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NEUROPHARMACOLOGY

Antiepileptic Drug Treatment of Nonconvulsive Seizures Induced by Experimental Focal Brain Ischemia

Walter Reed Army Institute of Research, Silver Spring, Maryland (A.J.W., F.C.T., X.M.L., J.A.H.); and Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland (J.E.M.)

Received March 26, 2004; accepted May 12, 2004.

	Abstract

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Nonconvulsive seizures (NCSs) after traumatic and ischemic brain injury are often refractory to antiepileptic drug therapy and are associated with a decline in patient outcome. We recently characterized an in vivo rat model of focal brain ischemia-induced NCS and here sought to evaluate potential pharmacological treatments. Electroencephalographic activity was recorded continuously for 24 h in freely behaving rats subjected to permanent middle cerebral artery occlusion (MCAo). Rats were treated with an antiepileptic drug from one of seven different drug classes at ED₅₀ and 2x ED₅₀ doses (as reported in other rat seizure models), delivered as a single i.v. injection 20 min post-MCAo. Vehicle-treated rats (n = 9) had an 89% incidence of NCS with an average number of NCS of 8.6 ± 1.9. The latency to onset of NCS was 32.5 ± 3.4 min post-MCAo with an average duration of 49.1 ± 8.2 s/event. The high doses of ethosuximide, gabapentin, fos-phenytoin, and valproate significantly reduced the incidence of NCS (11, 14, 14, and 38%, respectively), whereas midazolam, phenobarbital, and dextromethorphan had no significant effect at either dose. Across treatment groups, there was a low but significant correlation between the number of NCS events per animal and volume of brain infarction (r = 0.352). Antiepileptic drug therapy that prevented the occurrence of NCS also reduced mortality from 26 to 7%. Based on combined effects on NCS, infarction, neurological recovery, and mortality, ethosuximide and gabapentin were identified as having the best therapeutic profile.

Indeed, data suggest that the occurrence and duration of NCS in brain-injured patients are critical determinants of outcome (Young et al., 1996). Seizure duration and delay in time to seizure diagnosis and treatment were found to be independent predictors of patient outcome and significant variables increasing morbidity and mortality (Jordan, 1995; DeLorenzo et al., 1998). Seizures may exacerbate secondary injury by inducing glutamate excitotoxicity and/or enhancing the mismatch between energy supply and demand under ischemic conditions, leading to breakdown of ion gradients, mitochondrial damage, and eventually an irreversible state of injury. In both humans and rats, seizures have been shown to increase cerebral metabolism in this manner (Nevander et al., 1985; Meltzer et al., 2000). Even in nonischemic conditions, seizures have been associated with the induction of brain damage and long-lasting cognitive and neurological deficits (Young and Jordan, 1998; Vespa et al., 2003).

Treating NCS has proven difficult, particularly in cases of NCS lasting >30 min (nonconvulsive status epilepticus). For example, nonconvulsive status epilepticus is more refractory than generalized convulsive status epilepticus, with initial pharmacological treatments effective in only 15% of nonconvulsive status epilepticus cases compared with 55% in generalized convulsive status epilepticus (Treiman et al., 1998). In another study, nonconvulsive status epilepticus was controlled in only 25% of patients within 3 h after initiating treatment (Jordan, 1995). These clinical findings demonstrate the need to identify effective pharmacological interventions against brain injury-induced NCS.

Recently we described the occurrence of acute NCS, meeting the criteria established by Young et al. (1996) for defining clinical NCS in continuously monitored unanesthetized rats after MCAo, the first animal model identified with a high incidence (89%) of seizures induced by brain injury alone (Hartings et al., 2003). The MCAo model is widely used in pharmacological and molecular studies of brain injury and, importantly, mimics the EEG abnormalities observed in brain-injured patients, including seizures, spreading depolarizations, polymorphic delta activity, and periodic lateralized epileptiform discharges (Lu et al., 2001; Hartings et al., 2003). EEG analysis of MCAo injury has been used previously by our laboratory and others as a tool to assess functional recovery and neuroprotection in focal ischemic brain injury (Tortella et al., 1999; Williams et al., 2001, 2003; Williams and Tortella, 2002). Here, we focus specifically on NCS as a potential target for pharmacotherapy and neuroprotection using the MCAo brain injury model. We evaluated the efficacies of seven FDA-approved antiepileptic drugs at ED₅₀ and 2X ED₅₀ doses, as determined based on antiseizure efficacy in other rat seizure models. Our results show that gabapentin and ethosuximide have both antiseizure and neuroprotective properties and suggest these compounds as candidates for further evaluation in treating NCS associated with brain injury.

