Epilepsia, 32(6):778-782, 1991

Raven Press, Ltd., New York 0 International League Against Epilepsy

Long-Term Effects of Pilocarpine in Rats: Structural Damage of the Brain Triggers Kindling and Spontaneous Recurrent Seizures E. A. Cavalheiro, J. P. Leite, 2. A. Bortolotto, *W. A. Turski, *C. Ikonomidou, and *L. Turski Department of Neurology and Neurosurgery, Laboratory of Experimental Neurology, Escola Paulista de Medicina, Sao Paulo, Brazil; and *Department of Pharmacology, Medical School, Lublin, PoIand

Summary: Structural damage of the human brain (perinatal damage, cerebral trauma, head injury, cerebrovascular and degenerative diseases, intracranial tumor, metabolic diseases, toxins, drug-induced seizures) may lead to chronic epilepsy in survivors. Epidemiologic analyses show that a considerable time-delay occurs between the exposure of the brain to injury and the appearance of seizures. Such seizures are usually partial or mixed, may develop at any age, and are difficult to treat. In rats subjected to structural damage of the brain induced by sustained convulsions triggered by systemic administration of the cholinergic agent pilocarpine, spontaneous seizures may develop after a mean latency of 14-15 days. The mean frequency of spontaneous recurrent convulsions remains constant for several months. Evolution of these

convulsions proceeds through several electrographic and behavioral stages resembling kindling. Kindling may be otherwise induced in rodents by repeated systemic administration of convulsants or by repeated electrical stimulation of sensitive brain regions. These observations demonstrate that structural damage of the brain may lead to spontaneously recurrent convulsions (chronic epilepsy) in rats and that kindling may be involved in the evolution of such a condition. This finding suggests that kindling mechanisms underlie the development of epileptic foci from structural brain lesions. Such mechanisms may be involved in the etiology of some forms of epilepsy in humans. Key Words: Convulsions-Neurologic models-Pilocarpine-Kindling.

The mechanisms underlying epileptogenesis are not satisfactorily understood, although the etiology of human epilepsy has been a subject of intensive research for many years (Dichter and Ayala, 1987). The search for therapeutic approaches to epilepsy has been based on animal (rodent and primate) seizure models (Purpura et al., 1972). In the last century use of such models has led to the successful development of drugs active in human epilepsies (Meldrum, 1986). No drug has been introduced into human epilepsy therapy that was inactive against chemically (pentylenetetrazol, PTZ) or electrically

(maximal electroshock, MES) induced seizures in rodents (Janz, 1985). This historical perspective gives the impression that animal models of seizures satisfactorily resemble the human condition. This is certainly not true, and new animal models that better mimic human seizure disorders and are more useful for studying mechanisms of epilepsy are still needed. We report spontaneously recurrent seizures (chronic epilepsy) that occur in rats subjected to status epilepticus (SE) induced by high-dose treatment with the cholinergic agonist pilocarpine. This treatment leads unequivocally to epilepsy-related brain damage (Olney et al., 1986; Turski et al., 1989). Such seizures appear after a delay of 2 weeks, follow several behavioral stages resembling kindling during development, and persist for several months. Our experimental approach may serve as a model of epilepsy closely resembling the human condition.

Received November 1990; revision accepted February 1991. Dr. Turski's present address is Department of Neuropsychopharmacology, Schering AG, Sellerstr. 6-8, P.O. Box 65031 1, D-1000 Berlin, Germany. Address correspondence and reprint requests to Dr. E. A. Cavalheiro at Laboradrio de Neurologia Experimental, Escola Paulista de Medicina, R. Botucatu, 862, 04023 SBo Paulo, SP, Brazil.

