Animal models of epilepsy for the development of antiepileptogenic and disease-modifying drugs. A comparison of the pharmacology of kindling and post-status epilepticus models of temporal lobe epilepsy

Control of epilepsy has primarily focused on suppressing seizure activity by antiepileptic drugs (AEDs) after epilepsy has developed. AEDs have greatly improved the lives of people with epilepsy. However, the belief that AEDs, in addition to suppressing seizures, alter the underlying epileptogenic process and, in doing so, the course of the disease and its prognosis, is not supported by the current clinical and experimental data. An intriguing possibility is to control acquired epilepsy by preventing epileptogenesis, the process by which the brain becomes epileptic. A number of AEDs have been evaluated in clinical trials to test whether they prevent epileptogenesis in humans, but to date no drug has been shown to be effective in such trials. Thus, there is a pressing need for drugs that are truly antiepileptogenic to either prevent epilepsy or alter its natural course. For this purpose, animal models of epilepsy are an important prerequisite. There are various animal models with chronic brain dysfunctions thought to reflect the processes underlying human epilepsy. Such chronic models of epilepsy include the kindling model of temporal lobe epilepsy (TLE), post-status models of TLE in which epilepsy develops after a sustained status epilepticus, and genetic models of different types of epilepsy. Currently, the kindling model and post-status models, such as the pilocarpine or kainate models, are the most widely used models for studies on epileptogenic processes and on drug targets by which epilepsy can be prevented or modified. Furthermore, the seizures in these models can be used for testing of antiepileptic drug effects. A comparison of the pharmacology of chronic models with models of acute (reactive or provoked) seizures in previously healthy (non-epileptic) animals, such as the maximal electroshock seizure test, demonstrates that drug testing in chronic models of epilepsy yields data which are more predictive of clinical efficacy and adverse effects, so that chronic models should be used relatively early in drug development to minimize false positives. Interestingly, the pharmacology of elicited kindled seizures in fully kindled rats and spontaneous recurrent seizures in post-status models is remarkably similar. However, when these models are used for studying the antiepileptogenic effects of drugs, marked differences between models exist, indicating that the processes underlying epileptogenesis differ among models, even among different post-status models of TLE. A problem for clinical validation of TLE models is the lack of an AED, which effectively prevents epilepsy in humans. Thus, at present, it is not possible to judge which chronic model is best suited for developing new strategies in the search for antiepileptogenic and disease-modifying drugs, but rather a battery of models should be used to avoid false negative or positive predictions.     

Epileptogenesis in Experimental Models

Summary:  Epileptogenesis refers to a phenomenon in which the brain undergoes molecular and cellular alterations after a brain-damaging insult, which increase its excitability and eventually lead to the occurrence of recurrent spontaneous seizures. Common epileptogenic factors include traumatic brain injury (TBI), stroke, and cerebral infections. Only a subpopulation of patients with any of these brain insults, however, will develop epilepsy. Thus, there are two great challenges: (1) identifying patients at risk, and (2) preventing and/or modifying the epileptogenic process. Target identification for antiepileptogenic treatments is difficult in humans because patients undergoing epileptogenesis cannot currently be identified. Animal models of epileptogenesis are therefore necessary for scientific progress. Recent advances in the development of experimental models of epileptogenesis have provided tools to investigate the molecular and cellular alterations and their temporal appearance, as well as the epilepsy phenotype after various clinically relevant epileptogenic etiologies, including TBI and stroke. Studying these models will lead to answers to critical questions such as: Do the molecular mechanisms of epileptogenesis depend on the etiology? Is the spectrum of network alterations during epileptogenesis the same after various clinically relevant etiologies? Is the temporal progression of epileptogenesis similar? Work is ongoing, and answers to these questions will facilitate the identification of molecular targets for antiepileptogenic treatments, the design of treatment paradigms, and the determination of whether data from one etiology can be extrapolated to another.     

