Enhanced burst discharges in the CA1 area of the immature versus adult hippocampus: patterns and cellular mechanisms.

Burst discharges in the immature brain may contribute to its enhanced seizure susceptibility. The cellular mechanisms underlying burst discharges in the CA1 area of the immature versus adult hippocampus were investigated with simultaneous whole-cell and field-potential recordings. When GABAA receptors were blocked pharmacologically, bursts in CA1 were either graded or all-or-none (or mixed) as a function of electrical stimulation intensity. Most CA1 minislices from immature rats displayed all-or-none or mixed bursts, whereas the slices from adult rats predominantly elicited graded bursts. The frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were greater in CA1 pyramidal cells from the immature than the adult slices. The developmental differences in CA1 bursting were also detected in slices adjusted for maturational changes in brain volume (i.e., 350 µm thick for immature vs. 450 µm thick for adult rats). Neither N-methyl-d-aspartate (NMDA) nor group I metabotropic glutamate (mGlu1) receptor antagonists blocked the network-driven bursts in immature CA1, but an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker abolished them. Robust excitatory postsynaptic potentials (EPSPs) occurred after bursts in some immature CA1 slices (23%) but never in slices from the adult. The input-output (amount of current injected vs. number of action potentials generated) relationship was markedly greater in CA1 pyramidal cells in the immature compared with the adult hippocampus. These data suggest that the CA1 area of the immature brain is capable of generating network-driven bursts, which declines in adult rats. The increased propensity of burst generation in immature CA1 appears to involve a greater AMPA receptor-mediated synaptic network and an increased intrinsic spike-generating ability.NEW & NOTEWORTHY Burst discharges in the developing brain can provide valuable insights into epileptogenesis. We show that the immature hippocampal CA1 area is capable of generating all-or-none (i.e., network) bursts, which transitions to graded (i.e., nonnetwork) bursts in the mature brain via both synaptic and intrinsic mechanisms. Our results provide new clues to help understand possible mechanisms that may be shared in the immature and epileptic brain and how the normal brain becomes seizure prone (i.e., epileptogenesis).

Sensitivity of unilateral- versus bilateral-onset spike-wave discharges to ethosuximide and carbamazepine in the fluid percussion injury rat model of traumatic brain injury.

Unilateral-onset spike-wave discharges (SWDs) following fluid percussion injury (FPI) in rats have been used for nearly two decades as a model for complex partial seizures in human posttraumatic epilepsy (PTE). This study determined if SWDs with a unilateral versus bilateral cortical onset differed. In this experiment, 2-mo-old rats received severe FPI (3 atm) or sham surgery and were instrumented for chronic video-electrocorticography (ECoG) recording (up to 9 mo). The antiseizure drug, carbamazepine (CBZ), and the antiabsence drug, ethosuximide (ETX), were administered separately to determine if they selectively suppressed unilateral- versus bilateral-onset SWDs, respectively. SWDs did not significantly differ between FPI and sham rats on any measured parameter (wave-shape, frequency spectrum, duration, or age-related progression), including unilateral (∼17%) versus bilateral (∼83%) onsets. SWDs with a unilateral onset preferentially originated ipsilateral to the craniotomy in both FPI and sham rats, suggesting that the unilateral-onset SWDs were related to surgical injury and not specifically to FPI. ETX profoundly suppressed SWDs with either unilateral or bilateral onsets, and CBZ had no effect on either type of SWD. These results suggest that SWDs with either a unilateral or bilateral onset have a pharmacosensitivity similar to absence seizures and are very different from the complex partial seizures of PTE. Therefore, SWDs with a unilateral onset after FPI are not a model of the complex partial seizures that occur in PTE, and their use for finding new treatments for PTE could be counterproductive, particularly if their close similarity to normal brain oscillations is not acknowledged.NEW & NOTEWORTHY Unilateral-onset spike-wave discharges (SWDs) in rats have been used to model complex partial seizures in human posttraumatic epilepsy (PTE), compared to bilateral-onset SWDs thought to reflect human absence seizures. Here, we show that both unilateral- and bilateral-onset SWDs following traumatic brain injury are suppressed by the antiabsence drug ethosuximide and are unaffected by the antiseizure drug carbamazepine. We propose that unilateral-onset SWDs are not useful for studying mechanisms of, or treatments for, PTE.

