Refractory atypical absence seizures in rat: a two hit model

Epilepsy Research 62 (2004) 53–63

Refractory atypical absence seizures in rat: a two hit model Irina Serbanescua,b, Miguel A. Corteza,b,c, Colin McKerlied,e, O. Carter Snead IIIa,b,c,∗ a

Division of Neurology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ont., Canada M5G 1X8 b The Brain and Behavior Research Program, The Hospital for Sick Children, Toronto, Ont., Canada c Department of Pediatrics, The Hospital for Sick Children, Toronto, Ont., Canada d Integrative Biology Research Program, The Hospital for Sick Children, Toronto, Ont., Canada e Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont., Canada Received 18 April 2004; received in revised form 2 August 2004; accepted 4 August 2004

Abstract Medically refractory seizure disorders in children usually have malignant neurodevelopmental outcomes and often are associated with the presence of congenital cortical dysplasias in the brain. To date, there are no animal models of these disorders by which to test hypotheses of pathogenesis or to screen novel drugs for antiepileptic activity. In rats, treatment with the antimitotic agent methylazoxymethanol acetate (MAM) on gestational day (G) 15 produces a neuronal migration disorder similar to the cortical dysplasias seen in human brain. We sought to produce chronic, recurrent, medically refractory seizures by administration of the cholesterol biosynthesis inhibitor AY-9944 (AY) during postnatal development in rats exposed prenatally to MAM. Prenatal MAM and postnatal AY treatments resulted in spontaneous, recurrent atypical absence seizures that were characterized by bilaterally synchronous slow spike-and-wave discharges (SWD) with a frequency of 6 Hz. The MAM–AY-induced seizures were refractory to ethosuximide, sodium valproate, and the GABAB R antagonist CGP 35348, and were exacerbated by carbamazepine. Histological examination of brains from MAM-treated rats showed hippocampal heterotopias, in addition to atrophy and abnormalities of cortical lamination. The MAM–AY-treated rat represents a reproducible model of refractory atypical absence seizures in children with brain dysgenesis. © 2004 Elsevier B.V. All rights reserved. Keywords: MAM; AY-9944; Intractable absence seizures

1. Introduction

∗ Corresponding author. Tel.: +1 416 813 7851; fax: +1 416 813 6334. E-mail address: [email protected] (O.C. Snead III).

0920-1211/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2004.08.003

Although the prognosis for the majority of patients with epilepsy is favorable, up to 30% have medically refractory epilepsy, i.e. recurrent, spontaneous seizures that continue relentlessly in the face of appropriate therapy with antiepileptic drugs (Kwan and Brodie, 2000).

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The cognitive and psychosocial consequences of medically refractory seizures are onerous (Devinsky, 1999), particularly in children where continuous uncontrolled seizures usually herald a poor developmental outcome. For example, seizures are drug resistant in the vast majority of patients with Lennox–Gastaut syndrome (Sillinp¨aa¨ , 1995), an epilepsy syndrome notorious for its poor neurodevelopmental outcome. The neurobiological events that result in medically refractory epilepsy are not known, but intractable seizures often are associated with the presence of cortical malformations (Lee et al., 1997) which may occur in 30–40% of patients with refractory epilepsy (Farrell et al., 1992; Lee et al., 1997), as compared to 7–15% of all epilepsy patients (Brodtkorb et al., 1992; Lee et al., 1997) and 1% of the general population (Lee et al., 1997). These data raise the possibility that disorders of cortical development are a major contributing factor to the intractability of seizures in medically refractory epilepsy (Porter et al., 2002). For example, cortical malformations are particularly common in pediatric epilepsy syndromes notable for abysmal neurodevelopmental outcomes and intractable epilepsy, such as West’s syndrome (Meencke and Janz, 1984; Blume, 1988) and the Lennox–Gastaut syndrome (Blume, 1988). Recurrent seizures were found in 82% of 33 patients with cortical or subcortical heterotopias that led to unilateral or bilateral independent epileptiform discharges (Dubeau et al., 1995). Most cortical malformations seen in children with epilepsy are caused by disruptions in cell proliferation and neuronal migration (Mischel et al., 1995; Lee et al., 1997; Germano and Sperber, 1998), and include loss of cerebral lamination, clusters of ectopic neurons, and heterotopias (Dubeau et al., 1995; Germano and Sperber, 1998). In rats, prenatal treatment of the mother on gestational day 15 (G15) with the antimitotic agent methylazoxymethanol (MAM) produces a brain dysgenesis in the pups similar to that seen in human neuronal migration disorders. MAM acts by methylating pyrimidine bases within 2–24 h of administration (Colacitti et al., 1998) and affects selected neuronal populations depending on the time of administration (Johnston and Coyle, 1979). Rat pups born to mothers exposed to MAM on G15 exhibit ataxia, tremors, learning and memory impairments (Ramakers et al., 1993; Di Luca et al., 1995). These MAM-treated offspring are more susceptible to kainic acid, bicuculline, fluo-

