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Electrocorticographic characterization of chronic iron-induced epilepsy in rats
Neuroscience Letter,s, 1I0 (1990) 72 76 Elsevier Scientific Publishers Ireland Ltd.
72
NSL 06664
Electrocorticographic characterization of chronic iron-induced epilepsy in rats A k i y o s h i M o r i w a k i , Y u k i o H a t t o r i , N o b u y o s h i Nishida and Y a s u o Hori Department of Physiology, Okayama University Medical School, Okayama (Japan) (Received I August 1989; Revised version received 16 October 1989; Accepted 18 October 1989)
Key words: Iron-induced epilepsy; Cerebral cortex; Electrocorticogram; Isolated spike; Spike and wave complex; Rat Electrocorticograms were recorded from rats which were unilaterally injected with ferrous chloride solution into the sensorimotor cortex to induce chronic epileptic activity. All of the iron-injected rats showed isolated spikes near the injection site and in the contralateral cortex immediately after the injection. The injection produced 3 kinds of responses in the rats according to the frequency of the isolated spikes. Spike and wave complexes appeared bilaterally approximately 30 days or more after the injection in the rats in which the frequency of the isolated spikes was dominant on the side ipsilateral to the injection site or nearly equal on the two sides. These results suggest that there are at least 2 stages in the development of chronic iron-induced epilepsy.
Chronic or acute epileptiform activity has been reported to be induced by intracortical injection of FeC12 or FeCl3 solution [7, 9]. One of the distinctive features of ironinduced epilepsy is that epileptic activity lasts for three months or more. Little is known about the development of chronic iron-induced epilepsy, although electrocorticographic characteristics [8, 9] and extracellular activity of cortical neurons [3] have been reported in acute iron-induced epilepsy. There have been no reports concerning alterations in electrocorticograms (ECoGs) in chronic iron-induced epilepsy. In the present study, ECoGs were recorded from iron-induced epileptic rats for 12 months or more after a unilateral injection of FeCI2 solution into the sensorimotor cortex, and alterations of electrographic features of epilepsy were characterized in relation to the development and propagation of spike activity with time. Two hundred and fifty-five male Wistar rats weighing 230 to 280 g were used. The surgical procedures were essentially the same as those of Willmore et al. [8, 9]. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/ kg). The needle of a microsyringe was inserted through the small trephine hole which was made in the cranial bone over the left sensorimotor cortex at a point 1.5 mm rostral and 3.5 mm lateral to bregma. For experimental rats, 5/A of 100 mM FeCI2 Correspondence: A. Moriwaki, Department of Physiology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan. 0304-3940/90/$ 03.50 (~) 1990 Elsevier Scientific Publishers Ireland Ltd.
73
A
B
1 - 7 ~+,,,+.,,+,-~
C
~l,,++,.,x+~.
~,,,++.+.+w+~
2-7
3-7 J
4- 7 ~
~
~
~v~+,,+'~"4,'++++,'+'~++-'',
5- 7 ~
~,~'+'++++'++~
+'+'~'+'~¢~%~"+'~
6- 7 ~
++.,,,.,-+v,~,+~
~.~'~,++~
D
1-7 a-7 4
-
5--7 ~ 6-7
~
7
~ 200 pV
;~eo
Fig. 1. Electrocorticograms recorded from rats after an intracortical injection of FeC12 solution. A - C are the electrocorticographic tracings to show isolated spikes appearing ipsilaterally, bilaterally and contralaterally to the injection site, respectively, which were recorded 9 days after the injection. D is the electrocorticographic tracings to show spike and wave complexes recorded 46 days after the injection. In schematic diagram, a dot indicates the injection site and numbers 1 7 indicate the position of the recording electrodes.
