Racine Score Research Articles

The neuroprotective effect of perampanel in lithium-pilocarpine rat seizure model

PurposeStatus epilepticus (SE) causes irreversible neurodegeneration if not terminated quickly. Perampanel (PER), a potent AMPA receptor antagonist, has previously been shown to terminate seizures in the lithium-pilocarpine SE model. In the present study, we assessed whether PER would also prevent neuronal damage in this model. MethodsSE was induced in rats using lithium chloride and pilocarpine. Initiation of SE was defined as continuous seizures that exhibited as rearing accompanied by bilateral forelimb clonus (Racine score 4). Either PER (0.6, 2, or 6mg/kg) or diazepam (DZP, 10mg/kg) was administered intravenously 30min after SE initiation. Histopathological samples from treated and seizure-naive rats were taken one week after treatment and then stained with an anti–neuronal nuclei (NeuN) antibody. The sections were analyzed by using a pixel-counting algorithm to quantify the amount of staining in the CA1 subregion of the hippocampus, piriform cortex (Pir), and mediodorsal thalamic nucleus (MD). ResultsDZP administration did not suppress seizures or the degeneration of neurons in the examined areas. Seizures were terminated in 100% of rats treated with 6mg/kg PER (n=8) and in 47% (7/15) of rats treated with 2mg/kg PER, and neurons in the analyzed areas of these animals were preserved to the level seen in naive rats. In the eight animals in which 2mg/kg PER did not terminate the seizures, neuronal loss was partially attenuated in CA1 and Pir, and neurons were fully preserved in MD. Treatment with 0.6mg/kg PER did not terminate the seizures or significantly preserve neurons. The anti-seizure effect of PER correlated well with the degree of neuroprotection in each analyzed area. ConclusionsPER exhibited a strong neuroprotective effect in a drug-refractory SE model, and this effect was correlated with its attenuation of seizure.

Reevaluation of CNS Oxygen Toxicity Seizures in Male Sprague‐Dawley Rats

Prolonged breathing of hyperbaric oxygen (HBO2) results in central nervous system O2 toxicity (CNS‐OT; i.e., seizures, Sz's). The threat of CNS‐OT is what limits the use of HBO2 in hyperbaric, diving and submarine medicine. Past studies have identified CNS‐OT as the onset of visibly detectable tonic‐clonic Sz's preceded by increased cortical EEG activity. It has been our experience, however, that onset of 1st Sz in Sprague‐Dawley (SD) rats is variable between animals and, in some cases, difficult to confirm in real time. This is problematic because the latency time to seizures (LSz) is how the risk of CNS‐OT is evaluated in hyperbaric research. Consequently, we have begun using longer exposures to HBO2 to capture recurring changes in EEG activity and behaviors that can be analyzed offline to redefine the criteria for 1st Sz. We also wanted to know if subsequent Sz's increased in intensity. Accordingly, cortical EEGs were recorded in 27 adult male SD rats that were implanted with a DSI 4‐ET radio transmitter. At least 1 week following surgery, each rat was placed in a hyperbaric chamber and “dived” to 5 ATA O2. 71 Sz's were identified in 27 rats based on quantified changes in cortical EEG activity (NeuroScore 2.1) and our modified Racine scale of six Sz‐related behaviors. A cortical seizure (CSz) was defined as a series of EEG polyspikes that crossed a threshold of 200μV, lasted ≥15 seconds, and contained an increase in theta wave activity coupled with at least one of the Sz‐related behaviors (65% of the Sz were classified as CSz). Two other Sz types were also recorded: neurological seizures (NSz) that included only the EEG profile described above, but without any detectable behavior changes (14%); and behavioral seizure (BSz) that included one or more of the behavioral components without any detectable changes in cortical EEG activity (21%). LSz was calculated from the moment the chamber pressure reached 5 ATA O2 until the onset of CSz, NSz or BSz. The LSz to 1st seizure was 7.2 ± 5.3 min (n= 27), while the 2nd and 3rd Sz's occurred at 26.2 ± 29.88 min (n=25) and 40 ± 40.86 min (n=13), respectively. Remarkably, in between bouts of Sz's, animals exhibited normal behaviors (grooming, exploring, chewing, etc.) and normal EEG activity in 5 ATA O2. There was also a shift in the type of Sz between the 1st and 2nd Sz such that the fraction of BSz + NSz significantly increased from 22% to 44%. Moreover, intensity of the Sz increased progressively with each successive Sz based on our modified Racine score. We conclude that the majority of HBO2 Sz's in SD rats can be readily identified using a combination of defined EEG and behavioral criteria. However, not all Sz's during HBO2 fit both criteria. The occurrence of BSz suggests that neurological Sz's originate outside the cerebral cortex. The occurrence of NSz indicate that some Sz's will be missed in studies that do not measure EEG activity. The fact that 1st Sz is often underwhelming compared to subsequent Sz's suggests the LSz may be overestimated in some animals. To better understand the SD rat model of CNS‐OT, we recommend that future studies need to 1) implant telemetric leads into multiple cortical and subcortical areas to localize the site of Sz‐genesis; and 2) determine the degree of performance impairment during 1st, 2nd & 3rd Sz's.Support or Funding InformationONR N000141310405 & N000141612537