	Materials and Methods

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Surgical Procedures. Male Sprague-Dawley rats (270–330 g; Charles River Laboratories, Raleigh, VA) were used in all of the following experiments. Food and water were provided ad libitum pre- and postsurgery, and the animals were individually housed under a 12-h light/dark cycle. For all surgical procedures, anesthesia was induced by 5% halothane and maintained at 2% halothane delivered in oxygen, and body temperatures were maintained normothermic (37 ± 1°C) by means of a homeothermic heating system (Harvard Apparatus Inc., Holliston, MA). All procedures were approved by the Walter Reed Army Institute of Research Animal Care and Use Committee, and all research was conducted in compliance with the Animal Welfare Act, Guide for the Care and Use of Laboratory Animals (National Research Council), and other federal statutes and regulations relating to animals and experiments involving animals. Animals were maintained in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Indwelling i.v. cannulas (polyethylene-50) were placed into the right jugular vein of all animals for drug delivery, and EEG electrodes were implanted in the skull (see below). Electrodes consisted of stainless steel screws (0–80 x 1/8 in.) soldered to insulated nichrome wire (0.2 mm in diameter). The screws were implanted epidurally and fixed to the skull using dental acrylate cement (Tortella et al., 1997). Free ends of the wires were soldered to a multi-pin connector (March Electronics, West Hempstead, NY), also secured by dental acrylate.

After 2 to 3 days of recovery from the above-mentioned procedures, animals were subjected to permanent focal ischemia by using the filament method of MCAo as described previously (Tortella et al., 1999). Briefly, the right external carotid artery was isolated and its branches coagulated. A 3-0 uncoated monofilament nylon suture with rounded tip was introduced into the internal carotid artery via the external carotid artery and advanced approximately 22 mm from the carotid bifurcation until a slight resistance was observed, thus permanently occluding the origin of the middle cerebral artery.

General Procedures. On the day of MCAo surgery, animals were transferred from their home cages to custom-designed Plexiglas EEG recording chambers (Dragonfly Inc., Ridgeley, WV) equipped with multichannel gold contact swivel commutators (Plastics One, Roanoke, VA). The multi-pin connector on the rat skull was connected to the swivel system via a flexible shielded cable, allowing free movement of the animals during recordings. The swivel commutators were interfaced with a digital EEG amplifier and recording system (Harmonie software; Astro-Med, West Warwick, RI). Baseline EEG signals were then recorded for 30 min before MCAo surgery and continuously throughout the 24-h ischemic period after surgery. An i.v. bolus injection of vehicle or drug was given at 20 min postocclusion. Neurological scoring (see below) was performed before injury and 1 and 24 h post-MCAo. Animals not exhibiting maximal neurological deficit at 1 h postocclusion were excluded from the study. Animals that died before the 24-h endpoint were not included in the main analyses of antiepileptic drug effects on NCS; drug and vehicle groups thus consisted of seven to nine surviving rats (Table 1). Rectal temperatures were recorded before injury and 20 min, 1, 6, and 24 h postocclusion. At 24 h, rats were deeply anesthetized, euthanized by decapitation, and brains were harvested for quantification of infarction.

EEG Recording and Analysis. Bipolar recordings were made from each cerebral hemisphere to monitor EEG activity. Two electrodes were positioned bilaterally over parietal cortices 5 mm lateral to midline, at 0 and 4 mm posterior to bregma, by procedures described above. A fifth reference electrode was implanted posterior to lambda over the transverse sinus/cerebellum.

Continuous EEG recordings were obtained for 24 h after injury and were reviewed in entirety at a display resolution of 1 mm/s for detection of electrographic seizures. Subsequently, all seizure events were verified at a recording speed of 30 mm/s for scoring of NCS episodes. Our previous study showed that nearly all seizures in the acute phase (within 72 h) occur during this time epoch (Hartings et al., 2003) and are nonconvulsive in nature. Criteria for identifying NCS events were as follows: 1) the occurrence of repetitive spikes or spike-and-wave discharges recurring at frequencies >1 Hz, or continuous polyspiking; 2) spike amplitude greater than background activity; and 3) duration of continuous seizure activity (defined by 1 and 2) greater than 10 s. Seizures could be either generalized or focal, and consecutive seizure episodes were considered a single event if not separated by more than 10 s.