rothyl, and metrazol-induced seizures (de Feo et al., 1995; Baraban and Schwartzkroin, 1996; ChevassusAu-Louis et al., 1998; Germano and Sperber, 1998) and they show abnormal neuronal activity in the hippocampus and neocortex (Baraban and Schwartzkroin, 1995; Colacitti et al., 1999). However, no spontaneous seizures have been documented either by direct observation or by EEG in MAM-treated rats (Germano and Sperber, 1998). We sought to induce chronic, recurrent, medically refractory atypical absence seizures, in MAM-exposed rats by administering the cholesterol biosynthesis inhibitor AY-9944 (AY) (Fischer et al., 1972; Smith and Bierkamper, 1990; Cortez et al., 2001). We hypothesized that fetal treatment with MAM would increase the severity of the AY-induced seizures and make them refractory to the drugs that normally suppress them. We investigated the electrocorticographic (ECoG) changes in the MAM–AY-treated rats and conducted a pharmacological characterization of this animal model. We as well examined the histopathology of brains in MAM–AY-treated rats and compared those findings to the brain histology in control animals.

2. Materials and methods 2.1. Animals All these experiments were approved by the Ethics Committee of Lab Animal Services at the Hospital for Sick Children. Animals involved in this experimental design were treated according to guidelines for invasive surgical procedures. Timed, pregnant Long Evans hooded rats (G12) (n = 4) were obtained from Charles River (St. Constant, Quebec, Canada) and housed in the lab animal services facility of the Hospital for Sick Children in Toronto. The suckling rats were weaned at postnatal day (P) 21 and then housed in groups of three animals of the same gender per cage until surgery day (n = 32). All animals were maintained in a controlled environment at 12-h light:12-h dark with lights on at 06:00 h and given ad lib access to food and water. 2.2. Drugs AY-9944 (trans-1,4-bis[2-chloro-benzylaminomethyl]cyclohexane dihydrochloride), was a gift from

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Wyeth-Ayerst (Philadelphia, PA). CGP 35348 was a gift from Novartis (Basel, Switzerland). MAM was provided by Midwest Research Institute (Kansas City, KS). All other chemicals were obtained from standard commercial sources. 2.3. The AY model We have shown previously that postnatal treatment of rats with the cholesterol synthesis inhibitor, AY-9944 (AY) results in chronic, recurrent, life-long atypical absence seizures characterized by spontaneous, bilaterally synchronous, slow spike-and-wave discharges (SWD) that begin at postnatal day (P) 21, occur throughout all stages of sleep, and are associated with myoclonic jerks during sleep (Cortez et al., 2001). In the current experiments, the AY model was induced as described (Cortez et al., 2001). Briefly, after birth rats were given AY in a dose of 7.5 mg/kg subcutaneously every 6 days from P2 to P33 with no AY given thereafter. In control experiments, animals received equal volumes of saline subcutaneously at the same time points. ECoG recordings were then done from P55 onwards in both AY-treated and control animals. 2.4. Experimental design Eight pregnant rats were intraperitoneally (IP) injected at G15 with MAM (25 mg/kg) or the equivalent volume of saline for control (C). After birth, neonatal suckling rats were randomly treated with AY or an equivalent amount of saline every 6 days from P2 to P20 and no AY was administered afterwards. Body weights of all experimental animals were monitored throughout to determine any growth differences among the treatment groups. Animals were classified into MAM–AY, MAM–C, C–AY and C–C experimental groups for comparison. There was an equal distribution of male and female animals in all experimental groups. 2.5. Surgery and electrocorticography (ECoG) At P55, two frontal and two parietal monopolar epidural electrodes were implanted in all animals under pentobarbital anesthesia (35 mg/kg intraperitoneal injection). Electrodes were secured with dental cement and two screws attached to the parietal regions of the scalp. Following surgery, animals were returned to the animal facility for a 4-day recovery period. Subcuta-

