Seizure-Induced Neuronal Necrosis: Implications for Programmed Cell Death Mechanisms

Summary: Purpose : To determine definitively the morphology of neuronal death from lithium-pilocarpine (LPC)-and kainic acid (KA)-induced status epilepticus (SE), and to correlate this with markers of DNA fragmentation that have been associated with cellular apoptosis. Endogenous glutamate release is probably responsible for neuronal death in both seizure models, because neuronal death in both is N -methyl-D-aspartate receptor-mediated.
Methods : SE was induced for 3 hours in adult male Wistar rats with either LPC or KA, and 24 or 72 hours later the rats were killed. One group of rats had brain sections, stained with hematoxylin and eosin and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) technique, examined by light microscopy and by electron microscopy. A separate group of rats had DNA extracted from the same brain regions examined by electron microscopy in the first group. The extracted DNA was electrophoresed on an agarose gel with ethidium bromide and was examined for the presence or absence of internucleosomal DNA cleavage (DNA "laddering").
Results : Twenty-four and 72 hours after 3 hours of LPC- or KA-induced SE, neuronal death in the hippocampus, amygdala, and piriform, entorhinal, and frontal cortices was morphologically necrotic, in spite of DNA laddering in these regions 24 and 72 hours after SE and positive TUNEL staining in some of the regions 72 hours after SE. Ultrastructurally, necrotic neurons were dark and shrunken, with cytoplasmic vacuoles and pyknotic nuclei with small, irregular, dispersed chromatin clumps.
Conclusions : Our results, together with those of other reports, suggest that programmed cell death-promoting mechanisms are activated by SE in neurons that become necrotic rather than apoptotic and point to the possibility that such mechanisms may contribute to SE-induced neuronal necrosis.     

Kainic acid dose affects delayed cell death mechanism after status epilepticus

Kainic acid (KA)-induced status epilepticus (SE) produces hippocampal neuronal death, which varies from necrosis to apoptosis or programmed cell death (PCD). We examined whether the type of neuronal death was dependent on KA dose. Adult rats were induced SE by intraperitoneal injection of KA at 9 mg/kg (K9) or 12 mg/kg (K12). Hippocampal neuronal death was assessed by TUNEL staining, electron microscopy, and Western blotting of caspase-3 on days 1, 3 and 7 after SE induction. K12 rats showed higher a mortality rate and shorter latency to the onset of SE when compared with K9 rats. In both groups, acidophilic and pyknotic neurons were evident in CA1 at 24h after SE and neuronal loss developed from day 3. The degenerated neurons became TUNEL-positive on days 3 and 7 in K9 rats but not in K12 rats. Caspase-3 activation was detected on days 3 and 7 in K9 rats but was undetectable in K12 rats. Ultrastructural study revealed shrunken neurons exhibiting pyknotic nuclei containing small and dispersed chromatin clumps 24h after SE in CA1. No cells exhibited apoptosis. On days 3 and 7, the degenerated neurons were necrotic with high electron density and small chromatin clumps. There were no ultrastructural differences between the K9 and K12 groups. These results revealed that differences in KA dose affected the delayed cell death (3 and 7 days after SE); however, no effect was seen on the early cell death (24h after SE). Moderate-dose KA induced necrosis, while low-dose KA induced PCD.     

The temporal evolution of neuronal damage from pilocarpine-induced status epilepticus

The temporal evolution of irreversible neuronal damage from pilocarpine-induced seizures was studied by light microscopy. Neuronal cell death was judged on a 0–3 scale by estimating the percentage of acidophilic neurons in each of 23 brain regions. In addition, in the dorsal dentate hilus (CA4), quantitative cell counts of normal and acidophilic neurons were also performed. A few dead neurons (grade 0.5 damage) appeared in ventral hippocampal CA1 and CA3 regions after 20-min status epilepticus (SE). Slight-to-mild damage (grades 0.5–1.5) occurred in 14 and 12 brain regions after 40-min and 1-h SE respectively, and slight-to-moderate damage (grades 0.5–2.0) was found in 15 regions after 3-h SE. Twenty-four h and 72 h after 3-h SE, there was slight-to-severe damage (grade 0.5–3.0) in 22 and 21 regions respectively. Three-h SE produced more severe damage to 7 brain regions compared to 1-h SE, and 16 regions had more pronounced neuronal injury 24 h after rather than 0–4 h after 3-h SE. Eight brain regions had less damage 72 h compared to 24 h after SE, probably because of progressive neuronal lysis and dropout, but in mediodorsal and lateroposterior thalamic nuclei damage worsened from 24 to 72 h after SE. Neuronal cell counting revealed 20% acidophilic neurons in dorsal dentate hilus after 40-min SE and no difference between the 1-h and 3-h seizure groups (31% vs. 43% acidophilic neurons respectively). Among the 3 groups of rats with 3-h SE and varying recovery periods, the 24-h and 72-h recovery groups had higher percentages of acidophilic neurons (65% and 54% respectively) than the 0–4-h group (43%). Finally, the hippocampal CA2 region and dentate granule cell layer and the caudate-putamen, considered resistant to seizure-induced cell injury, were all damaged from SE lasting 40 min or more.     

