Programmed Neuronal Necrosis and Status Epilepticus

Summary:  We examined the mechanism of neuronal necrosis induced by hypoxia in dentate gyrus cultures or by status epilepticus (SE) in adult mice. Our observations showed that hypoxic necrosis can be an active process starting with early mitochondrial swelling and loss of the mitochondrial membrane potential, followed by cytochrome c release and caspase-9–dependent activation of caspase-3. This sequence of events (or program) was independent of protein synthesis and may be induced by energy failure and/or calcium overloading of mitochondria. We called this form of necrosis "programmed necrosis." After SE in adult mice, CA1 and CA3 pyramidal neurons displayed a necrotic morphology, associated with caspase-3 immunoreactivity and with double-stranded DNA breaks, suggesting that "programmed necrosis" may be involved in SE-induced neuronal loss.     

Programmed cell death in the lithium pilocarpine model: evidence for NMDA receptor and ceramide-mediated mechanisms

Ceramide is known to induce programmed cell death (PCD) in neural and non-neural tissues and to increase after kainic acid (KA) status epilepticus (SE). Ceramide increases have been shown to depend on NMDA receptor activation in the KA model, but these changes have not been studied in the lithium pilocarpine (LiPC) model. Thus, the purpose of this study was to determine if hippocampal ceramide levels increase after LiPC induced SE and if NMDA receptor blockade prevents PCD and any such ceramide increases. We found that LiPC induced SE resulted in ceramide increases and DNA fragmentation in the hippocampus of adult, P21, and P7 rats. The administration of MK-801, the NMDA receptor antagonist, in adults, 15min prior to pilocarpine, prevented ceramide increases, and DNA fragmentation.     

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.     

Death mechanisms in status epilepticus-generated neurons and effects of additional seizures on their survival

Status epilepticus (SE) increases neurogenesis in the subgranular zone (SGZ) of the adult dentate gyrus, but many of the newborn cells die, partly through caspase-induced apoptosis. Here we provide immunohistochemical evidence indicating that the caspase-evoked death of the new neurons involves the mitochondrial but not the death-receptor-mediated pathway. Cytochrome c released from mitochondria was found in a subset of progenitor cell progeny, while Fas ligand and tumor necrosis factor 1 receptor-associated domain as well as the mitochondria-related, caspase-independent apoptosis-inducing factor were not detected. We also show that additional seizures, induced at different stages during neuronal differentiation of progenitor cell progeny following SE, neither potentiate cell death mechanisms in the SGZ nor compromise the survival of the new cells. Thus, we found similar expression of cytochrome c, active caspase-3, caspase-cleaved PARP, and TUNEL/Hoechst-positive DNA fragmentation, as well as numbers of new cells in the SGZ in rats exposed to additional seizures at days 6 and 7 or days 33 and 34 following SE as in control animals only subjected to SE. We propose that the degree of survival of newly generated neurons is determined primarily by the initial SE insult and the ensuing pathology in the tissue environment, whereas spontaneous seizures play a minor role.     

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.     

Time Course of Apoptotic Cell Death within Mesencephalic Cell Suspension Grafts: Implications for Improving Grafted Dopamine Neuron Survival

The vast majority (90%) of embryonic mesencephalic dopamine (DA) neurons die following transplantation to the striatum. Recent reports indicate that at least a subpopulation of grafted cells undergo apoptotic cell death at early times following implantation. This study examines the temporal pattern and magnitude of apoptotic cell death following the implantation of mesencephalic cell suspension grafts. Two techniques, a modified terminal deoxynucleotide-mediated nucleotide end labeling (TUNEL) technique and cresyl violet staining, are used to assess apoptotic cell death by detection of its biochemical and morphological identifiers, respectively. Male, Fischer 344 rats were examined at 1, 4, 7, and 28 days following implantation of embryonic day 14 (E14) ventral mesencephalic cells to the DA-denervated striatum. Results indicate that the overwhelming majority of apoptotic cell death occurs within the first 7 days after transplantation. However, the impact of the apoptosis that occurs over the first week following grafting only appears to limit grafted tyrosine hydroxylase-immunoreactive (THir) neuron survival during the first 4 days. No significant differences between the survival rates of THir neurons at 4 days after grafting and at 28 days after grafting were found. Therefore, it appears that the critical interval during which an estimated 90% of grafted DA neurons die is during the first 4 days postimplantation and that a major contributor to this cell death is apoptosis.     

