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Time-dependent decrease in the effectiveness of antiepileptic drugs during the course of self-sustaining status epilepticus
Brain Research 814 Ž1998. 179–185
Research report
Time-dependent decrease in the effectiveness of antiepileptic drugs during the course of self-sustaining status epilepticus a,d,)
Andrey M. Mazarati
, Roger A. Baldwin d , Raman Sankar
a,b,c
, Claude G. Wasterlain
a,c,d
a
Department of Neurology, UCLA School of Medicine, Los Angeles, CA, USA Department of Pediatrics, UCLA School of Medicine, Los Angeles, CA, USA c Brain Research Institute, UCLA School of Medicine, Los Angeles, CA, USA d Neurology SerÕice, VA Medical Center, SepulÕeda, CA, USA
b
Accepted 13 October 1998
Abstract An animal model of self-sustaining status epilepticus ŽSSSE. induced in rats by brief intermittent perforant path stimulation ŽPPS. was examined with regard to the effects of two conventional antiepileptic drugs, diazepam and phenytoin. Thirty or sixty minutes PPS induced SSSE characterized by continuous behavioral and electrographic seizures lasting for hours. Both diazepam Ž10 mgrkg i.v.. and phenytoin Ž50 mgrkg i.v.. prevented the establishment of SSSE when administered 10 min prior to PPS. The injection of diazepam to seizing animals, 10 min after the end of 30 min PPS, was significantly less effective than pretreatment in attenuating SSSE. Administration of diazepam after 60 min PPS was characterized by a further decrease of its efficacy. Phenytoin was effective in aborting SSSE when injected 10 min after 30 min PPS. However, its efficacy was vastly decreased if injected 40 min after 30 min PPS, or 10 min after 60 min PPS. It is concluded that antiepileptic drugs, while highly effective in blocking the induction of SSSE, failed to affect its maintenance. SSSE induced by PPS is an advantageous animal model of refractory status epilepticus, which may be used in preclinical studies of novel antiepileptic drugs. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Self-sustaining status epilepticus; Antiepileptic drugs; Diazepam; Phenytoin; Refractoriness
1. Introduction Recently, we described a model of self-sustaining status epilepticus ŽSSSE. that develops as a result of brief intermittent electrical stimulation of the perforant path in rats w19x. We suggested that SSSE consists of two phases: the induction phase during which seizures depend entirely on the persistence of the epileptogenic stimuli and stop if the stimulation is discontinued. The second, maintenance phase, is characterized by self-perpetuation of seizure activity in the absence of stimulation. A similar dependence on the epileptogenic agent during the initiation of status epilepticus ŽSE., and the loss of this dependence later in the course of continuing seizures, have been described in the Li-pilocarpine model of SE w6x. The similarity of the data obtained from different animal models suggests that
this character of SE is widespread. It also implies differential pharmacological profiles of the two phases and raises the question of the possible correlation between self-maintenance of seizures and resistance to antiepileptic drugs. Indeed, clinical observations have suggested that the longer SE lasts, the more difficult it becomes to stop w29x. Experimental findings also showed a decrease in the efficacy of some antiepileptic drugs as a function of SE duration w9,21x. In the present study we examined the effectiveness of two antiepileptic drugs ŽAEDs. which are routinely employed in the treatment of status epilepticus, diazepam and phenytoin w26,28x, administered at different stages of SSSE. 2. Materials and methods 2.1. Animals and surgery
)
Corresponding author. VA Medical Center, 111N1, 16111 Plummer St., Sepulveda, CA, 91343, USA. Fax: q1-818-895-5801; E-mail: [email protected]
Under ketamine Ž60 mgrkg.rxylazine Ž15 mgrkg. anesthesia, male Wistar rats, 13–15 weeks old ŽSimonsen Labs, CA. were implanted with a bipolar stimulating elec-
0006-8993r98r$ - see front matterq 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 0 8 0 - 4
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trode into the angular bundle of the perforant path Ž4.5 mm left to and 1 mm anterior to lambda. and a bipolar recording electrode into the granule cell layer of the ipsilateral dentate gyrus Ž3.5 mm anterior to and 2.2 mm left to lambda.. The depth of both electrodes was optimized by finding the population spike of 2 mV or more, evoked from the dentate gyrus by stimuli delivered through the perforant path Žsingle square wave monophasic stimuli, 20 V, 0.1 ms..
calculated. The analysis was performed off-line by means of Monitor 8.1 software. In the experiments in which the drugs were injected before PPS, the zero point for calculations was taken as the end of PPS. In the experiments in which the drugs were injected after PPS, the zero time point was taken as the time of drug injection Ž10 or 40 min after the end of PPS. ŽFig. 1.. Data were analyzed by One-way ANOVA followed by the Student–Newman–Keuls ŽSNK. post hoc test. p - 0.05 was accepted as statistically significant.
2.2. Induction of SSSE 2.5. Terminology note Seven days after surgery, the animals underwent perforant path stimulation ŽPPS. in the awake state for 30 or 60 min with 10 s 20 Hz trains Ž0.1 ms square wave, 20 V. delivered every minute, together with 2 Hz continuous stimulation w19x. Seizure activity was monitored for 24 h by means of Monitor 8.1 software ŽStellate Systems. configured for automatic detection of seizures. Behavioral convulsions were quantified using the Racine scale for kindled seizures w22x. 2.3. Administration of antiepileptic drugs Diazepam Ž0.5, 5, 10 mgrkg i.v.. or phenytoin Ž50 mgrkg i.v.. were administered either 10 min prior to the beginning of, or 10 min after the end of 30 or 60 min of PPS. In a separate series of the experiments, phenytoin was administered 40 min after the end of 30-min PPS ŽFig. 1.. Control animals were treated with saline. Each group included 4–9 animals. 2.4. Analysis of SSSE SSSE was quantified by calculating total time spent in seizures, and the time of occurrence of the last seizure. In the experiments with drug administration after PPS, spike frequency Žnumber of spikes per 30 min epoch. was also
Fig. 1. Protocol used for drugs administration. PPS of 30 min ŽA. and 60 min ŽB. duration is indicated by gray bar. Diazepam ŽDZP. or phenytoin ŽPHT. were injected as indicated by arrows 10 min before PPS, or 10 min after the end of PPS, or ŽPHT. 40 min after the end of PPS. Thin horizontal line indicates absence of seizures; medium horizontal line indicates seizure activity during PPS; bold horizontal line indicates SSSE.
