183
Epilepsy Res., 11(1992) 183-191 Elsevier EPIRES 00470
Corpus callosotomy in the lithium-pilocarpine model of seizures and status epilepticus
E. HirschafbtC,O.C. Sneadayb, M. Vergnesc and F. Gillesavb “Division of Neurology and Department of Pathology, Children’s Hospital of Los Angeles,
‘Department of Neurology and Pathology,
University South California School of Medicine, Los Angeles, CA (USA) and YJPR 419 Centre de Neurochimie,
Strasbourg (France)
(Received 5 August 1991; accepted 25 January 1992) Key words: Corpus callosum; Pilocarpine; Lithium; Animal model; Cholinergic seizures
Section of the corpus callosum (SCC) is a useful surgical therapy in selected types of epilepsy, i.e., tonic, atonic, and intractable generalized convulsive seizures. Experimentally, the effects of SCC have been documented in animal models of focal seizures as well as generalized seizures. The object of this study was to determine the effect of SCC on behavioral and EEG symptomatology in the lithiurn-pilocarpine model of seizures and status epilepticus in the rat. SCC was well tolerated. Fifty-seven percent of SCC animals never developed status epilepticus, while all control animals developed status epilepticus. None of the SCC animals died after 24 h but 59% of control animals died within 24 h of status. Histology verified the extent of the SCC and demonstrated widespread brain damage in all animals who exhibited status epilepticus after 72 h. SCC was associated with a lesion of hippocampal commissure in 64% of animals in the SCC group. This protective effect was not related to lesion of the skull or the longitudinal sinus. The lesion of the hippocampal commissure may have contributed to the protective effect of SCC, since animals with an isolated lesion of the hippocampal commissure without SCC survived the status and showed an increased latency to seizure and status epilepticus. These data suggest that the lithium-pilocarpine model of status epilepticus may be useful in the study of the mechanism of efficacy of SCC in the treatment of epilepsy.
INTRODUCTION The cholinergic agonist pilocarpine (400 mg/kg) produces limbic seizures and status epilepticus accompanied by widespread brain damageu724. This epileptogenic effect of pilocarpine and other muscarinic cholinergic agonists is markedly potentiated by lithium7. Pretreatment with lithium 24 h Correspondence to: O.C. Snead III, M.D., Children’s Hospital of Los Angeles, Department of Neurology, Box 82, 4650 Sunset Boulevard, Los Angeles, CA 90027, USA.
before pilocarpine (30 mg/kg) results in EEG and behavioral changes similar to those seen with higher dose of pilocarpinegY2’. The initial seizures are mediated by muscarinic receptors, but during the evolution of status epilepticus NMDA receptors become involved”. The available literature suggests that section of the corpus callosum (SCC) and hippocampal commissure may have therapeutic benefit in selected types of generalized epilepsy in humans, including tonic, atonic, and intractable generalized convulsive seizures22. Purves et a1.21 reported that re-
0920-1211/92/$05.00 fQ 1992 Elsevier Science Publishers B.V. All rights reserved
184
peated bouts of generalized status epilepticus were eliminated with anterior SCC while Geoffroy et al.6 reported improvement in near status with complete SCC in severely retarded children. Experimentally, the effects of SCC have been documented in a number of animals models of seizures in cat, monkey and rat. These models include both focal seizures induced by amygdaloid kindling and metal implantation’ as well as generalized seizures such as those utilizing pentylenetetrazol” and penicillin’6 or the genetic model of absence in rats25. However, to date a protective effect of SCC similar to that observed in humans has not been reported in any experimental model of epilepsy2. Therefore there is currently no animal model of generalized seizures in which to investigate the mechanism of therapeutic efficacy of SCC in human epilepsy. The object of this study was to determine the effect of SCC on the behavioral and EEG symptomatology in the lithium-pilocarpine model of seizures and status epilepticus in rat.
MATERIALS
AND METHODS
Sprague-Dawley rats of lo-11 weeks (Harlan Company, Indianapolis) weighing 200-250 g at the time of surgery were used for all experiments. Animals were maintained on a 12-h day/night cycle and allowed free access to food and water. SCC was performed stereotactically under halothane anesthesia. The coordinates were derived from the atlas of Paxinos and Watson” with special reference to lambda. The skull was split in the midline. A pointed scalpel was then placed anteroposterior (AP) 2 mm, dorsoventral (DV) 4 mm. The scalpel was advanced anteriorly to AP 7 mm, positioned at DV 4.5 mm, advanced to AP 10 mm, positioned at DV 5 mm and advanced to AP 12 mm, where it was withdrawn, At the same surgery, four cortical electrodes were implanted. Depth recordings were obtained from bipolar twisted concentric enameled stainless steel electrodes (tip diameter 100 pm, vertical interelectrode distance 1 mm) placed in the dorsal hippocampus (AP) -4 mm, (ML) 2 mm, (DV) 4 mm and the region of the ventral pallidum and nucleus accumbens (AP) 0.7 mm, (ML) 2 mm, (DV) 8 mm,
reference bregma. There were three surgical control groups. (1) The first control group of animals had electrode implantation under anesthesia but no callosal section (unlesioned control group). (2) The second control group of animals had electrolytic lesion of the ventral hippocampal commissure (reference bregma AP -1.3 mm, DV 5 mm, ML 0 mm) and electrode implantation, but no callosal section. This control group was necessitated by the fact that complete SCC almost always damages the ventral hippocampal commissure as well. (3) The third control group of animals (sham-operated) had section of the skull and the longitudinal sinus in the midline and electrode implantation, but no callosal section. The surgery was well tolerated with all animals recovering. After 3-6 days of recovery, animals were treated with lithium chloride (3 mEq/kg) (Sigma, St. Louis) dissolved in water, injected intraperitoneally. Pilocarpine was administered 24 h later. The EEG was recorded from freely moving animals with a l&channel Grass polygraph 30 min prior to the subcutaneous injection of 30 mg/kg pilocarpine dissolved in saline. EEG and behavior were observed continuously for 6 h after pilocarpine injection in order to identify latency to first seizure, latency to onset of status epilepticus, latency to periodic epileptiform discharges, the duration of status and the time to recovery. The onset of seizure was characterized electrographically by the occurrence of generalized spikes and polyspikes for 20 s associated with sniffing, chewing, eye blinking and head bobbing. Status epilepticus was defined as forelimb clonus associated with continuous spikes of high amplitude. Periodic epileptiform discharge was defined as slow spike discharges punctuated by short periods of low voltage EEG, during this stage the animals were stuporous and exhibited intermittent myoclonic movement. The mortality at 24 h was noted and the survivors were recorded before decapitation at 25 or 72 h. For histology 25-pm coronal frozen sections were stained with cresyl violet and examined microscopically to ascertain the extent of the SCC and the presence of brain lesions related to the seizures. Statistical analysis between the four groups was performed using Wilcoxon’s signed ranks test.
