Limbic Status Epilepticus Research Articles

Locus Coeruleus and Neuronal Plasticity in a Model of Focal Limbic Epilepsy

A lesion of the noradrenergic nucleus Locus Coeruleus (LC) converts sporadic seizures evoked by microinfusion of bicuculline into the anterior piriform cortex (APC) of rats into limbic status epilepticus (SE). The purpose of this study was to evaluate the chronic effects of this new model of SE on the onset of secondary epileptogenesis. We further related the loss of noradrenaline (NE) with hippocampal mossy fiber sprouting. Male Sprague Dawley rats were treated with systemic saline or DSP-4 (a neurotoxin selective for noradrenergic terminals originating from the LC), microinfused with bicuculline into the APC three days later, and sacrificed after 45 days. Naïve and DSP-4 pretreated sham-operated rats served as respective controls. The following evaluations were performed: (a) monitoring of acute seizures and delayed occurrence of spontaneous recurrent seizures (SRS); (b) NE levels in the hippocampus, frontal and olfactory cortex; (c) occurrence of mossy fiber sprouting into the inner molecular layer of the dentate gyrus of the dorsal hippocampus. In 30% of rats lacking noradrenergic terminals, SE evoked from the APC was followed by SRS. Conversely, seizures evoked in intact rats did not result in chronic epileptogenesis. Seizures/SE did not modify NE levels as compared with baseline levels both in naïve and DSP-4-pretreated rats. Rats undergoing SE following DSP-4 + bicuculline developed SRS which were accompanied by hippocampal mossy fiber sprouting. Noradrenergic loss converts focally induced sporadic seizures into an epileptogenic SE, which is accompanied by mossy fiber sprouting within the dentate gyrus.

Temperature regulation is compromised in experimental limbic status epilepticus

Temperature dysregulation is well known in generalized convulsive status epilepticus but so far has not been reported in non-convulsive forms. In order to detect possible subtle alterations, we have analyzed the capability to compensate for external cooling in an animal model of limbic status epilepticus. Rats with electrically induced self-sustaining status epilepticus (SSSE) ( n = 6) as well as rats without electrical stimulation ( n = 6) were cooled for 3 h and then rewarmed for another hour. The time course of changes in epidural temperature in animals of both groups that underwent cooling and in control rats that were not cooled and not stimulated ( n = 6) was compared. In animals with limbic SSSE, temperature fell continuously and was significantly lower at all time points under cooling as compared with each of the two other groups. In animals that were not stimulated, temperature under cooling fell by 1 to 2 °C only and was not significantly different at any time point as compared with controls. The effect of cooling was reversible in both groups. The current data indicate that temperature homeostasis in limbic status epilepticus is markedly disturbed. This finding may suggest ictal involvement of primary thermoregulatory neurons in the anterior hypothalamus probably by spread of epileptic activity from temporo-mesial structures.

Levetiracetam: Antiepileptic Properties and Protective Effects on Mitochondrial Dysfunction in Experimental Status Epilepticus

To assess the anticonvulsant activity of the novel antiepileptic drug, levetiracetam (LEV) in a model of self-sustaining limbic status epilepticus, and to measure the consequence of LEV treatment on the pattern of mitochondrial dysfunction known to occur after status epilepticus (SE). The rat perforant pathway was stimulated for 2 h to induce self-sustaining status epilepticus (SSSE). Stimulated rats were assigned to one of three treatment groups, receiving intraperitoneal injections of saline, 200 mg/kg LEV, or 1,000 mg/kg LEV, 15 min into SSSE and at 3 times over the next 44-h period. All animals received diazepam after 3-h SSSE to terminate seizures. Forty-four hours later, the hippocampi were extracted and prepared for electrochemical high-performance liquid chromatography (HPLC), to measure reduced glutathione levels, and for spectrophotometric assays to measure activities of mitochondrial enzymes (aconitase, alpha-ketoglutarate dehydrogenase, citrate synthase, complex I, and complex II/III). These parameters were compared between treatment groups and with sham-operated rats. LEV administration did not terminate seizures or have any significant effect on spike frequency, although rats that received 1,000 mg/kg LEV did exhibit improved behavioral seizure parameters. Significant biochemical changes occurred in saline-treated stimulated rats compared with shams: with reductions in glutathione, alpha-ketoglutarate dehydrogenase, aconitase, citrate synthase, and complex I activities. Complex II/III activities were unchanged throughout. Rats that received 1,000 mg/kg LEV had significantly improved biochemical parameters, in many instances, comparable to sham control levels. Despite continuing seizures, administration of LEV (1,000 mg/kg) protects against mitochondrial dysfunction, indicating that in addition to its antiepileptic actions, LEV may have neuroprotective effects.