In the description of the anticonvulsant effect of AEDs, shortening of the time spent in seizures and time of the last seizure, must be distinguished from complete blockade of SSSE. Since SSSE is defined as ‘‘prolonged epileptic state that occurs when a single seizure lasts at least 30 min or intermittent seizures last at least 30 min without regaining the consciousness between seizures’’ w2x, we considered that SSSE was prevented or aborted by antiepileptic drug, when the last seizure occurred not later than 30 min after the injection. Otherwise, we described the effects of a drug Žif any. as attenuating, or shortening of SSSE duration. All experiments complied with the protocol approved by the Animal Care Committee of Sepulveda VAMC. 3. Results Thirty min PPS induced seizure activity that continued for 10.7 " 2.25 h after the end of stimulation ŽFig. 2., as has been described earlier w19x. Seizures recognized by the software were characterized by periods of synchronized activity with a spike frequency of 3 Hz or more, lasting from 10 s to 2 min, which recurred at varying intervals Ž5–30 min. and were accompanied by behavioral stage 4–5 seizures Žrearingrrearing and falling.. Between these events, animals continued to show paroxysmal activity, in the form of spikes with a frequency of 0.3–3 Hz accompanied by behavioral seizures of stages 1 through 3. Administration of diazepam was accompanied by motor disturbances which exacerbated as the dose of the drug increased. Diazepam at a dose of 0.5 mgrkg did not induce any obvious behavioral alterations. At 5 mgrkg, the animals showed some muscle relaxation and ataxia. The severity of these manifestations further increased at a dose of 10 mgrkg of diazepam. Phenytoin in a the tested dose did not induce obvious behavioral alterations. Administration of diazepam in doses of 0.5 mgrkg, 5 mgrkg Žnot shown. or 10 mgrkg ŽFig. 2A. before the beginning of PPS, effectively prevented the development of SSSE. For all doses, the time spent in seizures after the end of PPS did not exceed 5 min, and the last seizure occurred within 10 min after the end of PPS. Phenytoin Ž50 mgrkg. also blocked the establishment of SSSE ŽFig. 2A..
A.M. Mazarati et al.r Brain Research 814 (1998) 179–185
Fig. 2. The effects of diazepam ŽDZP. and phenytoin ŽPHT. on SSSE induced by 30 min PPS. ŽA. When injected before PPS, both drugs prevented the occurrence of SSSE. ŽB. Top: When administered 10 min after the end of PPS, diazepam attenuated, but not stopped SSSE, while phenytoin aborted SSSE ŽPHT, q10 min.. However, when given 40 min after the end of PPS, phenytoin failed to abort SSSE, although still shortened its duration ŽPHT, q40 min.. ) p- 0.05 vs. Control. ap0.05 vs. DZP in A Žunder conditions of pretreatment.. l p- 0.05 vs. PHTq10 min ŽOne-way ANOVAqSNK.. Bottom: time-course of spikes after diazepam and phenytoin administration after PPS. PPS is indicated by gray bar. Dashed and solid outlines indicate time frames when spike frequency for diazepam and phenytoin, respectively was significantly lower than in control ŽOne-way ANOVAqSNK..
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When administered 10 min after the end of 30 min PPS, diazepam in doses of 0.5 and 5 mgrkg had no seizure-protective effects Žnot shown.. After injection of diazepam in a dose of 10 mgrkg, electrographic seizures continued, although motor disturbances observed under these conditions, affected the behavioral expression of ongoing seizures. Total time spent in seizures was 95 " 22 min and the last seizure was observed at 140 " 32 min after the administration of diazepam, which was significantly longer than after diazepam injection before PPS. The time course of spikes was also affected by diazepam. In the effects of diazepam, two periods of significant suppression of spike frequency were observed: one immediately after drug administration, lasted for about 5 h, and the second one was observed between approximately 11 and 17 h of monitoring ŽFig. 2B.. Phenytoin Ž50 mgrkg. effectively aborted SSSE when injected after 30 min PPS: animals spent 6.3 " 2.5 min in seizures, and the last seizure occurred within 30 min after drug administration ŽFig. 2B.. Spike frequency was irreversibly suppressed by phenytoin starting from 20–30 min after its administration ŽFig. 2B.. In the next set of the experiments, we examined the effects of antiepileptic drugs on SSSE induced by 60 min PPS, which might induce more severe self-sustaining seizures. Indeed, although the time of last seizure after 30 and 60 min PPS did not differ significantly Ž644 " 134 and 730 " 147 min, respectively., after 60 min PPS, seizures occurred with higher incidence and the duration of individual seizures was longer. This resulted in significantly longer time spent in seizures Ž510 " 71 vs. 352 " 801, p - 0.05.. Both diazepam ŽFig. 3D. and phenytoin effectively prevented the establishment of SSSE when given prior to PPS Žlast seizure occurred no later than 10 min after injection, Fig. 3A.. Neither compound affected the frequency or time course of spikes when injected after 60 min PPS ŽFig. 3B.. However, when injected after PPS, both drugs failed to abort established SSSE, although both significantly reduced its duration. Time spent in seizures was 204 " 30 and 216 " 27 and time of the last seizure was 366 " 65 and 300 " 93 for diazepam and phenytoin, respectively Ž p - 0.05 vs. control, although all these measurements were significantly higher compared to those following pretreatment, Fig. 3B.. Neither of the compounds affected time course of spikes when injected after 60 min PPS ŽFig. 3B.. To differentiate between the effects of seizure duration and the length of PPS as a cause of increased refractoriness of SSSE established after 60 min PPS, we administered phenytoin 40 min after 30 min PPS ŽFig. 1A.. We chose phenytoin because of more pronounced difference in its effects after 30 and 60 min PPS, than that of diazepam. Delayed injection of phenytoin was poorly effective: time spent in seizures was 198 " 36 min and the last seizure was observed within 280 " 59 min Ž p - 0.05 compared with the effects of phenytoin injected 10 min after PPS.,
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although the duration of SSSE was still significantly shorter than in controls ŽFig. 2B..