Based on the onset/offset times of each NCS event as defined by the above-mentioned criteria, several descriptive parameters were computed for each treatment group. NCS/rat and total duration of NCS were calculated as the mean value of all animals in each group. Average duration NCS and latency of onset were calculated as the mean values from only those animals exhibiting NCS in each group. EEG recordings were also visually evaluated for other EEG abnormalities, including depressed baseline amplitude, focal slowing, polymorphic delta activity, periodic lateralized epileptiform discharges, and interictal spikes, sharp waves, polyspikes, or spike/slow-wave complexes.

Infarct Analysis and Neurological Scoring. Triphenyltetrazolium chloride was used to visualize and quantitate the area of brain infarction from seven coronal brain slices, which were integrated to obtain a final core infarct volume (Inquiry Digital Analysis System; Loats Assoc., Westminster, MD) (Tortella et al., 1999). Neurological scoring was based on a weighted 10-point scale, giving a positive score for each neurological deficit, including forelimb flexion, shoulder adduction, reduced resistance to lateral push, and contralateral circling (Tortella et al., 1999).

Statistical Analysis. Data are presented as the mean ± standard error of the mean. Infarct analysis, neurological scoring, and off-line EEG analysis were performed by an experimenter blinded to the treatment group. Infarct volume and neurological scores were evaluated by ANOVA followed by a Bonferroni post hoc test (adjusted for multiple comparisons) to compare individual treatments to the vehicle control group. Chi square test was used to assess treatment effects on NCS incidence, defined as the number of animals with and without identified NCS activity. Kruskal-Wallis nonparametric analysis was used to test effects of antiepileptic drug treatment on all other NCS parameters. The relationship between infarction and number of NCS was evaluated by Pearson's correlation, and Kaplan-Meier survival curves were used to assess differences in mortality. P values <0.05 were considered significant.

Compounds. The following antiepileptic drugs were used in this study: dextromethorphan (dextromethorphan hydrobromate monohydrate; Sigma-Aldrich, St. Louis, MO), ethosuximide (2-ethyl-2-methylsuccinimide; Sigma-Aldrich), fos-phenytoin (fos-phenytoin sodium; Parke-Davis, New York, NY), gabapentin ([1-(aminomethyl)-cyclohexan-acetic acid]; Sigma-Aldrich), midazolam (midazolam hydrochloride; Parenta Pharmaceuticals, West Columbia, SC], phenobarbital (phenobarbital sodium; Sigma-Aldrich), and valproate (valproate sodium; Sigma-Aldrich). Both ED₅₀ and twice (2 x) ED₅₀ doses were evaluated for each compound, as reported for effective antiseizure efficacy in other rat seizure models. All compounds were dissolved/diluted in a vehicle of 0.9% physiological saline (1 ml/kg). Injections were given i.v. to awake, freely behaving animals.

	Results

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EEG Analysis. Based on the time course of NCS occurrence described previously in the permanent MCAo model (Hartings et al., 2003), EEG activity was recorded continuously and NCS quantified over the 24-h period postinjury in both vehicle and antiepileptic drug-treated animals. Figure 1 displays an NCS discharge representative of those occurring in vehicle and antiepileptic drug-treated rats. NCS events initiated as rhythmic spike or sharp wave discharges (Fig. 1B) with amplitudes increasing above baseline activity (Fig. 1A) and developed into sustained, large-amplitude rhythmic spike, spike/wave, or polyspike discharges (Fig. 1C). At termination, the seizure pattern generally became arrhythmic (Fig. 1D) with increased polyspike occurrence and decreasing discharge amplitude. No overt motor convulsions were visually observed during the electrographic seizures.

View larger version (38K): [in this window] [in a new window]   Fig. 1. Representative NCS event after MCAo injury. A to D, expanded views of the regions indicated (a–d) on the slow-speed recording shown above. NCS were typically observed as distinct generalized events initiating as rhythmic spikes (B) above baseline activity (A). NCS activity rapidly progressed into rhythmic spike, spike/wave, or polyspike complexes (C) with regression into arrhythmic polyspikes (D) of decreasing amplitude and resolution.