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MATERIAL AND METHODS Thirty-two adult Wistar rats weighing 250-280 g were housed under environmentally controlled conditions (6:OO a.m./6:00 p.m. light/dark cycle; 2224°C) and permitted free access to food and water for 7 days before operation. For EEG recordings (Beckman model RM polygraph, time constant 0.03 s, high cutoff filter 15 Hz) bipolar twisted electrodes (tip diameter 100 pm, interelectrode distance 500 km) were stereotaxically positioned in the dorsal hippocampus (Turski et al., 1983) under sodium pentobarbital anesthesia (Nembutal; Ceva, Neuillysur-Seine, France; 50 mg/kg intraperitoneally , i.p.). Surface recordings were led from screws positioned bilaterally over the occipital cortex. Four to 5 days postoperatively, the rats were subjected to sustained convulsions induced by pilocarpine hydrochloride (Sigma, St. Louis, MO, U.S.A.; 380 mgl kg) (Turski et al., 1983). Seizures produced by pilocarpine in rodents provide an animal model of epilepsy that permits evaluation of morphologic sequelae of intractable convulsions (Turski et al., 1983; Olney et al., 1986). Methyl-scopolamine nitrate (Sigma; 1 mg/kg subcutaneously, s.c.) was administered 30 min before injection of pilocarpine to limit peripheral toxic effects (Turski et al., 1983). The criterion used to indicate convulsive response to pilocarpine was SE, defined as continuous seizures persisting for a period of at least 30 min before spontaneous termination (Olney et al., 1983; Turski et al., 1983). For continuous recording of EEG activity and for detection of spontaneous seizures, a videosupported EEG monitoring system was used. This system allowed simultaneous videotaping and EEG recording (on paper) in 2-4 subjects. The monitoring was discontinued for periods ranging from 15 to 30 min for daily care. The EEG monitoring was performed between August 1983 and March 1989. For morphologic examination of the brains by light microscopy and for correct location of implanted deep electrodes 120-125 days after administration

of pilocarpine, rats were anesthetized with an overdose of sodium pentobarbital and perfused with a fixative containing 10% acetic acid, 10% formaldehyde, and 80% methanol. The brains were allowed to fix in situ at 4°C for 24 h and then were removed and processed for paraffin embedding. Subsequently, serial coronal sections of the whole brain were cut 10-F.m thick, and every tenth section was mounted on a glass slide and stained either with cresyl violet or by the Fink and Heimer technique (Fink and Heimer, 1967). RESULTS Thirteen of 32 rats died in the course of sustained seizures induced by pilocarpine (380 mg/kg). Nineteen survivors were continuously monitored (for behavior or EEG) for 120 days (24 h/day) after receiving pilocarpine. Behavioral and electrographic features of pilocarpine-induced seizures (acute period) (Fig. 1) were similar to those reported previously (Turski et al., 1983, 1989). The treatment with pilocarpine (380 mg/kg) induced sequential behavioral (akinesia, facial automatisms, limbic seizures consisting of forelimb clonus with rearing, salivation, masticatory jaw movements, and falling) and EEG changes (significant theta rhythm and isolated spikes in hippocampus, synchronization of the activity in hippocampus and cortex, EEG seizures, SE). The animals that survived sustained seizures (19 of 32) were unresponsive to environment, akinetic, and comatose. This toxic period lasted for 3-5 days and was followed by progressive normalization of behavior. First spontaneous seizures (SRS) were monitored between 4 and 44 days after pilocarpine administration. The mean “seizure silent period” lasted for 14.8 3.0 days (mean 2 SEM, n = 19) (Fig. 1). During the “chronic period” (Fig. 1) SRS were observed with a frequency of 2-4 per animal per week. Cumulative analysis of SRS in the group of 19 animals over time showed constant frequency of 19.2

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FIG. 1. Sequence of temporal evolution of behavioral and EEG changes observed after structural damage to PILOCAFPINE the brain induced by sustained seizures triggered by pilocarpine (380 mgikg intraperitoneally) in rats. Acute period (24 h) corresponds to sustained seizures and status epilepticus induced by pilocarpine. Silent period (14.8 ? 3.0 days, n = 19) is characterized by progressive return to normal EEG and behavior and ends with first spontaneous recurrent seizure (SRS). Chronic period comprises the appearance of SRS. The initial period of the chronic phase, characterized by kindlinglike phenomena, may be defined as the subchronic period (12.8 2 1.5 days, n = 15). EXPERIMENTAL DESIGN

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? 1.8 seizures per 10 days during 120 days of observation (Fig. 2). Cumulative analysis of SRS over the light/dark cycle showed clear-cut increase in seizure frequency during the diurnal period in comparison with the nocturnal period (Fig. 3). The duration of SRS rarely exceeded 50-60 s and was replaced by depressed background activity with frequent EEG spikes (Fig. 4). Most SRS were characterized by bursts of spiking activity in the hippocampus that spread rapidly to cortical recordings. Only one SRS among all recorded in 7 animals in 120 days started in the cortex and subsequently spread to the hippocampus. No bursts of spiking activity restricted to the cortex were observed. No pattern of spiking activity was indicative of onset of SRS. The appearance of clusters of seizures (i.e., 3-7 seizures in 1 day) was noted in 4 animals. In terms of behavior, SRS were characterized by facial automatisms, head nodding, forelimb clonus, rearing, and falling. Evolution of spontaneous seizures in 15 animals (15 of 19) followed behavioral and electrographic stages of kindling (Fig. 4). In these animals, SRS was characterized by paroxysmal discharges localized to hippocampus without changes in cortical recordings (partial seizures) (Fig. 4A). Behavioral correlates of such seizures comprised an arrest reaction (akinesia, staring), followed by eye blinking, chewing, and head nodding (kindling stages 1 or 2) (Goddard, 1967; Racine, 1972). Subsequent seizures showed gradual EEG cortical synchronization with hippocampus and