185 TABLE I EEG and behavior results
Animals who exhibit seizure Animals who exhibit status epilepticus 24-h mortality Latency (mm) to first seizure Latency (min) to status epilepticus Latency (min) to periodic epileptiform discharges
Unlesioned
Sham-operated
Ventral hippocampal
see
control (n = 12)
control (n = 8)
lesion (n = 8)
(n = 14)
100%
100%
100%
100% 59% 24.0 + 2.0
100% 62% 30.8 + 2.3
100% 0%; 48.6 + 5.0*
35.4 + 2.0
40.5 + 2.6
55.3 k 4.9.
190.4 + 10.3
188.0 + 7.1
205.3 + 4.0
65%* 43%* 0% 36.8 + 3.6” (n = 9) 59.0 + 7.8*b (n = 6) 218.6 + 2.6*b (n = 6)
Results are expressed as means + SE. aDoes not include five animals who never had a seizure. bDoes not include eight animals who never developed status epilepticus. ‘P-CO.05.
RESULTS EEG and behavior In the unlesioned control group of animals (n = 12) with electrode implantation but no callosal section, the baseline EEG was normal (Fig. 1A). The animals exhibited signs of peripheral cholinergic stimulation with piloerection, salivation and tremor for the first 20 min after pilocarpine administra-
tion. Distinct seizures (Fig. 1B) were followed by status epilepticus (Fig. 1C) and by periodic epileptiform discharges (Fig. lD, Table I). After 6 h the EEG was still abnormal (Fig. 1E). Fifty-nine percent of the animals died within the first 24 h of status epilepticus. In those who survived longer than 24 h, the EEG showed spikes and polyspikes and wave discharge (Fig. 1F) without major motor symptoms. Simultaneous cortical and depth re-
Fig. 1. Serial EEG recordings from an unlesioned rat which received pilocarpine 24 h after lithium. R, right; L, left; F, frontal; P, parietal.
cordings (Fig. 3A,B) (n = 4) suggested that the seizure initially involved the region of the nucleus accumbens and the ventral pallidum and secondarily the hippocampus and the cortex. In the sham-operated control group of animals with lesion of the skull and of the longitudinal sinus (n = 8) all the animals exhibited seizures and status epilepticus. The mean latencies to the first seizure, to status epilepticus, and to the periodic epileptiform discharge were not significantly longer than in the unlesioned control group. Five of those
A
BASELINE
animals died within 24 h of status epiiepticus. In the 14 SCC animals treated with lithium and pilocarpine, the baseline EEG was normal (Fig. 3A), eight (57%) rats never developed status epilepticus, among them five (35%) never exhibited any seizure (significantly different from the unlesioned and the sham-operated control groups of animals). EEG (Fig. 2D) showed occasional synchronous spike and wave without abnormalities of behavior. In two complete SCC animals, EEG revealed a continuous sharp wave originating from
30min
LF-LP-----I VP-NAP B
BASELINE
LF-LP-
c LF_,J
., __
__ _,. I... -._y
BASELINE .__. W^L_X
VP-NA
3. (A) EEG recording of an unlesioned rat which received pilocarpine 24 h after lithium. Arrow, beginning of the seizure in the NA-VP 30 min after pilocarpine injection. (B) EEG recording of an unlesioned rat which received pilocarpine 24 h after lithium. Arrow, beginning of the seizure simultaneous in the hippocampus and in the cortex 25 min after pilocarpine injection. (C) EEG recording of an SCC rat which received pilocarpine 24 h after lithium. Arrow, rhythmic sharp waves in the NA-VP, 30 min after pilocarpine injection. L, left; F, frontal; P, parietal; NA, nucleus accumbens; VP, ventral pallidurn; HIP, hjppocampus. Fig.