Table 1 reports the number of NCS events per animal, total duration of NCS activity, average duration of individual NCS episodes, and latency to NCS onset postocclusion for vehicle- and drug-treated groups at low (ED₅₀) and high (2x ED₅₀) doses. The statistics of NCS occurrence in vehicle-treated animals was similar to that reported previously (Hartings et al., 2003); NCS occurred in 89% of animals with average values of 8.6 events/animal, 49-s duration/seizure, and onset time of 33 min postinjury (Table 1).

The incidence of NCS (percentage of animals with NCS) was significantly reduced by high-dose treatments of ethosuximide, gabapentin, fos-phenytoin, and valproate (Fig. 2; chi square analysis, P < 0.05). Ethosuximide, gabapentin, and fos-phenytoin were the most effective at treating NCS with only one animal in each of the 2x ED₅₀ groups exhibiting NCS. Moreover, the only ethosuximide-treated animal with NCS exhibited just one event, in contrast to eight and 14 NCS events in the gabapentin and fos-phenytoin animals, respectively.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Effect of antiepileptic drug treatment to reduce the incidence of NCS after permanent MCAo and 24-h recovery. *, P < 0.05; **, P < 0.01.

A significant difference in the number of NCS per rat and total duration of NCS was also measured between treatment groups (P < 0.05, Kruskal-Wallis nonparametric analysis). Post hoc analysis revealed that high doses of ethosuximide, gabapentin, and fos-phenytoin were associated with a significantly lower number of NCS per rat and total duration of NCS compared with the vehicle group. Of these, ethosuximide treatment provided the most effective seizure protection, with a 10-fold lower number of NCS per animal than gabapentin or fos-phenytoin treatment. Unlike ethosuximide and fos-phenytoin, however, gabapentin treatment also significantly reduced the number of NCS per rat at the low dose.

NCS onset times and average durations of individual NCS events shown in Table 1 reflect values computed from only those animals that exhibited NCS. No significant difference in these two parameters was measured between treatment groups (P > 0.05, Kruskal-Wallis). The average duration of individual NCS events ranged from 31 to 81 s in length, and the average time of onset ranged from 27 to 80 min from the time of occlusion.

Dextromethorphan and midazolam did not significantly reduce the incidence of NCS, NCS per rat, or total duration of NCS events. Although phenobarbital treatment reduced the NCS per rat and total duration, the effect was only observed at the low dose and phenobarbital did not reduce the incidence of NCS at either dose. Rather, the data suggest that these three compounds may contribute to EEG abnormalities and poor outcome. That is, two midazolam- and two phenobarbital-treated animals presented with recurrent interictal spikes or spike/waves (1–2/min) throughout the 24-h postinjury period. In addition, all midazolam animals that died before the 24-h endpoint (n = 5) were observed to have NCS, with a range from 6 to 24 events per animal. One animal from each of the phenobarbital- and dextromethorphan-treated groups was also excluded from the analysis due to the induction of recurrent arrhythmic polyspike activity postinjury.

Phenobarbital-treated animals exhibited reduced ambulation along with an onset of slow-wave EEG burst activity postinjection, a common phenomenon induced by barbiturates. Midazolam-treated animals exhibited a transient and mild sedation post-treatment. The high-dose treatments of dextromethorphan and fos-phenytoin were also associated with an immediate reduction in ambulation, although animals did not seem sedated. No other antiepileptic drug treatments had noticeable effects on behavior of the MCAo-injured animals.

Brain Injury. Vehicle-treated animals exhibited damage to striatal and cortical brain regions with infarct volumes of 200 ± 23 mm³ (22% hemispheric infarction) and demonstrated neurological impairments (6.5 ± 1.2 of 10) at 24-h postinjury. Significant differences in brain infarction (P < 0.05, ANOVA) and neurological scores (P < 0.05, ANOVA) were measured between treatment groups as shown in Table 2. Post hoc analysis revealed that gabapentin, phenobarbital, dextromethorphan, and ethosuximide treatments at 2x ED₅₀ doses were associated with significant reductions of brain infarction (P < 0.05, Bonferroni) compared with vehicle-treated animals (50, 36, 36, and 27% reduction, respectively). Significant neurological improvements were also measured with dextromethorphan, fos-phenytoin, and gabapentin (P < 0.05, Bonferroni). No significant differences in body temperature were measured between treatment groups (Table 3; P > 0.05, ANOVA). As reported previously with this model (Tortella et al., 1999; Williams et al., 2000), core body temperatures increased from baseline (37.2 ± 0.2°C) by 1 h post-MCAo (38.2 ± 0.4°C), remained elevated at 6 h (38.8 ± 0.2°C), and returned to within normal range by 24 h (37.5 ± 0.2°C) in vehicle-treated animals. All treatment groups exhibited similar changes in body temperature with the exception of the 2x ED₅₀ dose of fos-phenytoin. These animals exhibited a drop of 0.9°C from baseline at 1 h post-injury, as reported by others (Loscher et al., 1998).

