Contribution of protease-activated receptor 1 in status epilepticus-induced epileptogenesis.

Clinical observations and studies on different animal models of acquired epilepsy consistently demonstrate that blood-brain barrier (BBB) leakage can be an important risk factor for developing recurrent seizures. However, the involved signaling pathways remain largely unclear. Given the important role of thrombin and its major receptor in the brain, protease-activated receptor 1 (PAR1), in the pathophysiology of neurological injury, we hypothesized that PAR1 may contribute to status epilepticus (SE)-induced epileptogenesis and that its inhibition shortly after SE will have neuroprotective and antiepileptogenic effects. Adult rats subjected to lithium-pilocarpine SE were administrated with SCH79797 (a PAR1 selective antagonist) after SE termination. Thrombin and PAR1 levels and neuronal cell survival were evaluated 48h following SE. The effect of PAR1 inhibition on animal survival, interictal spikes (IIS) and electrographic seizures during the first two weeks after SE and behavioral seizures during the chronic period was evaluated. SE resulted in a high mortality rate and incidence of IIS and seizures in the surviving animals. There was a marked increase in thrombin, decrease in PAR1 immunoreactivity and hippocampal cell loss in the SE-treated rats. Inhibition of PAR1 following SE resulted in a decrease in mortality and morbidity, increase in neuronal cell survival in the hippocampus and suppression of IIS, electrographic and behavioral seizures following SE. These data suggest that the PAR1 signaling pathway contributes to epileptogenesis following SE. Because breakdown of the BBB occurs frequently in brain injuries, PAR1 inhibition may have beneficial effects in a variety of acquired injuries leading to epilepsy.

Cognitive impairment in temporal lobe epilepsy: role of online and offline processing of single cell information.

Cognitive impairment is a common comorbidity in temporal lobe epilepsy (TLE) and is often considered more detrimental to quality of life than seizures. While it has been previously shown that the encoding of memory during behavior is impaired in the pilocarpine model of TLE in rats, how this information is consolidated during the subsequent sleep period remains unknown. In this study, we first report marked deficits in spatial memory performance and severe cell loss in the CA1 layer of the hippocampus lower spatial coherence of firing in TLE rats. We then present the first evidence that the reactivation of behavior-driven patterns of activity of CA1 place cells in the hippocampus is intact in TLE rats. Using a template-matching method, we discovered that real-time (3-5 s) reactivation structure was intact in TLE rats. Furthermore, we estimated the entropy rate of short time scale (∼250 ms) bursting activity using block entropies and found that significant, extended temporal correlations exist in both TLE and control rats. Fitting a first-order Markov Chain model to these bursting time series, we found that long sequences derived from behavior were significantly enriched in the Markov model over corresponding models fit on randomized data confirming the presence of replay in shorter time scales. We propose that the persistent consolidation of poor spatial information in both real time and during bursting activity may contribute to memory impairments in TLE rats.

Altered short-term plasticity in the prefrontal cortex after early life seizures.

Seizures during development are a relatively common occurrence and are often associated with poor cognitive outcomes. Recent studies show that early life seizures alter the function of various brain structures and have long-term consequences on seizure susceptibility and behavioral regulation. While many neocortical functions could be disrupted by epileptic seizures, we have concentrated on studying the prefrontal cortex (PFC) as disturbance of PFC functions is involved in numerous co-morbid disorders associated with epilepsy. In the present work we report an alteration of short-term plasticity in the PFC in rats that have experienced early life seizures. The most robust alteration occurs in the layer II/III to layer V network of neurons. However short-term plasticity of layer V to layer V network was also affected, indicating that the PFC function is broadly influenced by early life seizures. These data strongly suggest that repetitive seizures early in development cause substantial alteration in PFC function, which may be an important component underlying cognitive deficits in individuals with a history of seizures during development.

[Relevance of basic research to clinical data: Good answers, wrong questions!]. / La recherche fondamentale est-elle utile à la clinique? Bonnes réponses, mauvaises questions !