Depletion of reduced glutathione precedes inactivation of mitochondrial enzymes following limbic status epilepticus in the rat hippocampus

The time course and critical determinants of mitochondrial dysfunction and oxidative stress following limbic status epilepticus (SE) were investigated in hippocampal sub-regions of an electrical stimulation model in rats, at time points 4–44 h after status. Mitochondrial and cytosolic enzyme activities were measured spectrophotometrically, and reduced glutathione (GSH) concentrations by HPLC, and compared to results from sham controls. The earliest change in any sub-region was a fall in GSH, appearing as early as 4 h in CA3 (−13%, p < 0.05), and persisting at all time points. This was followed by a transient fall in complex I activity (CA3, 16 h, −13%, p < 0.05), and later changes in aconitase (CA1,−18% and CA3, −22% at 44 h, p < 0.05). The activity of the cytosolic enzyme glyceraldehyde-3-phosphate-dehydrogenase was unaffected at all time points. It is known that GSH levels are dependent both on redox status, and on the availability of the precursor cysteine, in turn dependent on the cysteine/glutamate antiporter, for which extracellular glutamate concentrations are rate limiting. Both mechanisms are likely to contribute indirectly to GSH depletion following seizures. That a relative deficiency in GSH precedes later changes in the activities of complex I and aconitase in vulnerable hippocampal sub-regions, occurring within a clinically relevant therapeutic time window, suggests that strategies to boost GSH levels and/or otherwise reduce oxidative stress following seizures, deserve further study, both in terms of preventing the biochemical consequences of SE and the neuronal dysfunction and clinical consequences.

Status epilepticus induces time-dependent neuronal and astrocytic expression of interleukin-1 receptor type I in the rat limbic system

Interleukin-1β is rapidly synthesized by glia after the induction of seizures. Recent evidence shows that endogenous IL-1β has proconvulsant actions mediated by interleukin-1 receptor type I. This receptor also mediates interleukin-1β effects on neuronal susceptibility to neurotoxic insults.In this study, we investigated the basal and seizure-induced expression of interleukin-1 receptor type I in rat forebrain to identify the cells targeted by interleukin-1β during epileptic activity.Self-sustained limbic status epilepticus was induced in rats by electrical stimulation of the ventral hippocampus. Interleukin-1 receptor type I immunoreactivity was barely detectable in neurons in control brain tissue. During status epilepticus, interleukin-1 receptor type I was induced in the hippocampal neurons firstly, and several hours later in astrocytes localized in limbic and extralimbic areas. Neuronal interleukin-1 receptor type I expression in the hippocampus outlasted the duration of spontaneous electroencephalographic seizure and was not observed in degenerating neurons. Astrocytic expression occurred transiently, between six and 18 h after the induction of status epilepticus and was invariably found in regions of neuronal damage.These time-dependent, cell- and region-specific changes in interleukin-1 receptor type I expression during status epilepticus suggest that interleukin-1 receptor type I in neurons mediates interleukin-1β-induced fast changes in hippocampal excitability while interleukin-1 receptor type I receptors in astrocytes may mediate interleukin-1β effects on neuronal survival in hostile conditions.

Regulation of limbic status epilepticus by hippocampal galanin type 1 and type 2 receptors