Neuronal cell death in a rat model for mesial temporal lobe epilepsy is induced by the initial status epilepticus and not by later repeated spontaneous seizures

PURPOSE: To determine whether repeated seizures contribute to hippocampal sclerosis, we investigated whether cell loss in the (para) hippocampal region was related to the severity of chronic seizure activity in a rat model for temporal lobe epilepsy (TLE). METHODS: Chronic epilepsy developed after status epilepticus (SE) that was electrically induced 3-5 months before. The presence of neuronal damage was assessed by using Fluoro-Jade and dUTP nick end-labeling (TUNEL) of brain sections counterstained with Nissl. RESULTS: We found a negative correlation between the numbers of surviving hilar cells and the duration of the SE (r = -0.66; p < 0.01). In the chronic phase, we could discriminate between rats with occasional seizures (0.15 +/- 0.05 seizures per day) without progression and rats with progressive seizure activity (8.9 +/- 2.8 seizures/day). In both groups, the number of TUNEL-positive cells in parahippocampal regions was similar and higher than in controls. In the hippocampal formation, this was not significantly different from controls. Fluoro-Jade staining showed essentially the same pattern at 1 week and no positive neurons in chronic epileptic rats. CONCLUSIONS: Cell death in this rat model is related to the initial SE rather than to the frequency of spontaneous seizures. These results emphasize that it is of crucial importance to stop the SE as soon as possible to prevent extended cell loss and further progression of the disease. They also suggest that neuroprotectants can be useful during the first week after SE, but will not be very useful in the chronic epileptic phase.     

Circuit Mechanisms of Seizures in the Pilocarpine Model of Chronic Epilepsy: Cell Loss and Mossy Fiber Sprouting

We used the pilocarpine model of chronic spontaneous recurrent seizures to evaluate the time course of supragranular dentate sprouting and to assess the relation between several changes that occur in epilep tic tissue with different behavioral manifestations of this experimental model of temporal lobe epilepsy. Pilo carpine-induced status epilepticus (SE) invariably led to cell loss in the hilus of the dentate gyrus (DG) and to spontaneous recurrent seizures. Cell loss was often also noted in the DG and in hippocampal subfields CA1 and CA3. The seizures began to appear at a mean of 15 days after SE induction (silent period), recurred at variable frequencies for each animal, and lasted for as long as the animals were allowed to survive (325 days). The granule cell layer of the DG was dispersed in epileptic animals, and neo-Timm stains showed supra-and intragranular mossy fiber sprouting. Supragranular mossy fiber sprout ing and dentate granule cell dispersion began to appear early after SE (as early as 4 and 9 days, respectively) and reached a plateau by 100 days. Animals with a greater degree of cell loss in hippocampal field CAS showed later onset of chronic epilepsy (r= 0.83, p < 0.0005), suggest ing that CA3 represents one of the routes for seizure spread. These results demonstrate that the pilocarpine model of chronic seizures replicates several of the fea tures of human temporal lobe epilepsy (hippocampal cell loss, suprar and intragranular mossy fiber sprouting, den tate granule cell dispersion, spontaneous recurrent sei zures) and that it may be a useful model for studying this human condition. The results also suggest that even though a certain amount of cell loss in specific areas may be essential for chronic seizures to occur, excessive cell loss may hinder epileptogenesis.     

Mechanisms of Postecdysis‐Associated Programmed Cell Death of Peptidergic Neurons in Drosophila melanogaster

Crustacean cardioactive peptide (CCAP)‐expressing neurons undergo programmed cell death (PCD) within 24 hours after adult eclosion. A subset of the doomed CCAP neurons in the ventral nerve cord also expressed the neuropeptide bursicon and thus are referred to as bursCCAP neurons. In this study, we undertook comprehensive genetic and transgenic analyses to dissect the PCD mechanisms of bursCCAP neurons. Expression of a versatile caspase inhibitor, p35, blocked PCD of bursCCAP neurons, suggesting caspase‐dependent apoptosis. Further genetic analyses showed that Dronc/Dark and Drice are key caspases, but they are not sufficient to carry out the PCD fully. We did not find a role for other known caspases, Strica, Dredd, Damm, or Decay. Of interest, Dcp‐1 is required not for the death of bursCCAP neurons per se but for the removal of neural projections. DIAP1 is an important survival factor that inhibits premature death of bursCCAP neurons. We found that grim functions as a principal death inducer, whereas other death genes, hid, reaper, and sickle, show no endogenous function. Taken together with other studies, our work supports the role of grim as a major death inducer particularly for the removal of obsolete larval neurons during CNS metamorphosis. Results from the ectopic expression of the mutant grim lacking either N‐terminal IBM or internal GH3 domain indicated that both domains are necessary to induce CCAP cell death. J. Comp. Neurol. 521:3972–3991, 2013. © 2013 Wiley Periodicals, Inc.     