Spontaneous Recurrent Absence Seizure-like Events in Wild-Caught Rats.

Absence epilepsy is a heritable human neurological disorder characterized by brief nonconvulsive seizures with behavioral arrest, moderate-to-severe loss of consciousness (absence), and distinct spike-wave discharges (SWDs) in the EEG and electrocorticogram (ECoG). Genetic models of this disorder have been created by selectively inbreeding rats for absence seizure-like events with similar electrical and behavioral characteristics. However, these events are also common in outbred laboratory rats, raising concerns about whether SWD/immobility accurately reflects absence epilepsy as opposed to "normal" rodent behavior. We hypothesized that, if SWD/immobility models absence seizures, it would not exist in wild-caught rats due to the pressures of natural selection. To test this hypothesis, we compared chronic video/electrocorticogram recordings from male and female wild-caught (Brown-Norway [BN]) rats to recordings from laboratory outbred BN, outbred Long-Evans, and inbred WAG/Rij rats (i.e., a model of absence epilepsy). Wild-caught BN rats displayed absence-like SWD/immobility events that were highly similar to outbred BN rats in terms of spike-wave morphology, frequency, diurnal rhythmicity, associated immobility, and sensitivity to the anti-absence drug, ethosuximide; however, SWD bursts were less frequent and of shorter duration in wild-caught and outbred BN rats than the outbred Long-Evans and inbred WAG/Rij strains. We conclude that SWD/immobility in rats does not represent absence seizures, although they appear to have many similarities. In wild rats, SWD/immobility appears to represent normal brain activity that does not reduce survival in natural environments, a conclusion that logically extends to outbred laboratory rats and possibly to those that have been inbred to model absence epilepsy.SIGNIFICANCE STATEMENT Spike-wave discharges (SWDs), behavioral arrest, and diminished consciousness are cardinal signs of seizures in human absence epilepsy and are used to model this disorder in inbred rats. These characteristics, however, are routinely found in outbred laboratory rats, leading to debate on whether SWD/immobility is a valid model of absence seizures. The SWD/immobility events in wild-caught rats appear equivalent to those found in outbred and inbred rat strains, except for lower incidence and shorter durations. Our results indicate that the electrophysiological and behavioral characteristics of events underlying hypothetical absence epilepsy in rodent models are found in wild rats captured in their natural environment. Other criteria beyond observation of SWDs and associated immobility are required to objectively establish absence epilepsy in rat models.

Delayed Adjunctive Treatment of Organophosphate-Induced Status Epilepticus in Rats with Phenobarbital, Memantine, or Dexmedetomidine.

Organophosphate (OP) exposure induces status epilepticus (SE), a medical emergency with high morbidity and mortality. Current standard medical countermeasures lose efficacy with time so that treatment delays, in the range of tens of minutes, result in increasingly poor outcomes. As part of the Countermeasures Against Chemical Threats Neurotherapeutics Screening Program, we previously developed a realistic model of delayed treatment of OP-induced SE using the OP diisopropyl fluorophosphate (DFP) to screen compounds for efficacy in the termination of SE and elimination of neuronal death. Male rats were implanted for electroencephalogram (EEG) recordings 7 days prior to experimentation. Rats were then exposed to DFP, and SE was induced for 60 minutes and then treated with midazolam (MDZ) plus one of three antiseizure drugs (ASDs)-phenobarbital (PHB), memantine (MEM), or dexmedetomidine (DMT)-in conjunction with antidotes. EEG was recorded for 24 hours, and brains were stained with Fluoro-Jade B for quantification of degenerating neurons. We found that PHB + MDZ induced a prolonged suppression of SE and reduced neuronal death. MEM + MDZ treatment exacerbated SE and increased mortality; however, surviving rats had fewer degenerating neurons. DMT + MDZ significantly suppressed SE with only a minimal reduction in neuronal death. These data demonstrate that delayed treatment of OP-induced SE with other ASDs, when added to MDZ, can achieve greater seizure suppression with additional reduction in degenerating neurons throughout the brain compared with MDZ alone. The effect of a drug on the severity of seizure activity did not necessarily determine the drug's effect on neuronal death under these conditions. SIGNIFICANCE STATEMENT: This study assesses the relative effectiveness of three different delayed-treatment regimens for the control of organophosphate-induced status epilepticus and reduction of subsequent neuronal death. The data demonstrate the potential for highly effective therapies despite significant treatment delay and a potential disconnect between seizure severity and neuronal death.