4. Discussion The major finding of our study is that anticonvulsant efficacy of two drugs conventionally used for treatment of status epilepticus depends on the time of their administration. When given before PPS of either 30 or 60-min duration, both diazepam and phenytoin effectively prevent the establishment of SSSE. These effects were not due to the suppression of seizure activity during PPS. Total seizure time during PPS did not differ significantly among control and drug-treated groups ŽFig. 3C–E.. This suggests that injection of anticonvulsants prior to PPS blocked the mechanisms needed for the establishment of self-sustaining seizures. Administration of diazepam 10 min after 30 min PPS was significantly less effective in shortening SSSE compared to pre-treatment. The observed rapid disappearance of spikes after injection of diazepam, followed by their slow and partial return 7 to 11 h later may be related to redistribution of this highly lipid soluble compound w1x to adipose tissue with concomitant decrease in its brain concentration. Similar phenomenon is known to occur in humans, although at a much earlier time point w12x. Administration of diazepam following 60 min PPS showed further decrease in diazepam efficacy ŽFig. 4A.. Self-sustaining seizures continued on the average 6 h vs. 11 h on control, showing that late treatment failed to abort the maintenance phase of SSSE, although its severity was mitigated. The time dependent decrease in the efficacy of benzodiazepines, acting through GABA-Cly ionophore receptor complex w13,14,20x, in the treatment of experimental status epilepticus has been reported earlier w9,21x. Plastic changes in benzodiazepine receptors occurring during SE w9x accompanied by the reduced efficacy of GABA in opening Cly channels and diminished driving force for GABA receptor currents w8x, as well as the inversion of Cly equilibrium potential due to continuous depolarization and the resulting GABA-induced depolarization instead of hyperpolarization w24x may contribute to this phenomenon. Thus, we observed dramatic differences between the effects of diazepam on seizures depending on the time of its administration. While the induction of SSSE could be easily prevented with very low dose of diazepam
Fig. 4. Anticonvulsant efficacy of diazepam and phenytoin depends on the time of their administration. Time spent in seizures and time of the last seizure recorded under conditions of antiepileptic drugs injection after the end of PPS, are presented as percent of respective values in control groups. Notice that the efficacy of both diazepam ŽA. and phenytoin ŽB. decreased as they were injected after 60-min PPS vs. after 30-min PPS. The efficacy of phenytoin also decreased when it was injected 40 min after 30-min PPS Žindicated by asterisk, C..
Ž0.5 mgrkg., established SSSE could not be aborted by even 10 times higher dose. This is reminiscent of the finding by Kapur and MacDonald w9x that the dose of diazepam needed to stop Li-pilocarpine-induced SE increased 10 fold Ž4.2 to 40 mgrkg. when the time of its administration was delayed by 30 min. In contrast to diazepam, phenytoin was effective in aborting SSSE when given early after 30 min PPS. However, when injected at a late point after 30 min PPS, or after early 60 min PPS, the efficacy of phenytoin dramatically decreased ŽFig. 4B and C.. Animals posttreated with phenytoin in the 60 min PPS group continued to experience seizures for an average duration of 5 h, demonstrating the failure of another standard anticonvulsant to block the maintenance phase of SSSE. The anticonvulsant action of phenytoin is mediated through the blockade of voltagegated Naq channels w14,20x. Therefore, the observed decrease of the efficacy of phenytoin in blocking established SSSE, is independent of the SE-induced alterations in GABA receptor complex. The failure of two standard antiepileptic drugs to abort SSSE during its maintenance phase implies that seizures evolve certain mechanisms that cause refractoriness to antiepileptic drugs. The nature of the mechanisms respon-
Fig. 3. The effects of diazepam and phenytoin on SSSE induced by 60 min PPS. ŽA. When administered before PPS, both DZP and PHT prevented the establishment of SSSE. ŽB. Top: When injected 10 min after the end of PPS, neither of them aborted SSSE, although they shortened its duration. ) p - 0.05 vs. Control. ap - 0.05 vs. DZP and PHT, respectively in A Žunder conditions of pretreatment.. Bottom: Time-course of spikes after diazepam and phenytoin administration after PPS. No significant differences were observed among the groups ŽOne-way ANOVA.. All patterns on A and B are same as in Fig. 2. ŽC–E. Representative time course of seizures in a control animal ŽC. an animal pretreated with diazepam ŽD. or an animal with diazepam injected 10 min after PPS ŽE.. Each line represents 2 h of EEG monitoring. Each software-recognized seizure is shown by small black bar. PPS is indicated by gray bars on the top of each fragment. Injection of diazepam is indicated by an arrow in D and E. Notice that in the control animal self-sustaining seizures were observed for 17 h. In diazepam-pretreated rats, seizures occurred during PPS, but only a few seizures were observed after PPS and only within the first 20 min. In the diazepam-post-treated animal, self-sustaining seizures continued within 8 h.