Do proconvulsants modify or halt epileptogenesis? Pentylenetetrazole is ineffective in two rat models of temporal lobe epilepsy

In patients at risk of developing epilepsy after an initial precipitating injury to the brain, the epileptogenic latent period may offer a window of opportunity for initiating potential antiepileptogenic therapy in an attempt to prevent epilepsy from developing. One potential target for antiepileptogenesis is the development of neuronal hyperexcitability during the latent period. Surprisingly, some recent studies in models of temporal lobe epilepsy (TLE) have suggested that proconvulsant drugs could have favourable effects on epileptogenesis, resulting in the proposal of pursuing proconvulsant prophylaxis for epileptogenesis. In the present study, we evaluated this provocative hypothesis by experiments with the GABA(A) receptor antagonist pentylenetetrazole (PTZ) in two TLE models, the intrahippocampal kainate model and the lithium-pilocarpine model in rats. First, we repeatedly determined the PTZ seizure threshold by i.v. infusion of the convulsant during the latent period following intrahippocampal kainate. In line with recent experiments in the lithium-pilocarpine model, the PTZ seizure threshold was significantly decreased over several days following status epilepticus. We then studied whether prolonged infusion of a proconvulsant dose of PTZ at different times after kainate or pilocarpine affected the development of epilepsy. PTZ did not prevent the development of spontaneous recurrent seizures and did not decrease their frequency or severity, but exerted only a moderate disease-modifying effect in that spontaneous seizures in the kainate model were significantly shortened. These data indicate that administration of proconvulsant drugs such as PTZ during the latent period following SE is not a promising strategy for preventing epilepsy.     

Innate and adaptive immunity during epileptogenesis and spontaneous seizures: evidence from experimental models and human temporal lobe epilepsy

We investigated the activation of the IL-1 beta system and markers of adaptive immunity in rat brain during epileptogenesis using models of temporal lobe epilepsy (TLE). The same inflammatory markers were studied in rat chronic epileptic tissue and in human TLE with hippocampal sclerosis (HS). IL-1 beta was expressed by both activated microglia and astrocytes within 4 h from the onset of status epilepticus (SE) in forebrain areas recruited in epileptic activity; however, only astrocytes sustained inflammation during epileptogenesis. Activation of the IL-1 beta system during epileptogenesis was associated with neurodegeneration and blood-brain barrier breakdown. In rat and human chronic epileptic tissue, IL-1 beta and IL-1 receptor type 1 were broadly expressed by astrocytes, microglia and neurons. Granulocytes appeared transiently in rat brain during epileptogenesis while monocytes/macrophages were present in the hippocampus from 18 h after SE onset until chronic seizures develop, and they were found also in human TLE hippocampi. In rat and human epileptic tissue, only scarce B- and T-lymphocytes and NK cells were found mainly associated with microvessels. These data show that specific inflammatory pathways are chronically activated during epileptogenesis and they persist in chronic epileptic tissue, suggesting they may contribute to the etiopathogenesis of TLE.     

Refinement of a model of acquired epilepsy for identification and validation of biomarkers of epileptogenesis in rats

In rodent models in which status epilepticus (SE) is used to induce epilepsy, typically most animals develop spontaneous recurrent seizures (SRS). The SE duration for induction of epileptogenesis depends on the type of SE induction. In models with electrical SE induction, the minimum duration of SE to induce epileptogenesis in > 90% of animals ranges from 3–4 h. A high incidence of epilepsy is an advantage in the search of antiepileptogenic treatments, whereas it is a disadvantage in the search for biomarkers of epileptogenesis, because it does not allow a comparison of potential biomarkers in animals that either develop or do not develop epilepsy. The aim of this project was the refinement of an established SE rat model so that only ~ 50% of the animals develop epilepsy. For this purpose, we used an electrical model of SE induction, in which a self-sustained SE develops after prolonged stimulation of the basolateral amygdala. Previous experiments had shown that the majority of rats develop SRS after 4-h SE in this model so that the SE reduced duration to 2.5 h by administering diazepam. This resulted in epilepsy development in only 50% of rats, thus reaching the goal of the project. The latent period to onset of SRS wa s > 2 weeks in most rats. Development of epilepsy could be predicted in most rats by behavioral hyperexcitability, whereas seizure threshold did not differentiate rats that did and did not develop SRS. The refined SE model may offer a platform to identify and validate biomarkers of epileptogenesis.     