solution was injected into the cortex slowly. For control rats, an equal volume of saline was injected in the same way. Seven stainless-steel electrodes for E C o G recordings were implanted bilaterally in the cranial bone. The different electrodes were situated 3 m m rostral and 3 m m lateral, 3.5 m m lateral, and 4 m m caudal and 4 m m lateral to the bregma on both sides. The indifferent electrode was situated on the midline of the nasal bone. All the electrodes were fixed tightly to the bone with a dental resin. E C o G s were recorded immediately after the completion of the surgical procedure, and recordings were repeated three times a week for at least 12 months. Each recording session was continued for at least 3 h without interruption. The frequency of isolated spikes was determined from the isolated spikes recorded from the electrodes on the ipsilateral or contralateral cortex. Representative examples of electrocorticographic tracings obtained after an intracortical injection of FeCI2 solution are shown in Fig. 1. The epileptic discharges were
74
isolated spikes and spike and wave complexes, In all of the experimental rats, the isolated spikes with an amplitude of 20(~800/tV appeared ipsilaterally (Fig. IA), bilaterally (Fig. 1B), or contralaterally (Fig. I C) to the injection site within 1 h after the injection. In some of the experimental rats, the spike and wave complexes with an amplitude of 200-1000 #V and a spike frequency of 7-40 Hz appeared bilaterally approximately 30 days or more after the injection (Fig. 1D). The rats which showed only isolated spikes did not exhibit any abnormal behavior, but most of the rats which showed spike and wave complexes exhibited head nodding and vibrissa tremors during the period of spike and wave complexes. Saline-injected control rats showed no abnormalities in their ECoGs or behavior. Rats showing only isolated spikes were divided into three groups according to their spike frequency on the two sides of the cortex: group L, the spike frequency on the left side (injected side) was twice or more than that on the right side; group N, the frequency on the dominant side was less than twice that on the opposite side; group R, the frequency on the right side was twice or more than that on the left side. The ratios of spike frequency were unchanged in each rat in these groups. Rats showing spike and wave complexes were classified into group SW. Time-dependent changes in the isolated spike frequency on the two hemispheres in groups L, N, and R are shown in Fig. 2. The isolated spikes appeared asynchronously in groups L and R, but almost synchronously in group N. In groups L and R, the isolated spike frequency increased steeply up to 30 to 60 days after the injection, and thereafter was almost constant on both sides up to at least 90 days after
4
N
{...
2
,
0 0
30 60 9 0 0
30 60 9 0 0
0
30 60 90
Days after injection Fig. 2. Development in frequency of the isolated spikes in rats after an injection of FeClz solution. The number of isolated spikes per min in the cortex near the injection site (©) and on the contralateral homotopic cortex ( • ) were determined in electrocorticograms recorded after the injection. L, N and R indicate the groups of rats: group L, spike frequency on the left side was twice or more than that on the right side; group N, spike frequency on the dominant side was less than twice that on the opposite side; group R, spike frequency on the right side was twice or more than that on the left side. Each value represents the mean of 9-t7 different rats.
75 TABLE I CHANGES IN THE NUMBER OF RATS IN THE DIFFERENT GROUPS OF RATS Each value represents the number of rats in four groups from a total of 247 rats. L, N and R indicate the groups of rats described in Fig. 2. SW indicates the group of rats which showed spike and wave complexes. Number of rats
Days after injection
30 Days 90 Days
L
N
R
SW
71 47
67 30
104 101
5 69
the injection. The rats in group N also showed an increasing frequency up to 30 days after the injection, and then the increased frequency fell gradually. The number of rats in each group at two different stages after the injection is shown in Table I. The number of rats decreased in groups L and N at the progressive stages, while it was virtually unchanged in group R throughout the stages. The number of rats in group SW increased markedly from 30 to 90 days after the injection. The spike and wave complexes were observed in 28% of the iron-injected rats 90 days after the injection. Furthermore, 36% and 38 % of the rats showed spike and wave complexes 180 and 360 days after the injection, respectively (data not shown). The results