It has been well established that galanin is a potent endogenous anticonvulsant peptide. However, the role of galanin receptor subtypes in mediating anticonvulsant effects of the peptide is poorly understood. Using pharmacological, transgenic and antisense approaches, we examined the involvement of galanin receptors GalR1 and GalR2 in regulating seizures and associated neuronal degenerative changes. In the rat model of status epilepticus (SE) induced by electrical stimulation of perforant path, in vivo uncoupling of G protein coupled receptors (GPCR) through intrahippocampal administration of pertussis toxin (PTX) facilitated the initiation of SE, and increased the severity of the established SE. Injection of a non-selective GalR1/GalR2 agonist galanin (1-29) and a preferential GalR2 agonist galanin (2-11) into the hippocampus of PTX-pretreated rats revealed that while during early phase of SE galanin inhibited seizures predominantly through GalR1, GalR2 mediated anticonvulsant effects of the peptide during advanced stage of SE. GalR1 knockout mice showed increased severity of both pilocarpine- and perforant path stimulation -induced SE, compared to wild type (WT) littermates. In GalR1 knockout animals SE led to more severe and wider-spread hippocampal injury, than in WT. Focal downregulation of GalR2, which had been achieved in rats by intrahippocampal infusion of anti-GalR2 peptide nucleic acid (PNA) antisense, significantly increased the severity of perforant path stimulation- induced SE. Downregulation of GalR2 led to mild injury to hilar interneurons and potentiated seizure-induced hippocampal damage. In conclusion, both GalR1 and GalR2 mediate anticonvulsant effects of galanin. GalR1 and GalR2 exhibit differential effects on the initiation and the maintenance phases of SE. Activation of both galanin receptor subtypes exerts neuroprotective effects under conditions of excitotoxic injury.

Limbic Self‐sustaining Status Epilepticus in Rats Is Not Associated with Hyperthermia

To evaluate the impact of limbic status epilepticus on temperature. The perforant path in freely moving rats was stimulated electrically for 120 min to induce self-sustaining status epilepticus (SSSE). For 150 min after the end of stimulation, epidural temperature and electrographic and clinical seizure activity were assessed in animals with limbic and motor SSSE, as well as in animals without development of SE. Temperature in all animals with SSSE was elevated by 1.5+/-0.8 degrees C after the end of stimulation compared with baseline values (p<0.01). In animals with pure limbic SE, temperature decreased continuously to baseline values over the 150-min period of observation. In contrast, in animals with motor SSSE, temperature remained elevated during continuing epileptic activity and was still significantly higher 150 min after the end of stimulation compared with baseline (p<0.01). In animals that did not develop SSSE, temperature was not changed after the end of electrical stimulation and in the 150 min thereafter compared with baseline values. The results indicate that hyperthermia as seen in SE is the consequence of motor convulsions and not of epileptic activity itself, as seen in limbic SSSE.

Quantitative evaluation of central-type benzodiazepine receptors with [125I] Iomazenil in experimental epileptogenesisI. The rat kainate model of temporal lobe epilepsy

This study aimed at quantitatively evaluating hippocampal central-type benzodiazepine receptors (BZRs) in the kainate model of temporal lobe epilepsy (TLE) by in vitro autoradiography (ARG) using [125I] Iomazenil (IMZ) specific ligand for central-type BZRs. Kainate (1 μg/0.5 μl) was injected into the left amygdala to induce limbic status epilepticus. One, three, or six months after injection, in vitro ARG with [125I] IMZ and cell counts were performed in the hippocampal CA1–4 regions and dentate gyrus ipsilateral to the kainate injection site, and were compared with the vehicle-injected control group. In all kainate-treated rats, clear pyramidal neuron loss was observed in left hippocampal areas CA1–4. Compared with the control group, progressive reduction of [125I] IMZ binding was also observed. This resulted in a marked binding decrease paralleling pyramidal neuron loss in hippocampal areas CA1 (down to 83% of control), CA2 (76%), CA3 (75%), and CA4 (90%) at 6 months after kainate administration. Conversely, [125I] IMZ binding significantly increased in the dentate gyrus (up to 106% of control) at 1 month, but returned to nearly normal at 3–6 months. These results suggest that central-type BZR neuroimaging is useful in detecting hippocampal sclerosis in the mesial TLE, though central BZR alterations differ depending on hippocampal subfields and post-seizure time-courses.