KCC2 downregulation facilitates epileptic seizures

GABAA receptor-mediated inhibition depends on the maintenance of low level intracellular [Cl−] concentration, which in adult depends on neuron specific K+-Cl− cotransporter-2 (KCC2). Previous studies have shown that KCC2 was downregulated in both epileptic patients and various epileptic animal models. However, the temporal relationship between KCC2 downregulation and seizure induction is unclear yet. In this study, we explored the temporal relationship and the influence of KCC2 downregulation on seizure induction. Significant downregulation of plasma membrane KCC2 was directly associated with severe (Racine Score III and above) behavioral seizures in vivo, and occurred before epileptiform bursting activities in vitro induced by convulsant. Overexpression of KCC2 using KCC2 plasmid effectively enhanced resistance to convulsant-induced epileptiform bursting activities in vitro. Furthermore, suppression of membrane KCC2 expression, using shRNAKCC2 plasmid in vitro and shRNAKCC2 containing lentivirus in vivo, induced spontaneous epileptiform bursting activities in vitro and Racine III seizure behaviors accompanied by epileptic EEG in vivo. Our findings novelly demonstrated that altered expression of KCC2 is not the consequence of seizure occurrence but likely is the contributing factor.

Influences of pretreating with lipopolysaccharide the infant epilepsy rats on the gene expression of Toll-like receptor 4/high mobility group protein 1 gene and hippocampal sclerosis

Objective To investigate influences of pretreating with lipopolysaccharide (LPS) the infant epilepsy rats on the gene expression of Toll-like receptor 4/high mobility group protein 1 (TLR4/HMGB1) gene and astrocytes proliferation in the hippocampus in the chronic phase. Methods The rats on postnatal 21 day (21d) were randomly divided into control group, model Ⅰ group and model Ⅱ group.The model groups were induced into epilepsy model by using kainic acid (KA), and the model Ⅱ group was pretreated with intraperitoneal injection of LPS 2 hours before KA application.Behaviors of infant rats with epilepsy seizures were observed and given scores according to Racine score, and the gene expressions of TLR4 and HMGB1 at 3 h and 24 h after status epilepticus (SE) were detected by using fluorescence quantitative PCR, and astrocyte proliferation on 21 day after SE were detected by using immunohistochemistry, and the gial fibrillary acidic protein (GFAP) was detected. Results The TLR4 gene expressions at 3 h after SE in hippocampus of model group Ⅰ and model group Ⅱ were significantly higher than that of control group (t=4.806, 4.954, all P 0.05). The HMGB1 gene expressions at 3 h after SE in hippocampus of model group Ⅰ and model group Ⅱ were significantly higher than that of control group (t=3.626, 5.255, all P 0.05). The HMGB1 gene expressions at 24 h after SE in hippocampus of model group Ⅰ and model group Ⅱ were significantly higher than that of control group (t=4.046, 2.836, all P 0.05). The GFAP positive cells on 21 day after SE in hippocampus of control group were few, and were more significant in model group Ⅰ and model groupⅡ. Conclusions The expression of TLR4/HMGB1 gene in the hippocampus of infant epilepsy rats increased in acute phase, and LPS pretreatment can increase the expression of TLR4 gene in hippocampus of infant rats with epilepsy and the proliferation of astrocytes in the chronic phase. Key words: Lipopolysaccharide; Kainic acid; Epilepsy; Infant rat; High mobility group protein 1; Toll-like receptor 4

Beta Lactams Antibiotic Ceftriaxone Modulates Seizures, Oxidative Stress and Connexin 43 Expression in Hippocampus of Pentylenetetrazole Kindled Rats.