β-Amyloid induces apoptosis in human-derived neurotypic SH-SY5Y cells

Alzheimer's disease is primarily characterized by neurofibrillary tangles, senile plaques, and neurodegeneration. The major component of senile plaques is the β-amyloid peptide (βA4), which has been shown to be toxic to neurons in vitro. To date, the mechanism of βA4-induced neurotoxicity has not been determined in human-derived neurons. In this report, we present evidence that programmed cell death, or apoptosis, is involved in the neurotypic activity of βA4_1–40 and βA4_25–35 to the human-derived neurotypic cell line SH-SY5Y cells. The evidence for βA4-induced apoptosis includes: (1) labeling of cell nuclei for DNA nicked ends; (2) morphological changes in ultrastructure that are consistent with apoptosis (shrunken cells with pyknotic nuclei); (3) DNA laddering which can be blocked by aurintricarboxylic acid (ATA), an inhibitor of apoptosis; and (4) marginal release of intracellular lactate dehydrogenase (LDH), an indicator of necrosis. These results suggest that apoptosis is the major event involved in βA4-induced cytotoxicity in SH-SY5Y cells. A variety of reagents were tested to determine their activities against βA4-induced DNA laddering. Nerve growth factor and free radical scavengers were inactive in this system. While ATA blocked DNA laddering resulting from either βA4 or okadaic acid treatment, Congo red specifically attenuated only βA4-induced DNA fragmentation. These results suggest that compounds which bind fibrillar β-peptides can protect this human neurotypic cell line against apoptosis induced by βA4.     

Intranasal versus intravenous lorazepam for control of acute seizures in children: a randomized open-label study

Purpose: Intravenous lorazepam is considered the drug of first choice for control of acute convulsive seizures. However, resource or personnel constraints necessitate the study of alternative routes and medications. This study compared the efficacy and adverse effects of intranasal versus intravenous lorazepam in children aged 6–14 years who presented with acute seizures. Methods: This was a randomized open‐label study conducted at an Indian hospital from August 2008 to April 2009. One hundred forty‐one consecutive children aged 6–14 years who presented convulsing to the emergency room were included. After stabilization, the children were randomized to receive either intravenous or intranasal lorazepam (0.1 mg/kg, maximum 4 mg). The primary outcome measure was clinical seizure remission within 10 min of drug administration. The study was registered with clinicaltrials.gov (NCT00735527). Key Findings: Seventy patients were randomized to receive intravenous and 71 to receive intranasal lorazepam. The patients in the two groups were comparable at baseline. Clinical seizure remission within 10 min of drug administration was found in 80% of the intravenous group as compared to 83.1% of intranasal group. The lower limit of 95% confidence interval for effect size was approximately −9.7%, with an a priori cutoff for noninferiority of −10%. Significance: Intranasal administration of lorazepam is not found to be inferior to intravenous administration for termination of acute convulsive seizures in children.     

Neuronal stress and injury in C57/BL mice after systemic kainic acid administration

Kainate-induced seizures are widely studied as a model of human temporal lobe epilepsy due to behavioral and pathological similarities. While kainate-induced neuronal injury is well characterized in rats, relatively little data is available on the use of kainate and its consequences in mice. The growing availability of genetically altered mice has focused attention on the need for well characterized mouse seizure models in which the effects of specific genetic manipulations can be examined. We therefore examined the kainate dose–response relationship and the time-course of specific histopathological changes in C57/BL mice, a commonly used founder strain for transgenic technology. Seizures were induced in male C57/BL mice (kainate 10–40 mg/kg i.p.) and animals were sacrificed at various time-points after injection. Seizures were graded using a behavioral scale developed in our laboratory. Neuronal injury was assayed by examining DNA fragmentation using in situ nick translation histochemistry. In parallel experiments, we examined the expression an inducible member of the heat shock protein family, HSP-72, another putative marker of neuronal injury, using a monoclonal antibody. Seizure severity paralleled kainate dosage. At higher doses DNA fragmentation is seen mainly in hippocampus in area CA3, and variably in CA1, thalamus and amygdala within 24 h, is maximal within 72 h, and is largely gone by 7 days after administration of kainate. HSP-72 expression is also highly selective, occurring in limbic structures, and it evolves over a characteristic time-course. HSP-72 is expressed mainly in structures that also manifest DNA fragmentation. Using double-labeling techniques, however, we find essentially no overlap between neurons expressing HSP-72 and DNA fragmentation. These findings indicate that DNA fragmentation and HSP-72 expression are complementary markers of seizure-induced stress and injury, and support the notion that HSP-72 expression is neuroprotective following kainate-induced seizures.     

