Pentylenetetrazol seizures induce cell suffering but not death in the immature rat brain

To investigate whether long-term functional consequences of status epilepticus (SE) induced by pentylenetetrazol in 10-day-old rats correlated with cell injury and/or death, acid fuchsin and TUNEL staining were performed between 4 to 144 h after SE. Acid fuchsin stained hippocampus, amygdala and cerebral cortex at 24 h but not at 72 and 144 h. No DNA fragmentation was apparent at any time. Thus, immature neurons subjected to sustained seizures suffer transiently but survive probably by activating repair processes.     

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.     

Corticotropin-releasing factor – immunolabeled fibers in brain regions with localized kainate neurotoxicity

Rats treated with the neuroepileptic drug, kainic acid, exhibit a specific regional pattern of neurodegeneration 24 h following onset of acute limbic status epilepticus. At 24 h post-seizure, the areas undergoing neurodegeneration also exhibit substantial amounts of the neuropeptide corticotropin-releasing factor (CRF) which is not present under normal conditions. In experimental brains, CRF is localized immunocytochemically to cells and densely labeled fibers in areas with neurodegeneration. Networks of CRF fibers closely surround moribund neurons staining intensely for acid fuchsin. Acid fuchsin, an acidophilic dye, is used routinely as a marker for irreversible neuronal injury, and acid fuchsin-positive neurons are identified in specific areas affected by kainic neurotoxicity. Evidence exists in the literature that CRF functions in brain as a excitatory neurotransmitter/neuromodulator. Under certain pathological conditions (i.e., seizures, brain trauma, ischemia), it has been postulated that CRF could act as an neurotoxic agent. This study provides anatomical evidence that CRF may function following seizures as an neurotoxin because of the close proximity of CRF-labeled fibers to degenerating neurons. Received: 19 October 1998 / Revised: 3 February 1999 / Accepted: 24 March 1999     

Status epilepticus in epileptogenesis

There has been direct evidence of gamma-aminobutyric acidA receptor modification during status epilepticus. Neuropeptides galanin and neuropeptide Y were demonstrated to play a role in terminating status epilepticus. Many of the CA3 pyramidal neurons destined to die as a consequence of status epilepticus were demonstrated to diminish expression of the GluR2 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors. It was demonstrated that the pattern of cell loss due to status epilepticus is distinct in immature pups compared with adult rats. The genetic basis for susceptibility to neuronal loss during status epilepticus was described. There was increasing evidence of unique receptors and ion channels in the epileptic brain. The molecular studies of epileptic gamma-aminobutyric acidA receptors present on dentate granule cells of rats with temporal lobe epilepsy revealed altered gene and receptor expression before onset of recurrent spontaneous seizures. They also revealed insertion of new gamma-aminobutyric acidA receptors in the inhibitory synapses present on soma and proximal dendrites of dentate granule cells.     

Neuropathology of limbic status epilepticus induced by electrical stimulation of naive rats.

Neuronal damage in relation to the duration of seizure was studied in limbic status epilepticus (SE) induced by electric stimulation of naive rats. Adult Sprague-Dawley rats were stimulated at the right amygdala to induce SE. To stop the seizures, diazepam was given to different groups of rats at 0.5 h (n = 4), 1 h (n = 6), 2 h (n = 6), and 3-4 h (n = 8) of SE. Eighteen hours after the end of SE, the rats were perfusion fixed. Naive (n = 6) and sham-operated (n = 4) rats served as controls. Horizontal paraffin sections were stained with acid fuchsin and cresyl violet. Neuronal damage was absent after 30 min of SE. Status epilepticus of 1 h or longer duration regularly caused neuronal damage to the cerebral cortex, thalamus, hippocampus, amygdala, and pars reticulata of the substantia nigra. Damage in the cerebral cortex predominated in the entorhinal, temporal, and pyriform regions. In the hippocampus, the dentate hilus was most severely affected, followed by CA3 and CA1. Damage to the dentate granule layer was mild. Further studies of the pathophysiology of excitotoxicity may help to protect patients from sequels of status epilepticus such as neuronal damage and epilepsy.     

Resistance of immature hippocampus to morphologic and physiologic alterations following status epilepticus or kindling.

