Transformation of epilepsy treatment model: anti-epileptogenesis and disease-modifying therapy / 中华神经科杂志

Most anti-seizure medications do not change the course of epilepsy and are basically "symptomatic treatment". Even if a variety of new anti-seizure medications continue to come out, there are still more than 30% of patients develop drug-resistant epilepsy. Therefore, investigating new therapeutic targets and developing effective drugs to prevent or reverse the onset and progression of epilepsy are important goals of clinical and preclinical researches. Based on the current studies, to realize the transformation from anti-seizure to anti-epileptogenesis and disease-modifying therapy, it not only needs standardized animal models and biomarkers that can predict the epileptogenesis or progression but also needs sufficient patients, rigorous design schemes, and cutting-edge analysis methods to successfully transform preclinical research into clinical practice. There is no doubt that in the future, targeting various nerve injury pathways to achieve anti-epileptogenesis and disease-modifying therapy probably becomes a truly effective means of treating and preventing epilepsy.

Modeling of post-traumatic epilepsy and experimental research aimed at its prevention

Research on the prevention of post-traumatic epilepsy (PTE) has seen remarkable advances regarding its physiopathology in recent years. From the search for biomarkers that might be used to indicate individual susceptibility to the development of new animal models and the investigation of new drugs, a great deal of knowledge has been amassed. Various groups have concentrated efforts in generating new animal models of traumatic brain injury (TBI) in an attempt to provide the means to further produce knowledge on the subject. Here we forward the hypothesis that restricting the search of biomarkers and of new drugs to prevent PTE by using only a limited set of TBI models might hamper the understanding of this relevant and yet not preventable medical condition.

Role of Pre-Synaptic NMDA Receptors in the Modulation of Inhibitory Synaptic Transmission in Sensory-Motor and Visual Cortical Pyramidal Neurons in Brain Slices of Young Epileptic Mice

Background: Previous studies from animal models have shown that pre-synapticNMDA receptors (preNMDARs) are present in the cortex, but the role of inhibition mediated bypreNMDARs during epileptogenesis remains unclear. In this study, we wanted to observe thechanges in GABAergic inhibition through preNMDARs in sensory-motor and visual corticalpyramidal neurons after pilocarpine-induced status epilepticus.Methods: Using a pilocarpine-induced epileptic mouse model, sensory-motor and visualcortical slices were prepared, and the whole-cell patch clamp technique was used to recordspontaneous inhibitory post-synaptic currents (sIPSCs).Results: The primary finding was that the mean amplitude of sIPSC from the sensorymotorcortex increased significantly in epileptic mice when the recording pipette contained MK-801 compared to control mice, whereas the mean sIPSC frequency was not significantly different,indicating that post-synaptic mechanisms are involved. However, there was no significant presynapticinhibition through preNMDARs in the acute brain slices from pilocarpine-inducedepileptic mice.Conclusion: In the acute case of epilepsy, a compensatory mechanism of post-synapticinhibition, possibly from ambient GABA, was observed through changes in the amplitude withoutsignificant changes in the frequency of sIPSC compared to control mice. The role of preNMDARmediatedinhibition in epileptogenesis during the chronic condition or in the juvenile stagewarrants further investigation.

Expression and spatial distribution of P2X7 receptor in pilocarpine-induced epileptic rat hippocampus / 中南大学学报(医学版)

Objective:To investigate the dynamic expression and spatial distribution of P2X7 receptor in pilocarpine-induced epileptic rat hippocampus.Methods:Status epilepticus (SE) model of rats was established by intraperitoneal injection with chloride lithium and pilocarpine.Rat brain tissue and hippocampus were collected at 1,3,7,14,28 days after SE.The protein expression of P2X7 receptor in rat hippocampus was detected by Western blot.The distribution of P2X7 receptor in hippocampal sub-region was analyzed by immunohistochemistry.Results:Bilateral forelimb clonus appeared at (33.9±12.3 min after intraperitoneal injection with pilocarpine.The protein expression of P2X7 receptor was increased at 1d after SE,while it was decreased gradually from 3 d to minimum at 7 d,then it was elevated continuously to 28 d.Among them,the expression of P2X7 receptor was increased significantly at 1,14 and 28 d post-SE (P＜0.05).Immunohistochemical staining showed that P2X7 receptor was detected in all areas.The expression pattern of P2X7 receptor in hippocampal DG and CA3 area was consistent with protein expression,but its expression in hippocampal CA1 area was not significantly changed after SE.Conclusion:The expression of P2X7 receptor in post-SE hippocampus is in a time-dependent manner and spatial specificity.P2X7 receptor might be involved in the development of chronic epilepsy.