of this study indicate that isolated spikes appeared not only in the cortex near the injection site but also in the contralateral cortex. The appearance of isolated spikes in the cortex near the injection site is thought to be responsible for induction of the primary epileptic focus in the cortical tissue adjacent to the injection site, in which peroxidation of lipids is suggested to occur in neuronal plasma membranes of the cortex [5, 10]. The appearance of isolated spikes in the contralateral cortex seems to be responsible for the formation of the secondary epileptic focus. Tada et al. [4] also found that iron-induced spike activity transferred easily to the contralateral hemisphere. The formation of the secondary focus may be due to transcallosal propagation of metal ions and/or subcortical neuronal connections, as has been suggested in other experimental models of epilepsy [2, 6]. Spike and wave complexes appeared approximately 30 days or more after the injection, and the number of rats which showed spike and wave complexes increased up to 90 days after the injection. Concurrent with this period, decreases in the number of rats in groups L and N occurred, although the number of rats in group R was unchanged. The dominant side of isolated spike frequency in individual rats did not change. These results clearly indicate that spike and wave complex activity developed in the rats in groups L and N, but not in the rats in group R. In this study, it should be noted that in group N the isolated spike activity fell in the rats which did not show spike and wave complexes. These results suggest that another alteration occurs within the cortical tissue adjacent to the injection site, and that the alteration participates in the development of isolated spikes into spike and wave complexes. Thus,
76 retention of higher isolated spike activity in the injected hemisphere is p r o b a b l y essential for the d e v e l o p m e n t o f spike a n d wave complexes. In view of the electrocorticographic characteristics, it is likely that there are at least two stages in the d e v e l o p m e n t of chronic i r o n - i n d u c e d epilepsy. The first stage is a period from the time immediately after to a p p r o x i m a t e l y 30 days after the injection, at which time isolated spikes a p p e a r a n d the frequency of isolated spikes increases. The second stage is a period a p p r o x i m a t e l y 30 days or more after the injection, at which time spike a n d wave complexes appear a n d this is activity sustained. O u r previous findings [I], which indicated alterations in cyclic A M P generation in the cortex of rats showing isolated spikes or spike a n d wave complexes, support this hypothesis. At the t u r n i n g point of these epileptic stages, an improved alteration is likely to be responsible for the spike a n d wave complexes p r o b a b l y in the epileptic cortex. 1 Hattori, Y., Moriwaki, A., Hayashi, Y. and Hori, Y., Regional difference in responsivenessof norepinephrine-sensitivecyclic AMP-generating systems of rat cerebral cortex with iron-induced epileptic activity, J. Neurochem., in press. 2 Kudo, T. and Yamauchi, T., An ontogenic study of amygdala seizures induced by penicillin in rats, Exp. Neurol., 99 (1988) 531. 543. 3 Reid, S.A. and Sypert, G.W., Acute FeC13-inducedepileptogenicfoci in cats: electrophysiologicalanalyses, Brain Res., 188 (1980) 531 542. 4 Tada, T., Sakaki, T., Tanikake, T., Miyamoto, S., Utsumi, S., Yamamoto, H. and Hori, Y., A trial to produce acute and chronic focal epileptic models with subpial injection of ferrous chloride solution, Folia Psychiatr. Neurol. Jpn., 34 (1980) 367 368. 5 Triggs, W.J. and Willmore, L.J., In vivo lipid peroxidation in rat brain following intracortical Fe2÷ injection, J. Neurochem., 42 (1984) 976-980. 6 Trottier, S., Truchet, M. and Laroudie, C., Secondary ion microanalysis in the study of cobalt-induced epilepsy in the rat, Exp. Neurol., 76 (1982) 231 245. 7 Willmore, L.J., Hurd, R.W. and Sypert, G.W., Epileptiform activity initiated by pial iontophoresis of ferrous and ferric chloride on rat cerebral cortex, Brain Res., 152 (1978) 406~410. 8 Willmore, L.J., Sypert, G.W. and Munson, J.B., Recurrent seizures induced by cortical iron injection: a model of posttraumatic epilepsy, Ann. Neurol., 4 (1978) 329 336. 9 Willmore, L.J., Sypert, G.W., Munson, J.B. and Hurd, R.W., Chronic focal epileptiform discharges induced by injection of iron into rat and cat cortex, Science, 200 (1978) 1501 1503. 10 Willmore, L.J., Triggs, W.J. and Gray, J.D., The role of iron-induced hippocampal peroxidation in acute epileptogenesis,Brain Res., 382 (1986) 422 ~426.