Background and Purpose:This study aimed to investigate the effect of ceftriaxone on oxidative stress and gap junction protein (connexin 43, Cx-43) expression in pentylenetetrazole (PTZ) induced kindling model.Methods:Twenty four Sprague dawely rats were divided into 3 equal groups (a) normal group: normal rats. (b) PTZ kindled group: received PTZ at the dose of 50 mg/kg via intraperitoneal injection (i.p.) every other day for 2 weeks (c) ceftriaxone treated group: received ceftriaxone at the dose 200 mg\\kg/12 hrs via i.p. injection daily from the 6th dose of PTZ for 3 days. Racine score, latency before beginning the first myoclonic jerk and duration of the jerks used as parameters of behavioral assessment. Immunohistopathological study for Cx-43 expression in hippocampus and measurement of markers of oxidative stress (malondialdehyde [MDA], low reduced glutathione [GSH] and catalase [CAT]) in hippocampal neurons were done.Results:PTZ kindling was associated with behavioral changes (in the form high stage of Racine score, long seizure duration and short latency for the first jerk), enhanced oxidative stress state (as demonstrated by high MDA, low GSH and CAT) and up regulation of Cx43 in hippocampal regions. While, ceftriaxone treatment ameliorated, significantly, PTZ-induced convulsions and caused significant improvement in oxidative stress markers and Cx-43 expression in hippocamal regions (p < 0.05).Conclusions:These findings support the anticonvulsive effects of some beta-lactams antibiotics which could offer a possible contributor in the basic treatment of temporal lobe epilepsy. This effect might be due to reduction of oxidative stress and Cx43 expression.

Pilocarpine-induced status epilepticus in mice: A comparison of spectral analysis of electroencephalogram and behavioral grading using the Racine scale

Pilocarpine-induced status epilepticus (SE) is a widely used seizure model in mice, and the Racine scale has been used to index seizure intensity. The goal of this study was to analyze electroencephalogram (EEG) quantitatively using fast Fourier transformation (FFT) and statistically evaluate the correlation of electrographic seizures with convulsive behaviors. Simultaneous EEG and video recordings in male mice in a mixed genetic background were conducted and pilocarpine was administered intraperitoneally to induce seizures. The videos were graded using the Racine scale and the root-mean-square (RMS) power analysis of EEG was performed with Sirenia Seizure Pro software. We found that the RMS power was very weakly correlated with convulsive behavior induced by pilocarpine. Convulsive behaviors appeared long before electrographic seizures and showed a strong negative correlation with theta frequency activity and a moderate positive correlation with gamma frequency activity. Racine scores showed moderate correlations with RMS power across multiple frequency bands during the transition from first electrographic seizure to SE. However, there was no correlation between Racine scores and RMS power during the SE phase except a weak correlation with RMS power in the theta frequency. Our analysis reveals limitations of the Racine scale as a primary index of seizure intensity in status epilepticus, and demonstrates a need for quantitative analysis of EEG for an accurate assessment of seizure onset and severity.

078 — (TAM0117) Protection against convulsive seizure after hippocampal transplantation of mesenchymal cell adipose tissue in mice