Structural and Functional Alterations at Pre-Epileptic Stage Are Closely Associated with Epileptogenesis in Pilocarpine-induced Epilepsy Model

Pilocarpine-induced rat epilepsy model is an established animal model that mimics medial temporal lobe epilepsy in humans. The purpose of this study was to investigate neuroimaging abnormalities in various stages of epileptogenesis and to correlate them with seizure severity in pilocarpine-induced rat epilepsy model. Fifty male Sprague-Dawley rats were subject to continuous video and electroencephalographic monitoring after inducing status epilepticus (SE) and seizure severity was estimated by frequency and total durations of class 3 to 5 spontaneous recurrent seizures (SRS) by modified Racine's classification. The 7.0 Tesla magnetic resonance imaging (MRI) with high resolution flurodeoxyglucose positron emission tomography (FDG-PET) was performed at 3 hours, 1, 3, 7 days and 4 weeks after the initial insult. The initial SRS was observed 9.7±1.3 days after the pilocarpine injection. MRI revealed an abnormal T2 signal change with swelling in both hippocampi and amygdala in acute (day 1 after injection) and latent phases (days 3 and 7), in association with PET hypometabolism in these areas. Interestingly, the mean frequency of class 3 to 5 SRS was positively correlated with abnormal T2 signals in hippocampal area at 3 days. SRS duration became longer with more decreased glucose metabolism in both hippocampi and amygdala at 7 days after pilocarpine injection. This study indicates that development and severity of SRS at chronic phase could be closely related with structural and functional changes in hippocampus during the latent period, a pre-epileptic stage.

The Mechanism of Anti-Epileptogenesis by Levetiracetam Treatment is Similar to the Spontaneous Recovery of Idiopathic Generalized Epilepsy during Adolescence

OBJECTIVE: The anti-epileptogenic drug levetiracetam has anticonvulsant and anti-epileptogenesis effects. Synergy between cell death and inflammation can lead to increased levels of apoptosis inhibitory factors and brain-derived neurotrophic factor, aberrant neurogenesis and extended axon sprouting. Once hyperexcitation of the neural network occurs, spontaneous seizures or epileptogenesis develops. This study investigated whether the anti-epileptogenic effect of levetiracetam is due to its alternate apoptotic activity. METHODS: Adult male Noda epileptic rats were treated with levetiracetam or vehicle control for two weeks. mRNA quantification of Bax, Bcl-2 and GAPDH expression were performed from prefrontal cortex and hippocampus tissue samples. RESULTS: The levetiracetam-treated group showed a significant increase of Bax/Bcl-2 mRNA expression ratio in the prefrontal cortex than the control group, but no change in the Bax/Bcl-2 mRNA expression ratio in hippocampus. CONCLUSION: Idiopathic generalized epilepsy including childhood absence epilepsy develop at childhood and recover spontaneously during adolescence. The aberrant neural excitable network is pruned by a neural-maturing action. This study suggests the mechanism of acquired anti-epileptogenesis by levetiracetam treatment may be similar to spontaneous recovery of idiopathic generalized epilepsy during adolescence.

Antiapoptotic and neuroprotective role of Curcumin in Pentylenetetrazole (PTZ) induced kindling model in rat.