Neue Entwicklungen der Epileptogenese und therapeutische Perspektiven

Epileptogenesis describes the mechanisms of how epilepsies are generated. We have chosen four areas in which significant progress has been achieved in understanding epileptogenesis. Those are (1) inflammatory processes which play an increasingly important role for the generation of temporal lobe epilepsy with hippocampal sclerosis (TLE with HS), (2) disturbances of intrinsic properties of neuronal compartments, in particular acquired defects of ion channels of which those in dendrites are described here for TLE with HS, (3) epigenetic effects, which affect for example the methylation of promoters and secondarily can change the expression of specific genes in TLE with HS, and finally (4) the epileptogenesis of idiopathic epilepsies which are caused by inborn genetic alterations affecting mainly ion channels. Apart from aspects of basic research, we will describe clinical consequences and therapeutic perspectives.     

Mammalian Target of Rapamycin (mTOR) Inhibition: Potential for Antiseizure, Antiepileptogenic, and Epileptostatic Therapy

New epilepsy treatments are needed that not only inhibit seizures symptomatically (antiseizure) but also prevent the development of epilepsy (antiepileptogenic). The mammalian target of rapamycin (mTOR) pathway may mediate mechanisms of epileptogenesis and serve as a rational therapeutic target. mTOR inhibitors have antiepileptogenic and antiseizure effects in animal models of the genetic disease, tuberous sclerosis complex. The mTOR pathway is also implicated in epileptogenesis in animal models of acquired epilepsy and infantile spasms, although the effects of mTOR inhibitors are variable depending on the specific conditions and model. Furthermore, beneficial effects on seizures are lost when treatment is withdrawn, suggesting that mTOR inhibitors are "epileptostatic" in only stalling epilepsy progression during treatment. Clinical studies of rapamycin in human epilepsy are limited, but suggest that mTOR inhibitors at least have antiseizure effects in tuberous sclerosis patients. Further studies are needed to assess the full potential of mTOR inhibitors for epilepsy treatment.     

Various modifications of the intrahippocampal kainate model of mesial temporal lobe epilepsy in rats fail to resolve the marked rat-to-mouse differences in type and frequency of spontaneous seizures in this model

Temporal lobe epilepsy (TLE) is the most common type of acquired epilepsy in adults. TLE can develop after diverse brain insults, including traumatic brain injury, infections, stroke, or prolonged status epilepticus (SE). Post-SE rodent models of TLE are widely used to understand mechanisms of epileptogenesis and develop treatments for epilepsy prevention. In this respect, the intrahippocampal kainate model of TLE in mice is of interest, because highly frequent spontaneous electrographic seizures develop in the kainate focus, allowing evaluation of both anti-seizure and anti-epileptogenic effects of novel drugs with only short EEG recording periods, which is not possible in any other model of TLE, including the intrahippocampal kainate model in rats. In the present study, we investigated whether the marked mouse-to-rat difference in occurrence and frequency of spontaneous seizures is due to a species difference or to technical variables, such as anesthesia during kainate injection, kainate dose, or location of kainate injection and EEG electrode in the hippocampus. When, as in the mouse model, anesthesia was used during kainate injection, only few rats developed epilepsy, although severity or duration of SE was not affected by isoflurane. In contrast, most rats developed epilepsy when kainate was injected without anesthesia. However, frequent electrographic seizures as observed in mice did not occur in rats, irrespective of location of kainate injection (CA1, CA3) or EEG recording electrode (CA1, CA3, dentate gyrus) or dose of kainate injected. These data indicate marked phenotypic differences between mice and rats in this model. Further studies should explore the mechanisms underlying this species difference.     

