Seizure-induced Neuronal Death Research Articles

Seizure-induced neuronal death in the immature brain

The response of the developing brain to epileptic seizures and to status epilepticus is highly age-specific. Neonates with their low cerebral metabolic rate and fragmentary neuronal networks can tolerate relatively prolonged seizures without suffering massive cell death, but severe seizures in experimental animals inhibit brain growth, modify neuronal circuits, and can lead to behavioral deficits and to increases in neuronal excitability. Past infancy, the developing brain is characterized by high metabolic rate, exuberant neuronal and synaptic networks and overexpression of receptors and enzymes involved in excitotxic mechanisms. The outcome of seizures is highly model-dependent. Status epilepticus may produce massive neuronal death, behavioral deficits, synaptic reorganization and chronic epilepsy in some models, little damage in others. Long-term consequences are also highly age- and model-dependent. However, we now have some models which reliably lead to spontaneous seizures and chronic epilepsy in the vast majority of animals, demonstrating that seizure-induced epileptogenesis can occur in the developing brain. The mode cell death from status epilepticus is largely (but not exclusively) necrotic in adults, while the incidence of apoptosis increases at younger ages. Seizure-induced necrosis has many of the biochemical features of apoptosis, with early cytochrome release from mitochondria and capase activation. We speculate that this form of necrosis is associated with seizure-induced energy failure.

Oxidative Stress, Mitochondrial Dysfunction, and Epilepsy

Epilepsy is a common and heterogeneous neurological disorder arising from biochemical and molecular events that are incompletely understood. To effectively manage epilepsies, it is important to understand the mechanisms underlying both seizure-induced brain damage as well as seizure initiation. Oxidative stress is emerging as a mechanism that may play an important role in the etiology of seizure-induced neuronal death. Conversely, epileptic seizures are a common occurrence in mitochondrial diseases arising from defects in oxidative phosphorylation. This review focuses on the emerging role of oxidative stress and mitochondrial dysfunction both as a consequence and cause of epileptic seizures.

Formation of the Apaf-1/cytochrome c complex precedes activation of caspase-9 during seizure-induced neuronal death.

In this study we examine the in vivo formation of the Apaf-1/cytochrome c complex and activation of caspase-9 following limbic seizures in the rat. Seizures were elicited by unilateral intraamygdala microinjection of kainic acid to induce death of CA3 neurons within the hippocampus of the rat. Apaf-1 was found to interact with cytochrome c within the injured hippocampus 0-24 h following seizures by co-immunoprecipitation analysis and immunohistochemistry demonstrated Apaf-1/cytochrome c co-localization. Cleavage of caspase-9 was detected approximately 4 h following seizure cessation within ipsilateral hippocampus and was accompanied by increased cleavage of the substrate Leu-Glu-His-Asp-p-nitroanilide (LEHDpNA) and subsequent strong caspase-9 immunoreactivity within neurons exhibiting DNA fragmentation. Finally, intracerebral infusion of z-LEHD-fluoromethyl ketone increased numbers of surviving CA3 neurons. These data suggest seizures induce formation of the Apaf-1/cytochrome c complex prior to caspase-9 activation and caspase-9 may be a potential therapeutic target in the treatment of brain injury associated with seizures.

Zn2+‐induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia- and seizure-induced neuronal death. However, the intracellular signaling pathways involved in Zn2+-induced cell death are largely unknown. In the present study, we investigated the roles of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+-induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 microM), a Zn2+ ionophore, and Zn2+ (10 microM) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+-induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol-3 kinase had little effect on Zn2+-induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+-induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.

Cleavage of Bid May Amplify Caspase-8-Induced Neuronal Death Following Focally Evoked Limbic Seizures

The mechanism by which seizures induce neuronal death is not completely understood. Caspase-8 is a key initiator of apoptosis via extrinsic, death receptor-mediated pathways; we therefore investigated its role in mediating seizure-induced neuronal death evoked by unilateral kainic acid injection into the amygdala of the rat, terminated after 40 min by diazepam. We demonstrate that cleaved (p18) caspase-8 was detectable immediately following seizure termination coincident with an increase in cleavage of the substrate Ile-Glu-Thr-Asp (IETD)-p-nitroanilide and the appearance of cleaved (p15) Bid. Expression of Fas and FADD, components of death receptor signaling, was increased following seizures. In vivo intracerebroventricular z-IETD-fluoromethyl ketone administration significantly reduced seizure-induced activities of caspases 8, 9, and 3 as well as reducing Bid and caspase-9 cleavage, cytochrome c release, DNA fragmentation, and neuronal death. These data suggest that intervention in caspase-8 and/or death receptor signaling may confer protection on the brain from the injurious effects of seizures.