Kindling, a sub threshold chemical or electrical stimulation, increases seizure duration and enhances accompanied behavior until it reaches a sort of equilibrium state. The present study aimed to explore the effect of curcumin on the development of kindling in PTZ kindled rats and its role in apoptosis and neuronal damage. In a PTZ kindled Wistar rat model, different doses of curcumin (100, 200 and 300 mg/kg) were administrated orally one hour before the PTZ injections on alternate day during the whole kindling days. The following parameters were compared between control and experimental groups: the course of kindling, stages of seizures, Histopathological scoring of hippocampus, antioxidant parameters in the hippocampus, DNA fragmentation and caspase-3 expression in hippocampus, and neuron-specific enolase in the blood. One way ANOVA followed by Bonferroni post hoc analysis and Fischer’s Exact test were used for statistical analyses. PTZ, 30 mg/kg, induced kindling in rats after 32.0±1.4 days. Curcumin showed dose-dependent anti-seizure effect. Curcumin (300 mg/kg) significantly increased the latency to myoclonic jerks, clonic seizures as well as generalized tonic-clonic seizures, improved the seizure score and decreased the number of myoclonic jerks. PTZ kindling induced a significant neuronal injury, oxidative stress and apoptosis which were reversed by pretreatment with curcumin in a dose-dependent manner. Our study suggests that curcumin has a potential antiepileptogenic effect on kindling-induced epileptogenesis.

Post-Traumatic Ictogenesis and Epileptogenesis / 대한신경손상학회지

For ictogenesis, initial step is intrinsic bursts of pacemaker neurons and, through exaggerated circuits or networks, the involved neurons become hyperexcitable state. Hypersynchrony of hyperexcitable neurons can induce paroxysmal depolarization shift for developing seizure. The mechanism underlying the development of post-traumatic epilepsy still remains to be elucidated. By traumatic brain injury, breakdown of blood-brain barrier (BBB) may lead network changes, long-lasting epileptiform activity and eventual neurodegeneration. Recently the concept of inflammation and epileptogenesis is widely accepted. In the surgically resected brain tissue from refractory partial epilepsy patients, there are hallmarks of a chronic inflammatory state and, also, via animal experiments, we can find the role of inflammation in the genesis of seizure and epilepsy. Inflammatory mediators (IL-1b, TGF-beta1 and COX-2) are associated with the epileptogenic brain. They can reduce seizure threshold, induce neurodegeneration, neurogenesis, and synaptic plasticity, and also disregulate BBB permeability. The increase in knowledge about a role of inflammation in epileptogenesis may support the use of specific anti-inflammatory drugs for developing disease-modifying treatments that can interfere epileptogenesis.

Participación de los canales de calcio dependientes de voltaje en el desarrollo de la epilepsia / Participation of voltage-dependent calcium channels in the development of epilepsy

La epilepsia es una afección crónica producida por diferentes etiologías, caracterizada por la repetición de crisis debidas a una descarga excesiva de las neuronas cerebrales asociadas a síntomas clínicos o paraclínicos. Se debe a una despolarización rápida, en la membrana, de iones en una población de neuronas susceptibles, es decir, un cambio repentino en la carga intracelular negativa a positiva. Las causas más conocidas son: alteraciones genéticas, anoxia perinatal, traumatismos, tumores, malformaciones congénitas, alteraciones metabólicas, intoxicaciones farmacológicas, infecciones del sistema nervioso. Una crisis epiléptica es la aparición transitoria de signos y síntomas anormales causados por la actividad neuronal excesiva, mientras que la epilepsia se caracteriza por una permanente predisposición a generar crisis. En la despolarización de la membrana neuronal, los iones calcio desempeñan un papel importante debido a que son mensajeros intracelulares que regulan funciones como: liberación de neurotransmisores, neurosecreción, excitación neuronal, supervivencia de neuronas y regulación de expresión de genes. El ingreso de calcio a través de la membrana plasmática representa una forma para controlar el nivel intracelular de calcio. Se conoce poco sobre el mecanismo de entrada del calcio a la neurona pero un progreso notable representa la comprensión de la estructura, función y regulación de los canales de calcio dependientes de voltaje.