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neous Buprenorphine 0.05 mg/(kg day) was used for analgesia during the postoperative period. ECoG was recorded in animals in all four treatment groups at P60–P65 in order to assess baseline spike-and-wave (SWD) duration. All animals were placed freely moving, in individual, warm Plexiglas chambers (Harvard Apparatus, Holliston, MA) for a 20-min adaptation period before ECoG recordings in order to minimize movement artifact. One hour-long paper ECoG recording was obtained using a Grass Polysomnograph (Grass Instruments, Quincy, MA). All baseline and test recordings were performed from 10:00 to 14:00 h to minimize circadian variations (Wooley and Timiras, 1962; L¨oscher and Fielder, 1996; Cortez et al., 2001). SWD was scored in each animal only if two frontal and parietal electrode derivations demonstrated the distinct 7 Hz SWD morphology with an amplitude four times the baseline (Snead, 1988). 2.6. Pharmacological characterization Equal numbers of male and female MAM–AY-, MAM–C-, C–AY-, and C–C-treated rats were used in these experiments. All test drugs were administered IP after 1 h baseline ECoG and their effects assessed immediately following drug administration (Snead, 1995). We quantified the effect of antiabsence drugs with ethosuximide (100 mg/kg), valproic acid (sodium salt) (200 and 400 mg/kg), or the specific GABAB R antagonist CGP 35348 (100 mg/kg), on SWD duration. And similarly, we assessed the drug effect of carbamazepine (32 mg/kg), which is known to suppress simple and complex partial seizures, but to exacerbate typical and atypical absence seizures (Snead, 1995, 1988) 2.7. Histology At P70, rats from all four groups (n = 8) were anesthetized deeply with pentobarbital (65 mg/kg) and decapitated. Brains were immediately removed, stored at −80 ◦ C for 48 h and cut with a vibratome into 20␮m thick coronal sections. The sections were immediately mounted on gelatin-coated slides and air dried at room temperature for 24 h. The slides were stained with cresyl violet, and examined with an Olympus BX40 microscope for gross anatomy and brain architecture. Photographs of representative sections were taken with

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an Olympus camera. For histopathological evaluation, 120-day-old animals (n = 8) were euthanized by CO2 asphyxia. Brains were immediately removed and immersed in 10% neutral buffered formalin. Brains were then immersed in a minimum 10:1 volume of 10% neutral buffered formalin and stored at room temperature for a minimum of 48 h. Transverse sections of each brain were taken at the level of the forebrain, midbrain, and cerebellum. Tissue sections were processed, embedded in paraffin, sectioned at 5 ␮m, and stained with hematoxylin and eosin (H&E). 2.8. Data analysis AY-induced absence seizures were quantitated by inspection measuring SWD duration for consecutive 20-min epochs over a 1-h recording period (Snead, 1988). Animals were classified into MAM–AY (n = 8), MAM–C (n = 8), C–AY (n = 8) and C–C (n = 8) groups for comparison. For the pharmacological experiments, there were four animals in each treatment arm, including the control group. SWD was scored in each animal only if two frontal and parietal electrode derivations demonstrated the distinct 5–6 Hz SWD morphology and amplitude four to six times higher compared to baseline (Cortez et al., 2001). All data were expressed as arithmetic mean ± standard error of the mean (S.E.M.). Analysis of variance (ANOVA) for repeated measures was used to quantify the amplitude differences between ECoG baseline and SWD as a function of drug and time post-injection. Effectiveness for age pairing results was evaluated statistically using nonparametric Spearman correlation test with a probability (P) value of P < 0.05 chosen as an index of statistical significance.