NCS and Brain Infarction. There was no significant correlation between infarct volume and the number of NCS events for vehicle-treated animals. However, when data from all treatment groups were considered together, there was a low but positive correlation (Fig. 3; r = 0.352, P < 0.01). Specifically, a higher number of NCS events was associated with brain infarction values in excess of 20% (8.2 ± 1.5 NCS/animal) compared with animals with infarction values less than 20% (2.9 ± 0.7 NCS/animal) (P < 0.01, independent t test).

View larger version (17K): [in this window] [in a new window]   Fig. 3. A low but positive correlation (r = 0.352, P < 0.001) was observed between infarction and the number of NCS/animal. Infarction is reported as a percentage of hemispheric volume.

Mortality. Although animals that did not survive through the 24-h recovery period were excluded from the above-mentioned analysis, mortality data were examined across treatment groups and in relation to NCS activity recorded before death. Compared with vehicle rats (25% death, 3/12), mortality was lower for animals treated with gabapentin (22%, 4/18), dextromethorphan (16%, 3/19), valproate (11%, 2/18), ethosuximide (11%, 2/19), and phenobarbital (7%, 1/16) and higher after treatment with fos-phenytoin (30%, 6/20) and midazolam (27%, 6/22). Nonparametric distribution analysis of these data did not indicate a significant difference between treatment groups (Kaplan-Meier, P = 0.5739). However, drug-treated animals that exhibited NCS had a significantly higher mortality rate (26%, 22/84) than those that did not (7%, 4/58) (Fig. 4; P < 0.01, chi square).

View larger version (10K): [in this window] [in a new window]   Fig. 4. Comparison of mortality rate across treatment groups between animals that exhibited NCS activity versus those animals that did not exhibit NCS.

NCS and Hemorrhage. Twelve animals were excluded from the analysis due to the presence of hemorrhage at post-mortem examination. No significant difference in hemorrhage rate was observed between treatment groups. However, brain hemorrhage was associated with an 83% (10/12) incidence of NCS, 33.3 ± 6.8 NCS/animal, and a 58% (7/12) mortality rate.

Summary Analysis. Table 4 summarizes antiepileptic drug treatment effects. For NCS, infarction, and neurological recovery, a positive score (+) was assigned for a significant improvement at one or both doses compared with the vehicle group and a neutral score (O) was assigned for nonsignificant changes. Treatments that reduced mortality by >10% were also scored a + value. The final column, "Therapeutic Profile", is a summation of each of these scores. No effect (NE) was assigned to drugs that did not demonstrate protection against NCS.

	Discussion

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With the introduction of several new classes of antiepileptic drugs within the last decade, recent attention has focused on the potential use of antiepileptic drugs to treat brain injury (Trojnar et al., 2002; Calabresi et al., 2003). This strategy is based on the commonality of cellular mechanisms in seizure pathology and brain cell death, including glutamate excitotoxicity, ionic imbalances, oxidative stress, and inflammation. Clinically, Jordan (1999a) has reviewed the evidence and argued that seizures and acute brain injury work synergistically to worsen outcome. In accordance with this rationale, we have studied the brain seizures caused by focal ischemic injury and evaluated the efficacy of several classes of antiepileptic drugs after MCAo in the rat.

Table 4 summarizes the antiepileptic drug treatment effects found in this study. For each drug, scores were assigned based on the effect to attenuate NCS activity and improve brain injury outcome during the acute stage of MCAo injury. Based on these results, a summary "therapeutic profile" score was compiled [a no effect (NE) score was given to drugs that did not demonstrate protection against NCS] for each antiepileptic drug tested. Overall, gabapentin and ethosuximide emerged as the most therapeutically effective compounds followed by valproate and fos-phenytoin. In particular, gabapentin protected against NCS and brain injury at both high and low doses. At the high dose, ethosuximide was the most effective in preventing seizure activity and additionally improved mortality and brain injury outcomes. Fos-phenytoin and valproate also provided seizure protection, whereas midazolam, phenobarbital, and dextromethorphan provided no benefit or even exacerbated EEG abnormalities.

