilepsy Res. 14, 165. Masuda, Y., Y. Utsui, Y. Shiraishi, T. Karasawa, K. Yoshida and M. Shimizu, 1979, Relationships between plasma concentrations of diphenylhydantoin, phenobarbital, carbamazepine, and 3sulfamoylmethyl-l,2-benzisoxazole (AD-810), a new anticonvulsant agent, and their anticonvulsant or neurotoxic effects in experimental animals, Epilepsia 20, 623. McDonald, J.W., C.K. Chen, W.H. Trescher and M.V. Johnston, 1991, The severity of excitotoxic brain injury is dependent on brain temperature in immature rat, Neurosci. Lett. 126, 83. Panzenbeck, M.J., P. Coyle, R.G. Barton and J.P. Buyniski, 1989,
Phenytoin and MK-801 improve outcome in an occlusion/reperfusion model of cerebral ischemia in Fischer 344 rats, J. Cereb. Blood Flow Metab. 9 (Suppl. 1), S152. Rock, D.M., R.L. Macdonald and C.P. Taylor, 1989, Blockade of sustained repetitive action potentials in cultured spinal cord neurons by zonisamide (AD810, CI912), a novel anticonvulsant, Epilepsy Res. 3, 138. Sackellares, J.C., P.D. Donofrio, J.G. Wagner, B. Abou-Khalil, S. Berent and K. Aasved-Hoyt, 1985, Pilot study of zonisamide (1,2-benzisoxazole-3-methanesulfonamide) in patients with refractory partial seizures, Epilepsia 26, 206. Schauf, C.L., 1987, Zonisamide enhances slow sodium inactivation in Myxicola, Brain Res. 413, 185. Seino, M., H. Miyazaki and T. Ito, 1991, Zonisamide, in: New Antiepileptic Drugs, eds. F. Pisani, E. Perruca, G. Avanzini and A. Richens, Epilepsy Res. Suppl. 3, 169. Sherwood, N.M. and P.S. Timiras, 1970, A Stereotaxic Atlas of the Developing Rat Brain (University of California Press, Berkeley, CA) p. 15. Steen, P.A. and J.D. Michenfelder, 1978, Cerebral protection with barbiturates; relation to anesthetic effect, Stroke 9, 140. Suzuki, S., K. Kawakami, S. Nishimura, Y. Watanabe, K. Yagi, M. Seino and K. Miyamoto, 1992, Zonisamide blocks T-type calcium channel in cultured neurons of rat cerebral cortex, Epilepsy Res. 12, 21. Taft, W.C., G.L. Clifton, R.E. Blair and R.J. DeLorenzo, 1989, Phenytoin protects against ischemia-prodnced neuronal cell death, Brain Res. 483, 143. Tanaka, T., T. Fujita, S. Tanaka, K. Takano and Y. Yonemasu, 1992, Effect of anticonvulsants upon experimental limbic seizure status and regional cerebral blood flow in the hippocampus, No-ToShinkei 44, 234 [in Japanese]. Wasterlain, C.G., L.M. Adams, H. Hattori and P.H. Schwartz, 1992, Felbamate reduces hypoxic-ischemic brain damage in vivo, Eur. J. Pharmacol. 212, 275. Wielensky, A.J., P.N. Friel, L.M. Ojemann, C.B. Dodrill, K.B. McCormick and R.H. Levy, 1985, Zonisamide in epilepsy: a pilot study, Epilepsia 26, 212.