Rationale: The use of mesenchymal stem cells is an innovative and accessible strategy for the treatment of epileptic disorders because of their immunoregulatory mechanisms and trophic, proliferative, and antiapoptotic actions. The aim of this study was to evaluate the protective effect of mesenchymal cell adipose tissue (ATMC) transplanted into the hippocampus against convulsive seizure induced by maximum electroconvulsive shock (MES) and pentylenetetrazole (PTZ). The transplanted ATMC was tested using the same paradigm of pharmacological screening tests for antiepileptic drugs. Methods: The supraepididymal region of ATMC C57/B6 mice was isolated and cultured in DMEN low 10% SFB and used between 15 and 18 passages. The cells were transplanted into the hippocampus of adult male mice C57/B6 (PTZ: n= 8; MES: n= 12). Control groups (n = 6; n = 12) received the same volume of culture medium in the same location. Ten days after the surgery, PTZ or MES was applied to induce acute tonic–clonic seizures. The parameters used to evaluate the anticonvulsant activity of the ATMC were protection against seizure, reduction in the duration of each convulsive phase, and decreased mortality. Results: Immunofluorescence analysis confirmed the presence of ATMC in the hippocampus. Quantification of doublecortin (DCX) and FluoroJade B (FJB) did not differ between Sal-MES and ATMC-MES animals, and Iba1-stained cells indicated the presence of microglia at the site of injection that did not colocalize with ATMC. Animals transplanted with ATMC showed a significant reduction in the PTZ-induced seizure intensity (p b 0.05). Frequency of PTZ seizures classified by Racine's scores I to III and IV to V (Racine, R.J., Eletroencephalogr Clin Neurophysiol, 32:281, 1972) showed significant reduction in the ATMC group (p b 0.05, chi-square). The duration of tonic seizure and the mortality rate induced by MES were also reduced in the ATMC group (p b 0.05). Analysis of tonic seizure duration on days 3, 4, and 5 indicated significantly reduced seizure duration in the ATMC group (n= 12) when compared with the CTRL group (n= 12). The percentage of mortality was significantly reduced in the ATMC group compared with the control group (p b 0.05, chi-square). Discussion/conclusions: Our results indicated that the ATMC transplanted into the hippocampus had neuroprotective effects after seizures induced byMES and PTZ by reducing the duration andmortality rate in MES and frequency of seizures in the PTZ model. The immunohistochemistry analyses confirmed the presence of ATMC in the hippocampus of transplanted animals. These neuroprotective effects on seizures induced in both experimental models may be related to unknown inhibitory factors and immunomodulatory mechanisms assigned to ATMC present in the hippocampus. This study should contribute to the understanding of anticonvulsantmechanisms of ATMC, bringing strong therapeutic implications for the control of epileptic seizures.

Activation of Group 2 metabotropic glutamate receptors reduces behavioral and electrographic correlates of pilocarpine induced status epilepticus

Novel treatments for epilepsy are necessary because many epilepsy patients are resistant to medication. Metabotropic glutamate receptors (mGluRs), specifically mGluR 2 and 3, may serve as antiepileptic drug targets because of their role in controlling synaptic release. In this study, we administered a Group 2 mGluR agonist, LY379268, one of two mGluR2-specific positive allosteric modulators, BINA or CBiPES, or a cocktail of both BINA and LY379268 in a series of experiments using the pilocarpine model of SE. In one study, groups received treatments 15 min prior to pilocarpine, while in a second study groups received treatments after SE had been initiated to determine whether the drugs could reduce development and progression of SE. We measured bouts of stage 5 seizures, latency to the first seizure, and the maximum Racine score to characterize the seizure severity. We analyzed mouse EEG with implanted electrodes using a power analysis. We found that pretreatment and posttreatment with LY379268 was effective at reducing both behavioral correlates and power in EEG bandwidths associated with seizure, while CBiPES was less effective and BINA was ineffective. These data generally support continued development of mGluR2 pharmacology for novel antiepileptic drugs, though further study with additional drugs and concentrations will be necessary.

Vagus nerve stimulation inhibits seizure activity and protects blood–brain barrier integrity in kindled rats with cortical dysplasia