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sible for the occurrence of drug resistance is a subject for further studies. Correlating the occurrence of refractoriness with the establishment of the maintenance phase of SSSE may be useful. SE becomes self-sustaining after 15–30 min of stimulation. Resistance to diazepam occurs early after PPS, and may reflect the changes in GABA receptor function. Resistance to phenytoin requires longer duration of seizures. This suggests that maintenance of SSSE does not mean its refractoriness, but rather the evolves mechanisms which result in the failure of antiepileptic drugs. The potentiation of excitatory synapses, e.g., in a form of posttetanic andror long-term potentiation in the dentate gyrus that was reported to accompany SSSE w16x may contribute to this process. Another candidate for such mechanism may be translocation and autophosphorylation of calmodulin kinase II during of status epilepticus w27x, which may enhance glutamate release even in the face of blockade of voltage-gated Naq channels. This may restrict or even abolish the effectiveness of phenytoin. All the mentioned data imply that when conventional anticonvulsants fail to stop SSSE, another class of pharmacological agents, that would block the postsynaptic effects of excitatory amino acids may provide an alternative approach. Indeed, preliminary studies w17x showed that blockers of NMDA receptors ŽMK-801 and ketamine. aborted SSSE when given 10 min after 60 min PPS, and were effective in pilocarpine induced SE w23x. Other mechanisms may involve failure of neuromodulatory systems that normally control hippocampal excitability. For instance, SSSE depletes hippocampal stores of peptides dynorphin and galanin w15,18x which normally decrease glutamate release from dentate granule cells w5,30,31x. Furthermore, exogenous administration into the hilus of the dentate gyrus of ligands for kappa and galanin receptors both prevented and stopped established SSSE. Alternatively, the increase in the substance P immunoreactivity in the hippocampus during SSSE w11x may contribute to the presynaptic stimulation of glutamate release w7x. Development of animal models of refractory Žresistant to antiepileptic drugs. status epilepticus has both basic and practical significance. On one hand, studies of the mechanisms that underlie drug resistance are important for understanding the pathophysiology of status epilepticus. On the other hand, those models may be used for testing newly developed antiepileptic drugs and for comparison of their efficacy with standard anticonvulsants. The present experimental paradigm offers the first drug-free model of refractory SE, a condition which responds poorly to standard anticonvulsants and still carries a very high mortality w3,4,29x. This model may be used in evaluating alternate pharmacotherapy for refractory SE. Some very expensive agents are widely used for refractory SE Že.g., see Refs. w10,25x., but their efficacy has never been demonstrated neither under clinical, nor experimental conditions. Of course, the approximation of findings in
animal models to the clinical situation must be done with caution. While human data comparing the efficacy of diazepam at specific time points during the course of SE is not available, it is common clinical experience that SE becomes refractory to treatment with anticonvulsants with time. Comparison of the effectiveness of diazepam and phenytoin at certain time points in humans is complicated also due to the protocol for administering phenytoin which typically requires 20 min or more to deliver the typical full dose of the drug. The advantages of the present model include the possibility to control the severity of SSSE through the duration of epileptogenic stimulation and to control the refractoriness to anticonvulsants by manipulating the time of their administration in relation to PPS. Acknowledgements C.W. is supported by grant NS 11315 from NINDS and by the Research Service of VHA. R.S. is supported by CIDA NS 01792. References w1x P.A. Borea, D. Bonora, Brain receptor binding and lipophilic character of benzodiazepines, Biochem. Pharmacol. 32 Ž1983. 603–607. w2x Commission on Classification and Terminology of the International League Against Epilepsy, Proposal for revised clinical and electroencephalographic classification of epileptic seizures, Epilepsia 22 Ž1981. 489–501. w3x R.J. DeLorenzo, W.A. Hauser, A.R. Towne, J.G. Boggs, J.M. Pellock, L. Penberthy, L. Garnett, C.A. Fortner, D. Ko, A prospective, population-based epidemiologic study of status epilepticus in Richmond, Virginia, Neurology 46 Ž1996. 1029–1035. w4x R.J. DeLorenzo, J.M. Pellock, A.R. Towne, J.C. Boggs, Epidemiology of status epilepticus, J. Clin. Neurophysiol. 12 Ž1995. 316–325. w5x C.T. Drake, G.W. Terman, M.L. Simmons, T.A. Milner, K. D.D., P.A. Schwartzkroin, C. Chavkin, Dynorphin opioids present in dentate granule cells may function as retrograde inhibitory neurotransmitters, J. Neurosci. 14 Ž1994. 3736–3750. w6x R.S. Jope, R.A. Morrisett, O.C. Snead, Characterization of Lithium potentiation of pilocarpine-induced status epilepticus, Exp. Neurol. 91 Ž1986. 471–480. w7x I. Kangrga, M. Randic, Tachikinins and calcitonin-gene related peptide enhance release of glutamate and apartate from the rat spinal dorsal horn slices, J. Neurosci. 10 Ž1990. 1026–2038. w8x J. Kapur, D.A. Coulter, Experimental status epilepticus alters GABA A receptor function in CA1 pyramidal neurons, Ann. Neurol. 38 Ž1995. 893–900. w9x J. Kapur, R.L. MacDonald, Rapid seizure-induced reduction of benzodiazepine and Zn2q sensitivity of hippocampal dentate granule cell GABA A receptors, J. Neurosci. 17 Ž1997. 7532–7540. w10x M. Kuisma, R.O. Roine, Propofol in prehospital treatment of convulsive status epilepticus, Epilepsia 36 Ž1995. 1241–1243. w11x H. Liu, A.M. Mazarati, H. Katsumori, C.G. Wasterlain, Limbic seizures mediated by phenotypic switch in hippocampal principal neurons, Soc. Neurosci Abstr. 24 Ž1998. 2140. w12x C.T. Lombroso, Treatment of status epilepticus with diazepam, Neurology 16 Ž1966. 629–634. w13x R.L. MacDonald, K.M. Kelly, Antiepileptic drug mechanisms of action, Epilepsia 36 Ž1995. S2–S12.
