Inhibition of the multidrug transporter P-glycoprotein improves seizure control in phenytoin-treated chronic epileptic rats

PURPOSE: Overexpression of multidrug transporters such as P-glycoprotein (P-gp) may play a significant role in pharmacoresistance, by preventing antiepileptic drugs (AEDs) from reaching their targets in the brain. Until now, many studies have described increased P-gp expression in epileptic tissue or have shown that several AEDs act as substrates for P-gp. However, definitive proof showing the functional involvement of P-gp in pharmacoresistance is still lacking. Here we tested whether P-gp contributes to pharmacoresistance to phenytoin (PHT) by using a specific P-gp inhibitor in a model of spontaneous seizures in rats. METHODS: The effects of PHT on spontaneous seizure activity were investigated in the electrical post-status epilepticus rat model for temporal lobe epilepsy, before and after administration of tariquidar (TQD), a selective inhibitor of P-gp. RESULTS: A 7-day treatment with therapeutic doses of PHT suppressed spontaneous seizure activity in rats, but only partially. However, an almost complete control of seizures by PHT (93 +/- 7%) was obtained in all rats when PHT was coadministered with TQD. This specific P-gp inhibitor was effective in improving the anticonvulsive action of PHT during the first 3-4 days of the treatment. Western blot analysis confirmed P-gp upregulation in epileptic brains (140-200% of control levels), along with approximately 20% reduced PHT brain levels. Inhibition of P-gp by TQD significantly increased PHT brain levels in chronic epileptic rats. CONCLUSIONS: These findings show that TQD significantly improves the anticonvulsive action of PHT, thus establishing a proof-of-concept that the administration of AEDs in combination with P-gp inhibitors may be a promising therapeutic strategy in pharmacoresistant patients.     

Decreased expression of synaptic vesicle protein 2A, the binding site for levetiracetam, during epileptogenesis and chronic epilepsy

Purpose:   We previously showed that gene expression of synaptic vesicle protein 2A (SV2A), the binding site for the antiepileptic drug levetiracetam, is reduced during epileptogenesis in the rat. Since absence of SV2A has been associated with increased epileptogenicity, changes in expression of SV2A could have consequences for the progression of epilepsy. Therefore we investigated hippocampal SV2A protein expression of temporal lobe epilepsy (TLE) patients and in rats during epileptogenesis and in the chronic epileptic phase.
Methods:   SV2A immunocytochemistry and Western blot analysis were performed on the hippocampus of autopsy controls, patients that died from status epilepticus (SE), and pharmacoresistant TLE patients. In addition, in epileptic rats, SV2A expression was determined after SE during the acute, latent, and chronic epileptic phase.
Results:   In control tissue, presynaptic SV2A was expressed in all hippocampal subfields, with strongest expression in mossy fiber terminals. SV2A positive puncta were distributed in a patchy pattern over the somata and dendrites of neurons. SV2A decreased throughout the hippocampus of TLE patients with hippocampal sclerosis (HS), compared to autopsy control, SE, and non-HS tissue. In most rats, SV2A was already decreased in the latent period especially in the inner molecular layer and stratum lucidum. Similarly as in humans, SV2A was also decreased throughout the hippocampus of chronic epileptic rats, specifically in rats with a progressive form of epilepsy.
Discussion:   These data support previous findings that reduced expression of SV2A could contribute to the increased epileptogenicity. Whether this affects the effectiveness of levetiracetam needs to be further investigated.     