AimsThis study investigates the effects of vagus nerve stimulation (VNS) on seizure severity and blood–brain barrier (BBB) integrity in kindled rats with cortical dysplasia (CD). Main methodsPregnant rats were exposed to 145cGy of gamma-irradiation on day 17 of pregnancy. In offsprings, kindling was induced by giving subconvulsive doses of pentylenetetrazole. Left VNS was performed for 48h at output currents of 0.5 or 1mA. Horseradish peroxidase (HRP) was used to study the BBB permeability. Immunohistochemistry for occludin and P-glycoprotein (P-gp) was also performed. Key findingsKindled rats with CD exhibited seizures with mean Racine's scores of 3.57±1.2 during video EEG recording. Kindled animals with CD receiving VNS at 0.5 and 1.0mA did not exhibit either clinical or electrophysiological signs of seizure. Immunostaining for occludin, a tight junction protein, in hippocampus remained relatively intact in all groups. VNS-treated and -untreated kindled animals with CD revealed intense immunostaining for P-gp in hippocampal formation (P<0.01). Electron microscopic observations revealed frequent transport vesicles containing electron-dense HRP reaction products in the cytoplasm of brain capillary endothelial cells in both cerebral cortex and hippocampus of kindled animals with CD. Those which were exposed to 1mA VNS were observed to have brain capillary endothelial cells largely devoid of HRP reaction products in both cerebral cortex and hippocampus. SignificanceThe results of this study suggest that VNS therapy at 1mA inhibits seizure activity and protects BBB integrity by limiting the enhancement of transcellular pathway in kindled animals with CD.

Topiramate reduces blood–brain barrier disruption and inhibits seizure activity in hyperthermia-induced seizures in rats with cortical dysplasia

We investigated the effects of topiramate (TPM), a novel broad spectrum anticonvulsant, on seizure severity, survival rate and blood–brain barrier (BBB) integrity during hyperthermic seizures in rats with cortical dysplasia (CD). Offsprings of irradiated mothers were used in this study. To show the functional and morphological alterations in BBB integrity, quantitative analysis of Evans blue (EB) extravasation, immunohistochemistry and electron microscopic assessment of horseradish peroxidase (HRP) permeability were performed. Rats with CD exposed to hyperthermia exhibited seizures with mean Racine's scores of 3.92±1.2. Among the rats with CD pretreated with TPM, 21 of 24 rats showed no sign of seizure activity upon exposure to hyperthermia (p<0.01). The immunoreactivity of occludin, a tight junction protein, remained essentially unaltered in capillaries of hippocampus in all groups. In animals with CD exposed to hyperthermia, the significantly increased p-glycoprotein immunoreactivity in hippocampus (p<0.01) was slightly decreased by TPM pretreatment. Hyperthermic seizures increased BBB permeability to EB in animals with CD, but TPM pretreatment decreased the penetration of the tracer into the brain in these animals (p<0.01). Ultrastructurally frequent vesicles containing HRP reaction products were observed in capillary endothelial cells in cerebral cortex and hippocampus of rats with CD subjected to hyperthermia-induced seizures, and TPM pretreatment prevented the development of HRP reaction products in these animals. The results of this study suggest that TPM inhibits seizure activity and maintains BBB integrity in the course of febrile seizures in the setting of CD.

N w‐Propyl‐l‐arginine (L‐NPA) reduces status epilepticus and early epileptogenic events in a mouse model of epilepsy: behavioural, EEG and immunohistochemical analyses

We investigated the anticonvulsant and neurobiological effects of a highly selective neuronal nitric oxide synthase (nNOS) inhibitor, N (w) -propyl-l-arginine (L-NPA), on kainic acid (KA)-induced status epilepticus (SE) and early epileptogenesis in C57BL/6J mice. SE was induced with 20 mg/kg KA (i.p.) and seizures terminated after 2 h with diazepam (10 mg/kg, i.p). L-NPA (20 mg/kg, i.p.) or vehicle was administered 30 min before KA. Behavioural seizure severity was scored using a modified Racine score and electrographic seizure was recorded using an implantable telemetry device. Neuronal activity, activity-dependent synaptogenesis and reactive gliosis were quantified immunohistochemically, using c-Fos, synaptophysin and microglial and astrocytic markers. L-NPA treatment reduced the severity and duration of convulsive motor seizures, the power of electroencephalogram in the gamma band, and the frequency of epileptiform spikes during SE. It also reduced c-Fos expression in dentate granule cells at 2 h post-KA, and reduced the overall rate of epileptiform spiking (by 2- to 2.5-fold) in the first 7 days after KA administration. Furthermore, treatment with L-NPA suppressed both hippocampal gliosis and activity-dependent synaptogenesis in the outer and middle molecular layers of the dentate gyrus in the early phase of epileptogenesis (72 h post-KA). These results suggest that nNOS facilitates seizure generation during SE and may be important for the neurobiological changes associated with the development of chronic epilepsy, especially in the early stages of epileptogenesis. As such, it might represent a novel target for disease modification in epilepsy.