A.M. Mazarati et al.r Brain Research 814 (1998) 179–185 w14x R.L. MacDonald, B.S. Meldrum, General principles: principles of antiepileptic drugs action, in: R.H. Levy, R.H. Mattson, B.S. Meldrum ŽEds.., Antiepileptic Drugs, 4th edn., Raven Press, New York, 1995, pp. 61–77. w15x A. Mazarati, H. Liu, C. Wasterlain, U. Langel, U. Soomets, R. Sankar, D. Shin, Galanin modulates status epilepticus, Soc. Neurosci. Abstr. 24 Ž1998. 1937. w16x A. Mazarati, C. Wasterlain, Loss of hippocampal inhibition and enhanced LTP after self-sustaining status epilepticus, Epilepsia 38 Ž1997. 178, Suppl. 3. w17x A. Mazarati, C. Wasterlain, Pharmacological treatment of self-sustaining status epilepticus, Soc. Neurosci. Abstr. 23 Ž1997. 1687. w18x A.M. Mazarati, H. Liu, C.G. Wasterlain, Opioid peptide pharmacology and immunochemistry in an animal model of self-sustaining status epilepticus, Neuroscience 88 Ž1999. in press. w19x A.M. Mazarati, C.G. Wasterlain, R. Sankar, D. Shin, Self-sustaining status epilepticus after brief electrical stimulation of the perforant path, Brain Research 801 Ž1998. 251–253. w20x B.S. Meldrum, Update on the mechanism of action of antiepileptic drugs, Epilepsia 37 Ž1996. S4–S11, Suppl. 6. w21x R.A. Morrisett, R.S. Jope, O.C. Snead, Effects of drugs on the initiation and maintenance of status epilepticus induced by administration of pilocarpine to lithium-pretreated rats, Exp. Neurol. 97 Ž1987. 193–200. w22x R.J. Racine, Modification of seizure activity by electrical stimulation, Electroencephalogr. Clin. Neurophysiol. 32 Ž1972. 281–294. w23x A.C. Rice, R.J. DeLorenzo, The effect of NMDA receptor inhibition
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Research report
Time-dependent decrease in the effectiveness of antiepileptic drugs during the course of self-sustaining status epilepticus a,d,)
Andrey M. Mazarati
, Roger A. Baldwin d , Raman Sankar
a,b,c
, Claude G. Wasterlain
a,c,d
a
Department of Neurology, UCLA School of Medicine, Los Angeles, CA, USA Department of Pediatrics, UCLA School of Medicine, Los Angeles, CA, USA c Brain Research Institute, UCLA School of Medicine, Los Angeles, CA, USA d Neurology SerÕice, VA Medical Center, SepulÕeda, CA, USA
b
Accepted 13 October 1998
Abstract An animal model of self-sustaining status epilepticus ŽSSSE. induced in rats by brief intermittent perforant path stimulation ŽPPS. was examined with regard to the effects of two conventional antiepileptic drugs, diazepam and phenytoin. Thirty or sixty minutes PPS induced SSSE characterized by continuous behavioral and electrographic seizures lasting for hours. Both diazepam Ž10 mgrkg i.v.. and phenytoin Ž50 mgrkg i.v.. prevented the establishment of SSSE when administered 10 min prior to PPS. The injection of diazepam to seizing animals, 10 min after the end of 30 min PPS, was significantly less effective than pretreatment in attenuating SSSE. Administration of diazepam after 60 min PPS was characterized by a further decrease of its efficacy. Phenytoin was effective in aborting SSSE when injected 10 min after 30 min PPS. However, its efficacy was vastly decreased if injected 40 min after 30 min PPS, or 10 min after 60 min PPS. It is concluded that antiepileptic drugs, while highly effective in blocking the induction of SSSE, failed to affect its maintenance. SSSE induced by PPS is an advantageous animal model of refractory status epilepticus, which may be used in preclinical studies of novel antiepileptic drugs. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Self-sustaining status epilepticus; Antiepileptic drugs; Diazepam; Phenytoin; Refractoriness
1. Introduction Recently, we described a model of self-sustaining status epilepticus ŽSSSE. that develops as a result of brief intermittent electrical stimulation of the perforant path in rats w19x. We suggested that SSSE consists of two phases: the induction phase during which seizures depend entirely on the persistence of the epileptogenic stimuli and stop if the stimulation is discontinued. The second, maintenance phase, is characterized by self-perpetuation of seizure activity in the absence of stimulation. A similar dependence on the epileptogenic agent during the initiation of status epilepticus ŽSE., and the loss of this dependence later in the course of continuing seizures, have been described in the Li-pilocarpine model of SE w6x. The similarity of the data obtained from different animal models suggests that
this character of SE is widespread. It also implies differential pharmacological profiles of the two phases and raises the question of the possible correlation between self-maintenance of seizures and resistance to antiepileptic drugs. Indeed, clinical observations have suggested that the longer SE lasts, the more difficult it becomes to stop w29x. Experimental findings also showed a decrease in the efficacy of some antiepileptic drugs as a function of SE duration w9,21x. In the present study we examined the effectiveness of two antiepileptic drugs ŽAEDs. which are routinely employed in the treatment of status epilepticus, diazepam and phenytoin w26,28x, administered at different stages of SSSE. 2. Materials and methods 2.1. Animals and surgery
)
Corresponding author. VA Medical Center, 111N1, 16111 Plummer St., Sepulveda, CA, 91343, USA. Fax: q1-818-895-5801; E-mail: [email protected]
Under ketamine Ž60 mgrkg.rxylazine Ž15 mgrkg. anesthesia, male Wistar rats, 13–15 weeks old ŽSimonsen Labs, CA. were implanted with a bipolar stimulating elec-
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trode into the angular bundle of the perforant path Ž4.5 mm left to and 1 mm anterior to lambda. and a bipolar recording electrode into the granule cell layer of the ipsilateral dentate gyrus Ž3.5 mm anterior to and 2.2 mm left to lambda.. The depth of both electrodes was optimized by finding the population spike of 2 mV or more, evoked from the dentate gyrus by stimuli delivered through the perforant path Žsingle square wave monophasic stimuli, 20 V, 0.1 ms..
calculated. The analysis was performed off-line by means of Monitor 8.1 software. In the experiments in which the drugs were injected before PPS, the zero point for calculations was taken as the end of PPS. In the experiments in which the drugs were injected after PPS, the zero time point was taken as the time of drug injection Ž10 or 40 min after the end of PPS. ŽFig. 1.. Data were analyzed by One-way ANOVA followed by the Student–Newman–Keuls ŽSNK. post hoc test. p - 0.05 was accepted as statistically significant.