Epilepsy is defined as a chronic condition produced by different etiologies, characterized by the repetition of crises due to an excessive discharge of the cerebral neurons assoclated wlth cllnlcal symptoms. It responds to a fast ion depolarization in a population of abnormal neurons. Causes of epilepsy are: genetíc alteratíons, perínatal anoxía, traumatísms, tumors, congenital malformations, metabolic alterations, drug poisonings, and infections of the nervous system. Epileptic selzure Is the transitory occurrence of signs and abnormal symptoms caused by excessive or synchronous neurona! activity whereas, epilepsy Is characterized by a permaneni predisposition to genérate seizures. During depolarization of neurona! membrane, calcium ions play an important role because they are intracellular messengers that regúlate functions llke: neurotransmltter reléase, neurosecretlon, neuronal excltatlon, neuron survlval and gene expresslon regulatlon . The Influx of calcium through the plasmatlc membrane represents a way to control Intracellular calcium level. The mechanlsm of the entrance of calcium to the neuron Is llttle known, but understandlng the structure, functlon and regulatlon of voltage-gated calcium channels Is of remarkable progress.

Increased Seizure Susceptibility and Up-regulation of nNOS Expression in Hippocampus Following Recurrent Early-life Seizures in Rats

This study aimed to determine the long-term change of seizure susceptibility and the role of nNOS on brain development following recurrent early-life seizures in rats. Video-EEG recordings were conducted between postnatal days 50 and 60. Alterations in seizure susceptibility were assayed on day 22 or 50 using the flurothyl method. Changes in nNOS expression were determined by quantitative immunoblotting on day 50. On average, rats had 8.4+/-2.7 seizures during 10 daily 1 hr behavioral monitoring sessions. As adults (days 50-60), all rats displayed interictal spikes in the hippocampus and/or overlying cortex. Brief electrographic seizures were recorded in only one of five animals. Rats appeared to progress from a period of marked seizure susceptibility (day 22) to one of lessened seizure susceptibility (day 50). Up-regulation of nNOS expression following early-life recurrent seizures was observed on day 50. In conclusion, these data suggested that recurrent early-life seizures had the long-term effects on seizure susceptibility late in life and up-regulatory nNOS expression on the hippocampus during brain development, and nNOS appeared to contribute to the persistent changes in seizure susceptibility, and epileptogenesis.

Treatment of Epilepsy Associated with Brain Tumors

Epilepsy associated with brain tumors (EABT) is a multi-faceted disease that both oncological and epileptological concerns should be taken into consideration. Usually, it is characterized by chronic drug-resistant epilepsy with a low-grade brain tumor in the cerebrum. However, the distinction of typical EABT and simple brain tumors with short-term epilepsy is obscure. We need a working formulation based on the patient's burden in both oncological and epileptological aspects. The diagnosis of EABT is straightforward, but the treatment should be more complex. Medical treatment with anticonvulsants aloneseems tobe anoutdated remedy for EABT because of the risk of tumor growth and malignant progression in some patients as well as the expected favorable seizure control after surgery. Surgical treatment of EABT has resulted in seizure-free state in about 80% of patients. Complete resection of the tumor is an important prognostic factor in seizure control and probably also in tumor control. Recently, many authors emphasized a lesion-directed surgery aimed at a complete tumor removal in EABT. However, in some patients, especially in patients with dual pathology, electrophysiological studies have to be thoroughly applied. For the treatment of EABT in the temporal lobe, more sophisticated surgical strategy is required. A lesionectomy saving the uninterrupted hippocampus could be applied for selected patients. Further research is strongly needed for better understanding and treatment of EABT and low-grade glioma.

Effect of Diazoxide in Conunterating on Hyperexcitability by 4-Aminopyridine-induced Epileptiform Discharge Followed by Hypoxia in Young Rats / 대한소아신경학회지