Effect of carbamazepine on spontaneous recurrent seizures recorded from the dentate gyrus in rats with kainate-induced epilepsy.

OBJECTIVE: Animal models of chronic epilepsy with spontaneous recurrent seizures (SRSs) may be useful in the discovery and mechanistic analyses of antiseizure drugs (ASDs). Carbamazepine (CBZ), a widely used ASD with a well-defined mechanism, was analyzed in this proof-of-principle study to determine how a traditional ASD affects the properties of SRSs. METHODS: The effects of CBZ on electrographic SRSs recorded from the dentate gyrus were studied in freely behaving rats using a repeated, low-dose kainate model of acquired epilepsy with a repeated-measures, crossover protocol. RESULTS: Almost all seizure durations were >20 seconds. Both seizure likelihood and duration appeared to be similar between 1 and 8 hours after individual CBZ injections. CBZ-induced decreases in seizure frequency were not significant at 10 mg/kg; however, at 30 mg/kg, seizure frequency was significantly reduced for convulsive but not nonconvulsive seizures. At 100 mg/kg, CBZ strongly suppressed both convulsive and nonconvulsive seizures. Although CBZ had a dose-dependent effect on seizure frequency, CBZ did not affect seizure duration at any dose. The preceding interictal interval did not affect seizure duration; however, at 30 mg/kg CBZ, nearly all seizures were nonconvulsive when the interictal interval was <30 minutes (ie, during clusters). SIGNIFICANCE: Increased doses of CBZ (10-100 mg/kg) suppressed the frequency but not the duration of convulsive and nonconvulsive seizures in the repeated, low-dose kainate model. The repeated-measures, crossover protocol, which requires relatively few animals and compensates for progressive increases in seizure frequency during epileptogenesis after status epilepticus, allowed quantitative analyses of clinically relevant and translatable properties of SRSs.

Voluntary Control of Epileptiform Spike-Wave Discharges in Awake Rats.

Genetically inherited absence epilepsy in humans is typically characterized by brief (seconds) spontaneous seizures, which involve spike-wave discharges (SWDs) in the EEG and interruption of consciousness and ongoing behavior. Genetic (inbred) models of this disorder in rats have been used to examine mechanisms, comorbidities, and antiabsence drugs. SWDs have also been proposed as models of complex partial seizures (CPSs) following traumatic brain injury (post-traumatic epilepsy). However, the ictal characteristics of these rat models, including SWDs and associated immobility, are also prevalent in healthy outbred laboratory rats. We therefore hypothesized that SWDs are not always associated with classically defined absence seizures or CPSs. To test this hypothesis, we used operant conditioning in male rats to determine whether outbred strains, Sprague Dawley and Long-Evans, and/or the inbred WAG/Rij strain (a rat model of heritable human absence epilepsy) could exercise voluntary control over these epileptiform events. We discovered that both inbred and outbred rats could shorten the duration of SWDs to obtain a reward. These results indicate that SWD and associated immobility in rats may not reflect the obvious cognitive/behavioral interruption classically associated with absence seizures or CPSs in humans. One interpretation of these results is that human absence seizures and perhaps CPSs could permit a far greater degree of cognitive capacity than often assumed and might be brought under voluntary control in some cases. However, these results also suggest that SWDs and associated immobility may be nonepileptic in healthy outbred rats and reflect instead voluntary rodent behavior unrelated to genetic manipulation or to brain trauma.SIGNIFICANCE STATEMENT Our evidence that inbred and outbred rats learn to control the duration of spike-wave discharges (SWDs) suggests a voluntary behavior with maintenance of consciousness. If SWDs model mild absence seizures and/or complex partial seizures in humans, then an opportunity may exist for operant control complementing or in some cases replacing medication. Their equal occurrence in outbred rats also implies a major potential confound for behavioral neuroscience experiments, at least in adult rats where SWDs are prevalent. Alternatively, the presence and voluntary control of SWDs in healthy outbred rats could indicate that these phenomena do not always model heritable absence epilepsy or post-traumatic epilepsy in humans, and may instead reflect typical rodent behavior.