187
the region of the nucleus accumbens-ventral pallidum and a normal cortical EEG (Fig. 3C). When seizures did occur in the SCC group (n = 9) EEG and behavior symptoms were similar to those of the control groups of animals, showing bilateral synchronous spike and polyspike. The mean latency to the first seizure was significantly longer (P < 0.01) than in the unlesioned control. In the six (43%) SCC animals who exhibited status epilepticus, behavior was characterized by staring and occasional myoclonus but not the sustained bilateral forelimb clonus demonstrated by control groups. The latency to status epilepticus and periodic epileptiform discharges was also significantly longer in the SCC group than in the unlesioned and shamoperated controls (Table I). In 50% of the SCC animals the EEG was near normal 4 h after the injection of pilocarpine (Fig. 2E). None of the SCC animals died after 24 h. This was significantly different (P < 0.05) from the unlesioned and shamoperated control groups of animals. After 24 h, the
EEG was normal at this time in all SCC animals including those who exhibited status epilepticus (Fig. 2F). Histology revealed that the SCC was complete in six animals (Fig. 4), limited to the posterior twothirds in six, and to anterior two-thirds in two. None of the six animals with complete SCC developed status epilepticus and only two of these animals showed seizure activity. The ventral hippocampal commissure was totally sectioned in seven rats and partially in two animals. Complete SCC was associated with a lesion of the ventral hippocampal commissure in all but one animal. In that particular animal status epilepticus did not occur. In the control group of animals (n = 8) with lesion of the ventral hippocampal commissure all animals exhibited status epilepticus. The mean latency to the first seizure and to status epilepticus was significantly longer than in the unlesioned and the sham-operated controls (Table I). None of the animals with electrolytic lesion of the ventral hippo-
2/3 POSTERIOR
213 ANTERIOR
Fig. 4. Midline sagittal section of SCC animals. Reproduction from Pax&s and Watson (19). cc, corpus callosum; vhc, ventral hippocampal commissure. Coronal section shows (arrow) seaion of the corpus cabsum. D, do&, R, right. Ma~~tion x 7.3.
188 campal commissure died within 24 h of status epilepticus. This was signi~cantly different (P < 0.05) from the unlesioned and the sham-operated controls. After 24 h the EEG showed spikes and spike and wave. Histological examination demonstrated that the lesion of the hippocampal commissure was complete (Fig. 5). Histology Using cresyl violet stain 24 h after pilocarpine injection and light microscopy there was no evidence of cellular damage in the control groups or in the SCC group of animals. However, when the animals survived for more than 72 h, large and mostly symmetric regions of necrosis with beginning histiocytic response were present ventrolaterally in the forebrain of unlesioned and sham-operated controls, controls with lesions of the ventral hippocampal commissure and SCC animals who developed status epilepticus, but not in the SCC animals who had failed to develop status epilepticus (Fig. 6A). This damage was found predominantly in several brain regions, including piriform cortex, lateral olfactory tubercle, endopi~form nucleus and anterior ~ygd~oid area (Fig. 6B).
DISCUSSION The latency to onset of electrographic seizure and behavioral abnormality in control rats treated with lithium and pilocarpine was similar to that previously reported’*“. Also the success rate of inducing status epilepticus in our study, NO%, was comparable to that reported in the literatureRzO The mortality rate in this model differs from one study to another. Jope et al9 found the mortality to be almost 100% in the first 24 h while Persinger et al.” demonstrated an age-dependent mortality within the first 72 h following lithium-pil~a~ine induced status epilepticus with rats of 70 and 90 days, showing a mortality of 15% and 70% respectively. Since we used lo-11 week old animals, the mortality rate in the current study was similar to the latter study. These data demonstrate the protective effect of complete SCC against electrographic seizures, status epilepticus, and death in the lithium-pilocarpine model. This protective effect of SCC was not related to the lesion of the skull or the longitudinal sinus, but might involve the ~ppocampal commissure since complete SCC was associated with a le-
Fig. 5. Electrolytic lesion of the ventrat hippocampaI commissure and puncture hole in the corpus callosum in controi animals with hippocampal lesion but no WC. D, dorsal; R, right. Magnification x 10.
B Fig. 6. (A) Coronal section of a callosotomkd animal 72 h after piiocarpine injection. Callosotomy (arrow) protected this animal against status epilepticus. D, dorsal; R, right. (B) Coronal section of an uncallosotomized animal 72 h after pilocarpine injection. There is widespread necrosis (arrow) of piriform cortices, anterior cortical amygdaloid nuclei, and the ventral forebrain area.