Increased Bcl-w expression following focally evoked limbic seizures in the rat

Control of seizure-induced neuronal death may involve members of the Bcl-2 family of cell death regulating proteins. Bcl-w is a newly described anti-apoptotic member of this family that may confer neuroprotective effects. We therefore investigated Bcl-w expression in rat brain following focally evoked limbic seizures. Seizures were induced by unilateral microinjection of kainic acid into the amygdala of the rat and terminated after 40 min by diazepam. Constitutive Bcl-w expression was detected by Western blotting and immunohistochemistry. Bcl-w expression was increased 4–72 h following seizures within the injured hippocampus. Immunohistochemistry determined Bcl-w was predominantly expressed in neurons and seizures increased Bcl-w immunoreactivity within piriform cortex and surviving regions of the injured hippocampus. These data suggest Bcl-w may be involved in the modulation of seizure-induced brain injury.

Caspase-2 activation is redundant during seizure-induced neuronal death.

Seizure-induced neuronal death may be under the control of the caspase family of cell death proteases. We examined the role of caspase-2 in a model of focally evoked limbic seizures with continuous EEG recording. Seizures were elicited by microinjection of kainic acid into the amygdala of the rat and terminated after 40 min by diazepam. Caspase-2 was constitutively present in brain, mostly within neurons, and was detected in both cytoplasm and nucleus. Cleaved caspase-2 (12 kDa) was detected immediately following seizure termination within injured ipsilateral hippocampus, contiguous with increased Val-Asp-Val-Ala-Asp (VDVADase) activity, a putative measure of activated caspase-2. Expression of receptor interacting protein (RIP)-associated Ich-1-homologous protein with death domain (RAIDD) was increased following seizures, whereas expression of RIP and tumor necrosis factor receptor associated protein with death domain (TRADD), other components thought to be linked to the caspase-2 activation and signaling mechanism, were unchanged. Intracerebroventricular administration of z-VDVAD-fluoromethyl ketone blocked seizure-induced caspase-2 activity but did not alter caspase-8 activity and failed to affect DNA fragmentation or neuronal death. These data support activation of caspase-2 following seizures but suggest that parallel caspase pathways may circumvent deficits in caspase-2 function to complete the cell death process.

Adrenalectomy causes loss of zinc ions in zinc-enriched (ZEN) terminals and decreases seizure-induced neuronal death

Chelatable zinc ions from synaptic vesicles have been suggested to be involved in neuronal death caused by stroke, epilepsy and head trauma. Elevated glucocorticoid concentration exacerbates such neuron loss, while low levels protect. We have tested the notion that the neuroprotective effect of prior glucocorticoid reduction is mediated by a reduction of zinc ions contained in zinc-enriched (ZEN) synaptic vesicles. The level of vesicular zinc ions was evaluated by toluene sulfonamide quinoline (TSQ) fluorometry and zinc autometallography (ZnS AMG) 10 and 30 days, respectively, after adrenalectomy. The hippocampus showed significant vesicular zinc ion depletion following adrenalectomy. After the kainate injection, adrenalectomized rats showed proconvulsive seizure behavior, i.e. shortened latency to seizure onset time and increased seizure score. Additionally they showed decreased hippocampal CA3 neuronal death as compared to control animals. The present data suggest that zinc ions released from damaged ZEN terminals are involved in seizure-induced neuronal death.