3. Results 3.1. ECoG and behavioral characteristics of seizures in the MAM–AY-treated rat As indicated the total SWD duration was quantified in 20-min epoch over 1-h ECoG recordings. The ECoG recordings in C–C rats showed spontaneously recurrent SWD at 9 Hz lasting 1–2 s in duration. AY treatment during postnatal development both in C–AY and in MAM–AY animals resulted in a

marked exacerbation of SWD of slower frequency at 5–6 Hz. ECoG recordings in AY-treated animals were characterized by spontaneous, recurrent, bilaterally synchronous 5–6 Hz SWD that were significantly longer in duration than in C–C animals. SWD in the C–C group were 31 ± 40 versus 497 ± 225 in the C–AY group (mean ± S.E.M. s/h; P = 0.0014) (Fig. 1). As reported previously (Cortez et al., 2001), artifact free EEG-Video recordings demonstrated an imprecise correlation between the onset and offset of AY-induced epileptiform discharges and the onset and offset of ictal behavioral changes. In contrast to the abrupt onset and offset of the SWD in the AY-treated animals, there was a gradual onset and disappearance of the accompanying ictal behavioral changes that usually began after the onset of the SWD and outlasted the spike-andwave bursts. Staring, facial myoclonus and whisker twitching characterized ictal behavior in AY-treated animals. The complete immobility or “frozen stare” that typifies pharmacological and genetic rat models of typical absence seizures (Snead et al., 1999) was not observed in the AY-treated rats. Rather, the AY-treated animals showed an ability to move intermittently during the seizures. There were no postictal behavioral or ECoG changes. Spike and wave discharges in MAM–C animals were characterized by high amplitude irregular 5–7 Hz lasting up to 2 s in duration compared to the longer duration of SWD complexes in MAM–AY rats, although the frequency was about the same. The difference in spike and wave morphology was negligible when compared to the C–AY rat group. A higher SWD duration was found in MAM–AY rats (P = 0.0031) and C–AY rats (P = 0.0014) compared to C–C (31 ± 40 s/h). There was no significant difference in SWD duration between the MAM–AY rats (528 ± 300 s/h) and the C–AY rats (497 ± 225 s/h; P = 0.8306). Both MAM–AY and C–AY rats also had significantly greater SWD duration than MAM–C rats (197 ± 277 s/h; P = 0.0299) compared to MAM–AY, and (P = 0.0184) compared to C–AY. The MAM–C rats had significantly higher SWD duration than C–C rats (P = 0.0443). 3.2. Effect of pro- and antiabsence drugs on seizures in MAM–AY-treated rats The antiabsence drugs ethosuximide (ESM) and valproic acid (VPA) have been shown to suppress AY-

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Fig. 1. Baseline ECoG recordings at postnatal day 60 in C–C, C–AY, MAM–C and MAM–AY. showed bilaterally synchronous SWD (n = 8, each group). The SWD observed in C–C were brief and with a frequency of 9 Hz compared to those seen in MAM–AY, C–AY and MAM–C were spontaneous and recurrent and with a frequency of 5–6 Hz. The ictal behavior during SWD consisted of frozen stare, vibrissal twitching and facial myoclonus with the ability to move during seizures. Abbreviations: LF-P, left frontal–parietal; RF-P, right frontal–parietal; SWD, spike-and-wave discharge; MAM, methylazoxymethanol; AY, AY-9944; C, control.

