Characterization of the tetanus toxin model of refractory focal neocortical epilepsy in the rat

PURPOSE: To characterize in detail a model of focal neocortical epilepsy. METHODS: Chronic focal epilepsy was induced by injecting 25-50 ng of tetanus toxin or vehicle alone (controls) into the motor neocortex of rats. EEG activity was recorded from electrodes implanted at the injection site, along with facial muscle electromyographic (EMG) activity and behavioral monitoring intermittently for up to 5 months in some animals. Drug responsiveness was assessed by using the antiepileptic drugs (AEDs) diazepam (DZP) and phenytoin (PHT) delivered systemically, while 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), a competitive antagonist at AMPA receptors, was administered directly to the brain to investigate the potential benefits of focal drug delivery. RESULTS: Tetanus toxin induced mild behavioral seizures that persisted indefinitely in all animals. EEG spiking activity, occurring up to 80% of the time, correlated with clinical seizures consisting of interrupted behavioral activity, rhythmic bilateral facial twitching, and periods of abrupt motor arrest. Seizures were refractory to systemic administration of DZP and PHT. However, focal delivery of NBQX to the seizure site reversibly reduced EEG and behavioral seizure activity without detectable side effects. CONCLUSIONS: This study provides a long-term detailed characterisation of the tetanus toxin model. Spontaneous, almost continuous, well-tolerated seizures occur and persist, resembling those seen in neocortical epilepsy, including cortical myoclonus and epilepsia partialis continua. The seizures appear to be similarly resistant to conventional AEDs. The consistency, frequency, and clinical similarity of the seizures to refractory epilepsy in humans make this an ideal model for investigation of both mechanisms of seizure activity and new therapeutic approaches.     

Kainic acid induced hippocampal seizures in rats: comparisons of acute and chronic seizures using intrahippocampal versus systemic injections

Hyppocampal epilepsy is a recently defined syndrome occurring in 65% of all temporal lobe epilepsies as defined by: 1) electrographic (EEG) onset in the hippocampus (HC) prior to EEG seizures elsewhere, 2) post-resection hippocampal sclerosis and mossy fiber synaptic reorganizations and 3) relief of typical complex partial seizures after surgical resection of the hyppocampus. We used intrahippocampal kainic acid injections V² in rats at different developmental ages (postnatal 7 through adult) to develop long term spontaneous HC EEG spikes, EEG seizures, and behavioral seizures. Split-screen video/EEG monitoring demonstrated that this intrahippocampal kainic acid model produced progressive development of: 1) ipsilateral interictal spikes, 2) later polyspike complexes, 3) bilaterally-asynchronous EEG spiking, 4) unilateral HC EEG seizure onsets with occasional secondarily generalized spread to apposite HC and motor cortex to elicit complex partial seizures, and 5) in all seizing rats there was mossy fiber synaptic reorganization, even when injected at age 7 days. These results indicate that the intrahippocampal kainic acid injection model is similar to human hippocampal epilepsy.Supported by NIH Grants NS 02808, NS 31655 (T.L.B.); K08 NS 1603 (G.W.M.); and Fogarty Fellowship TWO 4959 (J.P.L.).     

Increases in NMDAR1 mRNA Levels are Involved in the Generation of Local Afterdischarges in Amygdaloid-Kindled Rats

Purpose : The Noda epileptic rat (NER), which was found in a colony of Crj:Wistar rats, shows spontaneous tonicxlonic convulsions at a frequency of approximately once per 30 h. However, weekly applied acoustic priming consistently induces tonic-clonic convulsion with sound stimuli. When NERs were given sound stimuli (95 dB, 8 kHz, 30 s) weekly from age 3 weeks, the percentage of animals with audiogenic seizures increased with age and was 100% after age 9 weeks. The audiogenic-response score to quantify the intensity of seizure responses also increased with age. Frontal cortical and hippocampal electroencephalograms (EEGs) showed low-voltage spike-and-wave complexes during tonic convulsions, which evolved into high-amplitude spike- or polyspike-and-wave complexes during clonic convulsion. Immediately after cessation of the seizures, the EEG showed a flattening or diffuse slowing. To elucidate the development of the seizure induction by acoustic priming, we examined the epileptiform discharges in the in-terictal EEG as a function of age.
Methods : The acoustic priming was applied to NERs from age 7 to 35 weeks. Cortical and hippocampal EEGs were recorded weekly during the interictal stage for 15 min after a 15-min habituation by using implanted electrodes.
Results : Sporadic spikes were observed predominantly in the hippocampus with age, although they were rarely synchronized in the cortex. Hippocampal spikes were observed in two (33.3%) of six animals at age 7–10 weeks, 23 (82.4%) of 28 animals at age 11–15 weeks, 12 (85.7%) of 14 animals at age 16–19 weeks, and 21 (100%) of 21 animals at age >20 weeks. This development of abnormal hippocampal discharges with acoustic priming was in line with the development of convulsive seizures.
Conclusions : Development of hippocampal excitation induced by acoustic priming is thought to lead to the induction of convulsive seizures in the NERs.     