Seizures in adult rats result in long-term deficits in learning and memory, as well as an enhanced susceptibility to further seizures. In contrast, fewer lasting changes have been found following seizures in rats younger than 20 days old. This age-dependency could be due to differing amounts of hippocampal neuronal damage produced by seizures at different ages. To determine if there is an early developmental resistance to seizure-induced hippocampal damage, we compared the effects of kainic acid (KA)-induced status epilepticus and amygdala kindling on hippocampal dentate gyrus anatomy and electrophysiology, in immature (16 day old) and adult rats. In adult rats, KA status epilepticus resulted in numerous silver-stained degenerating dentate hilar neurons, pyramidal cells in fields CA1 and CA3, and marked numerical reductions in CA3c pyramidal neuron counts (-57%) in separate rats. Two weeks following the last kindled seizure, some, but significantly less, CA3c pyramidal cell loss was observed (-26%). Both KA status epilepticus and kindling in duced mossy-fiber sprouting, as evidenced by ectopic Timm staining in supragranular layers of the dentate gyrus. In hippocampal slices from adult rats, paired-pulse stimulation of perforant path axons revealed a persistent enhancement of dentate granule-cell inhibition following KA status epilepticus or kindling. While seizures induced by KA or kindling in 16-day-old rats were typically more severe than in adults, the immature hippocampus exhibited markedly less KA-induced cell loss (-22%), no kindling-induced loss, no detectable synaptic rearrangement, and no change in dentate inhibition. These results demonstrate that, in immature rats, neither severe KA-induced seizures nor repeated kindled seizures produce the kind of hippocampal damage and changes associated with even less severe seizures in adults. The lesser magnitude of seizure-induced hippocampal alterations in immature rats may explain their greater resistance to long-term effects of seizures on neuronal function, as well as future seizure susceptibility. Conversely, hippocampal neuron loss and altered synaptic physiology in adults may contribute to increased sensitivity to epileptogenic stimuli, spontaneous seizures, and behavioral deficits.     

Temporal and spatial characterization of neuronal injury following lateral fluid-percussion brain injury in the rat

The pattern of neuronal injury following lateral fluid-percussion (FP) brain injury in the rat was systematically characterized at sequential time points to identify selectively vulnerable regions and to determine the temporal contribution of primary and delayed neuropathological events. Male Sprague-Dawley rats (n = 28) were killed 10 min, 2 h, 12 h, 24 h, 4 days, and 7 days following a lateral FP brain injury of moderate severity (2.2 atm), or 24 h after a sham injury. Brain sections were stained and analyzed using Nissl, acid fuchsin, and silver staining methods to identify regions with injured neurons or with visible lesions. Extensive numbers of acid fuchsin or silver-stained neurons were observed as early as 10 min after the FP brain injury in regions extending from the caudate/putamen to the pons. The frequency of injured neurons was greatest in the ipsilateral cortex, hippocampus, and thalamus, and a visible loss of Nissl-stained neurons was observed in these regions beginning at 12 h after the FP brain injury. Acid fuchsin-stained neurons were restricted to the same brain regions for all of the survival periods and gradually decreased in numbers between 24 h and 7 days after injury. These findings suggest that lateral FP brain injury in the rat produces a combination of focal cortical contusion and diffuse subcortical neuronal injury, which is present within minutes of the impact, progresses to a loss of neurons by 12 h, and does not markedly expand into other brain regions with survival periods up to 7 days. Furthermore, the acute onset and rapid evolution of the neuronal injury process may have important implications when considering a window of opportunity for pharmacological intervention. Received: 23 May 1995 / Revised, accepted: 15 September 1995     

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.     

Stimulation-induced status epilepticus: role of the hippocampal mossy fibers in the seizures and associated neuropathology

A study of seizure activity and neuronal cell death produced by intracerebroventricular kainic acid had suggested that seizures conveyed by the hippocampal mossy fibers are more damaging to CA3 pyramidal cells than seizures conveyed by other pathways. To test this idea, the effects of a unilateral mossy fiber lesion were determined on seizure activity and neuronal degeneration provoked by repetitive electrical stimulation of the hippocampal fimbria in unanesthetized rats. Fimbrial stimulation resulted in self-sustained status epilepticus accompanied by neuronal degeneration in several brain regions, including area CA3 of the hippocampal formation. A unilateral mossy fiber lesion more readily attenuated the electrographic and behavioral seizures provoked by fimbrial stimulation than those provoked by kainic acid. If status epilepticus developed in the presence of a mossy fiber lesion, denervated CA3 pyramidal cells were still destroyed, although similar lesions protect these neurons from kainic acid-induced status epilepticus. Thus the two models of status epilepticus employ somewhat different seizure circuitries and neurodegenerative mechanisms. Seizures which involve the mossy fiber projection are not necessarily more damaging to CA3 pyramidal cells than seizures which do not.     

In vivo and in vitro staining of acidophilic neurons as indicative of cell death following kainic acid-induced lesions in rat brain

Summary An in vivo method for positively staining dead neurons was developed and compared with an in vitro staining method using acid fuchsin. Neurons previously killed by intracerebral injections of kainic acid were selectively stained by trypan blue within 15 min of its injection in vivo into the central nervous system of rats. Such staining persisted for at least 4 days in vivo, and there was no evidence that trypan blue itself was toxic to the remaining cells. Intense staining of neurons with acid fuchsin in vitro was first observed in brain sections of rats killed about 6 h after kainic acid injection. This time was approximately 2–3 h before trypan blue, in vivo, was able to stain neurons. Thus, the loss of transport mechanisms (at least for trypan blue) apparently occurs subsequent to the development of basic products stainable with acidic dyes. At the earliest times, acid fuchsin stained neurons which had not yet lost Nissl substance, whereas the majority of trypan blue-stained neurons were not stained with Nissl dyes. After 24 h the majority of neurons stained with either acid fuchsin or trypan blue were Nissl-stain negative. The combination of staining with trypan blue in vivo with subsequent counterstaining of brain sections with acid fuchsin in vitro may have a potential use in the determination of the time of neuronal death in vivo.Supported by a grant from the Auckland Medical Research Foundation     