Mitochondrial superoxide production in kainate-induced hippocampal damage

The objective of this study was to determine the role of mitochondrial superoxide radical-mediated oxidative damage in seizure-induced neuronal death. Using aconitase inactivation as an index of superoxide production, we found that systemic administration of kainate in rats increased mitochondrial superoxide production in the hippocampus at times preceding neuronal death. 8-Hydroxy-2-deoxyguanosine, an oxidative lesion of DNA, was also increased in the rat hippocampus following kainate administration. Manganese(III) tetrakis(4-benzoic acid)porphyrin, a catalytic antioxidant, inhibited kainate-induced mitochondrial superoxide production, 8-hydroxy-2-deoxyguanosine formation and neuronal loss in the rat hippocampus. Kainate-induced increases of mitochondrial superoxide production and hippocampal neuronal loss were attenuated in transgenic mice overexpressing mitochondrial superoxide dismutase-2. We propose that these results demonstrate a role for mitochondrial superoxide production in hippocampal pathology produced by kainate seizures.

Seizure-induced neuronal death is associated with induction of c-Jun N-terminal kinase and is dependent on genetic background

Previous studies have shown that expression of c-Jun protein, as well as the c-Jun amino-terminal kinase (JNK) group of mitogen-activated protein kinases, may play a critical role in the pathogenesis of glutamate neurotoxicity. In order to define the molecular cascade that leads to c-Jun activation following excitotoxic injury and delineate whether induction of protein synthesis is related to cell death signaling cascades or those changes associated with increased seizure activity, we examined the expression of JNK-1, as well as its substrate, c-Jun and N-terminal phosphorylated c-Jun following kainic acid (KA) administration in two strains of mice. In the present study, we assessed the immunohistochemical expression of these proteins at time points between 2 h and 7 days, in excitotoxic cell death-resistant (C57BL/6) and -susceptible (FVB/N) mouse strains that were systemically injected with saline or kainic acid. No strain-related differences in the immunohistochemical expression of any of the proteins were observed in intact control mice. However, following KA administration, the magnitude and period of induction of JNK-1 protein was associated with impending cell death, while increased phosphorylation of c-Jun protein was associated with resistance to cell death. In contrast, expression of c-Jun protein does not appear to be a reliable indicator of impending cell death, as it was expressed in resistant and vulnerable subfields in mice susceptible to kainate injury. These results provide the first evidence that JNK-1 expression may be involved in producing the neuronal cell death response following excitotoxin-induced injury.

Accumulation of Zinc in Degenerating Hippocampal Neurons of ZnT3-Null Mice after Seizures: Evidence against Synaptic Vesicle Origin

In several brain injury models, zinc accumulates in degenerating neuronal somata. Suggesting that such zinc accumulation may play a causal role in neurodegeneration, zinc chelation attenuates neuronal death. Because histochemically reactive zinc is present in and released from synaptic vesicles of glutamatergic neurons in the forebrain, it was proposed that zinc translocation from presynaptic terminals to postsynaptic neurons may be the mechanism of toxic zinc accumulation. To test this hypothesis, kainate seizure-induced neuronal death was examined in zinc transporter 3 gene (ZnT3)-null mice, a strain that completely lacks histochemically reactive zinc in synaptic vesicles. Intraperitoneal injection of kainate induced seizures to a similar degree in wild type and ZnT3-null mice. Staining of hippocampal sections with a zinc-specific fluorescent dye, N-(6-methoxy-8-quinolyl)-p-carboxybenzoylsulfonamide, revealed that zinc accumulated in degenerating CA1 and CA3 neurons in both groups, indicating that zinc originated from sources other than synaptic vesicles. Injection of CaEDTA into the cerebral ventricle almost completely blocked zinc accumulation in ZnT3-null mice, suggesting that increases in extracellular zinc concentrations may be a critical event for zinc accumulation. Arguing against the possibility that zinc accumulation results from nonspecific breakdown of zinc-containing proteins, injection of kainate into the cerebellum did not induce zinc accumulation in degenerating granule neurons. Taken together, these results support the existing idea that zinc is released into extracellular space and then enters neurons to exert a cytotoxic effect. However, the origin of zinc is not likely to be synaptic vesicles, because zinc accumulation robustly occurs in ZnT3-null mice lacking synaptic vesicle zinc.

Free radical production correlates with cell death in an in vitro model of epilepsy.