What conclusions can be derived from experimental data on human epilepsies? This review discusses these issues, notably concerning human temporal lobe epilepsies (TLEs) and infantile epilepsies, where important advances have been achieved in both theory and the comprehension of epileptogenic mechanisms. A wide spectrum of human and animal data converge to show that the naive network transforms to one that generates seizures spontaneously. Thus, in TLE, experimental and human data suggest that the inaugurating status generates a sequence of events that lead to the sprouting of fibers and the formation of novel excitatory synapses. This reactive plasticity constitutes a basis for the generation of novel seizures by the epileptic network. Similarly, in vitro studies indicate that in immature hippocampal formation, the propagation of high- but not low-frequency seizures can transform a naive network into one that generates further seizures, thereby, giving an indication as to the types of seizure that are epileptogenic. In conclusion, it is suggested that although animal data cannot mimic human seizures in all their complex and variable etiologies, it provides essential indications on the mechanisms that enable seizure generation.

Antiepileptic drug treatment strategies in neonatal epilepsy.

The highest risk of seizures across the lifespan is in the neonatal period. The enhanced excitability of the immature brain compared to the mature brain is related to the sequential development and expression of essential neurotransmitter signaling pathways. During the neonatal period there is an overabundance of excitatory receptors, and γ-amino-butyric acid (GABA) is potentially depolarizing, as opposed to hyperpolarizing in the older brain. While this enhanced excitability is required for regulation of activity-dependent synapse formation and refining of synaptic connections that are necessary for normal brain development, enhanced excitability predisposes the immature brain to seizures. In addition to being common, neonatal seizures are very difficult to treat; antiepileptic drugs used in older children and adults are less efficacious, and possibly detrimental to brain development. In an effort to target the unique features of neurotransmission in the neonate, bumetanide, an NKCC1 inhibitor which reduces intraneuronal Cl(-) and induces a significant shift of EGABA toward more hyperpolarized values in vitro, has been used to treat neonatal seizures. As the understanding of the pathophysiology of genetic forms of neonatal epilepsy has evolved there have been a few successful attempts to pharmacologically target the mutated protein. This approach, while promising, is challenging due to the findings that the genetic syndromes presenting in infancy demonstrate genetic heterogeneity in regard to both the mutated gene and its function.

Protective effects of prenatal choline supplementation on seizure-induced memory impairment.

Choline is an essential nutrient for rats and humans, and its availability during fetal development has long-lasting cognitive effects (Blusztajn, 1998). We investigated the effects of prenatal choline supplementation on memory deficits associated with status epilepticus. Pregnant rats received a control or choline-supplemented diet during days 11-17 of gestation. Male offspring [postnatal day 29 (P29)-32] were tested for their ability to find a platform in a water maze before and after administration of a convulsant dose of pilocarpine at P34. There were no differences between groups in water maze performance before the seizure. One week after status epilepticus (P41-P44), animals that had received the control diet prenatally had a drastically impaired performance in the water maze during the 4 d testing period, whereas prenatally choline-supplemented rats showed no impairment. Neither the seizures nor the prenatal availability of choline had any effect on hippocampal choline acetyltransferase or acetylcholinesterase activities. This study demonstrates that prenatal choline supplementation can protect rats against memory deficits induced by status epilepticus.

Reduced neurogenesis after neonatal seizures.

Although neonatal seizures are quite common, there is controversy regarding their consequences. Despite considerable evidence that seizures may cause less cell loss in young animals compared with mature animals, there are nonetheless clear indications that seizures may have other potentially deleterious effects. Because it is known that seizures in the mature brain can increase neurogenesis in the hippocampus, we studied the extent of neurogenesis in the granule cell layer of the dentate gyrus over multiple time points after a series of 25 flurothyl-induced seizures administered between postnatal day 0 (P0) and P4. Rats with neonatal seizures had a significant reduction in the number of the thymidine analog 5-bromo-2'-deoxyuridine-5'-monophosphate- (BrdU) labeled cells in the dentate gyrus and hilus compared with the control groups when the animals were killed either 36 hr or 2 weeks after the BrdU injections. The reduction in BrdU-labeled cells continued for 6 d after the last seizure. BrdU-labeled cells primarily colocalized with the neuronal marker neuron-specific nuclear protein and rarely colocalized with the glial cell marker glial fibrillary acidic protein, providing evidence that a very large percentage of the newly formed cells were neurons. Immature rats subjected to a single seizure did not differ from controls in number of BrdU-labeled cells. In comparison, adult rats undergoing a series of 25 flurothyl-induced seizures had a significant increase in neurogenesis compared with controls. This study indicates that, after recurrent seizures in the neonatal rat, there is a reduction in newly born granule cells.