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longer duration of ictal events (secondarily generalized seizures) (Fig. 4B and C). Clonus of the forelimb and rearing with falling (kindling stage 4 or 5) (Racine, 1972) were the hallmarks of such seizures. Evolution of spontaneous kindling was variable in terms of both duration of paroxysmal discharges and sequence of behavioral stages in every individual animal, however. Several seizure stages of sim ilar or lesser intensity than the subsequent convulsion were monitored before final kindling stage 5 was observed. This initial “subchronic period” of kindling evolution took 7-22 days (12.8 k 1.5 days, mean k SEM, n = 15). Once stage 5 kindling was reached, most of the subsequent seizures were also generalized. Nevertheless, the duration of these seizures remained randomly variable, and behavioral intensity usually varied between stages 3 and 5 of kindling. In general, the quality of the subsequent seizure could not be predicted from the preceding one. In the remaining four animals, the first detected convulsion resembled higher stages of kindling (i.e., stage 3 , 4 or 5 ) and remained randomly variable for the period of observation. In these animals, the seizure silent period was longer (33 4.0 days, n = 4) than that in rats that progressed after kindling 1.1, n = 15). Tonic/clonic seizures stages (9.5 were observed in 3 animals (3 of 19). Morphologic analysis of the brains of rats monitored for 120 days showed a characteristic damage pattern involving the hippocampus, thalamus, amygdaloid complex, pyriform and entorhinal cortex, neocortex, and substantia nigra. The topography of the damage was similar to that described elsewhere (Turski et al., 1983; 1989; Olney et al., 1986; Cavalheiro, 1990).

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FIG. 2. Histogram of cumulative frequency of first spontaneous recurrent seizure (SRS) detected in the 19 rats over time. The total number of SRS was calculated for 10-day epochs. Epilepsia, Vol. 32, N o . 6, 1991

These studies demonstrate that structural damage of the brain (distributed mainly in the temporal lobe) may trigger SRS in rats. SRS occur after a considerable latency and show constant frequency in a 120-day period. Another hallmark of SRS is their temporal distribution during the diurnal phase of the light/dark cycle. Cavalheiro et al. (1982) previously showed that intrahippocampal administration of the excitatory amino acid kainic acid (KA) may trigger SRS in rats. Similar observations were also reported after systemic administration of KA (Cronin and Dudek, 1988). Significant differences between the SRS elicited by pilocarpine or by KA are apparent, however. First, SRS in rats subjected to KA do not appear to follow kindling stages during their early

SPONTANEOUS RECURRENT SEIZURES IN RATS

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FIG. 3. Histogram of distribution of first spontaneous recurrent seizure (SRS) detected in the 19 rats during the IighVdark cycle. The total number of SRS was calculated for 1-11epochs.

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development (Cavalheiro et al., 1982). This discrepancy may be explained in part by the method used in this early research, because continuous monitoring of the EEG ranged from 4 to 8 h/day. Second, in KA-treated rats SRS are observed for a period of 22-46 days and electrographic and behavioral seizures then disappear (Cavalheiro et al., 1982), whereas in the present experiments, SRS registered after pilocarpine treatment occurred with constant frequency during the entire 120-day observation period. A relationship between sleep and epilepsy is well documented. Although this relationship between sleep and SRS in pilocarpine-treated rats is supported by the observation on high frequency of SRS during the diurnal phase, our experimental design was not sufficient for detailed analysis (mainly because of a lack of electromyogram recordings). Analysis of the occurrence of SRS in relation to A