The antihelminthic moxidectin enhances tonic GABA currents in rodent hippocampal pyramidal neurons.

Macrocyclic lactones (MLs) are commonly used treatments for parasitic worm and insect infections in humans, livestock, and companion animals. MLs target the invertebrate glutamate-activated chloride channel that is not present in vertebrates. MLs are not entirely inert in vertebrates, though; they have been reported to have activity in heterologous expression systems consisting of ligand-gated ion channels that are present in the mammalian central nervous system (CNS). However, these compounds are typically not able to reach significant concentrations in the CNS because of the activity of the blood-brain barrier P-glycoprotein extrusion system. Despite this, these compounds are able to reach low levels in the CNS that may be useful in the design of novel "designer" ligand-receptor systems that can be used to directly investigate neuronal control of behavior in mammals and have potential for use in treating human neurological diseases. To determine whether MLs might affect neurons in intact brains, we investigated the activity of the ML moxidectin (MOX) at native GABA receptors. Specifically, we recorded tonic and phasic miniature inhibitory postsynaptic currents (mIPSCs) in ex vivo brain slices. Our data show that MOX potentiated tonic GABA currents in a dose-dependent manner but had no concomitant effects on phasic GABA currents (i.e., MOX had no effect on the amplitude, frequency, or decay kinetics of mIPSCs). These studies indicate that behavioral experiments that implement a ML-based novel ligand-receptor system should take care to control for potential effects of the ML on native tonic GABA receptors. NEW & NOTEWORTHY We have identified a novel mechanism of action in the mammalian central nervous system for the antihelminthic moxidectin, commonly prescribed to animals worldwide and currently being evaluated for use in humans. Specifically, moxidectin applied to rodent brain slices selectively enhanced the tonic GABA conductance of hippocampal pyramidal neurons.

Progression of convulsive and nonconvulsive seizures during epileptogenesis after pilocarpine-induced status epilepticus.

Although convulsive seizures occurring during pilocarpine-induced epileptogenesis have received considerable attention, nonconvulsive seizures have not been closely examined, even though they may reflect the earliest signs of epileptogenesis and potentially guide research on antiepileptogenic interventions. The definition of nonconvulsive seizures based on brain electrical activity alone has been controversial. Here we define and quantify electrographic properties of convulsive and nonconvulsive seizures in the context of the acquired epileptogenesis that occurs after pilocarpine-induced status epilepticus (SE). Lithium-pilocarpine was used to induce the prolonged repetitive seizures characteristic of SE; when SE was terminated with paraldehyde, seizures returned during the 2-day period after pilocarpine treatment. A distinct latent period ranging from several days to >2 wk was then measured with continuous, long-term video-EEG. Nonconvulsive seizures dominated the onset of epileptogenesis and consistently preceded the first convulsive seizures but were still present later. Convulsive and nonconvulsive seizures had similar durations. Postictal depression (background suppression of the EEG) lasted for >100 s after both convulsive and nonconvulsive seizures. Principal component analysis was used to quantify the spectral evolution of electrical activity that characterized both types of spontaneous recurrent seizures. These studies demonstrate that spontaneous nonconvulsive seizures have electrographic properties similar to convulsive seizures and confirm that nonconvulsive seizures link the latent period and the onset of convulsive seizures during post-SE epileptogenesis in an animal model. Nonconvulsive seizures may also reflect the earliest signs of epileptogenesis in human acquired epilepsy, when intervention could be most effective. NEW & NOTEWORTHY Nonconvulsive seizures usually represent the first bona fide seizure following a latent period, dominate the early stages of epileptogenesis, and change in severity in a manner consistent with the progressive nature of epileptogenesis. This analysis demonstrates that nonconvulsive and convulsive seizures have different behavioral outcomes but similar electrographic signatures. Alternatively, epileptiform spike-wave discharges fail to recapitulate several key seizure features and represent a category of electrical activity separate from nonconvulsive seizures in this model.