Mossy fiber sprouting after recurrent seizures during early development in rats.

In some children, epilepsy is a catastrophic condition, leading to significant intellectual and behavioral impairment, but little is known about the consequences of recurrent seizures during development. In the present study, we evaluated the effects of 15 daily pentylenetetrazol-induced convulsions in immature rats beginning at postnatal day (P) 1, 10, or 60. In addition, we subjected another group of P10 rats to twice daily seizures for 15 days. Both supragranular and terminal sprouting in the CA3 hippocampal subfield was assessed in Timm-stained sections by using a rating scale and density measurements. Prominent sprouting was seen in the CA3 stratum pyramidale layer in all rats having 15 daily seizures, regardless of the age when seizures began. Based on Timm staining in control P10, P20, and P30 rats, the terminal sprouting in CA3 appears to be new growth of axons and synapses as opposed to a failure of normal regression of synapses. In addition to CA3 terminal sprouting, rats having twice daily seizures had sprouting noted in the dentate supragranular layer, predominately in the inferior blade of the dentate, and had a decreased seizure threshold when compared with controls. Cell counting of dentate granule cells, CA3, CA1, and hilar neurons, with unbiased stereological methods demonstrated no differences from controls in rats with daily seizures beginning at P1 or P10, whereas adult rats with daily seizures had a significant decrease in CA1 neurons. Rats that received twice daily seizures on P10-P25 had an increase in dentate granule cells. This study demonstrates that, like the mature brain, immature animals have neuronal reorganization after recurrent seizures, with mossy fiber sprouting in both the CA3 subfield and supragranular region. In the immature brain, repetitive seizures also result in granule cell neurogenesis without loss of principal neurons. Although the relationship between these morphological changes after seizures during development and subsequent cognitive impairment is not yet clear, our findings indicate that during development recurrent seizures can result in significant alterations in cell number and axonal growth.

Effect of a single brief seizure on subsequent seizure susceptibility in the immature rat.

To determine whether a single seizure permanently affects the brain's susceptibility to further seizures, 27-day-old rats were subjected to a single seizure induced by either an electroshock or the administration of pentylenetetrazol. Three days following the seizure, the rats, along with age- and weight-matched control rats, underwent kindling. The rate of kindling did not differ between the control rats and those rats that had previously experienced a single electroshock or pentylenetetrazol-induced seizure. At the present time, there is no conclusive evidence that a single brief seizure results in permanently increased susceptibility to future convulsions.

A multicenter study of the efficacy of the ketogenic diet.

OBJECTIVE: To determine the efficacy of the ketogenic diet in multiple centers. DESIGN: A prospective study of the change in frequency of seizures in 51 children with intractable seizures who were treated with the ketogenic diet. SETTING: Patients were enrolled from the clinical practices of 7 sites. The diet was initiated in-hospital and the patients were followed up for at least 6 months. PATIENTS: Fifty-one children, aged 1 to 8 years, with more than 10 seizures per week, whose electroencephalogram showed generalized epileptiform abnormalities or multifocal spikes, and who had failed results when taking at least 2 appropriate anti-epileptic drugs. INTERVENTION: The children were hospitalized, fasted, and a 4:1 ketogenic diet was initiated and maintained. MAIN OUTCOME MEASURES: Frequency of seizures was documented from parental calendars and efficacy was compared with prediet baseline after 3, 6, and 12 months. The children were categorized as free of seizures, greater than 90% reduction, 50% to 90% reduction, or lower than 50% reduction in frequency of seizures. RESULTS: Eighty-eight percent of all children initiating the diet remained on it at 3 months, 69% remained on it at 6 months, and 47% remained on it at 1 year. Three months after initiating the diet, frequency of seizures was decreased to greater than 50% in 54%. At 6 months, 28 (55%) of the 51 initiating the diet had at least a 50% decrease from baseline, and at 1 year, 40% of those starting the diet had a greater than 50% decrease in seizures. Five patients (10%) were free of seizures at 1 year. Age, sex, principal seizure type, and electroencephalogram were not statistically related to outcome. CONCLUSION: The ketogenic diet is effective in substantially decreasing difficult-to-control seizures and can successfully be administered in a wide variety of settings.