Quantitative evaluation of central-type benzodiazepine receptors with [ 125I] Iomazenil in experimental epileptogenesis : I. The rat kainate model of temporal lobe epilepsy

This study aimed at quantitatively evaluating hippocampal central-type benzodiazepine receptors (BZRs) in the kainate model of temporal lobe epilepsy (TLE) by in vitro autoradiography (ARG) using [ 125I] Iomazenil (IMZ) specific ligand for central-type BZRs. Kainate (1 μg/0.5 μl) was injected into the left amygdala to induce limbic status epilepticus. One, three, or six months after injection, in vitro ARG with [ 125I] IMZ and cell counts were performed in the hippocampal CA1–4 regions and dentate gyrus ipsilateral to the kainate injection site, and were compared with the vehicle-injected control group. In all kainate-treated rats, clear pyramidal neuron loss was observed in left hippocampal areas CA1–4. Compared with the control group, progressive reduction of [ 125I] IMZ binding was also observed. This resulted in a marked binding decrease paralleling pyramidal neuron loss in hippocampal areas CA1 (down to 83% of control), CA2 (76%), CA3 (75%), and CA4 (90%) at 6 months after kainate administration. Conversely, [ 125I] IMZ binding significantly increased in the dentate gyrus (up to 106% of control) at 1 month, but returned to nearly normal at 3–6 months. These results suggest that central-type BZR neuroimaging is useful in detecting hippocampal sclerosis in the mesial TLE, though central BZR alterations differ depending on hippocampal subfields and post-seizure time-courses.

The role of norepinephrine in epilepsy: from the bench to the bedside

This article provides a brief review of the role of norepinephrine (NE) in epilepsy, starting from early studies reproducing the kindling model in NE-lesioned rats, through the use of specific ligands for adrenergic receptors in experimental models of epilepsy, up to recent advances obtained by using transgenic and knock-out mice for specific genes expressed in the NE system. Data obtained from multiple experimental models converge to demonstrate the antiepileptic role of endogenous NE. This effect predominantly consists in counteracting the development of an epileptic circuit (such as in the kindling model) rather than increasing the epileptic threshold. This suggests that NE activity is critical in modifying epilepsy-induced neuronal changes especially on the limbic system. These data encompass from experimental models to clinical applications as recently evidenced by the need of an intact NE innervation for the antiepileptic mechanisms of vagal nerve stimulation (VNS) in patients suffering from refractory epilepsy. Finally, recent data demonstrate that NE loss increases neuronal damage following focally induced limbic status epilepticus, confirming a protective effect of brain NE, which has already been shown in other neurological disorders.

Patterns of seizures, hippocampal injury and neurogenesis in three models of status epilepticus in galanin receptor type 1 (GalR1) knockout mice

The neuropeptide galanin exhibits anticonvulsant effects in experimental epilepsy. Two galanin receptor subtypes, GalR1 and GalR2, are present in the brain. We examined the role of GalR1 in seizures by studying the susceptibility of GalR1 knockout (KO) mice to status epilepticus (SE) and accompanying neuronal injury. SE was induced in GalR1 KO and wild type (WT) mice by Li-pilocarpine, 60 min electrical perforant path stimulation (PPS), or systemic kainic acid (KA). Seizures were analyzed using Harmonie software. Cell injury was examined by FluoroJade B- and terminal deoxynucleotidyl transferase-mediated uridine triphosphatenick end labeling; neurogenesis was studied using bromodeoxyuridine labeling. Compared with WT littermates, GalR1 KO showed more severe seizures, more profound injury to the CA1 pyramidal cell layer, as well as injury to hilar interneurons and dentate granule cells upon Li-pilocarpine administration. PPS led to more severe seizures in KO, as compared with WT mice. No difference in the extent of neuronal degeneration was observed between the mice of two genotypes in CA1 pyramidal cell layer; however, in contrast to WT, GalR1 KO developed mild injury to hilar interneurons on the side of PPS. KA-induced seizures did not differ between GalR1 KO and WT animals, and led to no injury to the hippocampus in either of experimental group. No differences were found between KO and WT mice in both basal and seizure-induced neuronal progenitor proliferation in all seizure types. Li-pilocarpine led to more extensive glia proliferation in GalR1 KO than in WT, and in both mouse types in two other SE models. In conclusion, GalR1 mediate galanin protection from seizures and seizure-induced hippocampal injury in Li-pilocarpine and PPS models of limbic SE, but not under conditions of KA-induced seizures. The results justify the development and use of GalR1 agonists in the treatment of certain forms of epilepsy.