Progressive, Seizure-Like, Spike-Wave Discharges Are Common in Both Injured and Uninjured Sprague-Dawley Rats: Implications for the Fluid Percussion Injury Model of Post-Traumatic Epilepsy.

Variable-duration oscillations and repetitive, high-voltage spikes have been recorded in the electrocorticogram (ECoG) of rats weeks and months after fluid percussion injury (FPI), a model of traumatic brain injury. These ECoG events, which have many similarities to spike-wave-discharges (SWDs) and absence seizures, have been proposed to represent nonconvulsive seizures characteristic of post-traumatic epilepsy (PTE). The present study quantified features of SWD episodes in rats at different time points after moderate to severe FPI, and compared them with age-matched control rats. Control and FPI-injured rats at 1 year of age displayed large-amplitude and frequent SWD events at frontal and parietal recording sites. At 3-6 months, SWDs were shorter in duration and less frequent; extremely brief SWDs (i.e., "larval") were detected as early as 1 month. The onset of the SWDs was nearly always synchronous across electrodes and of larger amplitude in frontal regions. A sensory stimulus, such as a click, immediately and consistently stopped the occurrence of the SWDs. SWDs were consistently accompanied by behavioral arrest. All features of SWDs in control and experimental (FPI) rats were indistinguishable. None of the FPI-treated rats developed nonconvulsive or convulsive seizures that could be distinguished electrographically or behaviorally from SWDs. Because SWDs have features similar to genetic absence seizures, these results challenge the hypothesis that SWDs after FPI reflect PTE.

Maternal Stress Combined with Terbutaline Leads to Comorbid Autistic-Like Behavior and Epilepsy in a Rat Model.

Human autism is comorbid with epilepsy, yet, little is known about the causes or risk factors leading to this combined neurological syndrome. Although genetic predisposition can play a substantial role, our objective was to investigate whether maternal environmental factors alone could be sufficient. We examined the independent and combined effects of maternal stress and terbutaline (used to arrest preterm labor), autism risk factors in humans, on measures of both autistic-like behavior and epilepsy in Sprague-Dawley rats. Pregnant dams were exposed to mild stress (foot shocks at 1 week intervals) throughout pregnancy. Pups were injected with terbutaline on postnatal days 2-5. Either maternal stress or terbutaline resulted in autistic-like behaviors in offspring (stereotyped/repetitive behaviors and deficits in social interaction or communication), but neither resulted in epilepsy. However, their combination resulted in severe behavioral symptoms, as well as spontaneous recurrent convulsive seizures in 45% and epileptiform spikes in 100%, of the rats. Hippocampal gliosis (GFAP reactivity) was correlated with both abnormal behavior and spontaneous seizures. We conclude that prenatal insults alone can cause comorbid autism and epilepsy but it requires a combination of teratogens to achieve this; testing single teratogens independently and not examining combinatorial effects may fail to reveal key risk factors in humans. Moreover, astrogliosis may be common to both teratogens. This new animal model of combined autism and epilepsy permits the experimental investigation of both the cellular mechanisms and potential intervention strategies for this debilitating comorbid syndrome.

Inhibition of the prostaglandin receptor EP2 following status epilepticus reduces delayed mortality and brain inflammation.