Free radical (FR) production, a major step in calcium-dependent neurodegeneration, has been linked to the generation of epileptiform activity and seizure-induced cell death. However, direct evidence of FR production in neurons during seizures has never been presented. Using hippocampal cultured slices we demonstrate that FRs are produced in CA3 but not CA1 pyramidal neurons during the rhythmic synchronous activity induced by the GABAA receptor antagonist bicuculline. The production of FRs (measured as changes in the fluorescence emission of dihydrorhodamine 123) was correlated with an increase in the baseline levels of intracellular calcium ([Ca2+]i) estimated by fluo-3 injected into individual neurons via a patch pipette. [Ca2+]i increased during spike bursting and returned to baseline levels after the burst termination in CA1, but not in CA3, pyramidal neurons where 'interburst' calcium concentrations progressively increased. Measurement of cell death, performed with propidium iodide 48 h after a 30-min exposure to bicuculline, revealed most prominent degeneration of pyramidal neurons in the CA3 pyramidal layer. The FR scavengers vitamin E and glutathione significantly reduced the seizure-induced neurodegeneration without supressing spontaneous epileptiform activity. These observations indicate that FR overproduction is related to seizure-induced neuronal death.

P75 Neurotrophin Receptor Expression Is Induced in Apoptotic Neurons After Seizure

Seizure causes neuronal cell loss in both animal models and human epilepsy. To determine the contribution of apoptotic mechanisms to seizure-induced neuronal cell death, rat brains were examined for the occurrence of terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL)-positive nuclei after pilocarpine-induced seizure. Numerous TUNEL-positive cells were observed throughout the postseizure hippocampus, piriform cortex, and entorhinal cortex. Combined TUNEL/NeuN immunocytochemistry demonstrated that the vast majority of TUNEL-positive cells were neurons. To identify components of the signal transduction cascade promoting postseizure apoptosis, the expression of the p75 neurotrophin receptor (p75NTR) was examined. Seizure-induced increases in p75NTR protein and mRNA were detected in hippocampus, piriform cortex, and entorhinal cortex. Immunohistochemical double labeling revealed almost complete correspondence between TUNEL-positive and p75NTR-expressing cells, suggesting that seizure-induced neuronal loss within the CNS occurs through apoptotic signaling cascades involving p75NTR.

Lithium-pilocarpine-induced status epilepticus produces necrotic neurons with internucleosomal DNA fragmentation in adult rats.

Prolonged and continuous epileptic seizures [status epilepticus (SE)] produce a widespread pattern of neuronal death, primarily in limbic brain regions. Because it has been suggested that seizure-induced neuronal death may be apoptotic in nature, we tested the hypothesis that lithium-pilocarpine-induced status epilepticus (LPCSE) produces apoptotic neurons. LPCSE lasting 3 h was induced in male Wistar rats which were allowed to recover for 24 or 72 h before perfusion-fixation. Neuronal death was assessed by light microscopy with the haematoxylin-and-eosin stain (H&E), with in situ DNA nick-end labelling (TUNEL stain), by electron microscopy, and by agarose gel electrophoresis of DNA extracted from vulnerable brain regions. Ultrastructurally, acidophilic neurons identified with H&E were dark, shrunken and necrotic in appearance, exhibiting pyknotic nuclei, irregular, dispersed chromatin clumps and cytoplasmic vacuolization. No cells with apoptotic features were seen. Acidophilic neurons were found in 21 out of 23 brain regions examined, and comprised 26-45% of the total number of neurons examined. A subset of these neurons (< 10% of the total number of neurons) were TUNEL-positive at 72 h, but not 24 h, after SE. Internucleosomal DNA cleavage (DNA 'laddering') was found in the six brain regions examined ultrastructurally 24 and 72 h after SE. These results indicate that, in adult rats, LPCSE produces neuronal injury with the appearance of necrosis rather than apoptosis. The necrotic neurons show nuclear pyknosis, chromatin condensation and internucleosomal DNA fragmentation, confirming the nonspecificity of these nuclear changes. Internucleosomal DNA cleavage and other programmed cell death mechanisms can be activated by SE in neurons which become necrotic.