Neuroprotective activity of antazoline against neuronal damage induced by limbic status epilepticus

Imidazoline drugs exert neuroprotective effects in cerebral ischaemia models. They also have effects against mouse cerebellar and striatal neuronal death induced by N-methyl- d-aspartate (NMDA) through the blockade of NMDA currents. Here, we investigated the effects of antazoline on NMDA toxicity and current in rat hippocampal neuronal cultures, and on an in vivo model of status epilepticus. In hippocampal cultures, antazoline (30 μM) decreased NMDA-mediated neurotoxicity and also blocked the NMDA current with voltage-dependent and fast-reversible action (inhibition by 85±3% at −60 mV). Status epilepticus was induced by injecting pilocarpine (200 nmol) directly into the right pyriform cortex of male adult rats. The rats then received immediately three consecutive i.p. injections at 30-min intervals of either PBS (control group) or antazoline at 10 mg/kg (low-dose group) or at 45 mg/kg (high-dose group). During the 6-h recording, status epilepticus lasted more than 200 min in all groups. In the high-dose group only, seizures completely ceased 1 h after the third injection of antazoline, then started again 1 h later. Rats were killed 1 week later, and Cresyl Violet-stained sections of their brain were analysed for damage quantification. On the ipsilateral side to the pilocarpine injection, pyriform cortex and hippocampal CA1 and CA3 areas were significantly protected in both antazoline-treated groups, whilst prepyriform and entorhinal cortices were only in the high-dose group. On the contralateral side to the pilocarpine injection, only the hippocampal CA3 area was significantly protected in the low-dose group, but all investigated structures were in the high-dose group. In conclusion, antazoline is a potent neuroprotective drug in different models of neuronal primary culture, as previously shown in striatal and cerebellar granule neurons [Neuropharmacology 39 (2000) 2244], and here in hippocampal neurons. Antazoline is also neuroprotective in vivo in the intra-pyriform pilocarpine-induced status epilepticus model.

A damage to locus coeruleus neurons converts sporadic seizures into self‐sustaining limbic status epilepticus

Various studies demonstrated that the neurotransmitter norepinephrine (NE) plays a relevant role in modulating seizures; in particular, a powerful effect consists in delaying the kindling of limbic areas such as the amygdala and hippocampus. Given the rich NE innervation of limbic regions, we selected a sensitive trigger area, the anterior piriform cortex, to test whether previous loss of noradrenergic terminals modifies sporadic seizures in rats. The damage to locus coeruleus terminals was produced by using the selective neurotoxin N-(-2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, 60 mg/kg i.p.). In intact rats, bicuculline (a GABA-A antagonist, 118 pmol) microinfused into this area produced sporadic seizures, while in rats previously injected with DSP-4, bicuculline determined long-lasting self-sustaining status epilepticus. In intact rats, sporadic seizures were accompanied by a marked increase in norepinephrine release in the contralateral piriform cortex, while in locus coeruleus-lesioned rats this phenomenon was attenuated. While bicuculline-induced sporadic seizures were prevented by the focal infusion of amino-7-phosphonoheptanoic acid (AP-7, a selective NMDA antagonist), or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide (NBQX, a selective non-NMDA antagonist), status epilepticus obtained in norepinephrine-lesioned rats was insensitive to AP-7 but was still inhibited by NBQX. By using fluorescent staining for damaged (Fluoro-Jade B) and intact (DAPI) neurons, as well as cresyl violet, we found that rats undergoing status epilepticus developed neuronal loss in various limbic regions. This study demonstrates a powerful effect of noradrenergic terminals in regulating the onset of limbic status epilepticus and its sensitivity to specific glutamate antagonists.

GluR5 kainate receptors, seizures, and the amygdala.