190
sion of the ventral hippocampal commissure in all but one animal. Moreover animals with an isolated lesion of the hippocampal commissure survived the status epilepticus and showed an increased latency to seizure and status epilepticus. Although a variety of pharmacologic treatments have been reported to alter lithium-pilocarpine induced status epilepticus’5, a protective effect of SCC in this model has not been reported previously; however, SCC has been reported to alter status epilepticus in other models. In a model of status epilepticus following stimulation of a kindled hippocampal focus in rats, McIntyre et a1.14showed that transection of the corpus callosum and the hip~ampal commissure increased the probability of developing status epilepticus while reducing its severity. In McIntyre’s study, animals with transection of the corpus callosum and the hippocampal commissure exhibited status epilepticus of short duration or only EEG abnormalities without motor components but SCC did not completely protect against seizure and status epilepticus. Since it has been shown that SCC may decrease or abolish the synchrony of epileptiform events, one would predict an inhibition of generalization of seizures in SCC animals”. For example bilateral implants of epileptogenic substances are known to produce synchronous and symmetrical spike and wave discharges in the presence of an intact corpus callosum, but this synchrony is disrupted if the corpus callosum is sectioned”**“. The corpus callosum has also been found to be the main pathway ensuring bilateral synchrony of the epileptic discharges in the feline generalized penicillin epilepsy model16 as well as in spontaneous absence seizures in ratsB. Our results suggest that the corpus callosum is not the primary structure responsible for synchronization in the lithium-pilocarpine model, since callosotomized animals never developed synchronous spikes or seizures. Alternative pathways such as anterior commissure, medial thalamus and midbrain reticular formation could be involved since those structures have been shown to be involved in the synchronization of discharges in other models’. There are additional data to support the hypothesis that the corpus callosum serves as a major pathway for the secondary generalization of focal
epileptiform discharges. In 1940, Erickson showed that complete disruption of the corpus callosum in monkeys prevents the spread of electrical induced a~erdischarges in the cortex to the opposite hemisphere. Also, kindling studies demonstrate that the integrity of the corpus callosum is necessary for secondary generalization of the kindled seizure26. The present study confirms the role of the corpus callosum in secondary generalization because animals with complete SCC did not develop generalized status epilepticus. In addition to mediating spread and synchronization of epileptiform discharges, interhemispheric connections serve both inhibitory and excitatory functions. Asanuma and Okuda’ demonstrated excitation in callosal pathways when recording from pyramidal tract cells in precruciate cortex during stimulation of the contralateral homotopic cortex but only with a restricted stimulus. Eidelberg4 recorded from a bulbar pyramidal electrode in cats while stimulating the ipsilateral or contralateral cortex and showed that activation of the callosal pathway served an inhibitory role. Wada et al. 13,26showed a facilitatory effect of SCC for up to 1 month in kindled cats. The results in our study suggest that the corpus callosum plays an excitatory more than an inhibitory role in the lithium-pilocarpine model of seizures and status epilepticus in rats. In the lithium-pilocarpine model in rats, Clifford et al3 have demonstrated that the earliest spikes and organized ictal discharges emanate from ventral forebrain structures such as nucleus accumbens and ventral pallidum and spread rapidly to involve the neocortex; this result is confirmed in the current experiment. These data give rise to the hypothesis that SCC inhibits the generalization of the seizures by preventing the spread of the discharges from the neocortex to the contralateral hemisphere via the corpus callosum and supresses the excitatory influence exerted by the neocortex of each hemisphere on the development of the epileptogenic processes in its contralateral homologue. Additional data are provided by Clifford et al.3 to support this hypothesis. Using 2-deoxyglucase quantitative autoradiography they also reported a higher rate of glucose consumption in the corpus callosum in the lithium-piiocarpine model
191
of status epilepticus. In conclusion, complete corpus callosotomy prevents the development of status epilepticus in the lithium-pilocarpine model of status epilepticus. Although these results should be confirmed in
other animal models of status epilepticus, they raise the possibility that the lithium-pilocarpine model may be used to study mechanisms of efficacy of SCC in the treatment of epilepsy.
REFERENCES
(1986) 554-570. 15 Morrisett, R.A., Jope, R.S. and Snead, OX., Effects of drugs on the initiation and maintenance of status epilepticus induced by administration of pilocarpine to lithium-pretreated rats, Exp. Neural., 97 (1987) 193-200. 16 Musgrave, J. and Gloor, P., The role of the corpus cahosum in bifateral interhe~sphe~c synchrony of spike and wave discharge in feline generalized penicillin epilepsy, Epilepsia, 21(1980) 369-378. 17 Mutani, R., Bergamini, L., Fariello, R. and Quattrocolo, G., Bilateral synchrony of epileptic discharge associated with chronic asymmetrical cortical foci, Electroenceph. Clin. Neurophysiol., 34 (1973) 53-59. 18 Ormandy, G.C., Jope, R.S. and Snead, O.C., Anticonvulsant action of MK-801 on the li~i~-pil~a~ine model of status epilepticus in rats, Exp. Neural., 106 (1989) 172-180. 19 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Orlando, FL, 1986. 20 Persinger, M.A., Makarec, K. and Bradley, J.C., Characteristics of limbic seizures evoked by peripheral injections of lithium and pilocarpine, Physiol. Behav., 44 (1988) 27-37. 21 Purves, S.J., Wada, J.A., Medsc, D., Woodhurst, W.B., Moyes, P.D., Strauss, E., Kosaka, B. and Li, D., Rest&s of anterior corpus cahosum section in 24 patients with medically intractable seixures, Neuro@y, 38 (1988) 1194-1201. 22 Spencer, S.S., Corpus callosum section and other disconnection procedures for medicdly intractable epilepsy, Epilepsia, 29 (Suppl.) (1988) S85S99. 23 Turski, L., Ikonomidou, C., Turski, W., Bortolotto, Z.A. and Cavafheiro, E.A., Review: Chohnergic mechanisms and epileptogenesis. The seizures induced by pilocarpine: a novel experimental model of intractable epilepsy, Syncipse, 3 (1989) 154-171. 24 Turski, W.A., CavaIheiro, E.A., Schwarx, M., Czucxwar, S., Kleirok, Z. and Turski, L., Limbic seizures produced by pilocarpine in rats: behavioral, electroencephalographic and neuropathological study, Behav. Brain Res., 9 (1983) 315-336. 25 Vergnes, M., Marescaux, C., Lannes, B., Depaubs, A., Micheietti, G. and Warter, J.M., Interhemispheric desynchronixation of spontaneous spike-wave discharges by corpus callosum transection in rats with petit mal like epilepsy, Epilepsy Res., 4 (1989) 8-13. 26 Wada, J.A. and Sato, M., The generalized convulsive seizure state induced by daily stimulation of the amygdala in split brain cats, Epikpsia, 16 (1975) 417-430.