Prostaglandin E2 is now widely recognized to play critical roles in brain inflammation and injury, although the responsible prostaglandin receptors have not been fully identified. We developed a potent and selective antagonist for the prostaglandin E2 receptor subtype EP2, TG6-10-1, with a sufficient pharmacokinetic profile to be used in vivo. We found that in the mouse pilocarpine model of status epilepticus (SE), systemic administration of TG6-10-1 completely recapitulates the effects of conditional ablation of cyclooxygenase-2 from principal forebrain neurons, namely reduced delayed mortality, accelerated recovery from weight loss, reduced brain inflammation, prevention of blood-brain barrier opening, and neuroprotection in the hippocampus, without modifying seizures acutely. Prolonged SE in humans causes high mortality and morbidity that are associated with brain inflammation and injury, but currently the only effective treatment is to stop the seizures quickly enough with anticonvulsants to prevent brain damage. Our results suggest that the prostaglandin receptor EP2 is critically involved in neuroinflammation and neurodegeneration, and point to EP2 receptor antagonism as an adjunctive therapeutic strategy to treat SE.

Valnoctamide enhances phasic inhibition: a potential target mechanism for the treatment of benzodiazepine-refractory status epilepticus.

Valnoctamide (VCD), a derivative of valproate, suppresses electrographic seizures in animal models of status epilepticus (SE), even when the seizures are resistant to benzodiazepines (BZDs). We therefore tested the effect of VCD on miniature inhibitory postsynaptic currents (mIPSCs) in CA1 pyramidal cells to determine if VCD acts directly on γ-aminobutyric acid (GABA)A receptors. Bath-applied VCD induced a specific, rapid, dose-dependent, and reversible slowing of the decay of mIPSCs (i.e., increased time constant) with no effect on their frequency or amplitude. This is similar to the effect of BZDs on mIPSCs, but the effect of VCD persisted in the presence of the BZD-binding site antagonist flumazenil, and was additive to the effect of the BZD, diazepam. These data suggest that VCD acts through a different binding site than that of BZDs, which likely accounts for its effect on BZD-refractory SE. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

When and how do seizures kill neurons, and is cell death relevant to epileptogenesis?

The effect of seizures on neuronal death and the role of seizure-induced neuronal death in acquired epileptogenesis have been debated for decades. Isolated brief seizures probably do not kill neurons; however, severe and repetitive seizures (i.e., status epilepticus) certainly do. Because status epilepticus both kills neurons and also leads to chronic epilepsy, neuronal death has been proposed to be an integral part of acquired epileptogenesis. Several studies, particularly in the immature brain, have suggested that neuronal death is not necessary for acquired epileptogenesis; however, the lack of neuronal death is difficult if not impossible to prove, and more recent studies have challenged this concept. Novel mechanisms of cell death, beyond the traditional concepts of necrosis and apoptosis, include autophagy, phagoptosis, necroptosis, and pyroptosis. The traditional proposal for why neuronal death may be necessary for epileptogenesis is based on the recapitulation of development hypothesis, where a loss of synaptic input from the dying neurons is considered a critical signal to induce axonal sprouting and synaptic-circuit reorganization. We propose a second hypothesis - the neuronal death pathway hypothesis, which states that the biochemical pathways causing programmed neurodegeneration, rather than neuronal death per se, are responsible for or contribute to epileptogenesis. The reprogramming of neuronal death pathways - if true - is proposed to derive from necroptosis or pyroptosis. The proposed new hypothesis may inform on why neuronal death seems closely linked to epileptogenesis, but may not always be.

Epilepsy therapy development: technical and methodologic issues in studies with animal models.