cDNA profiling of epileptogenesis in the rat brain

Symptomatic temporal lobe epilepsy typically develops in three phases: brain insult --> latency period (epileptogenesis) --> recurrent seizures (epilepsy). We hypothesized that remodeling of neuronal circuits underlying epilepsy is associated with altered gene expression during epileptogenesis. Epileptogenesis was induced by electrically triggered status epilepticus (SE) in rats. Animals were continuously monitored with video-EEG, and the hippocampus and temporal lobe were collected either during epileptogenesis (1, 4 and 14 days) or after the first spontaneous seizures (14 days) for cDNA array analysis. Altogether, 282 genes had altered expression, from which 87 were in the hippocampus and 208 in the temporal lobe (overlap in 13). Assessment of hippocampal gene expression during epileptogenesis indicated that 37 genes were altered in the 1-day group, 12 in the 4-day group and 14 in the 14-day epileptogenesis group. There were 42 genes with altered expression in the 14-day epilepsy group. In the temporal lobe, the number of genes with altered expression was 29 in the 1-day group, 155 in the 4-day group, 32 in the 14-day epileptogenesis group and 62 in the 14-day epilepsy group. Products of the altered genes are involved in neuronal plasticity, gliosis, organization of the cytoskeleton or extracellular matrix, cell adhesion, signal transduction, regulation of cell cycle, and metabolism. As most of these genes have not previously been implicated in epileptogenesis or epilepsy, these data open new avenues for understanding the molecular basis of epileptogenesis and provide new targets for rational development of anti-epileptogenic treatments for patients with an elevated risk of epileptogenesis after brain injury.     

Prolonged increase in rat hippocampal chemokine signalling after status epilepticus

Temporal lobe epilepsy (TLE) is one of the most common focal epilepsy syndromes. In a genome-wide expression study of the human TLE hippocampus we previously showed up-regulation of genes involved in chemokine signalling. Here we investigate in the rat pilocarpine model for TLE, whether changes in chemokine signalling occur during epileptogenesis and are persistent. Therefore we analysed hippocampal protein expression and cellular localisation of CCL2, CCL4, CCR1 and CCR5 after status epilepticus. We found increased CCL4 (but not CCL2) expression in specific populations of hilar astrocytes at 2 and 19 weeks after SE concomitant with a persistent up-regulation of its receptor CCR5. Our results show an early and persistent up-regulation of CCL4/CCR5 signalling during epileptogenesis and suggest that CCL4 signalling, rather than CCL2 signalling, could have a role in the epileptogenic process.     

Ketogenic diet exhibits neuroprotective effects in hippocampus but fails to prevent epileptogenesis in the lithium‐pilocarpine model of mesial temporal lobe epilepsy in adult rats

Purpose: Although the number of antiepileptic drugs (AEDs) is increasing, none displays neuroprotective or antiepileptogenic properties that could prevent status epilepticus (SE)–induced drug‐resistant epilepsy. Ketogenic diet (KD) and calorie restriction (CR) are proposed as alternative treatments in epilepsy. Our goal was to assess the neuroprotective or antiepileptogenic effect of these diets in a well‐characterized model of mesial temporal lobe epilepsy following initial SE induced by lithium‐pilocarpine in adult rats. Methods: Seventy‐five P50 male Wistar rats were fed a specific diet: normocalorie carbohydrate (NC), hypocalorie carbohydrate (HC), normocalorie ketogenic (NK), or hypocalorie ketogenic (HK). Rats were subjected to lithium‐pilocarpine SE, except six NC to constitute a control group for histology (C). Four rats per group were implanted with epidural electrodes to record electroencephalography (EEG) during SE and the next six following days. From the seventh day, the animals were video‐recorded 10 h daily to determine latency to epilepsy onset. Neuronal loss in hippocampus and parahippocampal cortices was analyzed 1 month after the first spontaneous seizure. Results: After lithium‐pilocarpine injection, neither KD nor CR modified SE features or latency to epilepsy. In hippocampal layers, KD or CR exhibited a neuroprotective potential without cooperative effect. Parahippocampal cortices were not protected by the diets. Conclusion: The antiepileptic effect of KD and/or CR is overwhelmed by lithium‐pilocarpine injection. The isolated protection of hippocampal layers induced by KD or CR or their association failed to modify the course of epileptogenesis.     