Effect of metal chelating agents on the direct and seizure-related neuronal death induced by zinc and kainic acid

The ability of metal chelating agents to affect seizure-induced neuronal death caused by intra-amygdaloid injections of kainic acid was investigated. N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), diethyldithiocarbamate (DEDTC) and diphenylthiocarbazone (dithizone), administered simultaneously or within 30 min of a kainate injection, all failed to affect the amount of neuronal loss in the ipsilateral hippocampus. This failure was not due to an inability to complex endogenous zinc as all these chelating agents quenched staining for endogenous zinc by the Timm method. However, the period for which this quenching occurred was short for DEDTC and dithizone (a maximum of 1.5 h) although it lasted for 8 h with TPEN. TPEN, but not DEDTC or dithizone prevented the neuronal loss caused by intra-hippocampal injections of zinc chloride. In the presence of diazepam to prevent seizures, co-injection of TPEN and kainate into the hippocampus also failed to prevent the direct cytotoxicity of kainate. Endogenous zinc, released from mossy fibres in the hippocampus by seizure activity, does not appear to modify seizure activity sufficiently to alter the extent of the resulting neuronal death.

Expression of heat shock protein-70 and limbic seizure-induced neuronal death in the rat brain.

The effect of MK-801, a non-competitive N-methyl-D-aspartate (NMDA) antagonist, on the kainic acid-induced expression of the inducible heat shock protein 70 kDa (HSP70) and on neuronal death in the rat hippocampus was investigated. HSP70 is expressed in approximately 80% of the pyramidal neurons in the CA1 field 1 day after kainic acid injection. The majority of these HSP70-immunopositive neurons exhibited swelling and a hollow appearance in the perikaryon, indicating that they had been injured following kainic acid-elicited limbic seizures. Four days after administration of kainic acid, 87% of the pyramidal neurons in the CA1 field were dead. When a single dose of MK-801 was administered 1 h before kainic acid injection, the number of rats suffering with seizures was reduced, the severity of limbic seizures was attenuated and seizure onset was delayed. Neither HSP70 expression on day 1 nor neuronal loss on day 4 in the CA1 pyramidal cell layer was observed in these animals. A considerable number of HSP70-immunopositive neurons was detected in the dentate hilus, however, and somewhat fewer in the CA3a and CA3c subfields on day 1. Severe neuronal damage in these regions followed on day 4. Interestingly, little HSP70 expression or neuronal loss was observed in the CA3b subfield in these same animals. When a single dose of MK-801 was given 4 h after kainic acid treatment, HSP70 expression was partially blocked; 18% of neurons expressed HSP70 on day 1 and 37% on day 4 in CA1 pyramidal neurons in comparison to the kainic acid controls. About 50% neuronal death was detected in the CA1 pyramidal cell layer 4 days after kainic acid treatment followed by MK-801. When the animals were treated with MK-801 4 h after kainic acid treatment followed by additional daily administration for 3 days, a negligible number of pyramidal neurons expressed HSP70, and the survival of pyramidal cells was significantly increased in the CA1 field. Limbic seizure-induced HSP70 expression not only indicates neuronal injury in the pyramidal cell layer of the hippocampus but also predicts delayed neuronal death, at least in the case of the CA1 field of animals that suffered stage IV-V seizures.

Kainic acid‐induced seizures and brain damage in the rat: Role of calcium homeostasis

Seizure activity induced by kainic acid (KA) and subsequent neuronal death are thought to be associated with an increase in cytoplasmic free calcium ([Ca2+]i) and can be prevented by N-methyl-D-aspartate (NMDA) antagonists. In addition to influx through receptor operated Ca2+ channels the increase in [Ca2+]i may be the result of an increased influx through voltage-operated calcium channels and/or release from intracellular deposits. It was therefore investigated whether compounds other than NMDA antagonists with known actions on the intracellular Ca2+ homeostasis had any protective effect against KA-induced neuronal death. Voltage-operated calcium channels in the cell membrane were blocked with the L-type ion channel antagonist, Nimodipine (1.0 mg/kg), and release of Ca2+ from internal stores was prevented with Dantrolene (10 mg/kg). Animals from two control groups injected with kainate (8 mg/kg) exhibited a survival rate of 67 and 53%, respectively. Countings of neurons in dorsal hippocampus showed subtotal or total loss in the CA1 and CA3 subregions. There were no significant differences concerning seizure and survival rates in the groups injected with kainate and treated with Dantrolene or Nimodipine and the control groups. The group treated with Dantrolene showed no neuropathological changes in the hippocampal CA3 region and only slight changes in the Ca1 region, while the neuron loss in the Nimodipine group did not differ from that of its control group. The results emphasize the importance of Dantrolene-sensitive Ca2+ release from intracellular stores for the development of seizure-induced neuronal death.