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sleep stages in rats subjected to pilocarpine must therefore await further studies. In the majority of rats (79%), the evolution of SRS resulting from pilocarpine treatment followed stages of kindling. The common characteristics of such seizures are: (a) a primary activation of the hippocampus (before cortex), (b) variable duration of discharges from one seizure to another that interferes with stereotyped progression until they are secondarily generalized, and (c) random duration of discharges after seizures become secondarily generalized. Other studies with electrical stimulation of the hippocampus (with neuronal lesions) in rats with SRS have shown a paradoxic increase in afterdischarge threshold (Cavalheiro et al., 1989). Electrical kindling differs from SRS in that the progression of seizure development is relatively linear and reaches a plateau (Goddard, 1967; Racine, 1972). Conversely, SRS show uneven progression; i.e., every seizure is not necessarily as intense as the previous seizure, although over time the development of kindling is clear. In a minority of rats (21%) secondarily generalized seizures were observed as the first epileptic event. In these animals, partial (kindling stage 1 and 2) and generalized (kindling stage 4 and 5) convulsions continued to appear randomly and progression of the condition did not parallel development of kindling. These animals showed considerably longer latencies to the first SRS, however, which may suggest another localization of the primary focus which could not be detected with hippocampal and cortical electrodes. These observations suggest that structural brain damage in rats may lead to development of chronic epilepsy. A kindling mechanism also may underlie, at least in part, the development of epileptic foci from structural brain lesions in the subchronic period of SRS. Some of these observations may have potential relevance to human partial epilepsy. Human partial seizures most commonly originate in the anterior hippocampal pes (Wieser, 1987) and are character-

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FIG. 4. Electrographic recordings of first spontaneous recurrent seizure (SRS) in a rat that showed kindlinglike characteristics during development of epileptic focus. A: First seizure registered 11 days after administration of pilocarpine. High-voltage fast activity preceded evolution of the seizure in the hippocampus. Cortical recording displayed no changes. The animal was akinetic and showed facial clonus (kindling stage 1). B: Third seizure registered 14 days after pilocarpine. Paroxysmal activity was faster and longer in the hippocampus. The animal showed eye blinking, chewing, and clonic forelimb movements (kindling stage 3). C: Eighth seizure recorded 21 days after pilocarpine. The seizure activity was synchronized in both recordings arld showed long duration. The animal displayed facial automatisms, clonus of the forelimbs, rearing, and falling (kindling stage 5). HPC, hippocampus; CX, cortex.

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E. A . CAVALHEIRO ET AL. ized by diversity in the length of discharges and behavioral symptoms during the chronic period (Engel and Cahan, 1986; Engel, 1988). Furthermore, the most common structural lesion known as hippocampal sclerosis and typical of human partial epilepsy (Meldrum and Corsellis, 1984) is associated with a decrease in seizure excitability (Cherlow et al., 1977). Very little is known about the evolution of epileptic foci between structural brain damage and the appearance of epileptic seizures in humans, however (Engel, 1987). Because SRS monitored in rats in the chronic period resemble some of the characteristic hallmarks of human partial epilepsy, development of epileptic foci in humans may follow some form of kindling. Acknowledgment: Supported by project grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), FundaFBo de Amparo a Pesquisa do Estado de SBo Paulo (FAPESP), Financiadora de Estudos e Projetos (FINEP) from the Brazil and Polish Academy of Sciences.

REFERENCES Cavalheiro EA. GAD-immunoreactive neurons are preserved in the hippocampus of rats with spontaneous recurrent seizures. Braz J Med Biol Res 1990;23:555-8. Cavalheiro EA, Riche DA, Le Gal La Salle G. Long-term effects of intrahippocampal kainic acid injection in rats: a method for inducing spontaneous recurrent seizures. Electroencephalogr Clin Neurophysiol 1982;53:581-9. Cavalheiro EA, Leite JP, Bortolotto ZA, Turski WA, Ikonomidou C, Turski L. Structural brain damage triggers kindling and spontaneously recurrent seizures. SOC Neurosci Absrr 1989;15:778. Cherlow DG, Dymon A, Crandall P, Walter R, Serafetinides E. Evoked response and after-discharge thresholds to electrical stimulation in temporal lobe epileptics. Arch Neurol 1977;34: 527-31. Cronin J, Dudek FE. Chronic seizures and collateral sprouting of dentate mossy fibers after kainic acid treatment in rats. Brain Res 1988;474:181-4. Dichter MA, Ayala GF. Cellular mechanisms of epilepsy: a status report. Science 1987;237: 157-64. Engel J Jr. New concepts of the epileptic focus. In: Wieser HG, Speckmann EJ, Engel J Jr, eds. The epileptic focus. London: Libbey, 1987:83-94. Engel J Jr. Brain metabolism and pathophysiology of human epilepsy. In: Dichter MA, ed. Mechanisms of epileptogenesis. The transition to seizure. New York: Plenum Press, 1988:1-15. Engel J Jr, Cahan L. Potential relevance of kindling to human partial epilepsy. In: Wada J, ed. Kindling 3. New York: Raven Press, 1986:37-54. Fink RP, Heimer L. Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res 1967;4:369-74. Goddard GV. Development of epileptic seizures through brain stimulation at low intensity. Nature 1967;214:1020-1.