72
NSL 06664
Electrocorticographic characterization of chronic iron-induced epilepsy in rats A k i y o s h i M o r i w a k i , Y u k i o H a t t o r i , N o b u y o s h i Nishida and Y a s u o Hori Department of Physiology, Okayama University Medical School, Okayama (Japan) (Received I August 1989; Revised version received 16 October 1989; Accepted 18 October 1989)
Key words: Iron-induced epilepsy; Cerebral cortex; Electrocorticogram; Isolated spike; Spike and wave complex; Rat Electrocorticograms were recorded from rats which were unilaterally injected with ferrous chloride solution into the sensorimotor cortex to induce chronic epileptic activity. All of the iron-injected rats showed isolated spikes near the injection site and in the contralateral cortex immediately after the injection. The injection produced 3 kinds of responses in the rats according to the frequency of the isolated spikes. Spike and wave complexes appeared bilaterally approximately 30 days or more after the injection in the rats in which the frequency of the isolated spikes was dominant on the side ipsilateral to the injection site or nearly equal on the two sides. These results suggest that there are at least 2 stages in the development of chronic iron-induced epilepsy.
Chronic or acute epileptiform activity has been reported to be induced by intracortical injection of FeC12 or FeCl3 solution [7, 9]. One of the distinctive features of ironinduced epilepsy is that epileptic activity lasts for three months or more. Little is known about the development of chronic iron-induced epilepsy, although electrocorticographic characteristics [8, 9] and extracellular activity of cortical neurons [3] have been reported in acute iron-induced epilepsy. There have been no reports concerning alterations in electrocorticograms (ECoGs) in chronic iron-induced epilepsy. In the present study, ECoGs were recorded from iron-induced epileptic rats for 12 months or more after a unilateral injection of FeCI2 solution into the sensorimotor cortex, and alterations of electrographic features of epilepsy were characterized in relation to the development and propagation of spike activity with time. Two hundred and fifty-five male Wistar rats weighing 230 to 280 g were used. The surgical procedures were essentially the same as those of Willmore et al. [8, 9]. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/ kg). The needle of a microsyringe was inserted through the small trephine hole which was made in the cranial bone over the left sensorimotor cortex at a point 1.5 mm rostral and 3.5 mm lateral to bregma. For experimental rats, 5/A of 100 mM FeCI2 Correspondence: A. Moriwaki, Department of Physiology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan. 0304-3940/90/$ 03.50 (~) 1990 Elsevier Scientific Publishers Ireland Ltd.
73
A
B
1 - 7 ~+,,,+.,,+,-~
C
~l,,++,.,x+~.
~,,,++.+.+w+~
2-7
3-7 J
4- 7 ~
~
~
~v~+,,+'~"4,'++++,'+'~++-'',
5- 7 ~
~,~'+'++++'++~
+'+'~'+'~¢~%~"+'~
6- 7 ~
++.,,,.,-+v,~,+~
~.~'~,++~
D
1-7 a-7 4
-
5--7 ~ 6-7
~
7
~ 200 pV
;~eo
Fig. 1. Electrocorticograms recorded from rats after an intracortical injection of FeC12 solution. A - C are the electrocorticographic tracings to show isolated spikes appearing ipsilaterally, bilaterally and contralaterally to the injection site, respectively, which were recorded 9 days after the injection. D is the electrocorticographic tracings to show spike and wave complexes recorded 46 days after the injection. In schematic diagram, a dot indicates the injection site and numbers 1 7 indicate the position of the recording electrodes.