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induced atypical absence seizures (Cortez et al., 2001). However, in the current experiments VPA had no significant effect on SWD duration in the rats that received prenatal MAM plus postnatal AY treatment. Similarly, SWD duration in ESM-treated MAM–AY animals was not significantly different from that of ESM-treated C–AY animals (P = 0.1664) (Fig. 2A). The GABAB receptor (GABAB R) antagonist, CGP 38348 has been shown to block SWD in the AY model (Smith and Fisher, 1996; Cortez et al., 2001), but in the MAM–AY rats, CGP 35348 had no significant effect on SWD duration (Fig. 2B). Carbamazepine (CBZ) exacerbates both typical (Snead et al., 1999) and atypical absence seizures (Cortez et al., 2001). There was an increase of up to 225% in SWD duration following CBZ treatment in MAM–AY rats (Fig. 2C). 3.3. Histopathological findings In transverse sections of midbrain from MAM exposed rats, the cortex and thalamus were histologically unremarkable. The neurons of the hippocampus were primarily round in shape and the molecular, pyramidal and polymorph layers were each apparent. However, there were segments of the hippocampus where the population of neurons in the granule cell layer was markedly reduced to a single layer with small shrunken neuronal bodies representing these atrophic segments of hippocampus. There was also bilateral and symmetrical dysmorphia of the granule cell layer of the dentate gyrus. The normally ordered layers of the dentate gyrus had a wavy appearance and the polymorph layer was

Fig. 2. (A) Antiepileptic drug effects on seizure severity in MAM–AY rats per 20-min interval, quantified over a 1-h recording period, n = 4 for each treatment. SWD duration showed no change after treatment with VPA 200 mg/kg (454 ± 150) (100% of baseline SWD), VPA 400 mg/kg (503 ± 225) (110% of baseline SWD). ESM 100 mg/kg (271 ± 137) suppressed up to 41% of SWD. The ESM effect is significantly weaker than that seen in animals treated with AY only. (B) SWD duration (mean ± S.D. s/h) at baseline (455 ± 275) and CGP 35348 treatment 100 mg/kg (474 ± 303), showed no significant difference (104% of baseline SWD). (C) CBZ 32 mg/kg (392 ± 269) exacerbated SWD by 225% compared to controls (265 ± 91). Similar effect was observed in the AY model (P < 0.05, one-way ANOVA). Abbreviations: SWD, spike-and-wave discharge; MAM, methylazoxymethanol; VPA, valproic acid; CGP, CGP 35348 (specific GABAB receptor antagonist); ESM, ethosuximide; CBZ, carbamazepine; AY, AY-9944.

characterized by moderate atrophy (data not shown). There also were a significant number of subependymal mononuclear cells within the third ventricle that contained large aggregates of irregular golden-brown granular material in 50% of the MAM exposed rats. The cresyl violet stained sections illustrated the nodular heterotopia shown in boxes at 4×, 10× and 40×

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Fig. 3. Photomicrographs of coronal hippocampal sections from Control and MAM-prenatally exposed animals. The cresyl violet stained sections illustrate the nodular heterotopia shown in boxes at 4×, 10× and 40× magnification. The arrows in the MAM section (middle) indicate the range of the nodular heterotopia within the hippocampus focusing on the CA1 region (tissue sections of 51 um). Abbreviations: LF-P, left frontal–parietal; RF-P, right frontal–parietal; SWD, spike-and-wave discharge; MAM, methylazoxymethanol; AY, AY-9944; C, control.

magnification. Tissues from MAM-exposed animals presented with a range of nodular heterotopias within the hippocampus focusing on the CA1 region (tissue sections of 51 um) (Fig. 3).

4. Discussion We report here the ECoG, pharmacological and histopathological characterization of the MAM–AY rat model with chronic and medically refractory atypical

absence seizures. This model was developed by exposing Long Evans hooded rats to MAM on G15, followed by treatment with AY-9944 during the first month of postnatal life. Our data indicate that (1) prenatal MAM treatment produces a neuronal migration disorder, which seems to target the hippocampus, as previously reported (Spatz and Laqueur, 1968; Singh, 1977; Chevassus-Au-Louis et al., 1998). (2) Rats exposed to prenatal MAM have significantly longer spike-andwave discharge duration than age-matched controls. (3) Prenatal treatment with MAM does not make