PURPOSE:The goal of the present study was to investigate the effects of diazoxide on 4-aminopyridine(4-AP)-induced hyperexcitability followed by normal oxygenation or an anoxia state in young rats. Also, we investigated the effects of carbamazepine(CBZP) on 4-AP-induced hyperexcitability followed by normal oxygenation or an anoxia state in young rats. METHODS:The visual cortex slices in this study were obtained from 13 to 18-days-old Sprague-Dawley rats. Extracellular recordings were performed to observe the induction of the epileptiform discharges perfused by artificial CSF containing 100 micrometer 4-AP with 7.5 mM K and the effects of 1 mM diazoxide and 50 micrometer CBZP followed by normal oxygenation or an anoxia state. RESULTS:Spontaneous epileptiform activities were observed in artificial CSF containing 100 micrometer 4-AP. The addition of diazoxide decreased the frequency of 4-AP- induced epileptiform activities followed by anoxia, but didnt block the 4-AP-induced epileptiform activities followed by anoxia. The addition of CBZP blocked the 4-AP-induced epileptiform activities followed by normal oxygenation. CONCLUSION:We observed that diazoxide did not counteract the epileptiform activities induced by 4-AP. Diazoxide inhibited the increased excitability followed an anoxia state in young rats. CBZP counteracted the epileptiform activities induced by 4-AP. Diazoxide may have limited utilities in the seizure therapy. Nevertheless, this could be of benefit during prolonged seizures where hypoxia becomes a significant factor.

Effect of Potassium, Bicuculline and CNQX on Epileptiform Activity Induced by Pilocarpine in the Rat Visual Cortex Slice

BACKGROUND: The purpose of this study was to investigate the effect of potassium, bicuculline (BIC) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on epileptiform activity induced by pilocarpine in the rat visual cortex slices. METHODS: In the rat visual cortex slices, we observed the change of pilocarpine-induced epileptiform discharges using extracellular recordings during perfusion of artificial cerebro-spinal fluid (ACSF) with various potassium concentrations ([K+], 2.5, 5, 7.5 and 10 mM) and ACSF with 10 micrometer BIC and 20 micrometer CNQX under 7.5 mM [K+]. RESULTS: Spontaneous interictal epileptiform activity induced by pilocarpine was observed in 5 mM or higher [K+] and ictal discharge was only detected in 7.5 mM [K+]. Increase of [K+] from 2.5 to 7.5 mM not only resulted in the increase of frequency and amplitude of epileptiform activity but also favored the transformation of pilocarpine-induced interictal activity into ictal activity in the rat visual cortex. However, in 10 mM [K+], the ictal discharge was unprovoked and interictal activity was provoked with decreased frequency and amplitude. The spontaneous ictal discharge was blocked but interictal activity was maintained with increased frequency and amplitude by BIC. Interictal and ictal activities were completely blocked by CNQX. CONCLUSIONS: These results suggested that the extracellular potassium concentration, GABA system, and non-NMDA mechanism seemed to be involved in the development and maintenance of pilocarpine-induced epileptiform activity in the rat visual cortex.

The Temporospatial Distribution of Glutamate Receptors and the Effect of MK-801 on Glutamate Receptors Activation in Kainate-induced Seizure Model: Quantitative Receptor Autoradiography of Ionotropic Glutamate Receptors

BACKGROUND: Excitotoxicity and epileptogenesis have often been associated with glutamate receptor activation. Some evidence indicates that selective down regulation of AMPA receptor may be the mechanism of delayed neuronal cell death in the hippocampus. METHODS: We used in situ hybridization to examine the hybridization density (HD) of NMDA and AMPA receptors on excitotoxicity and epileptogenesis in the hippocampus of the kainic acid (KA)-induced rat seizure model. Some Sprague-Dawley rats were injected with KA, and others with MK-801 prior to KA injection. The rats were killed at 8 hours or 4 weeks after KA or MK-801/KA injection. HD of [3H]MK-801 and [3H]AMPA bind-ing in subfields of the hippocampus was measured by an image analyzer. RESULTS: After 8 hours of KA injection, [3H]MK-801 binding was increased in CA1 and CA3, and decreased in dentate gyrus, and [3H]AMPA binding was decreased in all of CA1, CA3 and fascia dentata, and pretreatment of MK-801 did not affect [3H]AMPA binding in all of CA1, CA3 and dentate gyrus. After 4 weeks, both [3H]MK-801 and [3H]AMPA binding were prominently increased in inner molecular layer of dentate gyrus. CONCLUSIONS: Glutamate receptors, especially NMDA receptor, were associ-ated with excitotoxicity in the hippocampus but the selective down regulation of GluR2 subunit of AMPA receptor without NMDA receptor activation may not be sufficient to cause excitotoxic neuronal cell death in CA1 and CA3. In addition, the synaptic reorganization in inner molecular layer of dentate gyrus was proved to be chronically hyperex-citable in function and may contribute epileptogenesis.