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.     

Effectiveness of lidocaine infusion for status epilepticus in childhood: a retrospective multi-institutional study in Japan

We evaluated the usefulness of intravenous lidocaine therapy for managing of status epilepticus (SE) during childhood in a retrospective multi-institutional study. Questionnaires were sent to 28 hospitals concerning patients admitted for SE who were managed with lidocaine, assessing patient characteristics, treatment protocols and efficacy. In 279 treated patients, 261 SE occurrences at ages between 1 month and 15 years were analyzed. SE was classified as showing continuous, clustered, or frequently repeated seizures. Considering efficacy and side effects in combination, the usefulness of lidocaine was classified into six categories: extremely useful, useful, slightly useful, not useful, associated with deterioration, or unevaluated. In 148 SE cases (56.7%), lidocaine was rated as useful or extremely useful. Multivariate analysis indicated lidocaine was to be useful in SE with clustered and frequently repeated seizures, and SE attributable to certain acute illnesses, such as convulsions with mild gastroenteritis. Efficacy was poor when SE caused by central nervous system (CNS) infectious disease. Standard doses (approximately 2mg/kg as a bolus, 2mg/kg/h as maintenance) produced better outcomes than lower or higher doses. Poor responders to the initial bolus injection of lidocaine were less likely to respond to subsequent continuous infusion than good initial responders. We recommend lidocaine for use in SE with clustered or frequently repeated seizures, and in SE associated with benign infantile convulsion and convulsions with mild gastroenteritis. Lidocaine should be initiated with a bolus of 2mg/kg. If SE is arrested by the bolus, continuous maintenance infusion should follow; treatment should proceed to different measures when SE shows a poor response to the initial bolus of lidocaine.     

Neuronal trauma model: in search of Thanatos

Trauma to the nervous system triggers responses that include oxidative stress due to the generation of reactive oxygen species (ROS). DNA is a major macromolecular target of ROS, and ROS-induced DNA strand breaks activate poly(ADP-ribose)polymerase-1 (PARP-1). Upon activation PARP-1 uses NAD(+) as a substrate to catalyze the transfer of ADP-ribose subunits to a host of nuclear proteins. In the face of extensive DNA strand breaks, PARP-1 activation can lead to depletion of intracellular NAD(P)(H) pools, large decreases in ATP, that threaten cell survival. Accordingly, inhibition of PARP-1 activity after acute oxidative injury has been shown to increase cell survival. When NGF-differentiated PC12 cells, an in vitro neuronal model, are exposed to H(2)O(2) there is increased synthesis of poly ADP-ribose and decreases in intracellular NAD(P)(H) and ATP. Addition of the chemical PARP inhibitor 3-aminobenzamide (AB) prior to H(2)O(2) exposure blocks the synthesis of poly ADP-ribose and maintains intracellular NAD(P)(H) and ATP levels. H(2)O(2) injury is characterized by an immediate, necrotic cell death 2h after injury and a delayed apoptotic-like death 12-24h after injury. This apoptotic-like death is characterized by apoptotic membrane changes and apoptotic DNA fragmentation but is not associated with measurable caspase-3 activity. AB delays cell death beyond 24h and increases cell survival by approximately 25%. This protective effect is accompanied by significantly decreased necrosis and the apoptotic-like death associated with H(2)O(2) exposure. AB also restores caspase-3 which can be attributed to the activation of the upstream activator of caspase-3, caspase-9. Thus, the maintenance of intracellular ATP levels associated with PARP-1 inhibition shifts cell death from necrosis to apoptosis and from apoptosis to cell survival. Furthermore, the shift from necrosis to apoptosis may be explained, in part, by an energy-dependent activation of caspase-9.