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Janz D. Epilepsy: seizures and syndromes. In: Frey HH, Janz D, eds. Antiepileptic drugs. Handbook of experimental pharmucology, vol. 74. Berlin: Springer, 19853-34. Meldrum BS. Pharmacological approach to the treatment of epilepsy. In: Meldrum BS, Porter RJ, eds. New anticonvulsunt drugs. Current problems in epilepsy, vol. 4. London: Libbey, 1986:17-30. Meldrum BS, Corsellis JAN. Epilepsy. In: Hume Adams J, Corsellis JAN, Duchen LW, eds. Greenfield’s neuropathology. London: Arnold, 1984:921-50. Olney JW, deGubareff T, Labruyere J. Seizure-related brain damage induced by cholinergic agents. Nature 1983;301: 520-2. Olney JW, Collins RC, Sloviter RS. Excitotoxic mechanisms of epileptic brain damage. In: Delgado-Escueta AV, Ward AA Jr, Woodbury DM, Porter RJ, eds. Basic mechanisms ofthe epilepsies-molecular and cellular approaches. New York:. Raven Press, 1986:857-77. (Advances in neurology); vol. 44. Purpura DP, Penry JK, Tower D, Woodbury DM, Walter R, eds. Experimental models of epilepsy. New York: Raven Press, 1972. Racine RJ. Modification of seizure activity by electrical stimulation: 11. Motor seizure. Electroencephalogr Clin Neurophysiol 1972;32:281-94. Turski WA, Czuczwar SJ, Kleinrok Z, Turski L. Cholinomimetics produce seizures and brain damage in rats. Experientia 1983;39:1408-11. Turski L, Ikonomidou C, Turski WA, Bortolotto ZA, Cavalheiro EA. Cholinergic mechanisms and epileptogenesis. The seizures induced by pilocarpine: a novel experimental model of intractable epilepsy. Synapse 1989;3:154-71. Wieser HG. The phenomenology of limbic seizures. In: Wieser HG, Speckmann EJ, Engel J Jr, eds. The epileptic focus. London: Libbey, 1987:113-36.

RI?SUME Des ICsions structurelles du cerveau humain (aprks souffrance perinatale, traumatisme ckrtbral, maladies cCrCbro-vasculaires ou dCgCnCratives, tumeurs intracrbniennes, maladies mttaboliques, induction par substances toxiques ou par medicaments) peut entrainer une Cpilepsie chronique chez les survivants. Les analyses CpidCmiologiques montrent qu’il y a un long delai entre I’exposition du cerveau a la cause de la lesion e l’apparition des crises. De telles crises sont gCnCralement partielles ou mixtes, peuvent survenir h n’importe quel &ge, et sont de traitement difficile. Chez des rats, soumis a une Itsion structurelle du cerveau induite par des convulsions prolongCes aprks administration systCmique de pilocarpine, des crises spontantes peuvent survenir aprts une latence moyenne de 14 a 15 jours. La frkquence moyenne des convulsions rkcidivant spontankment reste constante pendant quelques mois. L’Cvolution de ces convulsions traverse plusieurs stades Clectrographiques et comportementaux qui ressemblent h ceux observCs dans le kindling. Le kindling peut &re induit chez les rongeurs par l’administration systkmique rCpttCe de produits convulsivants ou par une stimulation tlectrique rCpktte de regions sensibles du cerveau. Ces observations dtmontrent qu’une lCsion structurelle du cerveau peut provoquer chez le rat des convulsions rCcidivant de faqon spontante, c’est-h-dire une Cpilepsie chronique et que le kindling peut &re un mCcanisme d’un tel ttat. Ces donntes suggkrent qu’un mkcanisme de kindling sous-tend le dtveloppement de foyers Cpileptiques partir de ltsions cCrCbrales structurelles. De tels mCcanismes peuvent &re impliquCs dans I’Ctiologie de certaines formes d’kpilepsie humaine. (P. Genton, Marseille)