Factors Affecting Development of Epilepsy and Postoperative Recurrence of Epilepsy in Primary Brain Tumor

BACKGROUND: A small but significant proportion of patients with brain tumors continued to have seizures postoperatively. All of them could not be explained simply by the failure to adequately resect the tumor mass. We investigated factors influencing seizure recurrence in primary brain tumors. METHODS: We analyzed 435 patients treated with tumor surgery and examined the differences between epileptic seizure group (ESG) and non-epileptic seizure group (NESG). Among ESG, we selected 99 patients confirmed by pathology. We divided patients into chronic epileptic seizure group (CESG; duration of seizure attack >or=1 year) and acute epileptic seizure group (AESG; < 1 year). We also investigated the differences between two groups. RESULTS: Of 435 patients, 104 were ESG and 331 NESG. Among various factors, male, favorable neurological state, fronto-temporal lobe origin, astrocytoma, oligodendroglioma were statistically significant in ESG compared with NESG (p<0.05). Of 99 patients, 43 were CESG and 56 AESG. Among various factors, seizure recurrence rate without residual tumor or tumor recurrences was significantly higher in CESG than in AESG (p<0.05). On the other hand, the laboratory abnormalities, and the rate of residual tumor or tumor recurrences on follow-up MRI were significantly higher in AESG than in CESG (p<0.05). CONCLUSIONS: Among many factors, sex, neurological state, location and pathology of tumors were significantly related to seizure attacks in brain tumor. There were the differences of epileptogenesis between AESG and CESG. We suggest that patients with brain tumor and chronic epilepsy have to be investigated with extensive work-up including invasive electrophyosiologic studies.

Expression of c-myc mRNA in the Hippocampus of Pentylenetetrazol Kindling Rat

Kindling development is a good animal model of epilepsy and neural plasticity. It is induced by repeated subconvulsive electrical or chemical stimulations. This leads to progressive and permanent amplification of seizure activity resulting in permanent brain changes. Immediate early genes(IEGs) are proposed as the master switch for turning on molecular events in long term neural plasticity. The role of c-myc, an IEG, in the development of kindling is not known. This study was conducted to investigate the role of c-myc in the neural plastic changes underlying kinding. Among 115 adult male Spargue-Dawley rats, 51 were kindled by repeated administrations of subconvulsive doses(15-25mg/kg) of pentylenetetrazol(PTZ). Twenty-eight rats experienced various degree of convulsions induced by a single injection of convulsive dose(30-60mg/kg) of PTZ. Eighteen rats experienced mild or severe convulsions induced by a single injection of convulsive dose(30-60mg/kg) of PTZ. Eighteen rats experienced mild or severe convulsion by a single electroconvulsive shock(ECS). Eighteen rats received normal saline as a control group. Animals were sacrificed in 30 minutes, 1 hour and 48 hours after convulsion. C-myc mRNA levels in the hippocampus were quantified using slot-blot hybridization analysis. In the experiment of PTZ kindling, c-myc mRNA expression 30 minutes after convulsion was elevated about 3-8 times compared with controls. C-myc mRNA expression 1 hour after convulsion was elevated about 4 times at stage I, II, and V, ut was not elevated at stage III and IV. C-myc mRNA expression 48 hours after convulsion was elevated about 2-3 times compared with controls. In the experiment of PTZ-induced seizures, c-myc mRNA expression 30 minutes after convulsion was elevated 5-6 times compared with controls. C-myc mRNA expression 1 hour after convulsion was elevated 4-6 times. C-myc mRNA expression 48 hours after convulsion was elevated approximately 2 times. In the experiment of ECS-induced seizures, c-myc mRNA expression was elevated 4 times at 30 minutes and 1 hour after mild convulsion, but decreased at 30 minutes and 1 hour after severe convulsion compared with control. C-myc mRNA expression 48 hours after convulsion was elevated approximately 2 times. These results suggest that the enhanced expression of c-myc mRNA is a non-specific consequence in the development of PTZ kindling. In addition, c-myc does not seem to play an important role in turning on a molecular program underlying kindling.