Absence of tolerance to the anticonvulsant and neuroprotective effects of imidazenil against DFP-induced seizure and neuronal damage

The clinical use of diazepam or midazolam to control organophosphate (OP) nerve agent-induced seizure activity is limited by their unwanted effects including sedation, amnesia, withdrawal, and anticonvulsant tolerance. Imidazenil is an imidazo-benzodiazepine derivative with high intrinsic efficacy and selectivity for α2-, α3-, and α5- but low intrinsic efficacy for α1-containing GABAA receptors. We have previously shown that imidazenil is more efficacious than diazepam at protecting rats and mice from diisopropyl fluorophosphate (DFP)-induced seizures and neuronal damage without producing sedation. In the present study, we compared the tolerance liability of imidazenil and diazepam to attenuate the seizure activity and neurotoxic effects of DFP. Rats received protracted (14 days) oral treatment with increasing doses of imidazenil (1–4 mg/kg), diazepam (5–20 mg/kg), or vehicle. Eighteen hours after the last dose of the protracted treatment schedule, rats were tested for anticonvulsant tolerance after a 30 min pretreatment with a single test dose of imidazenil (0.5 mg/kg) or diazepam (5 mg/kg) prior to a DFP challenge (1.5 mg/kg). The anticonvulsant (modified Racine score scale) and neuroprotective (fluoro-jade B staining) effects of diazepam were significantly reduced in protracted diazepam-treated animals whereas the effects of imidazenil were not altered in protracted imidazenil-treated animals. The present findings indicate that protracted imidazenil treatment does not produce tolerance to its protective action against the neurotoxic effects of OP exposure.

Protective effect of 2,2′-dithienyl diselenide on kainic acid-induced neurotoxicity in rat hippocampus

In this study, we investigated the effects of 2,2'-dithienyl diselenide (DTDS), an organoselenium compound, against seizures induced by kainic acid (KA) in rats. Rats were pretreated with DTDS (50 or 100 mg/kg) by oral route 1 h before KA injection (10 mg/kg, intraperitoneal). Our results showed that DTDS (100 mg/kg) was effective in increasing latency for the onset of the first clonic seizure episode induced by KA, as well as in decreasing the appearance of seizures and the Racine's score. DTDS also caused a decrease in the excitatory electroencephalographic (EEG) changes, resulting from KA exposure in hippocampus and cerebral cortex of rats. Besides, elevated reactive species (RS) and carbonyl protein levels and Na(+), K(+)-ATPase activity in hippocampus of rats treated with KA were ameliorated by DTDS (50 and 100 mg/kg). Lastly, as evidenced by Cresyl-Violet stain, DTDS (100 mg/kg) elicited a protective effect against KA-induced neurodegeneration in rat hippocampus 7 days after KA injection. In conclusion, the present study showed that DTDS attenuated KA-induced status epilepticus in rats and the subsequent hippocampal damage.

Focused ultrasound-mediated suppression of chemically-induced acute epileptic EEG activity.

BackgroundEpilepsy is a common neurological disorder, which is attributed to uncontrollable abnormal hyper-excitability of neurons. We investigated the feasibility of using low-intensity, pulsed radiation of focused ultrasound (FUS) to non-invasively suppress epileptic activity in an animal model (rat), which was induced by the intraperitonial injection of pentylenetetrazol (PTZ).ResultsAfter the onset of induced seizures, FUS was transcranially administered to the brain twice for three minutes each while undergoing electroencephalographic (EEG) monitoring. An air-backed, spherical segment ultrasound transducer (diameter: 6 cm; radius-of-curvature: 7 cm) operating at a fundamental frequency of 690 KHz was used to deliver a train of 0.5 msec-long pulses of sonication at a repetitive rate of 100 Hz to the thalamic areas of the brain. The acoustic intensity (130 mW/cm2) used in the experiment was sufficiently within the range of safety guidelines for the clinical ultrasound imaging. The occurrence of epileptic EEG bursts from epilepsy-induced rats significantly decreased after sonication when it was compared to the pre-sonication epileptic state. The PTZ-induced control group that did not receive any sonication showed a sustained number of epileptic EEG signal bursts. The animals that underwent sonication also showed less severe epileptic behavior, as assessed by the Racine score. Histological analysis confirmed that the sonication did not cause any damage to the brain tissue.ConclusionsThese results revealed that low-intensity, pulsed FUS sonication suppressed the number of epileptic signal bursts using acute epilepsy model in animal. Due to its non-invasiveness and spatial selectivity, FUS may offer new perspectives for a possible non-invasive treatment of epilepsy.