Single injection of a low dose of pentylenetetrazole leads to epileptogenesis in an animal model of cortical dysplasia

Purpose:   Cortical dysplasia (CD) is one of the most frequent causes of pharmacoresistent focal epilepsy. Despite significant advances in various diagnostic and therapeutic methods, the basic mechanisms of higher susceptibility for seizures in patients with CD are unknown. Animal models of CD present with a lower threshold for seizure induction. The purpose of this study is to further characterize the animal model of in utero radiation-induced CD and to illustrate the effect of a late postnatal second hit (low dose of pentylenetetrazole, PTZ) on the development of spontaneous seizures.
Methods:   Pregnant Sprague–Dawley rats were irradiated on E17 (145 cGy; control group was left untreated). Litters were implanted with bifrontal epidural and hippocampal depth electrodes. After baseline electroencephalography (EEG) recording, animals received 30 mg/kg PTZ and were chronically monitored. Histopathology of the brains was verified.
Results:   No seizures were detected in animals that did not receive PTZ. PTZ-injected irradiated (XRT) rats showed severe prolonged, repetitive seizures during the acute period. During the chronic period, XRT rats had recurrent seizures and epileptiform spikes. PTZ-injected control animals exhibited milder and fewer acute seizures and did not show seizures during the chronic period. Histology was consistent with cortical and hippocampal dysplasia.
Conclusions:   This study shows that a single treatment with a low dose of PTZ renders XRT rats (but not age-matched controls) epileptic, exhibiting spontaneous epileptiform spikes and seizures on EEG. These results might mirror the natural history of patients with CD thought to be caused by prenatal/congenital or perinatal insults.     

Tetanus toxin-induced seizures in infant rats and their effects on hippocampal excitability in adulthood

A new experimental model of developmental epilepsy is reported. Behavioral and EEG features of seizures produced by unilateral intrahippocampal injection of tetanus toxin in postnatal day 9–11 rats, are described. Within 24–72 h of tetanus toxin injection, rat pups developed frequent and often prolonged seizures which included combinations of repetitive wet dog shakes, and wild running-jumping seizures. Intrahippocampal and cortical surface EEG recordings showed that coincident with these behaviors, electrographic seizures occurred not only in the injected hippocampus, but also in the contralateral hippocampus and bilaterally in the neocortex. Analysis of the interictal EEG revealed multiple independent spike foci. One week following tetanus toxin injection, the number of seizures markedly decreased; however, interictal spiking persisted. After injection rats were allowed to mature some were observed to have unprovoked behavioral seizures and/or epileptiform EEG activity. Mature animals were also studied using in vitro slice techniques. Recordings from hippocampal slices demonstrated spontaneous epileptiform burst discharges in the majority of rats which had tetanus toxin induced seizures as infants. These events occurred in area CA3 and consisted of interictal spikes and intracellularly recorded paroxysmal depolarization shifts (PDSs). On rarer occasions, electrographic seizures were recorded. The use of the tetanus toxin model in developing rats may facilitate a better understanding of the unique features of epileptogenesis in the developing brain and the consequences early-life seizures have on brain maturation and the genesis of epileptic conditions in later life.     

The Neuropathology of Hyperthermic Seizures in the Rat

Summary: Purpose: Single and repeated hyperthermic seizures were induced in rats beginning at age 22 days to determine the neuroanatomic consequences to the hippocampus and to compare these changes with those in the hippocampi of patients with temporal lobe epilepsy (TLE) experiencing febrile seizures.
Methods: Hyperthermic seizures were induced by placing rats in a bath of water at 45°C for 4 min. Seizures were visually observed, and some animals also were monitored electroen-cephalographically. Neurodegeneration was examined with a silver stain, whereas granule cell sprouting was detected with the Timm stain.
Results: In a majority of rats, hyperthermia-induced tonicclonic seizures ranged in duration from 30 s to 6 min; the seizure duration increased with the number of seizures. No neurodegeneration was detectable in these animals, although there was sprouting of granule cell collaterals into the inner molecular layer (IML) of the dentate. In a small number of animals, the short seizures evolved into status epilepticus, and neuronal degeneration was present in the hippocampus and other parts of the temporal lobe, and the mediodorsal thalamus.
Conclusions: This study confirms the relation between hyperthermia and seizure occurrence. It shows in particular that, as in the human, only prolonged seizures such as status epilepticus cause a pattern of neurodegeneration similar to that observed in human TLE.     