2.2. Induction of SSSE 2.5. Terminology note Seven days after surgery, the animals underwent perforant path stimulation ŽPPS. in the awake state for 30 or 60 min with 10 s 20 Hz trains Ž0.1 ms square wave, 20 V. delivered every minute, together with 2 Hz continuous stimulation w19x. Seizure activity was monitored for 24 h by means of Monitor 8.1 software ŽStellate Systems. configured for automatic detection of seizures. Behavioral convulsions were quantified using the Racine scale for kindled seizures w22x. 2.3. Administration of antiepileptic drugs Diazepam Ž0.5, 5, 10 mgrkg i.v.. or phenytoin Ž50 mgrkg i.v.. were administered either 10 min prior to the beginning of, or 10 min after the end of 30 or 60 min of PPS. In a separate series of the experiments, phenytoin was administered 40 min after the end of 30-min PPS ŽFig. 1.. Control animals were treated with saline. Each group included 4–9 animals. 2.4. Analysis of SSSE SSSE was quantified by calculating total time spent in seizures, and the time of occurrence of the last seizure. In the experiments with drug administration after PPS, spike frequency Žnumber of spikes per 30 min epoch. was also
Fig. 1. Protocol used for drugs administration. PPS of 30 min ŽA. and 60 min ŽB. duration is indicated by gray bar. Diazepam ŽDZP. or phenytoin ŽPHT. were injected as indicated by arrows 10 min before PPS, or 10 min after the end of PPS, or ŽPHT. 40 min after the end of PPS. Thin horizontal line indicates absence of seizures; medium horizontal line indicates seizure activity during PPS; bold horizontal line indicates SSSE.
In the description of the anticonvulsant effect of AEDs, shortening of the time spent in seizures and time of the last seizure, must be distinguished from complete blockade of SSSE. Since SSSE is defined as ‘‘prolonged epileptic state that occurs when a single seizure lasts at least 30 min or intermittent seizures last at least 30 min without regaining the consciousness between seizures’’ w2x, we considered that SSSE was prevented or aborted by antiepileptic drug, when the last seizure occurred not later than 30 min after the injection. Otherwise, we described the effects of a drug Žif any. as attenuating, or shortening of SSSE duration. All experiments complied with the protocol approved by the Animal Care Committee of Sepulveda VAMC. 3. Results Thirty min PPS induced seizure activity that continued for 10.7 " 2.25 h after the end of stimulation ŽFig. 2., as has been described earlier w19x. Seizures recognized by the software were characterized by periods of synchronized activity with a spike frequency of 3 Hz or more, lasting from 10 s to 2 min, which recurred at varying intervals Ž5–30 min. and were accompanied by behavioral stage 4–5 seizures Žrearingrrearing and falling.. Between these events, animals continued to show paroxysmal activity, in the form of spikes with a frequency of 0.3–3 Hz accompanied by behavioral seizures of stages 1 through 3. Administration of diazepam was accompanied by motor disturbances which exacerbated as the dose of the drug increased. Diazepam at a dose of 0.5 mgrkg did not induce any obvious behavioral alterations. At 5 mgrkg, the animals showed some muscle relaxation and ataxia. The severity of these manifestations further increased at a dose of 10 mgrkg of diazepam. Phenytoin in a the tested dose did not induce obvious behavioral alterations. Administration of diazepam in doses of 0.5 mgrkg, 5 mgrkg Žnot shown. or 10 mgrkg ŽFig. 2A. before the beginning of PPS, effectively prevented the development of SSSE. For all doses, the time spent in seizures after the end of PPS did not exceed 5 min, and the last seizure occurred within 10 min after the end of PPS. Phenytoin Ž50 mgrkg. also blocked the establishment of SSSE ŽFig. 2A..
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Fig. 2. The effects of diazepam ŽDZP. and phenytoin ŽPHT. on SSSE induced by 30 min PPS. ŽA. When injected before PPS, both drugs prevented the occurrence of SSSE. ŽB. Top: When administered 10 min after the end of PPS, diazepam attenuated, but not stopped SSSE, while phenytoin aborted SSSE ŽPHT, q10 min.. However, when given 40 min after the end of PPS, phenytoin failed to abort SSSE, although still shortened its duration ŽPHT, q40 min.. ) p- 0.05 vs. Control. ap0.05 vs. DZP in A Žunder conditions of pretreatment.. l p- 0.05 vs. PHTq10 min ŽOne-way ANOVAqSNK.. Bottom: time-course of spikes after diazepam and phenytoin administration after PPS. PPS is indicated by gray bar. Dashed and solid outlines indicate time frames when spike frequency for diazepam and phenytoin, respectively was significantly lower than in control ŽOne-way ANOVAqSNK..
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When administered 10 min after the end of 30 min PPS, diazepam in doses of 0.5 and 5 mgrkg had no seizure-protective effects Žnot shown.. After injection of diazepam in a dose of 10 mgrkg, electrographic seizures continued, although motor disturbances observed under these conditions, affected the behavioral expression of ongoing seizures. Total time spent in seizures was 95 " 22 min and the last seizure was observed at 140 " 32 min after the administration of diazepam, which was significantly longer than after diazepam injection before PPS. The time course of spikes was also affected by diazepam. In the effects of diazepam, two periods of significant suppression of spike frequency were observed: one immediately after drug administration, lasted for about 5 h, and the second one was observed between approximately 11 and 17 h of monitoring ŽFig. 2B.. Phenytoin Ž50 mgrkg. effectively aborted SSSE when injected after 30 min PPS: animals spent 6.3 " 2.5 min in seizures, and the last seizure occurred within 30 min after drug administration ŽFig. 2B.. Spike frequency was irreversibly suppressed by phenytoin starting from 20–30 min after its administration ŽFig. 2B.. In the next set of the experiments, we examined the effects of antiepileptic drugs on SSSE induced by 60 min PPS, which might induce more severe self-sustaining seizures. Indeed, although the time of last seizure after 30 and 60 min PPS did not differ significantly Ž644 " 134 and 730 " 147 min, respectively., after 60 min PPS, seizures occurred with higher incidence and the duration of individual seizures was longer. This resulted in significantly longer time spent in seizures Ž510 " 71 vs. 352 " 801, p - 0.05.. Both diazepam ŽFig. 3D. and phenytoin effectively prevented the establishment of SSSE when given prior to PPS Žlast seizure occurred no later than 10 min after injection, Fig. 3A.. Neither compound affected the frequency or time course of spikes when injected after 60 min PPS ŽFig. 3B.. However, when injected after PPS, both drugs failed to abort established SSSE, although both significantly reduced its duration. Time spent in seizures was 204 " 30 and 216 " 27 and time of the last seizure was 366 " 65 and 300 " 93 for diazepam and phenytoin, respectively Ž p - 0.05 vs. control, although all these measurements were significantly higher compared to those following pretreatment, Fig. 3B.. Neither of the compounds affected time course of spikes when injected after 60 min PPS ŽFig. 3B.. To differentiate between the effects of seizure duration and the length of PPS as a cause of increased refractoriness of SSSE established after 60 min PPS, we administered phenytoin 40 min after 30 min PPS ŽFig. 1A.. We chose phenytoin because of more pronounced difference in its effects after 30 and 60 min PPS, than that of diazepam. Delayed injection of phenytoin was poorly effective: time spent in seizures was 198 " 36 min and the last seizure was observed within 280 " 59 min Ž p - 0.05 compared with the effects of phenytoin injected 10 min after PPS.,
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although the duration of SSSE was still significantly shorter than in controls ŽFig. 2B..