solution was injected into the cortex slowly. For control rats, an equal volume of saline was injected in the same way. Seven stainless-steel electrodes for E C o G recordings were implanted bilaterally in the cranial bone. The different electrodes were situated 3 m m rostral and 3 m m lateral, 3.5 m m lateral, and 4 m m caudal and 4 m m lateral to the bregma on both sides. The indifferent electrode was situated on the midline of the nasal bone. All the electrodes were fixed tightly to the bone with a dental resin. E C o G s were recorded immediately after the completion of the surgical procedure, and recordings were repeated three times a week for at least 12 months. Each recording session was continued for at least 3 h without interruption. The frequency of isolated spikes was determined from the isolated spikes recorded from the electrodes on the ipsilateral or contralateral cortex. Representative examples of electrocorticographic tracings obtained after an intracortical injection of FeCI2 solution are shown in Fig. 1. The epileptic discharges were
74
isolated spikes and spike and wave complexes, In all of the experimental rats, the isolated spikes with an amplitude of 20(~800/tV appeared ipsilaterally (Fig. IA), bilaterally (Fig. 1B), or contralaterally (Fig. I C) to the injection site within 1 h after the injection. In some of the experimental rats, the spike and wave complexes with an amplitude of 200-1000 #V and a spike frequency of 7-40 Hz appeared bilaterally approximately 30 days or more after the injection (Fig. 1D). The rats which showed only isolated spikes did not exhibit any abnormal behavior, but most of the rats which showed spike and wave complexes exhibited head nodding and vibrissa tremors during the period of spike and wave complexes. Saline-injected control rats showed no abnormalities in their ECoGs or behavior. Rats showing only isolated spikes were divided into three groups according to their spike frequency on the two sides of the cortex: group L, the spike frequency on the left side (injected side) was twice or more than that on the right side; group N, the frequency on the dominant side was less than twice that on the opposite side; group R, the frequency on the right side was twice or more than that on the left side. The ratios of spike frequency were unchanged in each rat in these groups. Rats showing spike and wave complexes were classified into group SW. Time-dependent changes in the isolated spike frequency on the two hemispheres in groups L, N, and R are shown in Fig. 2. The isolated spikes appeared asynchronously in groups L and R, but almost synchronously in group N. In groups L and R, the isolated spike frequency increased steeply up to 30 to 60 days after the injection, and thereafter was almost constant on both sides up to at least 90 days after
4
N
{...
2
,
0 0
30 60 9 0 0
30 60 9 0 0
0
30 60 90
Days after injection Fig. 2. Development in frequency of the isolated spikes in rats after an injection of FeClz solution. The number of isolated spikes per min in the cortex near the injection site (©) and on the contralateral homotopic cortex ( • ) were determined in electrocorticograms recorded after the injection. L, N and R indicate the groups of rats: group L, spike frequency on the left side was twice or more than that on the right side; group N, spike frequency on the dominant side was less than twice that on the opposite side; group R, spike frequency on the right side was twice or more than that on the left side. Each value represents the mean of 9-t7 different rats.
75 TABLE I CHANGES IN THE NUMBER OF RATS IN THE DIFFERENT GROUPS OF RATS Each value represents the number of rats in four groups from a total of 247 rats. L, N and R indicate the groups of rats described in Fig. 2. SW indicates the group of rats which showed spike and wave complexes. Number of rats
Days after injection
30 Days 90 Days
L
N
R
SW
71 47
67 30
104 101
5 69
the injection. The rats in group N also showed an increasing frequency up to 30 days after the injection, and then the increased frequency fell gradually. The number of rats in each group at two different stages after the injection is shown in Table I. The number of rats decreased in groups L and N at the progressive stages, while it was virtually unchanged in group R throughout the stages. The number of rats in group SW increased markedly from 30 to 90 days after the injection. The spike and wave complexes were observed in 28% of the iron-injected rats 90 days after the injection. Furthermore, 36% and 38 % of the rats showed spike and wave complexes 180 and 360 days after the injection, respectively (data not shown). The results