Caspase-dependent programmed cell death pathways are not activated in generalized seizure-induced neuronal death

Activation of the caspase-dependent cell death pathways has been shown in focal seizures, but whether this occurs in prolonged generalized seizures is not known. We investigated whether the initiator caspase in the extrinsic pathway, caspase-8, or the intrinsic pathway, caspase-9, is activated during the first 24 h following lithium-pilocarpine-induced status epilepticus, when neuronal death is maximal and widespread. The thymuses of rats given methamphetamine were used as positive controls for caspase-3-activated cellular apoptosis. Following methamphetamine treatment, caspase-9 but not caspase-8 was activated in thymocytes. However, 6 or 24 h following status epilepticus, none of 26 brain regions studied showed either caspase-8 or -9 activation by immunohistochemistry, western blotting and enzyme activity assays. Our results provide evidence against the activation of the extrinsic and intrinsic caspase pathways in generalized seizures, which produce morphologically necrotic neurons with internucleosomal DNA cleavage (DNA laddering), a programmed process. In contrast, there is increasing evidence that caspase-independent programmed mechanisms play a prominent role in seizure-induced neuronal death.     

Intracerebral injection of caspase-3 inhibitor prevents neuronal apoptosis after kainic acid-evoked status epilepticus

In the aftermath of prolonged continuous seizure activity (status epilepticus, SE), neuronal cell death occurs in the brain regions through which the seizure propagates. Recent studies have implicated apoptotic processes in this seizure-related injury. Because activation of caspase-3-like cysteine proteases plays a crucial role in mammalian neuronal apoptosis, we explored the possibility that activation of caspase-3 is involved in the neuronal apoptotic cell death that occurs in rat brain following SE induced by systemic kainic acid. Caspase-3 activity was determined immunocytochemically using CM1 antibodies specific for catalytically active subunit (p17) of the enzyme. We found an induction of caspase-3 activity in rhinal cortex and amygdala at 24 h after SE. To determine whether activation of caspase-3-like proteases is a necessary component of the injury process, we delivered a caspase-3 inhibitor, z-DEVD-fmk, into the lateral ventricle prior to, and following SE. z-DEVD-fmk treatment substantially attenuated apoptotic cell death after SE, both in hippocampus and rhinal cortex, as evaluated by analysis of internucleosomal DNA fragmentation and neuronal nuclear morphology. Our findings implicate caspase-3 cysteine protease in the neurodegenerative response to SE and suggest that this degeneration can be attenuated by inhibition of caspase-3-like enzyme activity.     

Status epilepticus triggers caspase-3 activation and necrosis in the immature rat brain

The mode and mechanism of neuronal death induced by status epilepticus (SE) in the immature brain have not been fully characterized. In this study, we analyzed the contribution of neuronal necrosis and caspase-3 activation to CA1 damage following lithium-pilocarpine SE in P14 rat pups. By electron microscopy, many CA1 neurons displayed evidence of early necrosis 6 hours following SE, and the full ultrastructural features of necrosis at 24-72 hours. Caspase-3 was activated in injured (acidophilic) neurons 24 hours following SE, raising the possibility that they died by caspase-dependent "programmed" necrosis.     