Pathology and pathophysiology of the amygdala in epileptogenesis and epilepsy

Acute brain insults, such as traumatic brain injury, status epilepticus, or stroke are common etiologies for the development of epilepsy, including temporal lobe epilepsy (TLE), which is often refractory to drug therapy. The mechanisms by which a brain injury can lead to epilepsy are poorly understood. It is well recognized that excessive glutamatergic activity plays a major role in the initial pathological and pathophysiological damage. This initial damage is followed by a latent period, during which there is no seizure activity, yet a number of pathophysiological and structural alterations are taking place in key brain regions, that culminate in the expression of epilepsy. The process by which affected/injured neurons that have survived the acute insult, along with well-preserved neurons are progressively forming hyperexcitable, epileptic neuronal networks has been termed epileptogenesis. Understanding the mechanisms of epileptogenesis is crucial for the development of therapeutic interventions that will prevent the manifestation of epilepsy after a brain injury, or reduce its severity. The amygdala, a temporal lobe structure that is most well known for its central role in emotional behavior, also plays a key role in epileptogenesis and epilepsy. In this article, we review the current knowledge on the pathology of the amygdala associated with epileptogenesis and/or epilepsy in TLE patients, and in animal models of TLE. In addition, because a derangement in the balance between glutamatergic and GABAergic synaptic transmission is a salient feature of hyperexcitable, epileptic neuronal circuits, we also review the information available on the role of the glutamatergic and GABAergic systems in epileptogenesis and epilepsy in the amygdala.     

Behavioral and histological assessment of the effect of intermittent feeding in the pilocarpine model of temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is the most resistant type of epilepsy. Currently available drugs for epilepsy are not antiepileptogenic. A novel treatment for epilepsy would be to block or reverse the process of epileptogenesis. We used intermittent feeding (IF) regimen of the dietary restriction (DR) to study its effect on epileptogenesis and neuroprotection in the pilocarpine model of TLE in rats. The effect of IF regimen on the induction of status epilepticus (SE), the duration of latent period, and the frequency, duration, severity and the time of occurrence of Spontaneous Recurrent Seizures (SRS) were investigated. We also studied the effect of IF regimen on hippocampal neurons against the excitotoxic damage of prolonged SE (about 4 h) induced by pilocarpine. The animals (Wistar, male, 200–250 g) were divided into four main groups: AL–AL (ad libitum diet throughout), AL–IF (PfS) [IF post-first seizure], AL–IF (PSE) [IF post-SE] and IF–IF (IF diet throughout), and two AL and IF control groups. SE was induced by pilocarpine (350 mg/kg, i.p.) and with diazepam (6 mg/kg, i.p.) injected after 3 h, the behavioral signs of SE terminated at about 4 h (AL animals, n = 29, 260.43 ± 8.74 min; IF animals, n = 19, 224.32 ± 20.73 min). Behavioral monitoring was carried out by 24 h video recording for 3 weeks after the first SRS. Rat brains were then prepared for histological study with Nissl stain and cell counting was done in CA1, CA2 and CA3 regions of the hippocampus. The results show that the animals on IF diet had significantly less SE induction and significantly longer duration of latent period (the period of epileptogenesis) was seen in IF–IF group compared to the AL–AL group. The severity of SRS was significantly more in AL–IF (PfS) compared to the AL–IF (PSE) group. These results indicate that IF diet can make rats resistant to the induction of SE and can prolong the process of epileptogenesis. The results of the histological study show that the number of pyramidal neurons was statistically less in CA1, CA2 and CA3 of the hippocampus in the experimental groups compared to the control groups. However, IF regimen could not protect the hippocampal neurons against the excitotoxic injury caused by a prolonged SE. We conclude that IF regimen can significantly influence various behavioral characteristics of pilocarpine model of TLE. Further studies can elaborate the exact mechanisms as well as its possible role in the treatment of human TLE.     

Mammalian target of rapamycin (mTOR) inhibition as a potential antiepileptogenic therapy: From tuberous sclerosis to common acquired epilepsies