The amygdala is a critical brain region for limbic seizure activity, but the mechanisms underlying its epileptic susceptibility are obscure. Several lines of evidence implicate GluR5 (GLU(K5)) kainate receptors, a type of ionotropic glutamate receptor, in the amygdala's vulnerability to seizures and epileptogenesis. GluR5 mRNA is abundant in temporal lobe structures including the amygdala. Brain slice recordings indicate that GluR5 kainate receptors mediate a portion of the synaptic excitation of neurons in the rat basolateral amygdala. Whole-cell voltage-clamp studies demonstrate that GluR5 kainate receptor-mediated synaptic currents are inwardly rectifying and are likely to be calcium permeable. Prolonged activation of basolateral amygdala GluR5 kainate receptors results in enduring synaptic facilitation through a calcium-dependent process. The selective GluR5 kainate receptor agonist ATPA induces spontaneous epileptiform bursting that is sensitive to the GluR5 kainate receptor antagonist LY293558. Intra-amygdala infusion of ATPA in the rat induces limbic status epilepticus; in some animals, recurrent spontaneous seizures occur for months after the ATPA treatment. Together, these observations indicate that GluR5 kainate receptors have a unique role in triggering epileptiform activity in the amygdala and could participate in long-term plasticity mechanisms that underlie some forms of epileptogenesis. Accordingly, GluR5 kainate receptors represent a potential target for antiepileptic and antiepileptogenic drug treatments. Most antiepileptic drugs do not act through effects on glutamate receptors. However, topiramate at low concentrations causes slow inhibition of GluR5 kainate receptor-mediated synaptic currents in the basolateral amygdala, indicating that it may protect against seizures, at least in part, through suppression of GluR5 kainate receptor responses.

Can the Long-Term Outcome After Status Epilepticus Be Modified?

Phenobarbital and MK-801, but Not Phenytoin, Improve the Long-Term Outcome of Status Epilepticus Prasad A, Williamson JM, Bertram EH Ann Neurol 2002;51:175–181 To examine the effect of therapy on status epilepticus (SE) acutely and on long-term outcome, we compared three drugs with three different mechanisms. Phenobarbital, MK-801, and phenytoin were administered at 1, 2, and 4 hours after initiation of limbic status epilepticus by “continuous” hippocampal stimulation in rats. We evaluated the effects of these drugs on the course of SE and the subsequent development of chronic epilepsy. Phenobarbital and MK-801 were superior to phenytoin in suppressing SE and in preventing chronic epilepsy. There was no benefit if treatment was given 2 hours after the initiation of SE. Phenobarbital was most effective in suppressing electrographic seizure activity, but MK-801 had a slightly wider window for the prevention of chronic epilepsy. Early treatment, rather than electrographic suppression of SE, correlated with prevention of chronic epilepsy. This study shows that the drugs administered, which have different mechanisms of action, have clear differences in altering the outcomes. The findings suggest that studies of SE treatment should examine the effect of therapy on SE itself, as well as the long-term benefits of each treatment. The use of N-methyl-D-aspartate receptor antagonists should be considered early in the treatment of SE. Seizure-Induced Memory Impairment is Reduced by Choline Supplementation Before or After Status Epilepticus Holmes GL, Yang Y, Liu Z, Cermak JM, Sarkisian MR, Stafstrom CE, Neill JC, Blusztajn JK Epilepsy Res 2002;48:3–13 Prenatal choline supplementation can protect rats against cognitive deficits induced by status epilepticus induced by the cholinergic agent pilocarpine [J. Neurosci. 20 (2000) 1]. In the present day, we have extended this novel finding by investigating the effects of pre- and postnatal choline supplementation in memory deficits associated with status epilepticus induced with kainic acid (KA). In the first experiment pregnant rats received a normal, choline-supplemented, or choline deficient diet starting on the 11th day of gestation and continuing until postnatal (P) 7. At P42, rats were given a convulsant dosage of KA. Two weeks following the KA-induced status epilepticus rats underwent testing of visual-spatial memory using the Morris water maze test. Rats receiving supplemental choline performed better in the water maze than the deficient and control groups. Moreover, the activity of hippocampal choline acetyltransferase was 18% lower in the choline deficient animals as compared with the other two groups. In the second experiment we administered KA to P35 rats that had been given a normal diet. Following the status epilepticus the rats were given a choline-supplemented or control diet for 4 weeks and then tested in the water maze. Rats receiving choline supplementation performed far better than rats receiving a regular diet. This study demonstrates that choline supplementation prior to or following KA-induced status epilepticus can protect rats from memory deficits induced by status epilepticus.