of this study indicate that isolated spikes appeared not only in the cortex near the injection site but also in the contralateral cortex. The appearance of isolated spikes in the cortex near the injection site is thought to be responsible for induction of the primary epileptic focus in the cortical tissue adjacent to the injection site, in which peroxidation of lipids is suggested to occur in neuronal plasma membranes of the cortex [5, 10]. The appearance of isolated spikes in the contralateral cortex seems to be responsible for the formation of the secondary epileptic focus. Tada et al. [4] also found that iron-induced spike activity transferred easily to the contralateral hemisphere. The formation of the secondary focus may be due to transcallosal propagation of metal ions and/or subcortical neuronal connections, as has been suggested in other experimental models of epilepsy [2, 6]. Spike and wave complexes appeared approximately 30 days or more after the injection, and the number of rats which showed spike and wave complexes increased up to 90 days after the injection. Concurrent with this period, decreases in the number of rats in groups L and N occurred, although the number of rats in group R was unchanged. The dominant side of isolated spike frequency in individual rats did not change. These results clearly indicate that spike and wave complex activity developed in the rats in groups L and N, but not in the rats in group R. In this study, it should be noted that in group N the isolated spike activity fell in the rats which did not show spike and wave complexes. These results suggest that another alteration occurs within the cortical tissue adjacent to the injection site, and that the alteration participates in the development of isolated spikes into spike and wave complexes. Thus,
76 retention of higher isolated spike activity in the injected hemisphere is p r o b a b l y essential for the d e v e l o p m e n t o f spike a n d wave complexes. In view of the electrocorticographic characteristics, it is likely that there are at least two stages in the d e v e l o p m e n t of chronic i r o n - i n d u c e d epilepsy. The first stage is a period from the time immediately after to a p p r o x i m a t e l y 30 days after the injection, at which time isolated spikes a p p e a r a n d the frequency of isolated spikes increases. The second stage is a period a p p r o x i m a t e l y 30 days or more after the injection, at which time spike a n d wave complexes appear a n d this is activity sustained. O u r previous findings [I], which indicated alterations in cyclic A M P generation in the cortex of rats showing isolated spikes or spike a n d wave complexes, support this hypothesis. At the t u r n i n g point of these epileptic stages, an improved alteration is likely to be responsible for the spike a n d wave complexes p r o b a b l y in the epileptic cortex. 1 Hattori, Y., Moriwaki, A., Hayashi, Y. and Hori, Y., Regional difference in responsivenessof norepinephrine-sensitivecyclic AMP-generating systems of rat cerebral cortex with iron-induced epileptic activity, J. Neurochem., in press. 2 Kudo, T. and Yamauchi, T., An ontogenic study of amygdala seizures induced by penicillin in rats, Exp. Neurol., 99 (1988) 531. 543. 3 Reid, S.A. and Sypert, G.W., Acute FeC13-inducedepileptogenicfoci in cats: electrophysiologicalanalyses, Brain Res., 188 (1980) 531 542. 4 Tada, T., Sakaki, T., Tanikake, T., Miyamoto, S., Utsumi, S., Yamamoto, H. and Hori, Y., A trial to produce acute and chronic focal epileptic models with subpial injection of ferrous chloride solution, Folia Psychiatr. Neurol. Jpn., 34 (1980) 367 368. 5 Triggs, W.J. and Willmore, L.J., In vivo lipid peroxidation in rat brain following intracortical Fe2÷ injection, J. Neurochem., 42 (1984) 976-980. 6 Trottier, S., Truchet, M. and Laroudie, C., Secondary ion microanalysis in the study of cobalt-induced epilepsy in the rat, Exp. Neurol., 76 (1982) 231 245. 7 Willmore, L.J., Hurd, R.W. and Sypert, G.W., Epileptiform activity initiated by pial iontophoresis of ferrous and ferric chloride on rat cerebral cortex, Brain Res., 152 (1978) 406~410. 8 Willmore, L.J., Sypert, G.W. and Munson, J.B., Recurrent seizures induced by cortical iron injection: a model of posttraumatic epilepsy, Ann. Neurol., 4 (1978) 329 336. 9 Willmore, L.J., Sypert, G.W., Munson, J.B. and Hurd, R.W., Chronic focal epileptiform discharges induced by injection of iron into rat and cat cortex, Science, 200 (1978) 1501 1503. 10 Willmore, L.J., Triggs, W.J. and Gray, J.D., The role of iron-induced hippocampal peroxidation in acute epileptogenesis,Brain Res., 382 (1986) 422 ~426.