1 Asanuma, H. and Okuda, O., Effects of transcaffosai volleys on pyramidal tract cell activity of cat, /. Neurophysiol., 25 (1962) 198-208. 2 Blume, W.T., Corpus callosum section for seizure control: rationale and review of experimental and cEnicaI data, Cleveland Clin. Q., 51(1984) 319-332. 3 Clifford, D.B., Olney, J.W., Maniotis, A., Collins, C. and Zorumsky, F., The functional anatomy and pathology of lit~um-pil~a~ine and high-dose pilocarpine seizures, Neuroscience, 23 (1987) 953-958. 4 Eidelberg, E., CaIIosal and non callosal connections between the sensory-motor cortices in cat and monkey, Electroenceph. Cfk. Neurophysiol., 26 (1%9) 557-564. 5 Erickson, T.C., Spread of the epileptic discharge: an experimental study of the after-discharge induced by electrical stimulation of the cerebral cortex, Arch. Neural. Psychiatry, 43 (1940) 429-452. 6 Geoffroy, G., Lassonde, M., DesIisIe, F. and Decarie, M., Corpus callosotomy for control of intractable epilepsy in children, Neurology, 33 (1983) 891-897. 7 Honchar, M.P., Olney, J.W. and Sherman, W.R., Systemic choiinergic agents induce scixures and brain damage in lithium-treated rats, Science, 220 (1983) 323-325. 8 Isaacson, R.L., Schwartz, H., Per&f, N. and Pinson, L., The role of the corpus callosum in the establishment of areas of secondary epileptiform activity, Epilepsy, 12 (1971) 133-141. 9 Jope, R.S., Morrisett, R. and Snead, O.C., Characterization of lithium potentiation of pilocarpine-induced status epiIepticus in rats, Exp. Neural., 91(1986) 4X-480. 10 Marcus, M.E. and Watson, C.W., Symmetrical epileptogenie foci in monkey cerebral cortex, Arch. Neural., 19 (1968) 99-116. 11 Marcus, M.E. and Watson, C.W., Bilateral synchronous spike wave electrographic patterns in the cat, Arch. Neurol., 14 (1966) 601-610. 12 Marcus, E.M., Watson, C.V. and Jacobson, S., Role of the corpus cahosum in bilateral synchronous discharges induced by intravenous ~ntylene~~ol, Neurology, 19 (1%9) 309. 13 McCaughram, J.A., Corcoran, M.E. and Wada, J.A., Role of the forebrain commissure in amygdaloid kindling in rats, Epilepsia, 19 (1978) 19-33. 14 McIntyre, D.C., Stokes, K.A. and Edson, N., Status epiIepticus following stimulation of a kindled bippocampal focus in intact and commissurotomised rats, Exp. Neural., 94
Epilepsy Res., 11(1992) 183-191 Elsevier EPIRES 00470
Corpus callosotomy in the lithium-pilocarpine model of seizures and status epilepticus
E. HirschafbtC,O.C. Sneadayb, M. Vergnesc and F. Gillesavb “Division of Neurology and Department of Pathology, Children’s Hospital of Los Angeles,
‘Department of Neurology and Pathology,
University South California School of Medicine, Los Angeles, CA (USA) and YJPR 419 Centre de Neurochimie,
Strasbourg (France)
(Received 5 August 1991; accepted 25 January 1992) Key words: Corpus callosum; Pilocarpine; Lithium; Animal model; Cholinergic seizures
Section of the corpus callosum (SCC) is a useful surgical therapy in selected types of epilepsy, i.e., tonic, atonic, and intractable generalized convulsive seizures. Experimentally, the effects of SCC have been documented in animal models of focal seizures as well as generalized seizures. The object of this study was to determine the effect of SCC on behavioral and EEG symptomatology in the lithiurn-pilocarpine model of seizures and status epilepticus in the rat. SCC was well tolerated. Fifty-seven percent of SCC animals never developed status epilepticus, while all control animals developed status epilepticus. None of the SCC animals died after 24 h but 59% of control animals died within 24 h of status. Histology verified the extent of the SCC and demonstrated widespread brain damage in all animals who exhibited status epilepticus after 72 h. SCC was associated with a lesion of hippocampal commissure in 64% of animals in the SCC group. This protective effect was not related to lesion of the skull or the longitudinal sinus. The lesion of the hippocampal commissure may have contributed to the protective effect of SCC, since animals with an isolated lesion of the hippocampal commissure without SCC survived the status and showed an increased latency to seizure and status epilepticus. These data suggest that the lithium-pilocarpine model of status epilepticus may be useful in the study of the mechanism of efficacy of SCC in the treatment of epilepsy.
INTRODUCTION The cholinergic agonist pilocarpine (400 mg/kg) produces limbic seizures and status epilepticus accompanied by widespread brain damageu724. This epileptogenic effect of pilocarpine and other muscarinic cholinergic agonists is markedly potentiated by lithium7. Pretreatment with lithium 24 h Correspondence to: O.C. Snead III, M.D., Children’s Hospital of Los Angeles, Department of Neurology, Box 82, 4650 Sunset Boulevard, Los Angeles, CA 90027, USA.