The association between seizure clustering and convulsive status epilepticus in patients with intractable complex partial seizures

PURPOSE: We examined the association between seizure clustering and convulsive status epilepticus (SE) in patients with intractable complex partial seizures, to identify whether patients whose seizures typically cluster are at high risk for convulsive SE (CSE). METHODS: Seventy-six patients with intractable complex partial epilepsy who underwent presurgical evaluation in the Montefiore Epilepsy Management Unit from 1993 to 1997 were contacted and interviewed about typical seizure frequency and distribution and history of CSE. Seizure clustering was defined as three or more complex partial seizures within a 24-h period, with return to baseline between seizures. RESULTS: Of the 76 patients contacted, 21 (28%) had experienced at least one episode of CSE, and 36 (47%) typically experienced clustered seizures. SE occurred in 16 (44%) of 36 patients with clustered seizures, and in five (12.5%) of 40 patients with nonclustered seizures (p < 0.002). Of 53 patients with temporal lobe epilepsy, CSE occurred in 13 (50%) of 26 patients with clustered seizures, and four (14.8%) of 27 patients with nonclustered seizures (p < 0.006). CONCLUSIONS: Patients with intractable complex partial or localization-related epilepsy who typically experience seizure clustering are at a significantly higher risk for CSE than are patients with nonclustered seizures.     

Development of tolerance to levetiracetam in rats with chronic epilepsy

Purpose: Pharmacoresistance is a major problem in the treatment of epilepsy. We showed previously that pharmacoresistance, at least partially, is due to an up-regulation of the multidrug transporter (MDT) P-glycoprotein (P-gp): inhibition of P-gp improves seizure control in phenytoin-treated epileptic rats (poststatus epilepticus rat model for temporal lobe epilepsy). Since it has been suggested that levetiracetam (LEV) is no substrate for MDTs, we hypothesized that LEV would more adequately control seizures in this rat model.
Methods: Chronic epileptic rats were treated repeatedly with LEV (2-week interval; different dosages) via continuous infusion using osmotic minipumps, 5–6 months after electrically induced status epilepticus. The anticonvulsive effects were determined by video-EEG monitoring and the concentration of LEV was measured in plasma and brain homogenates using gas chromatography.
Results: LEV adequately entered the epileptic brain and dose-dependently suppressed spontaneous seizures in chronic epileptic rats for 3–4 days. Hereafter, seizure frequency increased, while LEV plasma levels did not change. Seizure behavior was less severe throughout the whole treatment. LEV did not affect seizure duration. After a withdrawal period of 2 weeks all rats initially responded again to LEV.
Conclusions: The initial seizure control by LEV supports the observation that LEV is not impeded by MDTs. However, the failure to control seizures for a longer period of time indicates the development of tolerance to this drug. This poses another problem in the treatment of this kind of epilepsy. Whether tolerance may be prevented by intermittent administration of LEV should be further investigated.     

Resistance to phenobarbital extends to phenytoin in a rat model of temporal lobe epilepsy

PURPOSE: Most patients who are resistant to the first antiepileptic drug (AED) treatment are also resistant to a treatment with a second or third AED, indicating that patients who have an inadequate response to initial treatment with AEDs are likely to have refractory epilepsy. Animal models of refractory epilepsy are important tools to study mechanisms of AED resistance and develop new treatment strategies for counteracting resistance. We have recently described a rat model of temporal lobe epilepsy (TLE), in which spontaneous recurrent seizures (SRS) develop after a status epilepticus induced by sustained electrical stimulation of the basolateral amygdala. Prolonged treatment of epileptic rats with phenobarbital (PB) resulted in two subgroups, PB responders and PB nonresponders. METHODS: In the present study we examined if rats with PB-resistant seizures are also resistant to phenytoin (PHT), using continuous EEG/video recording of spontaneous seizures. RESULTS: First, a new group of 15 epileptic rats was produced and selected by treatment with PB into responders (8 rats) and nonresponders (6 rats), respectively. During subsequent treatment with PHT, the doses of PHT had to be individually adjusted for each rat to avoid toxicity. Treatment with PHT led to complete seizure control in two animals and a >50% reduction of seizure frequency in three other rats, which were considered PHT responders. In nine of the remaining rats, PHT did not exert any clear anticonvulsant effect, so that these rats were considered nonresponders. Plasma levels of PHT did not differ significantly between responders and nonresponders. When comparing the PB and PHT nonresponder groups, five of the six PB-resistant rats (83%) were also resistant to PHT, demonstrating that rats that have an inadequate response to initial treatment with PB are likely to be also resistant to treatment with a second AED. CONCLUSIONS: The AED-resistant rats of our model meet the definition of pharmacoresistance in animal models, that is, persistent seizure activity not responding to at least two AEDs at maximum tolerated doses. This new model of pharmacoresistant TLE may be useful in the targeted development of new therapies for refractory epilepsy.     