Mitochondrial dysfunction associated with neuronal death following status epilepticus in rat

Status epilepticus (SE) in humans and animal models results in significant cerebral damage and an increased risk of subsequent seizures, associated with a characteristic pattern of neuronal loss particularly affecting the hippocampus. Seizure related cell death is considered to be excitotoxic, but studies have been limited, concentrating on terminal events rather than initial mechanisms. We have studied the biochemical events in the first few days following SE. Self-sustaining limbic SE was induced in adult rats using perforant path stimulation, and animals were allowed to recover. Biochemical studies were performed at 16, 44 h and 8 days following SE, using spectrophotometric enzyme assays and HPLC on regional brain homogenates compared with those from sham-operated controls. Haematoxylin and eosin histology was also undertaken at each time point. Brain aconitase and α-ketoglutarate dehydrogenase (αKDH) activity were both significantly ( P<0.05) reduced by approximately 20% in the first 16–44 h following status, but had returned to normal by 8 days. These enzymes are part of the tri-carboxylic acid (Krebbs) cycle in the mitochondrial matrix, and are known to be sensitive to free radical, especially peroxynitrite damage. There was a similar decrease in reduced glutathione levels. Histological studies confirmed evidence of acute neuronal damage up to 44 h, and neuronal loss by 8 days. This is the first in vivo demonstration of this pattern of mitochondrial dysfunction and loss of brain glutathione following SE. The pattern of abnormalities is consistent with reversible mechanisms being involved in excitotoxic cell damage. This, together with the timing of changes, suggests new avenues for therapeutic intervention.

Regional accumulation of 14C-zonisamide in rat brain during kainic acid-induced limbic seizures.

Zonisamide (ZNS) is an antiepileptic drug developed in Japan. Various experimental studies have investigated the effects of ZNS. However, the mechanism of action of ZNS against limbic seizures and secondary generalization is not well-known. We studied ictal regional accumulation of ZNS in the rat brain during kainic acid (KA)-induced limbic status epilepticus. Fourteen male Wistar rats underwent a stereotactic operation. For recording the electroencephalogram (EEG), electrodes were placed in the left amygdala (LA), left dorsal hippocampus, and over the left sensorimotor cortex. For microinjection, a stainless steel cannula was also inserted into the LA. Seven days after surgery, rats were anesthetized and a catheter was inserted into the femoral vein. The animals were immobilized and allowed to recover from anesthesia for at least two hours. In eight rats, 1.0 microL (1.0 microg) of KA was injected into the LA, and 1.0 microL of phosphate buffer solution was injected into the LA in six control rats. Sixty minutes after injection, 14C-ZNS was administered intravenously, and an autoradiographic study was done. During limbic status epilepticus, only seizures in the sensorimotor cortex were markedly attenuated a few minutes after 14C-ZNS administration. Additionally, high uptake of 14C-ZNS was noted ipsilaterally in the sensorimotor cortex, parietal cortex and thalamus (lateral portion). In control rats, no EEG change was seen, and distribution of 14C-ZNS was rather homogeneous. These results suggested that ZNS suppresses secondary generalization of limbic seizures by a direct effect on the cerebral cortex.

GABAA receptor antisense epilepsy: Histological changes following infusion of antisense oligodeoxynucleotide to GABAA receptor γ2 subunit into rat hippocampus

A deficiency of neuronal inhibition mediated by γ-aminobutyric acid (GABA) via the GABAA receptor complex has been hypothesised to be a central factor in epileptogenesis. Intrahippocampal infusion of antisense oligodeoxynucleotide (ODN) to the GABAA receptor γ2 subunit in rats leads to electrographic limbic status epilepticus. In this model, epileptic phenomena are accompanied by loss of hippocampal neurones. The purpose of the present study was to investigate the time-course of morphological changes following hippocampal antisense 'knockdown' of the GABAA receptor γ2 subunit. γ2 subunit antisense ODN was infused continuously into the right hippocampus for periods between 1 and 5 days. After about 4 days of infusion, pronounced neurodegenerative changes were consistently observed within the ipsilateral hippocampus. In general, marked loss of CA3 pyramidal cells was found. The notion that the histological changes induced by the antisense ODN were specific to the applied ODN sequence was supported by the finding that a mismatch control ODN did not induce neurodegenerative changes, except for a small lesion in the immediate vicinity of the infusion site. Extensive ipsilateral hippocampal infiltration with monocytes and macrophages was a feature of antisense ODN infusion, but was considerably less pronounced after the infusion of control ODN. Immunocytochemistry using an antibody labeling glial fibrillary acidic protein (GFAP), revealed marked astroglial hypertrophy/proliferation after 4 days of antisense treatment, i.e., coincident with the development of neurodegeneration, in the ipsilateral hippocampus. At this time GFAP-immunoreactivity was also evident in the contralateral hippocampus, indicating contralateral spread of seizure activity. [Neurol Res 2001; 23: 39-46]

Diagnosis and treatment of nonconvulsive status epilepticus.