GABA is the principal inhibitory neurotransmitter in the CNS, and modulating its actions has long been a strategy for the treatment of epilepsy. Gabapentin is a cyclic analog of GABA commonly used to treat neuropathic pain and as an adjunctive therapy for refractory epilepsy (Czuczwar and Patsalos, 2001). Recent evidence suggests gabapentin modulates both potassium (Kir3) and calcium (N-type) channels through GABA_B receptor coupling in hippocampal cells (Bertrand et al., 2003). Although the role of calcium channels has been widely studied in brain injury, this is the first in vivo report of neuroprotective effects with gabapentin. It should be noted that calcium channel (L-type) antagonists have failed to show clinical efficacy for the treatment of brain injury (Horn and Limburg, 2001), although drugs targeting other types of calcium channels (i.e., N, P/Q, or T) have not been thoroughly evaluated in clinical trials (Calabresi et al., 2003).

The succinimide ethosuximide has been shown to modulate T-type calcium channel function (Danober et al., 1998). Therapeutically, ethosuximide is effective at blocking the generalized spike/wave discharges occurring in absence (petit mal) epilepsy, a form of nonconvulsive seizure. These seizures are thought to result from dysfunction in cortico-cortical and/or thalamocortical feedback loops (Danober et al., 1998). Particularly, thalamocortical and thalamic reticular neurons generate 7- to 12-Hz rhythmic activity through T-type calcium channel mechanisms, but this rhythm can shift to paroxysmal absence-like 3-Hz discharges when inhibitory mechanisms are disrupted. Alternatively, ethosuximide may disrupt absence seizures by acting on cortical neurons that also express T-type calcium channels (Manning et al., 2004). Ethosuximide treatment was also associated with a significant neuroprotective effect and low mortality rate. Ethosuximide is a very safe clinical drug with low toxicity at effective antiseizure doses. In other reports, ethosuximide has shown neuroprotective effects after oxygen/glucose deprivation in hippocampal slices (Rekling, 2003), but before the present study has not been reported as a neuroprotective agent in vivo.

Valproate-treated animals also had a lower incidence of NCS at the high dose. Valproate has been used since the 1960s for treatment of epilepsy and has become the most widely prescribed antiepileptic drug worldwide (Perucca, 2002). Valproate increases brain GABA levels and also inhibits sodium channel function. However, reports have indicated that the anticonvulsant actions of valproate are not related to GABA effects (Bernasconi et al., 1985), making it likely that the antiseizure effects of valproate are through modulation of sodium channel activity. We found that the high dose of valproate significantly reduced the incidence of NCS with no significant effect on MCAo infarct outcome. This is consistent with the failure of sodium channel blockers in the clinical treatment of stroke. Similarly, valproate has antiseizure properties in neocortical slices (Yang et al., 2000), but no neuroprotective effect after oxygen/glucose deprivation in hippocampal slices (Rekling, 2003).

Fos-phenytoin is a water-soluble disodium phosphate ester of the hydantoin drug phenytoin, a commonly used antiepileptic drug. Fos-phenytoin has been shown to block repetitive neuronal firing through voltage-dependent modulation of sodium channels (Calabresi et al., 2003). Both fos-phenytoin and phenytoin have shown neuroprotective properties in focal ischemic brain injury (Rataud et al., 1994), but no significant neuroprotection effect was reported after phase III clinical stroke trials. In the current study, the high dose of fos-phenytoin significantly reduced NCS incidence but did not yield significant neuroprotection.

Glutamate receptors have also been widely studied as neuroprotective and antiepileptic targets. Dextromethorphan is a clinically available nonopioid antitussive shown to have a low affinity for the glutamatergic N-methyl-D-aspartate receptor (Loscher and Honack, 1993) and is known to have neuroprotective and antiseizure properties in experimental models (Tortella and Musacchio, 1986; Tortella et al., 1999). In the present study, the high dose of dextromethorphan offered neuroprotection and improved neurological recovery but did not significantly attenuate NCS activity. Although higher doses were not evaluated, acute toxicity has been noted with doses of dextromethorphan as low as 12.5 mg/kg in normal rats, including sedation and induction of brain seizures (Tortella et al., 1999).