Medial septal stimulation increases seizure threshold and improves cognition in epileptic rats

Background

Temporal lobe epilepsy is most prevalent among focal epilepsies, and nearly one-third of patients are refractory to pharmacological intervention. Persistent cognitive and neurobehavioral comorbidities also occur due to the recurrent nature of seizures and medication-related side effects.

Anticonvulsive effect of a selective mGluR8 agonist (S)-3,4-dicarboxyphenylglycine (S-3,4-DCPG) in the mouse pilocarpine model of status epilepticus

Summary:  Purpose: We sought to investigate the anticonvulsive and neuroprotective effect of a selective metabotropic glutamate receptor 8 (mGluR8) agonist ( S )-3,4-dicarboxyphenylglycines ( S -3,4-DCPG) on pilocarpine-induced status epilepticus (PISE) and subsequent loss of hilar neurons in the dentate gyrus after systemic (intravenous) or local (intracerebroventricular) administration. We compared the difference in granular cell responses after paired-pulse stimulation of the perforant pathway and the sensitivity to local injection of S -3,4-DCPG into the stratum granulosum in the control and mice at 2 months after PISE.
Methods: We used intravenous, intracerebroventricular, or intrahippocampal administration of S -3,4-DCPG to mice with status epilepticus or temporal lobe epilepsy and neurophysiologic recording of somatic field excitatory postsynaptic potential (sfEPSP) and population spike (PS) of granular cells in response to perforant-pathway stimulation or S -3,4-DCPG treatment.
Results: Intracerebroventricular (1.91 μmol) but not systemic administration of S -3,4-DCPG (at doses of 12.5, 50, 100, 200, 400, 800, and 1,200 mg/kg) could control PISE with no neuroprotective effect. In epileptic mice, mGluR8-mediated inhibition of fEPSPs was reduced significantly in granular cell bodies.
Conclusions: At doses ranging from 12.5 to 1,200 mg/kg, intravenous administration of S -3,4-DCPG may not be effective in controlling status epilepticus. Down-regulation of mGluR8 may be related to reduced S -3,4-DCPG–mediated inhibition and the subsequent occurrence of spontaneously recurrent seizures.     

Seizure frequency in pilocarpine-treated rats is independent of circadian rhythm

Pilocarpine-induced status epilepticus (SE) results in chronic spontaneous recurrent seizures resembling human temporal lobe epilepsy. In this and other experimental models, behaviorally monitored seizure frequency was suggested to vary in a circadian fashion, and to increase with time. We re-addressed those hypotheses using continuous video-electroencephalography (EEG) telemetry in rats with SE at 30 days of age. In 11 chronically epileptic animals monitored up to 300 days after SE in a fixed 12 h light/dark cycle, we found that seizure frequency did not correlate with circadian rhythm.     

Effects of fluoxetine and TFMPP on spontaneous seizures in rats with pilocarpine-induced epilepsy