Cyclothiazide induces seizure behavior in freely moving rats

We have previously demonstrated that cyclothiazide (CTZ) is a potent convulsant drug inducing robust epileptiform activity in hippocampal neurons both in vitro and in vivo. Here we further establish an animal model for CTZ-induced behavioral seizures in freely moving rats. Microinjection of CTZ into the left ventricle dose-dependently induced robust seizure behaviors within 3 h after administration. At a dose of 0.75 μmol, CTZ induced Racine score IV–V seizure behaviors in 71% (n = 14) of the rats were tested. In addition, CTZ also induced epileptiform EEG activity accompanying behavioral seizures. The convulsant action of CTZ on both behavior and EEG was blocked by pretreatment with clinical anticonvulsant drug diazepam (n = 5). In conclusion, our results demonstrate that CTZ is capable of inducing behavioral seizures in intact animals. Since CTZ acts on both GABAergic and glutamatergic systems, this new animal epilepsy model will be useful for anticonvulsant drug testing and general epilepsy research.

Different phases of afterdischarge during rapid kindling procedure in mice

The basic mechanisms of hippocampal networks in epileptogenesis are not entirely understood. To help achieve a better understanding of these mechanisms, we studied the extra-cellular electrically evoked responses in the hippocampi of mice during rapid kindling. Kindling protocol was achieved by stimulating the dorsal right hippocampus six times daily for four days using bipolar electrodes to produce sub-convulsive electrical discharges. Motor responses and analyzed electroencephalographic recordings showed progression from partial complex seizures to generalized seizures associated with different consecutive patterns within the afterdischarges. A spike-wave pattern appeared immediately after stimulation in combination with a poly-spike complex superimposed over the wave (AD1). AD1 was followed by a poly-spike complex (AD2), which was followed by a progressive modification of repetitive spikes (AD3). An ictal depression event was observed at the end of each AD3. Theta oscillations were observed at stage 1-2 of kindling, while beta/gamma oscillations appeared within AD2, associated with stage 4-5 from Racine's score. Benzodiazepine, a GABA (A) agonist (Diazepam) administered at non-sedative doses and only on days 3 and 4 of kindling, limited beta and gamma frequency bands and the progression of seizure severity, suggesting that the failure of GABA (A) agonism mediates the propagation or generalization of seizures. We conclude that different phases of afterdischarge occur during kindling and that high frequencies mediate generalization of seizures.

Impact of receptor changes on treatment of status epilepticus

Neurotransmitter receptors play a key role in the pathogenesis and current treatment strategies for status epilepticus (SE). Benzodiazepines are commonly used to treat SE exert their anticonvulsant action by stimulating γ-aminobutyric type A receptors (GABAA receptors). These drugs are quite effective in terminating SE in approximately 55–66% of patients, but there is a residual group of patients with seizures resistant to benzodiazepeines. The mechanisms underlying initiation and maintenance of prolonged SE were explored.

Increased severity of chemically induced seizures in mice with partially deleted Vitamin D receptor gene

Vitamin D is a neuroactive steroid hormone with multiple functions in the brain. Numerous clinical and experimental data link various Vitamin D-related dysfunctions to epilepsy. Here, we study the role of Vitamin D receptors (VDRs) in experimental epilepsy in mice. To examine this problem, we assessed the seizure profiles in VDR knockout mice following a systemic injection of pentylenetetrazole (70 mg/kg). Overall, compared to the wild-type (WT) 129S1 mice ( n = 10 in each group), the VDR knockout group significantly demonstrated shorter latencies to the onset, higher Racine scores and increased mortality rates. Our findings suggest that VDRs modulate seizure susceptibility in mice, and that the Vitamin D/VDR endocrine system may be involved in the pathogenesis of epilepsy.