The search for new treatments for seizures, epilepsies, and their comorbidities faces considerable challenges. This is due in part to gaps in our understanding of the etiology and pathophysiology of most forms of epilepsy. An additional challenge is the difficulty in predicting the efficacy, tolerability, and impact of potential new treatments on epilepsies and comorbidities in humans, using the available resources. Herein we provide a summary of the discussions and proposals of the Working Group 2 as presented in the Joint American Epilepsy Society and International League Against Epilepsy Translational Workshop in London (September 2012). We propose methodologic and reporting practices that will enhance the uniformity, reliability, and reporting of early stage preclinical studies with animal seizure and epilepsy models that aim to develop and evaluate new therapies for seizures or epilepsies, using multidisciplinary approaches. The topics considered include the following: (1) implementation of better study design and reporting practices; (2) incorporation in the study design and analysis of covariants that may influence outcomes (including species, age, sex); (3) utilization of approaches to document target relevance, exposure, and engagement by the tested treatment; (4) utilization of clinically relevant treatment protocols; (5) optimization of the use of video-electroencephalography (EEG) recordings to best meet the study goals; and (6) inclusion of outcome measures that address the tolerability of the treatment or study end points apart from seizures. We further discuss the different expectations for studies aiming to meet regulatory requirements to obtain approval for clinical testing in humans. Implementation of the rigorous practices discussed in this report will require considerable investment in time, funds, and other research resources, which may create challenges for academic researchers seeking to contribute to epilepsy therapy discovery and development. We propose several infrastructure initiatives to overcome these barriers.

Issues related to development of antiepileptogenic therapies.

Several preclinical proof-of-concept studies have provided evidence for positive treatment effects on epileptogenesis. However, none of these hypothetical treatments has advanced to the clinic. The experience in other fields of neurology such as stroke, Alzheimer's disease, or amyotrophic lateral sclerosis has indicated several problems in the design of preclinical studies, which likely contribute to failures in translating the positive preclinical data to the clinic. The Working Group on "Issues related to development of antiepileptogenic therapies" of the International League Against Epilepsy (ILAE) and the American Epilepsy Society (AES) has considered the possible problems that arise when moving from proof-of-concept antiepileptogenesis (AEG) studies to preclinical AEG trials, and eventually to clinical AEG trials. This article summarizes the discussions and provides recommendations on how to design a preclinical AEG monotherapy trial in adult animals. We specifically address study design, animal and model selection, number of studies needed, issues related to administration of the treatment, outcome measures, statistics, and reporting. In addition, we give recommendations for future actions to advance the preclinical AEG testing.

Epilepsy biomarkers.

A biomarker is defined as an objectively measured characteristic of a normal or pathologic biologic process. Identification and proper validation of biomarkers of epileptogenesis (the development of epilepsy) and ictogenesis (the propensity to generate spontaneous seizures) might predict the development of an epilepsy condition; identify the presence and severity of tissue capable of generating spontaneous seizures; measure progression after the condition is established; and determine pharmacoresistance. Such biomarkers could be used to create animal models for more cost-effective screening of potential antiepileptogenic and antiseizure drugs and devices, and to reduce the cost of clinical trials by enriching the trial population, and acting as surrogate markers to shorten trial duration. The objectives of the biomarker subgroup for the London Workshop were to define approaches for identifying possible biomarkers for these purposes. Research to identify reliable biomarkers may also reveal underlying mechanisms that could serve as therapeutic targets for the development of new antiepileptogenic and antiseizure compounds.

A once-per-day, drug-in-food protocol for prolonged administration of antiepileptic drugs in animal models.

PURPOSE: Convenient and effective methods for administering potential antiepileptic drugs (AEDs) chronically should facilitate many experiments in animal models of chronic epilepsy with spontaneous recurrent seizures. This proof-of-principle study aimed to optimize a once-per-day, drug-in-food protocol by testing the effect of carbamazepine (CBZ) on the frequency of convulsive seizures in rats with kainate-induced epilepsy. METHODS: Adult male rats were given repeated low-dose kainate injections until convulsive status epilepticus persisted for >3 h. After the rats developed spontaneous recurrent seizures, food pellets with CBZ (30, 100, or 300 mg/kg/day) were provided once per day in three 2-week trials (n = 7-9 rats) involving 5 days of CBZ or control treatment, separated by two recovery days within a trial. The total amount of food provided and consumed per day corresponded to a normal caloric diet (60 g/kg/day). KEY FINDINGS: When provided once per day, all animals ate the CBZ-containing food irregularly but continuously throughout the 24-h day. With this daily feeding protocol, CBZ significantly reduced the frequency of spontaneous convulsive seizures in a dose-dependent manner. It is important to note that the effect of CBZ was consistent across the 5 days and throughout each day of the trials. With food administered at 9:00 a.m., and blood assayed at 5:00 p.m., higher food levels of CBZ resulted in higher plasma concentrations of CBZ. SIGNIFICANCE: This AED-in-food protocol is simple, efficient, inexpensive, reliable, and noninvasive; it allows easier long-term drug administration and is less stressful and more humane than other methods of AED administration.