PURPOSE: Fluoxetine is a selective serotonin [5-hydroxytryptamine (5-HT)] reuptake inhibitor (SSRI) commonly used to treat depression. Some uncontrolled clinical studies have reported that SSRIs increase seizures, but animal experiments with evoked-seizure models have suggested that SSRIs at therapeutic doses decrease seizure susceptibility. We tested the hypothesis that fluoxetine and trifluoromethylphenylpiperazine (TFMPP, a nonselective 5-HT-receptor agonist) reduce the frequency of spontaneous motor seizures in pilocarpine-treated rats. METHODS: Fluoxetine (20 mg/kg) and TFMPP (5 mg/kg) were administered to rats with pilocarpine-induced epilepsy. Phenobarbital (PB; 10 mg/kg) was a positive control, and saline (i.e., 0.5 ml) controlled for the injection protocol. Each rat received each treatment (intraperitoneally) once per day for 5 consecutive days with 1 week between treatments. Rats were continuously video-monitored for the last 72 h of each treatment. RESULTS: When compared with saline over the entire 72-h observation period, PB and fluoxetine treatment, but not TFMPP, reduced the spontaneous-seizure rate. Plots of magnitude of the drug effect as a function of seizure frequency after saline treatment revealed larger drug effects for fluoxetine and PB in the rats with the highest control seizure rate. When the data from the five rats with the highest seizure frequency in saline were analyzed for the first 6 h after treatment, TFMPP also significantly reduced seizure frequency. CONCLUSIONS: Animal models with spontaneous seizures can be used to screen potential antiepileptic drugs, and fluoxetine and TFMPP reduce spontaneous seizures in the pilocarpine model of temporal lobe epilepsy.     

Behavioral and Histopathological Analysis of Domoic Acid Administration in Marmosets

Summary:  Purpose: To induce status epilepticus (SE) followed by the subsequent onset of spontaneous recurrent seizures, thus characterizing a new model of temporal lobe epilepsy in a nonhuman primate.
Methods: Male and female marmosets ( Callithrix jacchus ) (n = 18), ages between 2 and 8 years, were injected with domoic acid (0.5–4 mg/kg, i.p.) or saline, and behaviorally assessed with regard to the presence of acutely induced seizures and for ≤6 months for spontaneous seizures. Injection of doses ranging from 3.5 to 4 mg/kg either did not induce SE or resulted in fatal SE. Even a 5-min SE duration (SE blockade resulting from diazepam injection) proved lethal to marmosets within 1 h of domoate administration, regardless of intensive care and monitoring of the animals. Animals injected with doses ranging from 0.5 to 3 mg/kg that developed only a few minor convulsive signs were allowed a 6-month survival period for the assessment of spontaneous epileptic events. At the end of the experiment, 6-month period, or acute intoxication associated with SE induction, animals were deeply anesthetized and had their brains subjected to histologic processing for Nissl and delta-FosB.
Results: For the animals injected with domoate that did not develop SE (i.e., those that survived), we could not detect any behavioral signs of spontaneous epileptic seizures in the 6-month observation period, and only minor indications of neuropathologic changes (i.e., neuronal death) over Nissl-stained sections, as well as some small changes in the staining for delta-FosB in a few of the animals.
Conclusions: Systemic administration of domoic acid to marmosets is not effective for the generation of a model of chronic temporal lobe epilepsy. Administration of domoic acid at doses that do not lead to SE also did not lead to the development of temporal lobe epilepsy or clear-cut behavioral changes over a 6-month period.     

Electrical stimulation-induced seizures in rats: a "dose-response" study on resultant neurodegeneration

Perforant pathway stimulation (PPS) is used to study temporal lobe epilepsy in rodents. High-frequency PPS induces acute seizures, which can lead to neuron death and spontaneous epilepsy. However, the minimum duration of PPS that induces neurodegeneration in naive rodents is unknown. Freely moving Sprague-Dawley rats received one episode of continuous, bilateral PPS (range 1-180 min). Simultaneous recording from the hippocampal granule cell layer confirmed the presence of epileptiform activity and showed precisely when seizure activity was terminated by anesthesia. Fluoro-Jade B staining, 1-7 days after PPS, determined neuronal degeneration. Thirty-five minutes of continuous PPS produced no apparent neuron death anywhere in the brain. The minimum duration that caused neurodegeneration, which was confined to the dentate hilus, was 40 min. These data indicate that, in freely moving naive rats: (1) 40 min of PPS-induced seizure activity is the threshold for brain cell death, and (2) dentate hilar neurons are the most vulnerable to PPS. Further studies are warranted to determine the threshold of epileptogenic neurodegeneration.     