Increased hippocampal CA3 vulnerability to low-level kainic acid following lateral fluid percussion injury.

This study was designed to determine whether a secondary increase in neuronal activity induced by a low dose of kainic acid (KA), a glutamate analogue, exacerbates the anatomical damage in hippocampal regions following a mild lateral fluid percussion (LFP) brain injury. KA (9 mg/kg) was injected intraperitoneally in LFP-injured rats (n = 16) 1 h post-trauma. The neuronal loss in the CA3, CA4, and hilar regions at 7 days was quantified by two-dimensional cell counts. Hippocampal activation 15 min following KA injection was assessed by measuring local glucose metabolic rates (lCMR(glc)). Following LFP + KA, the ipsilateral side exhibited a 62.7%, 75.7%, and 52.1% decrease in the number of CA3, CA4 and hilar neurons, respectively, compared to naive rats (n = 3). These CA3 and CA4 neuronal counts were also significantly decreased compared to LFP + saline (n = 5) and sham + KA (n = 9) groups. The median Racine Score, used to rate the severity of behavioral seizures, was 4 in LFP + KA and 2 in sham + KA groups (p < 0.015), suggesting a reduction in seizure threshold following injury. lCMRglc in CA3 following LFP + KA was 121.8 +/- 2.0 (mean +/- SE) ipsilaterally and 71.5 +/- 5.4 contralaterally (p < 0.0012). No changes were found in the BBB permeability as measured by [(14)C]aminoisobutyric acid in CA3, CA4, and hilar regions. We conclude that the presence of low-level KA 1 h after LFP dramatically increases the extent of hippocampal activation and induces a striking loss of ipsilateral CA3 and CA4 pyramidal neurons. Neuronal excitation during a time of cellular vulnerability may trigger or amplify the cycle of secondary damage in functionally impaired, but potentially viable, tissue.

The intracerebral administration of phenytoin using controlled-release polymers reduces experimental seizures in rats

Purpose: An alternative strategy for the treatment of intractable seizures may be to administer anticonvulsants directly into the brain near the site of a seizure focus using controlled-release polymers. We describe the pharmacokinetics of a phenytoin–ethylene-vinyl acetate (EVAc) controlled-release polymer and report the reduction of seizures in a cobalt-induced rat model of epilepsy with the intracerebral delivery of phenytoin using surgically implanted polymers. Methods: In the pharmacokinetics study, the drug release rate of 50%-loaded phenytoin–EVAc polymers ( n=3) was determined in vitro over 15 weeks initially and then several months later (over a 2-week period after 1 year of in vivo release). In the efficacy study, 85 rats underwent implantation of skull-mounted cortical electrodes for electrocorticography (ECoG) and then underwent application of cobalt chloride to the cerebral cortex for the induction of seizures. Rats in the treatment group ( n=9) underwent surgical implantation of phenytoin–EVAc polymers and rats in the control group ( n=10) underwent implantation of empty EVAc polymers. In the morbidity study, the potential histologic pathology of the intracerebral delivery of increasing doses of phenytoin from the polymer (10, 20, 30, and 50% loading) was assessed. Results: Phenytoin was released in vitro from EVAc polymers in a controlled fashion with an initial release of 0.20% of the total loaded dose per week and a continued release of 0.70% of the total loaded dose per week after 365 days of implantation in the brain. The intracerebral controlled-release of phenytoin resulted in a statistically significant reduction in seizure activity in the treatment group as evidenced by lower Racine scores. The four groups of rats ( n=5 per group) that underwent intracerebral implantation of 10, 20, 30, or 50%-loaded phenytoin–EVAc polymers displayed expected average weight gain and normal behavior over 365 days. One rat in the 50% group, however, died 354 days after polymer implantation for undetermined reasons. Conclusions: The intracerebral delivery of phenytoin using an EVAc polymer, which will release this drug for a calculated period of 3.5 years, resulted in a significant reduction in seizures in a rat model of cobalt-induced epilepsy by both behavioral and ECoG criteria. In rats, the long-term interstitial delivery of phenytoin in the brain was not associated with any deleterious effects.