Long-lasting neuronal apoptotic cell death in regions with severe ischemia after photothrombotic ring stroke in rats

Apoptotic and necrotic cell death may act in concert in focal cerebral ischemia. This study explored the temporal and spatial pattern of apoptosis and necrosis in a novel photothrombotic ring stroke model with or without spontaneous reperfusion. Adult male Wistar rats were subjected to a ring-shaped laser irradiation beam simultaneously with intravenous erythrosin B infusion. The presence and attributes of apoptosis and necrosis in the anatomically well-defined cortical region at risk and ring-lesion region were verified under light microscopy with TUNEL, Hoechst 33342, and hematoxylin and eosin staining. Cells exhibiting apoptotic morphology with chromatin condensation and apoptotic bodies and necrotic ghost appearance were observed. The occurrence of apoptosis and necrosis in the ischemic regions was confirmed by electron microscopy and gel electrophoresis, in which DNA isolated from the lesion area revealed both a ladder and a smear. Double staining with TUNEL and the cell markers NeuN, glial fibrillary acidic protein, and ED-1 revealed that the majority of apoptotic cells were of neuronal origin. Cells exhibiting pyknosis/eosinophilia, apoptosis, or ghost appearance were quantified by stereological means. In subregions with severe ischemia, the peak appearance of apoptotic cells started earlier, i.e., at 24 h, than the peak of necrotic cells, and the high concentration of the apoptotic cells remained as long as that of necrotic cells, i.e., until 72 h post-ischemia. The ratio of apoptotic to necrotic cells was approximately 1:2. Therefore, apoptosis may be an important contributor to neuronal cell death in brain regions with severely reduced blood flow after thrombo-embolic stroke.     

经鼻给予TGFβ1对癫痫持续状态大鼠海马神经元的保护作用

目的探讨经鼻腔给予TGFβ1(transforming growth factor beta1,TGFβ1)对氯化锂-匹罗卡品所致癫痫持续状态(status epilepticus,SE)大鼠海马神经元的保护作用及其潜在的机制。方法健康雄性SD大鼠60只,随机分为转化生长因子(TGF)组、匹罗卡品(Pilo)组和正常对照组(control)。建立氯化锂-匹罗卡品癫痫持续状态模型。应用TUNEL染色、Fluoro-Jade B(FJB)荧光染色法分别观察各组大鼠海马神经元的原位凋亡及变性死亡情况。采用免疫组化方法检测凋亡相关基因caspase-3的蛋白表达。结果 SE后24h、48h、72h,TGF组大鼠海马FJB、TUNEL、caspase-3阳性细胞均较Pilo组显著减少(P<0.05);72h最为明显(P<0.01)。结论经鼻(IN)给予TGFβ1可以显著抑制或减轻癫痫持续状态大鼠海马神经元的变性与凋亡,从而发挥神经保护作用。其潜在的神经保护机制可能涉及下调caspase-3蛋白表达。     

Methamphetamine-induced neuronal necrosis: the role of electrographic seizure discharges

We have evidence that methamphetamine (METH)-induced neuronal death is morphologically necrotic, not apoptotic, as is currently believed, and that electrographic seizures may be responsible. We administered 40 mg/kg i.p. to 12 male C57BL/6 mice and monitored EEGs continuously and rectal temperatures every 15 min, keeping rectal temperatures <41.0 °C. Seven of the 12 mice had repetitive electrographic seizure discharges (RESDs) and 5 did not. The RESDs were often not accompanied by behavioral signs of seizures—i.e., they were often not accompanied by clonic forelimb movements. The 7 mice with RESDs had acidophilic neurons (the H&E light-microscopic equivalent of necrotic neurons by ultrastructural examination) in all of 7 brain regions (hippocampal CA1, CA2, CA3 and hilus, amygdala, piriform cortex and entorhinal cortex), the same brain regions damaged following generalized seizures, 24 h after METH administration. The 5 mice without RESDs had a few acidophilic neurons in 4 of the 7 brain regions, but those with RESDs had significantly more in 6 of the 7 brain regions. Maximum rectal temperatures were comparable in mice with and without RESDs, so that cannot explain the difference between the two groups with respect to METH-induced neuronal death. Our data show that METH-induced neuronal death is morphologically necrotic, that EEGs must be recorded to detect electrographic seizure activity in rodents without behavioral evidence of seizures, and that RESDs may be responsible for METH-induced neuronal death.     