Two classes of GABA drugs that increase the affinity of GABA for the GABA_A-type receptor are benzodiazepines and barbiturates. Although widely used in the treatment of epilepsy, neither of the drugs tested from these classes (phenobarbital and midazolam) were effective at blocking NCS. Midazolam treatment actually increased the average number of seizures per animal and induced continuous interictal spikes in some animals. Treatment of nonconvulsive status epilepticus with midazolam has also been associated with an increased risk of death in elderly patients (Litt et al., 1998). The high dose of phenobarbital was associated with a reduction in brain infarction, but similar to midazolam, induced continuous polyspiking in one animal.

Although NCS in rat MCAo does not qualify as status epilepticus, these seizures model important aspects of the clinical nonconvulsive status epilepticus condition in that they occur predominantly as generalized events (focal with secondary generalization) without overt motor manifestations in brain injury subjects. In the current study, we observed a high incidence of NCS, which was associated with a low but positive correlation to infarct volume. The presence of NCS was also associated with a higher mortality rate within the 24-h recovery period. Clinically, both mortality and outcome have been related to NCS occurrence and duration. For instance, mortality was found to be significantly higher for brain injury patients with nonconvulsive status epilepticus compared with those with brain injury alone (Young et al., 1996; Litt et al., 1998) and even higher in patients for whom nonconvulsive status epilepticus persists >10 h (Young et al., 1996). We also noted that brain hemorrhage was associated with a 3.5-fold increase in the number of NCS events per animal and 2.3-fold higher mortality rate. Although these effects were not statistically significant, they are consistent with the clinical finding that seizures occur in a greater proportion of patients with intracranial hemorrhage than with ischemic stroke alone (Vespa et al., 2003).

Identification of optimal antiepileptic drug compounds may offer a safe and effective way to prophylactically treat brain injury-induced NCS without adversely affecting non-NCS patients. Toxicity in the form of sedation or ataxia has been reported at the following doses for the antiepileptic drugs effective in this study: ethosuximide, 400 mg/kg (Leite and Cavalheiro, 1995); gabapentin, 50 mg/kg (Lado et al., 2001); valproate, 365 mg/kg (Shenoy et al., 1982); and fos-phenytoin, 50 mg/kg (Loscher et al., 1998). Using these data, we can define protective index values by the minimal toxic dose as reported in the literature divided by the effective dose to block NCS as reported here, yielding the following values: gabapentin, 2.0; ethosuximide, 1.6; valproate, 1.2; and fos-phenytoin, 1.0. These values suggest a narrow but "safe" therapeutic dose range to treat brain injury-induced NCS for each drug except possibly fos-phenytoin.

In summary, continuous EEG monitoring of the ischemic rat brain has allowed us to compare and contrast the efficacy of several clinically available antiepileptic drugs for the treatment of MCAo-induced NCS in rats. Effective attenuation of NCS was obtained using relatively high doses (2x the reported ED₅₀) of the following antiepileptic drugs in order of efficacy: ethosuximide > gabapentin > fos-phenytoin > valproate. Effective blockade of NCS activity was also shown to reduce mortality by 73%. Gabapentin and ethosuximide had the best overall therapeutic profile, including both antiseizure and neuroprotection properties within a safe therapeutic dose range. Importantly, neither of these compounds has previously been reported to possess neuroprotective properties in vivo. These data may help guide clinical treatment strategies to improve brain trauma outcome and improve survivability, particularly in the presence of brain seizure activity.

	Acknowledgements

 
We thank Minori Kinjo, Brad Cunningham, Chelyse Stefanik, and Brandon Lebow for expert technical support.

	Footnotes

 
This project was supported by government funding. Previous versions of these data have been published in abstract form (Williams et al., Int Neurotrauma Soc Abstr 2003; Williams et al., Soc Neurosci Abstr 2003).

Our views do not necessarily purport or reflect the position of the Department of the Army or the Department of Defense (para 4-3, AR 360-5).

doi:10.1124/jpet.104.069146.

ABBREVIATIONS: NCS, nonconvulsive seizure; MCAo, middle cerebral artery occlusion; EEG, electroencephalographic; ANOVA, analysis of variance.

Address correspondence to: Dr. Anthony J. Williams, Department of Applied Neurobiology, Division of Neurosciences, Walter Reed Army Institute of Research, Silver Spring, MD 20910. E-mail: anthony.williams{at}na.amedd.army.mil

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