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AY-induced atypical absence seizures more severe, but confers medical refractoriness upon these seizures. Hence, MAM–AY rats have atypical absence seizures that are intractable to treatment with antiabsence drugs. Our laboratory has previously provided pharmacological and developmental characterization of a model of atypical absence, in Long Evans hooded rats and C3H mice with the cholesterol inhibitor AY-9944 during development (Cortez et al., 2000; Cortez et al., 2001). The AY-induced seizure represents a valid, clinically relevant animal model of atypical absence seizures, which differs from the standard rat models of typical absence epilepsy (Marescaux et al., 1984; Snead, 1988; Jando et al., 1995; Vadasz et al., 1995; Snead et al., 1999) much in the same way that atypical absence seizures in Lennox–Gastaut syndrome differ from the seizures that characterize typical absence epilepsy in children (Bare et al., 1998). In the AY model, the SWD bursts are more frequent and prolonged than those reported for the standard genetic rat or mouse models of typical absence epilepsy (Snead et al., 1999). The epileptiform discharges in the AY model are characterized by slow spike-and-waves at 5–6 Hz (Cortez et al., 2001) in contrast to the classic rat and mouse models of typical absence where the spike-andwave frequency is 8–9 Hz (Snead et al., 1999). The slow SWD in the AY model is reminiscent of that seen in clinical atypical absence seizures (Blume, 1988). As well, the involvement of hippocampal circuitry in addition to thalamocortical circuitry, the occurrence of SWD during sleep, and the associated myoclonus define this model as one of spontaneous, recurrent, atypical absence seizures (Cortez et al., 2001). Hippocampal involvement is an important defining feature of atypical absence seizures because limbic circuitry is silent during SWD bursts in animal models of typical absence seizures (Vergnes et al., 1987; Banerjee et al., 1993). Finally, the behavioral changes associated with the spike-and-wave discharges in the AY model are not of abrupt onset and offset and thus mirror those seen in clinical atypical absence seizures (Bare et al., 1998). In clinical and experimental typical absence seizures, there is a sudden cessation of motor activity with complete immobility that correlates with the onset of SWD and lasts throughout the SWD burst (Blume, 1988). However, in clinical atypical absence seizures (Bare et al., 1998) and in the AY model (Cortez et al., 2001), the onset and offset of ictal behavioral arrest

is gradual and does not comport precisely with bursts of SWD and there is purposeful movement during the SWD. The behavior of the MAM–AY rat is consistent with atypical absence seizures that were refractory to standard antiepileptic drugs. AY-induced seizures in MAM-exposed rats were resistant to treatment with ethosuximide, and valproic acid, were not blocked by the GABAB R antagonist CGP 35348 but they were exacerbated by carbamazepine and a GABAB R agonist (−) baclofen (data not shown). Thus, MAM exposure during fetal development rendered the AY-induced seizures refractory to conventional antiabsence drugs. MAM has been used since 1968 to induce structural abnormalities in the rat brain that are similar to those seen in human brain dysgeneses (Spatz and Laqueur, 1968). Fetal MAM exposure on G15 has been reported to produce hippocampal and cortical heterotopias, reduction of cortical thickness, and ectopic neurons in Wistar and Spague–Dawley rats (Kabat et al.,1985; Collier and Ashwell, 1993; Germano and Sperber, 1998; Baraban et al., 2000). Given the similarity between the pathological features induced by MAM and those observed in patients with intractable epilepsy due to cortical migrational abnormalities (Porter et al., 2002) there has been an interest in the seizure susceptibility of MAM-treated rats. Decreased seizure threshold to bicuculline (de Feo et al., 1995), electroconvulsive shock (Collier and Ashwell, 1993) and kainic acid (Germano and Sperber, 1998) have been reported in MAM-treated Wistar or Sprague–Dawley rats. Intracellular recordings have shown abnormal firing patterns in MAM-induced heterotopic neurons (Colacitti et al., 1999). No spontaneous seizures have been documented either by direct observation or by EcoG (Germano and Sperber, 1998). However, MAM exposed rats have been shown to have dramatically reduced sensitivity to commonly prescribed antiepileptic drugs to 4-aminopyridine-induced interictal epileptiform bursting in in vitro hippocampal slice preparations (Smyth et al., 2002). In epileptic patients, cortical malformations are usually associated with early-onset seizures that prove intractable to antiepileptic drug therapy (Meencke and Janz, 1984; Farrell et al., 1992; Porter et al., 2002). Hence, the MAM–AY in vivo model mimics this scenario and comports with the MAM in vitro modeling data referable to refractory partial seizures (Smyth et al.,