癫痫持续状态后海马神经元凋亡的形态学证据及意义

癫痫持续状态（ｓｔａｔｕｓｅｐｉｅｐｔｉｃｕｓ，ＳＥ）可造成选择性神经元损害。近来研究发现这些脑组织中神经元死亡可能是通过凋亡（ａｐｏｐｔｏｓｉｓ）机制。腹腔注射贝美格（Ｂｅｍｅｇｒｉｄｅ）诱导ＳＥ，研究了鼠ＳＥ后脑损害中的神经元凋亡现象，以ＨＥ染色、电镜、荧光（ＡｃｒｉｄｉｎｅＯｒａｎｇｅ，ＡＯ）细胞核染色方法观察细胞形态，以流式细胞术及凝胶电冰鉴定ＤＮＡ片断。ＳＥ后２４小时，海马中的易损神经元死亡具有凋亡特征：①光镜及电镜观察到细胞核内染色体凝聚和边及；②阳性ＡＯ着色细胞核；③流式细胞术及凝胶电冰显示具有ＤＮＡ片断。而且有Ⅰ型和Ⅱ型两种形式的凋亡。因此ＳＥ引起海马损害中神经元有通过调亡机制发生死亡．     

Latency to onset of status epilepticus determines molecular mechanisms of seizure-induced cell death

The molecular mechanisms mediating degeneration in response to neuronal insults, including damage evoked by prolonged seizure activity, show substantial variability across laboratories and injury models. Here we investigate the extent to which the proportion of cell death occurring by apoptotic vs. necrotic mechanisms may be shifted by changing the temporal parameters of the insult. In initial studies with continuous seizures (status epilepticus, SE), signs of apoptotic degeneration were most clearly observed when SE occurred following a long latency (>86 min) after injection of kainic acid as compared with a short-latency SE (<76 min). Therefore, in this study we directly compared short- with long-latency SE for the expression of molecular markers for apoptosis and necrosis in an especially vulnerable brain region (rhinal cortex). Molecular markers of apoptosis (DNA fragmentation, cleavage of ICAD, an inhibitor of "caspase-activated DNase" (CAD), and prevalence of a caspase-generated fragment of alpha-spectrin) were detected following long-latency SE. Short-latency SE resulted in expression of predominantly necrotic features of cell death, such as "non-ladder" pattern of genomic DNA degradation, prevalence of a calpain-generated alpha-spectrin fragment, and absence of ICAD cleavage. Silver staining revealed no significant difference in the extent and spatial distribution of degeneration between long- or short-latency SE. These data indicate that the latency to onset of SE determines the extent to which apoptotic or necrotic mechanisms contribute to the degeneration following SE. The presence of a long latency period, during which multiple brief seizure episodes may occur, favors the occurrence of apoptotic cell death. It is possible that the absence of such "preconditioning" period in short-latency SE favors predominantly necrotic profile.     

海人酸致癎大鼠海马CA_3区神经元线粒体与细胞核损伤及caspase-3表达的研究

目的观察海人酸(KA)诱导的癫疒间持续状态(SE)大鼠海马CA3区神经元线粒体与细胞核超微结构的损伤及caspase-3表达的变化。方法用KA诱导大鼠SE 2 h。分别于SE终止后第3 h、12 h、24 h取海马CA3区制作切片,光镜下观察神经元的变化,电镜下观察线粒体和细胞核的超微结构;免疫组化方法检测相同部位caspase-3的表达变化。结果光镜下SE后24 h神经元出现排列散乱、胞体皱缩、胞浆红染以及胞核固缩。电镜下SE后3 h,可见线粒体嵴肿胀及膜的崩解;SE后24 h细胞核染色质明显边聚。Caspase-3平均阳性细胞数及灰度值于SE后12 h较正常对照组显著增加(均P<0.05);24 h出现极显著增加(均P<0.01)。结论SE后早期海马神经元线粒体损伤可能是神经元损伤的关键环节。     

Ultrastructural identification of dentate granule cell death from pilocarpine-induced seizures