4. Discussion The major finding of our study is that anticonvulsant efficacy of two drugs conventionally used for treatment of status epilepticus depends on the time of their administration. When given before PPS of either 30 or 60-min duration, both diazepam and phenytoin effectively prevent the establishment of SSSE. These effects were not due to the suppression of seizure activity during PPS. Total seizure time during PPS did not differ significantly among control and drug-treated groups ŽFig. 3C–E.. This suggests that injection of anticonvulsants prior to PPS blocked the mechanisms needed for the establishment of self-sustaining seizures. Administration of diazepam 10 min after 30 min PPS was significantly less effective in shortening SSSE compared to pre-treatment. The observed rapid disappearance of spikes after injection of diazepam, followed by their slow and partial return 7 to 11 h later may be related to redistribution of this highly lipid soluble compound w1x to adipose tissue with concomitant decrease in its brain concentration. Similar phenomenon is known to occur in humans, although at a much earlier time point w12x. Administration of diazepam following 60 min PPS showed further decrease in diazepam efficacy ŽFig. 4A.. Self-sustaining seizures continued on the average 6 h vs. 11 h on control, showing that late treatment failed to abort the maintenance phase of SSSE, although its severity was mitigated. The time dependent decrease in the efficacy of benzodiazepines, acting through GABA-Cly ionophore receptor complex w13,14,20x, in the treatment of experimental status epilepticus has been reported earlier w9,21x. Plastic changes in benzodiazepine receptors occurring during SE w9x accompanied by the reduced efficacy of GABA in opening Cly channels and diminished driving force for GABA receptor currents w8x, as well as the inversion of Cly equilibrium potential due to continuous depolarization and the resulting GABA-induced depolarization instead of hyperpolarization w24x may contribute to this phenomenon. Thus, we observed dramatic differences between the effects of diazepam on seizures depending on the time of its administration. While the induction of SSSE could be easily prevented with very low dose of diazepam
Fig. 4. Anticonvulsant efficacy of diazepam and phenytoin depends on the time of their administration. Time spent in seizures and time of the last seizure recorded under conditions of antiepileptic drugs injection after the end of PPS, are presented as percent of respective values in control groups. Notice that the efficacy of both diazepam ŽA. and phenytoin ŽB. decreased as they were injected after 60-min PPS vs. after 30-min PPS. The efficacy of phenytoin also decreased when it was injected 40 min after 30-min PPS Žindicated by asterisk, C..
Ž0.5 mgrkg., established SSSE could not be aborted by even 10 times higher dose. This is reminiscent of the finding by Kapur and MacDonald w9x that the dose of diazepam needed to stop Li-pilocarpine-induced SE increased 10 fold Ž4.2 to 40 mgrkg. when the time of its administration was delayed by 30 min. In contrast to diazepam, phenytoin was effective in aborting SSSE when given early after 30 min PPS. However, when injected at a late point after 30 min PPS, or after early 60 min PPS, the efficacy of phenytoin dramatically decreased ŽFig. 4B and C.. Animals posttreated with phenytoin in the 60 min PPS group continued to experience seizures for an average duration of 5 h, demonstrating the failure of another standard anticonvulsant to block the maintenance phase of SSSE. The anticonvulsant action of phenytoin is mediated through the blockade of voltagegated Naq channels w14,20x. Therefore, the observed decrease of the efficacy of phenytoin in blocking established SSSE, is independent of the SE-induced alterations in GABA receptor complex. The failure of two standard antiepileptic drugs to abort SSSE during its maintenance phase implies that seizures evolve certain mechanisms that cause refractoriness to antiepileptic drugs. The nature of the mechanisms respon-
Fig. 3. The effects of diazepam and phenytoin on SSSE induced by 60 min PPS. ŽA. When administered before PPS, both DZP and PHT prevented the establishment of SSSE. ŽB. Top: When injected 10 min after the end of PPS, neither of them aborted SSSE, although they shortened its duration. ) p - 0.05 vs. Control. ap - 0.05 vs. DZP and PHT, respectively in A Žunder conditions of pretreatment.. Bottom: Time-course of spikes after diazepam and phenytoin administration after PPS. No significant differences were observed among the groups ŽOne-way ANOVA.. All patterns on A and B are same as in Fig. 2. ŽC–E. Representative time course of seizures in a control animal ŽC. an animal pretreated with diazepam ŽD. or an animal with diazepam injected 10 min after PPS ŽE.. Each line represents 2 h of EEG monitoring. Each software-recognized seizure is shown by small black bar. PPS is indicated by gray bars on the top of each fragment. Injection of diazepam is indicated by an arrow in D and E. Notice that in the control animal self-sustaining seizures were observed for 17 h. In diazepam-pretreated rats, seizures occurred during PPS, but only a few seizures were observed after PPS and only within the first 20 min. In the diazepam-post-treated animal, self-sustaining seizures continued within 8 h.