Prediction of postoperative seizure control by hippocampal event-related potentials

PURPOSE: In spite of unequivocal results of the presurgical evaluation, between 10 and 30% of patients with medial temporal lobe epilepsy (MTLE) do not become seizure free by temporal lobe surgery. Because event-related potentials (ERPs) recorded within the hippocampal formation have been shown to be sensitive to the epileptogenic process, we examined whether ERPs can help to improve the prediction of postoperative seizure control. METHODS: We recorded ERPs to words from bilateral intrahippocaampal electrodes by using a visual word-recognition paradigm in 70 patients with unilateral hippocampal pathology and related these measurements to seizure outcome after temporal lobe surgery. RESULTS: Words elicited N400 potentials, which were reduced in amplitude on repetition on the side contralateral to hippocampal sclerosis. This contralateral repetition effect, however, was significantly diminished in the group of patients who experienced seizure recurrence after the operation. Contralateral repetition effects thus permitted correct prediction of postoperative seizure control in 94% of all patients. CONCLUSIONS: Recording ERPs to words within the medial temporal lobes can improve the prediction of postoperative seizure control. Reduced repetition effects contralateral to the side of hippocampal sclerosis may indicate bilateral epileptogenicity.     

Only certain antiepileptic drugs prevent seizures induced by pilocarpine

Seizures produced in rats by systemically administered pilocarpine (PILO) provide a model for studying the generation and spread of convulsive activity in the forebrain. PILO, 380 mg/kg, induces a sequence of behavioral and electroencephalographic alterations indicative of motor limbic seizures and status epilepticus which is followed by widespread damage to the limbic forebrain resembling that occurring subsequent to prolonged intractable seizures in humans. The present study was undertaken to determine whether clinically utilized antiepileptic drugs share an ability to suppress seizures and brain damage elicited by PILO in rats. Clonazepam, ED₅₀ 0.35 mg/kg (0.25–0.49), phenobarbital, 23.4 mg/kg (18.5–29.6), and valproic acid, 286 mg/kg (202–405), prevented the buildup of limbic seizures and protected against seizure-related brain damage. Pretreatment with trimethadione, 179 mg/kg (116–277), resulted in a moderate protection against PILO-induced seizures, whereas carbamazepine, 10–50 mg/kg, and diphenylhydantoin, 10–200 mg/kg, blocked neither convulsions nor brain damage produced by the drug. Surprisingly, ethosuximide, 196 mg/kg (141–272), and acetazolamide, 505 mg/kg (332–766), both lowered the threshold for seizures induced by PILO and converted a non-convulsant dose of PILO, 200 mg/kg, into a convulsant one. These results indicate that only certain anticonvulsant drugs elevate the threshold for PILO-induced seizures and prevent the occurrence of epilepsy-related brain damage. The resistance of seizures produced by PILO in rats to antiepileptic drugs reaffirms the clinically obvious lack of effective treatments for limbic convulsions.     

Expression and cellular distribution of major vault protein: a putative marker for pharmacoresistance in a rat model for temporal lobe epilepsy

PURPOSE: Because drug transporters might play a role in the development of multidrug resistance (MDR), we investigated the expression of a vesicular drug transporter, the major vault protein (MVP), in a rat model for temporal lobe epilepsy. METHODS: By using real-time polymerase chain reaction (PCR) analysis and immunocytochemistry, we quantified MVP mRNA and protein from the dentate gyrus (DG) and parahippocampal cortex (PHC) taken from EEG-monitored rats at 1 week after electrically induced status epilepticus (SE) and at 5-9 months after SE, when rats exhibit spontaneous seizures. RESULTS: Within 1 week after SE, MVP mRNA levels increased in both DG and PHC compared with those in controls. In chronic epileptic rats, MVP mRNA was still significantly upregulated in the PHC, whereas in the DG, the expression returned to control levels. MVP protein increased within 1 day after SE in reactive microglial cells within most limbic regions; the hippocampus showed the highest expression at 1 week after SE. In chronic epileptic rats, MVP protein expression was largely decreased in most brain regions, but it was still high, especially in the piriform cortex. The occurrence of SE was a prerequisite for increased MVP expression, because no increase was found in electrically stimulated rats that did not exhibit SE. CONCLUSIONS: MVP expression is upregulated in chronic epileptic rats and may contribute to the development of pharmacoresistance.     