Surgery for intractable seizures in infancy and early childhood.

Epilepsy surgery is becoming an increasingly used therapy for infants and young children with severe, medically intractable seizures. As in older children and adults, the presurgical evaluations of possible candidates typically consist of a detailed history, neurologic and neuropsychologic examination, and anatomic and functional neuroimaging. The "gold standard" test, however, is the recording of ictal events using simultaneous EEG and video monitoring. While temporal lobe resection is the most commonly performed surgery in older children and adults, nontemporal lobe resections, corpus callosotomies, and hemispherectomies are more commonly performed in younger children. Antiepileptic drugs remain the mainstay of treatment of children with epilepsy. However, the clinician should consider surgery early in the course of the catastrophic seizure disorders of childhood: infantile spasms, Sturge-Weber syndrome, and Rasmussen's encephalitis.

Effects of neonatal seizures on subsequent seizure-induced brain injury.

BACKGROUND: Although seizures are very common in neonates and are often the harbinger of poor neurologic outcome, there is controversy regarding the degree of brain damage induced by seizures during early development. Here, we evaluated the effect of neonatal seizures on subsequent brain injury induced by status epilepticus. METHODS: Twenty-five seizures were induced by the inhalant flurothyl in neonatal rats during the first 5 days of life. Flurothyl reliably produced generalized seizures with concomitant electroencephalographic changes and a low mortality rate. During adolescence or early adulthood, animals were subjected to status epilepticus using either kainic acid or perforant path stimulation. RESULTS: Although flurothyl-induced neonatal seizures did not cause cell death, animals that had neonatal seizures had significantly more severe brain injury after both kainic acid and perforant path stimulation than did animals without a history of neonatal seizures. CONCLUSIONS: Neonatal seizures increase the susceptibility of the developing brain to subsequent seizure-induced injury.

Beneficial effects of enriched environment following status epilepticus in immature rats.

BACKGROUND: There is increasing evidence that enriching the environment can improve cognitive and motor deficits following a variety of brain injuries. Whether environmental enrichment can improve cognitive impairment following status epilepticus (SE) is not known. OBJECTIVE: To determine whether the environment in which animals are raised influences cognitive function in normal rats and rats subjected to SE. METHODS: Rats (n = 100) underwent lithium-pilocarpine-induced SE at postnatal (P) day 20 and were then placed in either an enriched environment consisting of a large play area with toys, climbing objects, and music, or in standard vivarium cages for 30 days. Control rats (n = 32) were handled similarly to the SE rats but received saline injections instead of lithium-pilocarpine. Rats were then tested in the water maze, a measure of visual-spatial memory. A subset of the rats were killed during exposure to the enriched or nonenriched environment and the brains examined for dentate granule cell neurogenesis using bromodeoxyuridine (BrdU) and phosphorylated cyclic AMP response element binding protein (pCREB) immunostaining, a brain transcription factor important in long-term memory. RESULTS: Both control and SE rats exposed to the enriched environment performed significantly better than the nonenriched group in the water maze. There was a significant increase in neurogenesis and pCREB immunostaining in the dentate gyrus in both control and SE animals exposed to the enriched environment compared to the nonenriched groups. Environmental enrichment resulted in no change in SE-induced histologic damage. CONCLUSIONS: Exposure to an enriched environment in weanling rats significantly improves visual-spatial learning. Even following SE, an enriched environment enhances cognitive function. An increase in neurogenesis and activation of transcription factors may contribute to this enhanced visual-spatial memory.

Comparison of valproate and phenobarbital treatment after status epilepticus in rats.