Most current treatments for epilepsy are symptomatic therapies that suppress seizures but do not affect the underlying course or prognosis of epilepsy. The need for disease-modifying or "antiepileptogenic" treatments for epilepsy is widely recognized, but no such preventive therapies have yet been established for clinical use. A rational strategy for preventing epilepsy is to target primary signaling pathways that initially trigger the numerous downstream mechanisms mediating epileptogenesis. The mammalian target of rapamycin (mTOR) pathway represents a logical candidate, because mTOR regulates multiple cellular functions that may contribute to epileptogenesis, including protein synthesis, cell growth and proliferation, and synaptic plasticity. The importance of the mTOR pathway in epileptogenesis is best illustrated by tuberous sclerosis complex (TSC), one of the most common genetic causes of epilepsy. In mouse models of TSC, mTOR inhibitors prevent the development of epilepsy and underlying brain abnormalities associated with epileptogenesis. Accumulating evidence suggests that mTOR also participates in epileptogenesis due to a variety of other causes, including focal cortical dysplasia and acquired brain injuries, such as in animal models following status epilepticus or traumatic brain injury. Therefore, mTOR inhibition may represent a potential antiepileptogenic therapy for diverse types of epilepsy, including both genetic and acquired epilepsies.     

Efficacy of current antiepileptics to prevent neurodegeneration in epilepsy models

Results of experiments performed in animal epilepsy models and human epilepsy during the past decade indicate that the epileptic brain is not a stable neuronal network, but undergoes modifications caused by the underlying etiology and/or recurrent seizures. In many forms of epilepsy, such as temporal lobe epilepsy, the underlying etiologic factor triggers a cascade of events (epileptogenesis) leading to spontaneous seizures and cognitive decline. In some patients, the condition progresses, due in part to recurrent seizures. The current treatment of epilepsy focuses exclusively on preventing or suppressing seizures, which are symptoms of the underlying disease. Now, however, we are beginning to understand the underlying neurobiology of the epileptic process, as well as factors that might predict the risk of progression in individual patients. Thus, there are new opportunities to develop neuroprotective and antiepileptogenic treatments for patients who, if untreated, would develop drug-refractory epilepsy associated with cognitive decline. These treatments might improve the long-term outcome and quality-of-life of patients with epilepsy. Here we review the available data regarding the neuroprotective effects of antiepileptic drugs (AEDs) at different phases of the epileptic process. Analysis of published data suggests that initial-insult modification and prevention of the progression of seizure-induced damage are candidate indications for treatment with AEDs. An understanding of the molecular mechanisms underlying the progression of epileptic process will eventually show what role AEDs have in the neuroprotective and antiepileptogenic treatment regimen.     

Effect of lamotrigine treatment on epileptogenesis: an experimental study in rat

Prevention of epileptogenesis in patients with acute brain damaging insults like status epilepticus (SE) is a major challenge. We investigated whether lamotrigine (LTG) treatment started during SE is antiepileptogenic or disease-modifying. To mimic a clinical study design, LTG treatment (20 mg/kg) was started 2 h after the beginning of electrically induced SE in 14 rats and continued for 11 weeks (20 mg/kg per day for 2 weeks followed by 10 mg/kg per day for 9 weeks). One group of rats (n = 14) was treated with vehicle. Nine non-stimulated rats with vehicle treatment served as controls. Outcome measures were occurrence of epilepsy, severity of epilepsy, and histology (neuronal loss, mossy fiber sprouting). Clinical occurrence of seizures was assessed with 1-week continuous video-electroencephalography monitoring during the 11th (i.e. during treatment) and 14th week (i.e. after drug wash-out) after SE. LTG reduced the number of electrographic seizures during SE to 43% of that in the vehicle group (P < 0.05). In the vehicle group, 93% (13/14), and in the LTG group, 100% (14/14) of the animals, developed epilepsy. In both groups, 64% of the rats had severe epilepsy (seizure frequency >1 per day). The mean frequency of spontaneous seizures, seizure duration, or behavioral severity of seizures did not differ between groups. The severity of hippocampal neuronal damage and density of mossy fiber sprouting were similar. In LTG-treated rats with severe epilepsy, however, the duration of seizures was shorter (34 versus 54s, P < 0.05) and the behavioral seizure score was milder (1.4 versus 3.4, P < 0.05) during LTG treatment than after drug wash-out. LTG treatment started during SE and continued for 11 weeks was not antiepileptogenic but did not worsen the outcome. These data, together with earlier studies of other antiepileptic drugs, suggest that strategies other than Na(+)-channel blockade should be explored to modulate the molecular cascades leading to epileptogenesis after SE.