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sible for the occurrence of drug resistance is a subject for further studies. Correlating the occurrence of refractoriness with the establishment of the maintenance phase of SSSE may be useful. SE becomes self-sustaining after 15–30 min of stimulation. Resistance to diazepam occurs early after PPS, and may reflect the changes in GABA receptor function. Resistance to phenytoin requires longer duration of seizures. This suggests that maintenance of SSSE does not mean its refractoriness, but rather the evolves mechanisms which result in the failure of antiepileptic drugs. The potentiation of excitatory synapses, e.g., in a form of posttetanic andror long-term potentiation in the dentate gyrus that was reported to accompany SSSE w16x may contribute to this process. Another candidate for such mechanism may be translocation and autophosphorylation of calmodulin kinase II during of status epilepticus w27x, which may enhance glutamate release even in the face of blockade of voltage-gated Naq channels. This may restrict or even abolish the effectiveness of phenytoin. All the mentioned data imply that when conventional anticonvulsants fail to stop SSSE, another class of pharmacological agents, that would block the postsynaptic effects of excitatory amino acids may provide an alternative approach. Indeed, preliminary studies w17x showed that blockers of NMDA receptors ŽMK-801 and ketamine. aborted SSSE when given 10 min after 60 min PPS, and were effective in pilocarpine induced SE w23x. Other mechanisms may involve failure of neuromodulatory systems that normally control hippocampal excitability. For instance, SSSE depletes hippocampal stores of peptides dynorphin and galanin w15,18x which normally decrease glutamate release from dentate granule cells w5,30,31x. Furthermore, exogenous administration into the hilus of the dentate gyrus of ligands for kappa and galanin receptors both prevented and stopped established SSSE. Alternatively, the increase in the substance P immunoreactivity in the hippocampus during SSSE w11x may contribute to the presynaptic stimulation of glutamate release w7x. Development of animal models of refractory Žresistant to antiepileptic drugs. status epilepticus has both basic and practical significance. On one hand, studies of the mechanisms that underlie drug resistance are important for understanding the pathophysiology of status epilepticus. On the other hand, those models may be used for testing newly developed antiepileptic drugs and for comparison of their efficacy with standard anticonvulsants. The present experimental paradigm offers the first drug-free model of refractory SE, a condition which responds poorly to standard anticonvulsants and still carries a very high mortality w3,4,29x. This model may be used in evaluating alternate pharmacotherapy for refractory SE. Some very expensive agents are widely used for refractory SE Že.g., see Refs. w10,25x., but their efficacy has never been demonstrated neither under clinical, nor experimental conditions. Of course, the approximation of findings in
animal models to the clinical situation must be done with caution. While human data comparing the efficacy of diazepam at specific time points during the course of SE is not available, it is common clinical experience that SE becomes refractory to treatment with anticonvulsants with time. Comparison of the effectiveness of diazepam and phenytoin at certain time points in humans is complicated also due to the protocol for administering phenytoin which typically requires 20 min or more to deliver the typical full dose of the drug. The advantages of the present model include the possibility to control the severity of SSSE through the duration of epileptogenic stimulation and to control the refractoriness to anticonvulsants by manipulating the time of their administration in relation to PPS. Acknowledgements C.W. is supported by grant NS 11315 from NINDS and by the Research Service of VHA. R.S. is supported by CIDA NS 01792. References w1x P.A. Borea, D. Bonora, Brain receptor binding and lipophilic character of benzodiazepines, Biochem. Pharmacol. 32 Ž1983. 603–607. w2x Commission on Classification and Terminology of the International League Against Epilepsy, Proposal for revised clinical and electroencephalographic classification of epileptic seizures, Epilepsia 22 Ž1981. 489–501. w3x R.J. DeLorenzo, W.A. Hauser, A.R. Towne, J.G. Boggs, J.M. Pellock, L. Penberthy, L. Garnett, C.A. Fortner, D. Ko, A prospective, population-based epidemiologic study of status epilepticus in Richmond, Virginia, Neurology 46 Ž1996. 1029–1035. w4x R.J. DeLorenzo, J.M. Pellock, A.R. Towne, J.C. Boggs, Epidemiology of status epilepticus, J. Clin. Neurophysiol. 12 Ž1995. 316–325. w5x C.T. Drake, G.W. Terman, M.L. Simmons, T.A. Milner, K. D.D., P.A. Schwartzkroin, C. Chavkin, Dynorphin opioids present in dentate granule cells may function as retrograde inhibitory neurotransmitters, J. Neurosci. 14 Ž1994. 3736–3750. w6x R.S. Jope, R.A. Morrisett, O.C. Snead, Characterization of Lithium potentiation of pilocarpine-induced status epilepticus, Exp. Neurol. 91 Ž1986. 471–480. w7x I. Kangrga, M. Randic, Tachikinins and calcitonin-gene related peptide enhance release of glutamate and apartate from the rat spinal dorsal horn slices, J. Neurosci. 10 Ž1990. 1026–2038. w8x J. Kapur, D.A. Coulter, Experimental status epilepticus alters GABA A receptor function in CA1 pyramidal neurons, Ann. Neurol. 38 Ž1995. 893–900. w9x J. Kapur, R.L. MacDonald, Rapid seizure-induced reduction of benzodiazepine and Zn2q sensitivity of hippocampal dentate granule cell GABA A receptors, J. Neurosci. 17 Ž1997. 7532–7540. w10x M. Kuisma, R.O. Roine, Propofol in prehospital treatment of convulsive status epilepticus, Epilepsia 36 Ž1995. 1241–1243. w11x H. Liu, A.M. Mazarati, H. Katsumori, C.G. Wasterlain, Limbic seizures mediated by phenotypic switch in hippocampal principal neurons, Soc. Neurosci Abstr. 24 Ž1998. 2140. w12x C.T. Lombroso, Treatment of status epilepticus with diazepam, Neurology 16 Ž1966. 629–634. w13x R.L. MacDonald, K.M. Kelly, Antiepileptic drug mechanisms of action, Epilepsia 36 Ž1995. S2–S12.
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