Clinical and neuroimaging features of good and poor seizure control patients with mesial temporal lobe epilepsy and hippocampal atrophy

Summary: Purpose: Hippocampal atrophy (HA) and signal changes, detected at magnetic resonance imaging, have been associated with intractable seizures. Such a relation has been established by tertiary centers, where the prevalence of more severe cases tends to be higher. We evaluated the clinical and imaging variables that may have relevance to seizure control in patients with mesial temporal lobe epilepsy (MTLE) and HA. Methods: MTLE patients from the outpatient clinic of University of São Paulo School of Medicine at Ribeirão Preto were evaluated with protocols for the temporal lobe. Patients were considered to have good seizure control (GC; n = 42 ) if they had three of fewer seizures per year. Patients with pharmacoresistance and who did not fit the criteria for GC were considered to have poor seizure control (PC; n = 44 ). We made group comparisons and correlations of clinical data and hippocampal volume (HV) with seizure frequency. Results: No statistical differences were observed between the GC and PC groups in the following parameters: age at the time of study, age at the time of the initial precipitating injury (IPI) or first epileptic seizure, epilepsy duration and follow‐up, and family history of epilepsy. No differences were found in HV between GC (male, 2.04 ± 0.60 cc ; female, 2.00 ± 0.70 cc ) and PC (male, 2.26 ± 0.47 cc ; female, 2.15 ± 0.48 cc ) groups. Regression analysis indicated no correlation between seizure frequency and HV (p = 0.33) . Conclusions:These findings suggest that the intensity of HA does not have a direct correlation with seizure frequency in patients with MTLE with HA and that the detection of HA in MTLE patients does not mean an unequivocal indication of intractability.     

Selective and persistent upregulation of mdr1b mRNA and P-glycoprotein in the parahippocampal cortex of chronic epileptic rats

There is recent evidence that increased expression of multidrug transporters, such as P-glycoprotein (P-gp), may lead to reduced antiepileptic drug (AED) concentrations in the brain, shortly after status epilepticus (SE), thereby suggesting a possible mechanism for drug-resistance. To get insights on whether increased P-gp expression is a consequence of the initial insult, or evolves more gradually as a result of recurrent spontaneous seizures, we used a rat model of temporal lobe epilepsy in which spontaneous seizures develop after an electrically induced SE. We investigated the temporal and region-specific expression of two isoforms of the multidrug resistance gene (mdr1a and mdr1b, both encoding for P-gp) in two regions within the temporal lobe (the dentate gyrus (DG) and the parahippocampal cortex (PHC)). Using real-time PCR, we found that the mdr1b isoform was increased in the temporal lobe, 1 week after SE; however, this increase was reversible in dentate gyrus while it persisted in the parahippocampal cortex of chronic epileptic rats. Mdr1b upregulation was related to the occurrence of spontaneous seizures, since this isoform was unchanged in rats that were stimulated, but that did not develop SE (non-SE). The mdr1a isoform was transiently upregulated in the dentate gyrus. P-gp immunostaining was enhanced in endothelial and glia-like cells, 1 week after SE. In chronic epileptic rats, the number of strongly P-gp positive glia-like cells was much lower than 1 week after SE, and it was mainly present in the most ventral part of the temporal lobe. These cells were in close appostion to strongly stained blood vessels. These findings show that both mdr1a and mdr1b are induced by SE, although the increase in mdr1b isoform was more persistent. More importantly, increased P-gp expression is still present in chronic epileptic rats.