OBJECTIVE: To investigate the long-term effects of two widely used antiepileptic medications, valproate and phenobarbital, on learning and behavior in the kainic acid (KA) model of epilepsy. BACKGROUND: Prior clinical and animal studies have demonstrated that phenobarbital administered during development may result in subsequent cognitive impairment. It is unclear whether these adverse effects of phenobarbital extend to other antiepileptic drugs. METHODS: A convulsant dose of KA was administered to rats on postnatal day (P) 35. From P36-75 rats received daily injections of phenobarbital (PH), valproate (VPA), or saline and spontaneous seizure frequency was monitored with video recordings. After tapering of the drugs, the rats were tested in the water maze (a measure of visuospatial memory) and handling test (a measure of emotionality). Brains were then analyzed for histologic lesions. RESULTS: KA caused status epilepticus in all the rats. In the PH- and saline-treated groups, there was impaired learning in the water maze, increased emotionality, recurrent seizures, and histologic lesions in the hippocampal areas CA3, CA1, and dentate hilus. However, VPA-treated rats had no spontaneous seizures, abnormalities in handling, or deficits in visuospatial learning, and had fewer histologic lesions than animals receiving KA alone. CONCLUSIONS: The long-term consequences of AED treatment during development are related to the drug used. VPA treatment after KA-induced status epilepticus prevents many of the neurologic sequelae typically seen after KA.

Effects of serial administration of kainic acid on the developing brain.

The purpose of this study was to determine whether the repeated administration of kainic acid at 24-hr intervals results in pharmacological kindling. Rather than developing an increased intensity of seizures, this method of serial administration of kainic acid resulted in the development of tolerance to its effects.

Anticonvulsant action and long-term effects of gabapentin in the immature brain.

The anticonvulsant action and the long-term effects on learning, memory and behavior of the new generation antiepileptic drug gabapentin (GBP) were investigated in immature animals. Kainic acid (KA) was administered to rats on postnatal day (P) 35. Animals were treated with GBP or saline from P36 to P75 and spontaneous seizure frequency was monitored. After tapering the drug, the rats were tested in the water maze and open field test. Brains were then analyzed for histological lesions. Animals treated with GBP following KA-induced status epilepticus had a reduced incidence of spontaneous recurrent seizures, a better pathology score, and less aggressiveness compared to saline-treated controls. Effectiveness of GBP on seizure threshold was tested using flurothyl inhalation in 10 separate age groups of animals ranging from the newborn period to adulthood. Furthermore, GBP plasma concentration peaks were determined in all age groups. At all ages, GBP pre-treated animals demonstrated a higher seizure threshold. Plasma GBP concentrations did not significantly change with age. These data suggest that acute administration of a single therapeutic dose of GBP increases the seizure threshold at all ages studied, while chronic treatment following the status reduces spontaneous seizure frequency and cell damage and has no long-term adverse consequences on cognitive processes during development.

Basic fibroblast growth factor is highly neuroprotective against seizure-induced long-term behavioural deficits.

Basic fibroblast growth factor has been reported to protect neurons of various structures from excitotoxic damage. To study the effects of basic fibroblast growth factor on seizure-induced brain damage we infused the growth factor into the lateral ventricles of 35-day-old rats receiving convulsant dosages of kainic acid. Artificial cerebrospinal fluid or basic fibroblast growth factor at dosages of 0.5 ng/h or 2.5 ng/h was infused into the lateral ventricle continuously for seven days starting two days before and continuing for five days after the animals had kainic acid-induced status epilepticus. At age 80 days the animals underwent behavioural testing using the water maze, open field, and handling tests and at age 95 days were tested for seizure threshold using flurothyl inhalation. Neither artificial cerebrospinal fluid or basic fibroblast growth factor modified the latency or duration of the acute seizures following kainic acid. However, rats infused with 2.5 ng/h, but not 0.5 ng/h of basic fibroblast growth factor, had fewer spontaneous recurrent seizures, a higher seizure threshold, better performance in the handling, open field and water maze test, and less cell loss in the hippocampus when compared to rats receiving artificial cerebrospinal fluid or 0.5 ng/h of basic fibroblast growth factor. These results show that basic fibroblast growth factor has a dose-related neuroprotective effect against seizure-induced long-term behavioural deficits when administered by osmotic pump prior to seizure onset. This neuroprotective effect is not related to an anticonvulsant effect.

Neuroprotective effects of brain-derived neurotrophic factor in seizures during development.