Nonconvulsive status epilepticus (SE) is not uncommon and comprises at least one-third of all cases of SE. However, nonconvulsive SE consists of very different syndromes, a common feature being the difficulty in making the diagnosis. In this review, nonconvulsive SE is divided into typical absence SE, complex partial SE, nonconvulsive SE in patients with learning difficulties (including electrical SE during sleep, atypical absence SE and tonic SE), and nonconvulsive SE in coma. These conditions have different prognoses and treatments. The diagnosis of these conditions is critically dependent on EEG. When the EEG demonstrates typical ictal patterns, the diagnosis is usually straightforward. However, in many circumstances the EEG has to be differentiated from encephalopathic patterns, and this differentiation can prove troublesome, although the clinical and electrographic response to treatment can prove helpful. Nonconvulsive SE in patients with learning difficulties possibly provides the greatest diagnostic difficulty; the clinical presentation can be subtle resulting in the diagnosis being frequently missed. Whether the neuronal damage that occurs in convulsive SE and in animal models of limbic SE also occurs in nonconvulsive SE in humans is still a matter of debate. There are critical differences between the animal models and the human condition. Indeed, the prognosis of nonconvulsive SE is usually dependent on the underlying aetiology rather than the persistence of electrographic discharges. Because of these doubts, a more conservative approach to the treatment of particular types of nonconvulsive SE (those with a better prognosis) has been taken in this article. Thus, in most instances, oral benzodiazepines for the treatment of typical absence SE and complex partial SE are recommended. In some circumstances intravenous medication is necessary, but in neither condition is anaesthetic coma recommended. This contrasts with nonconvulsive SE in coma in which a more aggressive approach is suggested. Until there are more relevant animal models, and controlled trials of conservative versus more aggressive treatment, treatment regimens for nonconvulsive SE will remain largely speculative.

Synaptic connections from multiple subfields contribute to granule cell hyperexcitability in hippocampal slice cultures.

Limbic status epilepticus and preparation of hippocampal slice cultures both produce cell loss and denervation. This commonality led us to hypothesize that morphological and physiological alterations in hippocampal slice cultures may be similar to those observed in human limbic epilepsy and animal models. To test this hypothesis, we performed electrophysiological and morphological analyses in long-term (postnatal day 11; 40-60 days in vitro) organotypic hippocampal slice cultures. Electrophysiological analyses of dentate granule cell excitability revealed that granule cells in slice cultures were hyperexcitable compared with acute slices from normal rats. In physiological buffer, spontaneous electrographic granule cell seizures were seen in 22% of cultures; in the presence of a GABA(A) receptor antagonist, seizures were documented in 75% of cultures. Hilar stimulation evoked postsynaptic potentials (PSPs) and multiple population spikes in the granule cell layer, which were eliminated by glutamate receptor antagonists, demonstrating the requirement for excitatory synaptic transmission. By contrast, under identical recording conditions, acute hippocampal slices isolated from normal rats exhibited a lack of seizures, and hilar stimulation evoked an isolated population spike without PSPs. To examine the possibility that newly formed excitatory synaptic connections to the dentate gyrus contribute to granule cell hyperexcitability in slice cultures, anatomical labeling and electrophysiological recordings following knife cuts were performed. Anatomical labeling of individual dentate granule, CA3 and CA1 pyramidal cells with neurobiotin illustrated the presence of axonal projections that may provide reciprocal excitatory synaptic connections among these regions and contribute to granule cell hyperexcitability. Knife cuts severing connections between CA1 and the dentate gyrus/CA3c region reduced but did not abolish hilar-evoked excitatory PSPs, suggesting the presence of newly formed, functional synaptic connections to the granule cells from CA1 and CA3 as well as from neurons intrinsic to the dentate gyrus. Many of the electrophysiological and morphological abnormalities reported here for long-term hippocampal slice cultures bear striking similarities to both human and in vivo models, making this in vitro model a simple, powerful system to begin to elucidate the molecular and cellular mechanisms underlying synaptic rearrangements and epileptogenesis.