The development of recurrent seizures after continuous intrahippocampal infusion of methionine sulfoximine in rats: A video-intracranial electroencephalographic study

Glutamine synthetase is deficient in astrocytes in the epileptogenic hippocampus in human mesial temporal lobe epilepsy (MTLE). To explore the role of this deficiency in the pathophysiology of MTLE, rats were continuously infused with the glutamine synthetase inhibitor methionine sulfoximine (MSO, 0.625 μg/h) or 0.9% NaCl (saline control) unilaterally into the hippocampus. The seizures caused by MSO were assessed by video-intracranial electroencephalogram (EEG) monitoring. All (28 of 28) of the MSO-treated animals and none (0 of 12) of the saline-treated animals developed recurrent seizures. Most recurrent seizures appeared in clusters of 2 days' duration (median; range, 1 to 12 days). The first cluster was characterized by frequent, predominantly stage I seizures, which presented after the first 9.5 h of infusion (median; range, 5.5 to 31.7 h). Subsequent clusters of less-frequent, mainly partial seizures occurred after a clinically silent interval of 7.1 days (median; range, 1.8 to 16.2 days). The ictal intracranial EEGs shared several characteristics with recordings of partial seizures in humans, such as a distinct evolution of the amplitude and frequency of the EEG signal. The neuropathology caused by MSO had similarities to hippocampal sclerosis in 23.1% of cases, whereas 26.9% of the animals had minimal neuronal loss in the hippocampus. Moderate to severe diffuse neuronal loss was observed in 50% of the animals. In conclusion, the model of intrahippocampal MSO infusion replicates key features of human MTLE and may represent a useful tool for further studies of the cellular, molecular and electrophysiological mechanisms of this disorder.     

Monoamine variability in the chronic model of atypical absence seizures

Purpose:   We studied the variability of the slow-spike-and-wave discharges (SSWDs) derived from AY-9944 (AY) treatment during brain development of Long-Evans hooded (LEh) rats.
Methods:   Although all LEh rats received the standard dose of AY (7.5 mg/kg), we have observed an intersubject variability of the total SSWD duration at postnatal day (P) 55. Therefore, we set out to investigate the underlying brain levels of norepinephrine (NE), dopamine (DA), and serotonin (5-HT) and its metabolite (5-HIAA), as determined by high-performance liquid chromatography (HPLC) analyses from four different brain regions: thalamus (Th), frontoparietal cortex (Cx), hippocampus (Hp), and brainstem (Bs).
Results:   All brains were obtained after two baseline electrocorticographic (ECoG) recordings with characteristic chronic, recurrent, bilaterally synchronous 4–6 Hz SSWD, at P 55 (336.25 ± 97.23 s/h) and P60 (494.50 ± 150.36 s) (r = 0.951, r² = 0.904, p < 0.005, Pearson product). The thalamic NE levels and the brainstem NE, DA, and 5HT levels were all significantly correlated with baseline SSWD duration at P55 and P60 (p < 0.01, Pearson product).
Conclusion:   Our data indicate that brain monoamine levels may determine the intersubject variability of SSWD duration in AY rats with chronic atypical absence seizures.     

Efficacy of Thyrotropin-Releasing Hormone in the Treatment of Intractable Epilepsy

Purpose : The ictal EEG and magnetoencephalogram (MEG) for gelastic seizures were recorded in a 4-year-old girl with tuberous sclerosis. The sites of origin for the seizure activities were investigated by using an equivalent current dipole (ECD) with the MEG.
Methods : EEG and MEG were recorded simultaneously under the administration of diazepam (DZP). The MEG recording was performed on a system consisting of an array of 64 sensors uniformly distributed over the patient's whole head (CTF, Canada), and the estimated ECDs were superimposed on the magnetic resonance imaging (MRI) images (Siemens, 1.5 Tesla).
Results : Two laughing attacks lasting 5 s each were documented. The ictal EEG showed gradually increasing 11–Hz rhythmic α activities with dominance over the frontocentral areas bilaterally, followed by irregular spike-and-wave discharges. The ictal MEG detected bilateral frontal rhythmic sharp waves before the appearance of the activities on the EEG. The estimated ECDs were localized in the deep white matter of the right frontal lobe on the MRI. However, those dipoles did not coincide with the locations of her cortical tubers.
Conclusions : Although gelastic seizures accompanied with hypothalamic hamartomas are well known, several reports have suggested a temporal or frontal lobe origin for gelastic seizures. In this patient, the ECD indicated that the seizures originated in the frontal lobe, although ictal scalp EEG recordings could not determine the precise focus. Thus, in cases in which the use of ictal scalp EEG fails to show the sites of origin for the seizures, it is recommended that the origins be estimated by using the non-invasive method of ictal MEG analysis.     

A potential model of pediatric posttraumatic epilepsy

Preclinical models of pediatric posttraumatic epilepsy (PTE) are lacking. We hypothesized that traumatic brain injury (TBI), induced by controlled cortical impact, in immature rats would cause electroencephalographic (EEG) epileptiform activity and behavioral seizures. TBI or sham craniotomy was performed on postnatal day 17. Using video-EEG monitoring 4–11 months post-TBI, most TBI rats (87.5%) showed EEG spiking and one had spontaneous, recurrent seizures. Controls showed neither EEG spikes nor electrographic/behavioral seizures. Late seizures were rare after TBI, but EEG spiking was common and may represent a surrogate for PTE.