before pilocarpine (30 mg/kg) results in EEG and behavioral changes similar to those seen with higher dose of pilocarpinegY2’. The initial seizures are mediated by muscarinic receptors, but during the evolution of status epilepticus NMDA receptors become involved”. The available literature suggests that section of the corpus callosum (SCC) and hippocampal commissure may have therapeutic benefit in selected types of generalized epilepsy in humans, including tonic, atonic, and intractable generalized convulsive seizures22. Purves et a1.21 reported that re-
0920-1211/92/$05.00 fQ 1992 Elsevier Science Publishers B.V. All rights reserved
184
peated bouts of generalized status epilepticus were eliminated with anterior SCC while Geoffroy et al.6 reported improvement in near status with complete SCC in severely retarded children. Experimentally, the effects of SCC have been documented in a number of animals models of seizures in cat, monkey and rat. These models include both focal seizures induced by amygdaloid kindling and metal implantation’ as well as generalized seizures such as those utilizing pentylenetetrazol” and penicillin’6 or the genetic model of absence in rats25. However, to date a protective effect of SCC similar to that observed in humans has not been reported in any experimental model of epilepsy2. Therefore there is currently no animal model of generalized seizures in which to investigate the mechanism of therapeutic efficacy of SCC in human epilepsy. The object of this study was to determine the effect of SCC on the behavioral and EEG symptomatology in the lithium-pilocarpine model of seizures and status epilepticus in rat.
MATERIALS
AND METHODS
Sprague-Dawley rats of lo-11 weeks (Harlan Company, Indianapolis) weighing 200-250 g at the time of surgery were used for all experiments. Animals were maintained on a 12-h day/night cycle and allowed free access to food and water. SCC was performed stereotactically under halothane anesthesia. The coordinates were derived from the atlas of Paxinos and Watson” with special reference to lambda. The skull was split in the midline. A pointed scalpel was then placed anteroposterior (AP) 2 mm, dorsoventral (DV) 4 mm. The scalpel was advanced anteriorly to AP 7 mm, positioned at DV 4.5 mm, advanced to AP 10 mm, positioned at DV 5 mm and advanced to AP 12 mm, where it was withdrawn, At the same surgery, four cortical electrodes were implanted. Depth recordings were obtained from bipolar twisted concentric enameled stainless steel electrodes (tip diameter 100 pm, vertical interelectrode distance 1 mm) placed in the dorsal hippocampus (AP) -4 mm, (ML) 2 mm, (DV) 4 mm and the region of the ventral pallidum and nucleus accumbens (AP) 0.7 mm, (ML) 2 mm, (DV) 8 mm,
reference bregma. There were three surgical control groups. (1) The first control group of animals had electrode implantation under anesthesia but no callosal section (unlesioned control group). (2) The second control group of animals had electrolytic lesion of the ventral hippocampal commissure (reference bregma AP -1.3 mm, DV 5 mm, ML 0 mm) and electrode implantation, but no callosal section. This control group was necessitated by the fact that complete SCC almost always damages the ventral hippocampal commissure as well. (3) The third control group of animals (sham-operated) had section of the skull and the longitudinal sinus in the midline and electrode implantation, but no callosal section. The surgery was well tolerated with all animals recovering. After 3-6 days of recovery, animals were treated with lithium chloride (3 mEq/kg) (Sigma, St. Louis) dissolved in water, injected intraperitoneally. Pilocarpine was administered 24 h later. The EEG was recorded from freely moving animals with a l&channel Grass polygraph 30 min prior to the subcutaneous injection of 30 mg/kg pilocarpine dissolved in saline. EEG and behavior were observed continuously for 6 h after pilocarpine injection in order to identify latency to first seizure, latency to onset of status epilepticus, latency to periodic epileptiform discharges, the duration of status and the time to recovery. The onset of seizure was characterized electrographically by the occurrence of generalized spikes and polyspikes for 20 s associated with sniffing, chewing, eye blinking and head bobbing. Status epilepticus was defined as forelimb clonus associated with continuous spikes of high amplitude. Periodic epileptiform discharge was defined as slow spike discharges punctuated by short periods of low voltage EEG, during this stage the animals were stuporous and exhibited intermittent myoclonic movement. The mortality at 24 h was noted and the survivors were recorded before decapitation at 25 or 72 h. For histology 25-pm coronal frozen sections were stained with cresyl violet and examined microscopically to ascertain the extent of the SCC and the presence of brain lesions related to the seizures. Statistical analysis between the four groups was performed using Wilcoxon’s signed ranks test.
185 TABLE I EEG and behavior results
Animals who exhibit seizure Animals who exhibit status epilepticus 24-h mortality Latency (mm) to first seizure Latency (min) to status epilepticus Latency (min) to periodic epileptiform discharges
Unlesioned
Sham-operated
Ventral hippocampal
see
control (n = 12)
control (n = 8)
lesion (n = 8)
(n = 14)
100%
100%
100%
100% 59% 24.0 + 2.0
100% 62% 30.8 + 2.3
100% 0%; 48.6 + 5.0*
35.4 + 2.0
40.5 + 2.6
55.3 k 4.9.
190.4 + 10.3
188.0 + 7.1
205.3 + 4.0
65%* 43%* 0% 36.8 + 3.6” (n = 9) 59.0 + 7.8*b (n = 6) 218.6 + 2.6*b (n = 6)
Results are expressed as means + SE. aDoes not include five animals who never had a seizure. bDoes not include eight animals who never developed status epilepticus. ‘P-CO.05.