Cell loss in the hippocampal formation is a common event in patients with temporal lobe epilepsy. The belief that dentate granule neurons are relatively resistant to excitotoxic injury has recently been challenged both, in epileptic patients and in animal models of temporal lobe epilepsy. The nature of dentate granule cell damage in epilepsy has been reported as either apoptotic, necrotic or both. The lack of a consensus on this topic stems from use of different animal models and different experimental techniques for characterizing the apoptotic/necrotic process. Using electron microscopy for defining the, nature of cell loss and one of the main animal models of status epilepticus (SE) we have focussed on the nature of the degenerative process in dentate granule cells. Ultrastructural morphological changes of these cells were evaluated 2.5-48 h after pilocarpine-induced status epilepticus. A variety of morphologies ranging from apoptosis to necrosis, could be seen at 2.5 h after SE onset and continued at least over the following 48 h. Some cells displayed coalescence of chromatin against nuclear membranes. In such cases however, chromatin did not have well-defined edges (as it should, if it were apoptosis). Condensation of cytoplasm. present in both processes was also frequently found. Neither obvious apoptotic budding-off of cytoplasm nor typical membrane-bound apoptotic bodies were found. Our results indicate that in the dentate granule cell layer pilocarpine-induced SE promotes a degenerative process in which apoptotic and necrotic features overlap.     

锂-匹罗卡品致大鼠癫癎持续状态后脑内神经元的凋亡

探讨癫持续状态 (StatusEpilepticus,SE)时细胞凋亡的发生及其与海马硬化的关系。采用锂 匹罗卡品诱发大鼠SE模型 ,在SE的不同时点采大鼠脑标本 ,利用TUNEL染色方法检测大鼠海马皮质神经元的凋亡出现情况。结果发现 ,正常对照组大鼠大脑皮质可见散在的TUNEL阳性细胞 ,海马区未见TUNEL阳性细胞。SE1h ,皮质TUNEL阳性细胞数即开始增加 ,SE后 8h ,海马区开始出现TUNEL阳性细胞 ,SE后 1d ,大脑皮质TUNEL阳性细胞数开始明显增加 ,海马区也可见到较多TUNEL阳性细胞。SE后 5d ,皮质及海马的TUNEL阳性细胞数达到高峰。 7d时皮质及海马TUNEL阳性数均明显下降。结果提示 ,SE可引起神经元凋亡 ,5d时达到高峰 ,7d时已明显下降。神经元凋亡与SE引起的迟发性神经元死亡有关 ,并参与了海马硬化的形成。     

Dissociation of the immunoreactivity of synaptophysin and GAP-43 during the acute and latent phases of the lithium-pilocarpine model in the immature and adult rat

RATIONALE: Lithium-pilocarpine-induced status epilepticus (SE) generates neuronal lesions in the limbic forebrain, cerebral cortex and thalamus that lead to circuit reorganization and spontaneous recurrent seizures. The process of reorganization in regions with neuronal damage is not fully clarified. METHODS: In the present study, we evaluated by immunohistochemistry the early reorganization during the latent period with two neuronal markers, synaptophysin and growth-associated protein 43 (GAP-43) in rats subjected to SE at PN21 and as adults. RESULTS: Synaptophysin immunoreactivity increased between 24 h and 3 weeks post-SE in regions with severe and rapidly occurring neuronal loss, namely thalamus, amygdala, piriform and entorhinal cortices. GAP-43 expression decreased at 1 and 3 weeks in the same regions. The immunoreactivity of synaptophysin and GAP-43 increased in the inner molecular layer of dentate gyrus from 24 h after SE, and decreased in the outer molecular layer from 72 h after SE. These changes likely result from the death of hilar neurons and the reduction of the input from the entorhinal cortex. In 21-day-old rats that experience less SE-induced neuronal loss, increased immunoreactivity of synaptophysin was only found in piriform and entorhinal cortex while no changes occurred in GAP-43 expression. CONCLUSION: These findings suggest that there is an age-related relation between the extent and rapidity of the process of neuronal death and the expression of these markers. Synaptophysin appears to be a more sensitive marker of plasticity induced by SE than GAP-43.     

匹罗卡品致痫大鼠海马calpain活性变化和其对神经元死亡的影响

目的探讨在匹罗卡品致痫的癫痫持续状态（SE）大鼠模型中,钙蛋白酶在大鼠海马组织中的活性,及钙蛋白酶对神经元坏死、凋亡产生的影响。方法雄性成年wistar大鼠,应用匹罗卡品致痫产生SE后60min后终止发作,24h后取材,行HE染色及tunel染色,观察海马神经元的坏死及凋亡情况,以及western blot检测钙蛋白酶1（μ-calpain）的活性。结果癫痫持续状态后24h,海马组织HE染色神经元数量减少,tunel阳性细胞数增加,钙蛋白酶1出现76ku条带。结论大鼠癫痫持续状态后24h,钙蛋白酶1在海马组织神经元活性增加,海马神经元出现坏死及凋亡。钙蛋白酶1与神经元的死亡存在着正相关。     

Inhibition of apoptosis by hyperbaric oxygen in a rat focal cerebral ischemic model.