Finding a better drug for epilepsy: preclinical screening strategies and experimental trial design.

The antiepileptic drugs (AEDs) introduced during the past two decades have provided several benefits: they offered new treatment options for symptomatic treatment of seizures, improved ease of use and tolerability, and lowered risk for hypersensitivity reactions and detrimental drug-drug interactions. These drugs, however, neither attenuated the problem of drug-refractory epilepsy nor proved capable of preventing or curing the disease. Therefore, new preclinical screening strategies are needed to identify AEDs that target these unmet medical needs. New therapies may derive from novel targets identified on the basis of existing hypotheses for drug-refractory epilepsy and the biology of epileptogenesis; from research on genetics, transcriptomics, and epigenetics; and from mechanisms relevant for other therapy areas. Novel targets should be explored using new preclinical screening strategies, and new technologies should be used to develop medium- to high-throughput screening models. In vivo testing of novel drugs should be performed in models mimicking relevant aspects of drug refractory epilepsy and/or epileptogenesis. To minimize the high attrition rate associated with drug development, which arises mainly from a failure to demonstrate sufficient clinical efficacy of new treatments, it is important to define integrated strategies for preclinical screening and experimental trial design. An important tool will be the discovery and implementation of relevant biomarkers that will facilitate a continuum of proof-of-concept approaches during early clinical testing to rapidly confirm or reject preclinical findings, and thereby lower the risk of the overall development effort. In this review, we overview some of the issues related to these topics and provide examples of new approaches that we hope will be more successful than those used in the past.

Continuous electroencephalographic monitoring with radio-telemetry in a rat model of perinatal hypoxia-ischemia reveals progressive post-stroke epilepsy.

The development of acquired epilepsy after a perinatal hypoxic-ischemic (HI) insult was investigated in rats. After unilateral carotid ligation with hypoxia on postnatal day 7, cortical electroencephalographic and behavioral seizures were recorded with continuous radio-telemetry and video. Chronic recordings were obtained between 2 and 12 months of age in freely behaving HI-treated and sham control rats. The hypotheses were that the acquired epilepsy is directly associated with an ischemic infarct (i.e., no lesion, no epilepsy), and the resultant epilepsy is temporally progressive. Every HI-treated rat with a cerebral infarct developed spontaneous epileptiform discharges and recurrent seizures (100%); in contrast, no spontaneous epileptiform discharges or seizures were detected with continuous monitoring in the HI-treated rats without infarcts. The initial seizures at 2 months generally showed focal onset and were nonconvulsive. Subsequent seizures had focal onsets that propagated to the homotopic contralateral cortex and were nonconvulsive or partial; later seizures often appeared to have bilateral onset and were convulsive. Spontaneous epileptiform discharges were initially lateralized to ipsilateral neocortex but became bilateral over time. The severity and frequency of the spontaneous behavioral and electrographic seizures progressively increased over time. In every epileptic rat, seizures occurred in distinct clusters with seizure-free periods as long as a few weeks. The progressive increase in seizure frequency over time was associated with increases in cluster frequency and seizures within each cluster. Thus, prolonged, continuous seizure monitoring directly demonstrated that the acquired epilepsy after perinatal HI was progressive with seizure clusters and was consistently associated with a cerebral infarct.