Although the immature brain is highly susceptible to seizures, it is more resistant to seizure-induced neuronal loss than the adult brain. The developing brain contains high levels of neurotrophins which are involved in growth, differentiation and survival of neurons. To test the hypothesis that neurotrophins may protect the developing brain from seizure-induced neuronal loss, brain-derived neurotrophic factor up-regulation was blocked by intracerebroventricular infusion of an 18mer antisense oligodeoxynucleotide sequence to brain-derived neurotrophic factor in 19-day-old rats using micro-osmotic pumps. Control rats were infused with sense or missense oligodeoxynucleotide. Status epilepticus was induced by intraperitoneal administration of kainic acid 24 h after the start of oligodeoxynucleotide infusion. Seizure duration was significantly increased in the antisense oligodeoxynucleotide plus kainic acid group compared to groups that received kainic acid alone or kainic acid plus sense or missense oligodeoxynucleotide. There was no difference between groups in the latency to forelimb clonus. A twofold increase in brain-derived neurotrophic factor levels was observed in the hippocampus 20 h following kainic acid-induced seizures. This kainic acid-induced increase was absent in animals receiving infusion of antisense oligodeoxynucleotide to brain-derived neurotrophic factor at time of seizure induction. Hippocampi of rats in this group (antisense oligodeoxynucleotide plus kainic acid) showed a loss of CA1 and CA3 pyramidal cells and hilar interneurons. This neuronal loss was not dependent upon seizure duration since animals injected with diazepam to control seizure activity in the antisense plus kainic acid group also showed similar neuronal loss. Administration of kainic acid or infusion of antisense alone did not produce any cell loss in these regions. Induction of seizures at postnatal day 20, in the presence or absence of antisense oligonucleotide, did not produce an impairment in learning and memory when tested 15 days later in the Morris water maze. The hippocampi of these animals did not show any synaptic reorganization as assessed by growth-associated protein-43 immunostaining and Timm staining. Our findings confirm prior studies demonstrating that seizures in the immature brain are associated with little, if any, cell loss. However, when seizure-induced increase in brain-derived neurotrophic factor is blocked, seizures do result in neuronal loss in the developing brain. Thus, brain-derived neurotrophic factor appears to provide protection against kainic acid seizure-induced neuronal damage in the developing brain.

Consequences of recurrent seizures during early brain development.

It is well documented that prolonged seizures (status epilepticus) can cause neuronal injury and result in synaptic reorganization in certain brain regions. However, the effect of recurrent, relatively short seizures in young animals on subsequent brain development is not known. To study the consequences of recurrent seizures on the developing brain, we subjected immature rats to a total of 50 flurothyl-induced seizures from postnatal day 11 until day 23. Immunohistochemistry for c-fos was performed to characterize the pattern of neuronal activation following the seizures. Cell counting of dentate granule cells, CA3, CA1, and hilar neurons, using unbiased stereological methods, and the silver impregnation method were used to evaluate neuronal death following the recurrent seizures. Timm and Golgi staining were performed four weeks after the 50th seizure to evaluate the effects of recurrent seizures on synaptic organization. Our results show that recurrent flurothyl-induced seizures progressively increased excitability of the brain, as revealed by a dramatic increase in the extent and intensity of c-fos immunostaining. While no cell loss was detected in the hippocampus with either Cresyl Violet or silver stains, animals experiencing multiple daily seizures developed increased mossy fiber sprouting in both the supragranular layer of the dentate gyrus and the infrapyramidale layer of the CA3 region. Golgi staining confirmed that there was an increase in mossy fibers in the pyramidal cell layer. Our results suggest that serial recurrent seizures in the immature brain can lead to significant changes in mossy fiber distribution even though the seizures do not cause significant hippocampal cell loss.

Effect of kainate-induced seizures on tissue trace element concentrations in the rat.

It has been shown that epileptics have lower mean blood concentration of manganese than do controls but the cause of this abnormality has not been determined. In order to investigate the effects of seizures on manganese distribution in the body, rats were treated with kainic acid to produce spontaneous seizures which were quantitated for number and severity. Manganese, zinc, copper and iron concentrations were determined in blood, brain, liver, heart and kidney. Kainate-treated animals ate more food but gained less weight than controls. Liver and kidney manganese concentrations were significantly higher in kainate-treated animals than in controls. Blood manganese concentration showed a significant negative correlation with seizure index while heart manganese concentration showed a significant positive correlation with seizure index. None of the other trace elements showed a significant correlation between trace element concentration and seizure index in any of the tissues, although iron concentration was lower in brain and copper concentration was lower in kidney of kainate-treated animals than in their appropriate controls. These data show that manganese concentrations are generally elevated in tissues of kainate-treated animals. This increased manganese concentration may be related to the increased energy demand of these animals.