RESULTS EEG and behavior In the unlesioned control group of animals (n = 12) with electrode implantation but no callosal section, the baseline EEG was normal (Fig. 1A). The animals exhibited signs of peripheral cholinergic stimulation with piloerection, salivation and tremor for the first 20 min after pilocarpine administra-
tion. Distinct seizures (Fig. 1B) were followed by status epilepticus (Fig. 1C) and by periodic epileptiform discharges (Fig. lD, Table I). After 6 h the EEG was still abnormal (Fig. 1E). Fifty-nine percent of the animals died within the first 24 h of status epilepticus. In those who survived longer than 24 h, the EEG showed spikes and polyspikes and wave discharge (Fig. 1F) without major motor symptoms. Simultaneous cortical and depth re-
Fig. 1. Serial EEG recordings from an unlesioned rat which received pilocarpine 24 h after lithium. R, right; L, left; F, frontal; P, parietal.
cordings (Fig. 3A,B) (n = 4) suggested that the seizure initially involved the region of the nucleus accumbens and the ventral pallidum and secondarily the hippocampus and the cortex. In the sham-operated control group of animals with lesion of the skull and of the longitudinal sinus (n = 8) all the animals exhibited seizures and status epilepticus. The mean latencies to the first seizure, to status epilepticus, and to the periodic epileptiform discharge were not significantly longer than in the unlesioned control group. Five of those
A
BASELINE
animals died within 24 h of status epiiepticus. In the 14 SCC animals treated with lithium and pilocarpine, the baseline EEG was normal (Fig. 3A), eight (57%) rats never developed status epilepticus, among them five (35%) never exhibited any seizure (significantly different from the unlesioned and the sham-operated control groups of animals). EEG (Fig. 2D) showed occasional synchronous spike and wave without abnormalities of behavior. In two complete SCC animals, EEG revealed a continuous sharp wave originating from
30min
LF-LP-----I VP-NAP B
BASELINE
LF-LP-
c LF_,J
., __
__ _,. I... -._y
BASELINE .__. W^L_X
VP-NA
3. (A) EEG recording of an unlesioned rat which received pilocarpine 24 h after lithium. Arrow, beginning of the seizure in the NA-VP 30 min after pilocarpine injection. (B) EEG recording of an unlesioned rat which received pilocarpine 24 h after lithium. Arrow, beginning of the seizure simultaneous in the hippocampus and in the cortex 25 min after pilocarpine injection. (C) EEG recording of an SCC rat which received pilocarpine 24 h after lithium. Arrow, rhythmic sharp waves in the NA-VP, 30 min after pilocarpine injection. L, left; F, frontal; P, parietal; NA, nucleus accumbens; VP, ventral pallidurn; HIP, hjppocampus. Fig.
187
the region of the nucleus accumbens-ventral pallidum and a normal cortical EEG (Fig. 3C). When seizures did occur in the SCC group (n = 9) EEG and behavior symptoms were similar to those of the control groups of animals, showing bilateral synchronous spike and polyspike. The mean latency to the first seizure was significantly longer (P < 0.01) than in the unlesioned control. In the six (43%) SCC animals who exhibited status epilepticus, behavior was characterized by staring and occasional myoclonus but not the sustained bilateral forelimb clonus demonstrated by control groups. The latency to status epilepticus and periodic epileptiform discharges was also significantly longer in the SCC group than in the unlesioned and shamoperated controls (Table I). In 50% of the SCC animals the EEG was near normal 4 h after the injection of pilocarpine (Fig. 2E). None of the SCC animals died after 24 h. This was significantly different (P < 0.05) from the unlesioned and shamoperated control groups of animals. After 24 h, the
EEG was normal at this time in all SCC animals including those who exhibited status epilepticus (Fig. 2F). Histology revealed that the SCC was complete in six animals (Fig. 4), limited to the posterior twothirds in six, and to anterior two-thirds in two. None of the six animals with complete SCC developed status epilepticus and only two of these animals showed seizure activity. The ventral hippocampal commissure was totally sectioned in seven rats and partially in two animals. Complete SCC was associated with a lesion of the ventral hippocampal commissure in all but one animal. In that particular animal status epilepticus did not occur. In the control group of animals (n = 8) with lesion of the ventral hippocampal commissure all animals exhibited status epilepticus. The mean latency to the first seizure and to status epilepticus was significantly longer than in the unlesioned and the sham-operated controls (Table I). None of the animals with electrolytic lesion of the ventral hippo-
2/3 POSTERIOR
213 ANTERIOR
Fig. 4. Midline sagittal section of SCC animals. Reproduction from Pax&s and Watson (19). cc, corpus callosum; vhc, ventral hippocampal commissure. Coronal section shows (arrow) seaion of the corpus cabsum. D, do&, R, right. Ma~~tion x 7.3.
188 campal commissure died within 24 h of status epilepticus. This was signi~cantly different (P < 0.05) from the unlesioned and the sham-operated controls. After 24 h the EEG showed spikes and spike and wave. Histological examination demonstrated that the lesion of the hippocampal commissure was complete (Fig. 5). Histology Using cresyl violet stain 24 h after pilocarpine injection and light microscopy there was no evidence of cellular damage in the control groups or in the SCC group of animals. However, when the animals survived for more than 72 h, large and mostly symmetric regions of necrosis with beginning histiocytic response were present ventrolaterally in the forebrain of unlesioned and sham-operated controls, controls with lesions of the ventral hippocampal commissure and SCC animals who developed status epilepticus, but not in the SCC animals who had failed to develop status epilepticus (Fig. 6A). This damage was found predominantly in several brain regions, including piriform cortex, lateral olfactory tubercle, endopi~form nucleus and anterior ~ygd~oid area (Fig. 6B).