The hypothesis was tested that hyperbaric oxygen therapy (HBO) reduced brain infarction by preventing apoptotic death in ischemic cortex in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and subsequently were exposed to HBO (2.5 atmospheres absolute) for 2 h, at 6 h after reperfusion. Rats were killed and brain samples were collected at 24, 48, 72 h, and 7 days after reperfusion. Neurologic deficits, infarction area, and apoptotic changes were evaluated by clinical scores, 2,3,7-triphenyltetrazolium chloride staining, caspase-3 expression, DNA fragmentation assay, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL)-hematoxylin and eosin (H&E) costaining. In MCAO/R without HBO treatment animals, DNA fragmentation was observed in injured cortex at 24, 48, and 72 h but not in samples at 7 days after reperfusion. Double labeling of brain slides with NeuN and caspase-3 demonstrated neurons in the injured cortex labeled with caspase-3. TUNEL+H&E costaining revealed morphologic apoptotic changes at 24, 48, and 72 h after reperfusion. Hyperbaric oxygen therapy abolished DNA fragmentation and reduced the number of TUNEL-positive cells. Hyperbaric oxygen therapy reduced infarct area and improved neurologic scores at 7 days after reperfusion. One of the molecular mechanisms of HBO-induced brain protection is to prevent apoptosis, and this effect of HBO might preserve more brain tissues and promote neurologic functional recovery.     

癫痫持续发作对大鼠海马神经元的影响

目的 研究实验性癫痫大鼠在癫痫发作持续不同时间对海马神经元的影响。方法24只SD大鼠随机分成4组：诱发火鼠瘢痫持续状态（status cpilepticus，SE）〈10、10～30、〉30min组及正常对照组。于电镜下观察各组海马神经元超微结构变化；采用免疫组化方法检测凋亡相关基因bcl-2、bax的蛋白表达及通过流式细胞技术检测细胞凋亡情况。结果SE〈10min组海马神经元所受影响不大．SE10～30min组海马神经元具有明显凋亡特征．SE〉30min组多数海马神经元呈坏死性改变。结论大鼠SE对海马神经元损伤有凋亡和坏死两种不同形式．这与癫痫发作的持续时间密切相关。     

Increased caspase 3 and Bax immunoreactivity accompany nuclear GAPDH translocation and neuronal apoptosis in Parkinson's disease

In situ end labeling combined with YOYO staining was used to mark apoptotic DNA fragmentation and chromatin condensation respectively in human postmortem brain sections. Increased numbers of apoptotic neuronal nuclei were identified in the Parkinson's disease (PD) nigra compared with age-matched controls. Caspase 3 and Bax showed increased immunoreactivity in melanized neurons of the PD nigra compared with controls. Importantly, GAPDH nuclear accumulation was also observed in the PD nigra, suggesting apoptotic rather than necrotic cell death. Interestingly, both Lewy bodies and the intranuclear Marinesco's bodies were GAPDH immunoreactive in the PD brain.     

Expression time course and spatial distribution of activated caspase-3 after experimental status epilepticus: contribution of delayed neuronal cell death to seizure-induced neuronal injury

Pilocarpine-induced status epilepticus (PCSE) is a widely used model to study neurodegeneration in limbic structures after prolonged epileptic seizures. However, mechanisms mediating neuronal cell death in this model require further characterization. Examining the expression time course and spatial distribution of activated caspase-3, we sought to determine the role of apoptosis in PCSE-mediated neuronal cell death. Expression of activated caspase-3, predominantly located in neurons, was detected 24 h (amygdala; piriform and temporal cortex) and 7 days (hippocampus; amygdala; piriform, temporal and parietal cortex; thalamus) after PCSE with strongest induction being observed in the amygdala, the piriform cortex, and the hippocampus. Further analysis revealed TUNEL positivity (24 h and 7 days after SE) and a significant, progressive neuronal cell loss in all brain regions displaying caspase-3 activation. Corresponding to high levels of activated caspase-3 expression, neuronal cell loss was most pronounced in the amygdala, piriform cortex, and dorsal CA-1 hippocampus. These results demonstrate that apoptosis contributes significantly to PCSE-induced neuronal cell death.