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2002). The correlation of cortical malformations with refractory seizures is not applicable to our study, because the histological evaluation found no changes consistent with cortical malformations. Common strategies used currently for both the development of antiepileptic drugs and the investigation of the basic pathogenesis of epilepsy entail the use of animal models of seizures that are known to respond to currently marketed antiepileptic drugs (White, 2002). There are a number of problems that have emerged with this approach. First, the question arises about the utility of existing models in discovering novel and efficacious antiepileptic drugs. The use of standard animal models of seizures in adult rodents (i.e. electroshock, pentylenetetrazole, kindling, strychnine, etc.) to screen for anticonvulsant efficacy and spectrum of seizures against which the potential antiepileptic drug is effective has become a self-fulfilling prophecy. Putative antiepileptic drugs shown to be efficacious against seizures in these animal model screens have proven to be possessed of a clinical efficacy that is similar to other drugs that have been predicted to be effective by the animal models. The data to support this supposition are that the rates of remission in patients who receive an established AED are similar to those who are treated with a new AED (Brodie et al., 1995, 1999; Kwan and Brodie, 2000). Therefore, the likelihood that new drugs developed with existing strategies that utilize standard animal models will be beneficial for the 30% of patients with medically refractory epilepsy is remote. The second problem that exists with current strategies of antiepileptic drug development is that it is unlikely that the use of the standard models alluded to above will give rise to new and blinding insights into clinically relevant pathogenic processes involved in epileptogenesis. There are at least three reasons for this concern. First, acute animal models of seizures lend themselves to hypothesis testing that addresses the underlying seizure events and not epileptogenesis, the latter requiring an animal to have spontaneous, recurrent seizures over a long period of time. Second, the fundamental question of what makes epilepsy refractory, cannot be addressed with an animal model that responds to known antiepileptic drugs. Finally, existing models that utilize adult animals do not address the unique problems of medically refractory epilepsy in children where it is highly likely that the refractori-

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ness of the epilepsy is related to a perturbation in brain development. Selection criterion for laboratory models of intractable epilepsy has been proposed (L¨oscher, 1997). These criteria include similarity to the clinical condition, paroxysmal EEG abnormalities that are associated with the behavioral ictal event in the animal, resistance of the seizures to standard AEDs, and the ability to use the putative model for long term studies on anticonvulsant drug efficacy. The MAM–AY model meets all of these criteria. An additional advantage is that the MAM–AY model is uniquely applicable to the investigation of refractory epilepsy in children. Specifically, the MAM–AY model of medically refractory epilepsy is particularly relevant to the Lennox–Gastaut syndrome in children. The ictal event in the MAM–AY model consists of spontaneous recurrent, medically refractory atypical absence seizures that occur in the presence of a congenitally dysplastic brain, a finding observed in 75% of patients with the Lennox–Gastaut syndrome (Zifkin, 1990; Sillinp¨aa¨ , 1995). Recent data concerning the molecular basis of drug-resistant seizures (Remy et al., 2003; Siddiqui et al., 2003) are not relevant to refractory atypical absence seizures in the Lennox–Gastaut syndrome. Those recently published data involve partial seizures in adult patients rather than the refractory primary generalized seizures seen in Lennox–Gastaut patients with cortical dysplasias, a clinical condition to which the MAM–AY model is directly relevant.

Acknowledgements This project was performed in part using MAM, a compound provided by the National Cancer Institute’s Chemical Carcinogen Reference Standards Repository operated under contract by Midwest Research Institute No. N02-CB-07008. The authors thank the technical staff of Laboratory Animal Services for the care taken during the chronic experiments, Dick Liu for excellent technical support, Craig Fleming for recovery and preparation of tissue for histological evaluation, and Marilyn McLaughlin for assistance in preparation of the manuscript. The authors would like to thank Sonia Cheung for assistance in the preliminary MAM EEG recordings.

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