DISCUSSION The latency to onset of electrographic seizure and behavioral abnormality in control rats treated with lithium and pilocarpine was similar to that previously reported’*“. Also the success rate of inducing status epilepticus in our study, NO%, was comparable to that reported in the literatureRzO The mortality rate in this model differs from one study to another. Jope et al9 found the mortality to be almost 100% in the first 24 h while Persinger et al.” demonstrated an age-dependent mortality within the first 72 h following lithium-pil~a~ine induced status epilepticus with rats of 70 and 90 days, showing a mortality of 15% and 70% respectively. Since we used lo-11 week old animals, the mortality rate in the current study was similar to the latter study. These data demonstrate the protective effect of complete SCC against electrographic seizures, status epilepticus, and death in the lithium-pilocarpine model. This protective effect of SCC was not related to the lesion of the skull or the longitudinal sinus, but might involve the ~ppocampal commissure since complete SCC was associated with a le-
Fig. 5. Electrolytic lesion of the ventrat hippocampaI commissure and puncture hole in the corpus callosum in controi animals with hippocampal lesion but no WC. D, dorsal; R, right. Magnification x 10.
B Fig. 6. (A) Coronal section of a callosotomkd animal 72 h after piiocarpine injection. Callosotomy (arrow) protected this animal against status epilepticus. D, dorsal; R, right. (B) Coronal section of an uncallosotomized animal 72 h after pilocarpine injection. There is widespread necrosis (arrow) of piriform cortices, anterior cortical amygdaloid nuclei, and the ventral forebrain area.
190
sion of the ventral hippocampal commissure in all but one animal. Moreover animals with an isolated lesion of the hippocampal commissure survived the status epilepticus and showed an increased latency to seizure and status epilepticus. Although a variety of pharmacologic treatments have been reported to alter lithium-pilocarpine induced status epilepticus’5, a protective effect of SCC in this model has not been reported previously; however, SCC has been reported to alter status epilepticus in other models. In a model of status epilepticus following stimulation of a kindled hippocampal focus in rats, McIntyre et a1.14showed that transection of the corpus callosum and the hip~ampal commissure increased the probability of developing status epilepticus while reducing its severity. In McIntyre’s study, animals with transection of the corpus callosum and the hippocampal commissure exhibited status epilepticus of short duration or only EEG abnormalities without motor components but SCC did not completely protect against seizure and status epilepticus. Since it has been shown that SCC may decrease or abolish the synchrony of epileptiform events, one would predict an inhibition of generalization of seizures in SCC animals”. For example bilateral implants of epileptogenic substances are known to produce synchronous and symmetrical spike and wave discharges in the presence of an intact corpus callosum, but this synchrony is disrupted if the corpus callosum is sectioned”**“. The corpus callosum has also been found to be the main pathway ensuring bilateral synchrony of the epileptic discharges in the feline generalized penicillin epilepsy model16 as well as in spontaneous absence seizures in ratsB. Our results suggest that the corpus callosum is not the primary structure responsible for synchronization in the lithium-pilocarpine model, since callosotomized animals never developed synchronous spikes or seizures. Alternative pathways such as anterior commissure, medial thalamus and midbrain reticular formation could be involved since those structures have been shown to be involved in the synchronization of discharges in other models’. There are additional data to support the hypothesis that the corpus callosum serves as a major pathway for the secondary generalization of focal
epileptiform discharges. In 1940, Erickson showed that complete disruption of the corpus callosum in monkeys prevents the spread of electrical induced a~erdischarges in the cortex to the opposite hemisphere. Also, kindling studies demonstrate that the integrity of the corpus callosum is necessary for secondary generalization of the kindled seizure26. The present study confirms the role of the corpus callosum in secondary generalization because animals with complete SCC did not develop generalized status epilepticus. In addition to mediating spread and synchronization of epileptiform discharges, interhemispheric connections serve both inhibitory and excitatory functions. Asanuma and Okuda’ demonstrated excitation in callosal pathways when recording from pyramidal tract cells in precruciate cortex during stimulation of the contralateral homotopic cortex but only with a restricted stimulus. Eidelberg4 recorded from a bulbar pyramidal electrode in cats while stimulating the ipsilateral or contralateral cortex and showed that activation of the callosal pathway served an inhibitory role. Wada et al. 13,26showed a facilitatory effect of SCC for up to 1 month in kindled cats. The results in our study suggest that the corpus callosum plays an excitatory more than an inhibitory role in the lithium-pilocarpine model of seizures and status epilepticus in rats. In the lithium-pilocarpine model in rats, Clifford et al3 have demonstrated that the earliest spikes and organized ictal discharges emanate from ventral forebrain structures such as nucleus accumbens and ventral pallidum and spread rapidly to involve the neocortex; this result is confirmed in the current experiment. These data give rise to the hypothesis that SCC inhibits the generalization of the seizures by preventing the spread of the discharges from the neocortex to the contralateral hemisphere via the corpus callosum and supresses the excitatory influence exerted by the neocortex of each hemisphere on the development of the epileptogenic processes in its contralateral homologue. Additional data are provided by Clifford et al.3 to support this hypothesis. Using 2-deoxyglucase quantitative autoradiography they also reported a higher rate of glucose consumption in the corpus callosum in the lithium-piiocarpine model
191
of status epilepticus. In conclusion, complete corpus callosotomy prevents the development of status epilepticus in the lithium-pilocarpine model of status epilepticus. Although these results should be confirmed in
other animal models of status epilepticus, they raise the possibility that the lithium-pilocarpine model may be used to study mechanisms of efficacy of SCC in the treatment of epilepsy.
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