Topiramate reduces neuronal injury after experimental status epilepticus

Prolonged seizures are associated with injury to vulnerable neurons, particularly in the hippocampus. Identification of compounds that attenuate injury after prolonged seizures could be of value in the management of refractory status epilepticus. We hypothesized that topiramate, an anticonvulsant with multiple mechanisms of action, would attenuate hippocampal neuronal injury when given after experimental status epilepticus. Limbic status epilepticus was induced in adult male Wistar rats for 140 min by unilateral hippocampal electrical stimulation. Rats then received intraperitoneal injections of either vehicle (n=6) or topiramate at 20 mg/kg (n=6), 40 mg/kg (n=7) or 80 mg/kg (n=7). Three days later, hippocampal sections were processed for neuronal degeneration using a silver impregnation stain. Seizure-induced damage was assessed by measuring the density of silver staining in hippocampal regions CA1, CA3 and dentate hilus. Administration of topiramate at each dose was associated with a significant reduction in staining density bilaterally in area CA1 and the dentate hilus. Reduction in staining density in area CA3 was seen contralateral to the side of stimulation at the two higher topiramate doses only. The results indicate that administration of topiramate after experimental status epilepticus can attenuate seizure-induced hippocampal neuronal injury.     

Selective death of hippocampal CA3 pyramidal cells with mossy fiber afferents after CRH-induced status epilepticus in infant rats

Previous studies of CRH-induced status epilepticus in infant rats demonstrated neuronal loss in several limbic structures, including the CA3 region of the hippocampus. The goal of the present study was to identify the neurons affected by CRH-induced seizures and determine whether they formed synapses with afferent axon terminals. Clusters of neurons in the CA3 region of the hippocampus were osmiophilic when viewed in thick sections. Semi-thin 2-μ sections of the pyramidal cell layer contained dark, shrunken neurons with apical and basal dendrites among normal appearing pyramidal cells. Electron microscopy revealed degenerating pyramidal cells with intact cell membranes and electron dense nuclei and cytoplasm. The shrunken dendrites of these cells had spines and were postsynaptic to large immature-appearing mossy fibers. Thus, CA3 pyramidal neurons that are linked via mossy fibers to the tri-synaptic excitatory hippocampal circuit die subsequent to CRH-induced status epilepticus. The shrunken appearance and selective loss of these neurons are incompatible with necrosis as the mechanism of degeneration.     

Neuropathology of limbic status epilepticus induced by electrical stimulation of naive rats

Abstract

Neuronal damage in relation to the duration of seizure was studied in limbic status epilepticus (SE) induced by electric stimulation of naive rats. Adult Sprague-Dawley rats were stimulated at the right amygdala to induce SE. To stop the seizures, diazepam was given to different groups of rats at 0.5 h (n = 4), 7 h fn = 6), 2 h (n = 6), and 3-4 h (r\ = 8) of SE. Eighteen hours after the end of SE, the rats were perfusion fixed. Naive (n = 6) and sham-operated (n = 4) rats served as controls. Horizontal paraffin sections were stained with acid fuchsin and cresyl violet. Neuronal damage was absent after 30 min of SE. Status epilepticus of I h or longer duration regularly caused neuronal damage to the cerebral cortex, thalamus, hippocampus, amygdala, and pars reticulata of the substantia nigra. Damage in the cerebral cortex predominated in the entorhinal, temporal, and pyriform regions. In the hippocampus, the dentate hilus was most severely affected, followed by CA3 and CAT. Damage to the dentate granule layer was mild. Further studies of the pathophysiology of excitotoxicity may help to protect patients from sequels of status epilepticus such as neuronal damage and epilepsy. [Neurol Res 1999; 21: 399-403]     

Resistance of the immature hippocampus to seizure-induced synaptic reorganization.

Temporal lobe epilepsy is a common form of epilepsy in human adults and is associated with a unique pattern of damage in the hippocampus. The damage includes cell loss of the CA3 and CA4 areas and synaptic growth (sprouting) of mossy fibers in the supragranular layer of the dentate gyrus. Experimental evidence indicates that in adult rats the excitatory amino acid, kainic acid, induces a similar pattern of changes in hippocampal circuitry associated with alterations in perforant path excitation and inhibition. It has been suggested that, in humans, this type of damage may be a result of seizures early in life. In this study we examined the effects of kainic acid-induced status epilepticus on synaptic reorganization and paired-pulse electrophysiology in developing rats and adults. Kainic acid induced more severe seizures in 15-day-old rat pups than in adults. In contrast to adult rats, these seizures did not produce CA3/CA4 neuronal loss, mossy fiber sprouting or changes in paired-pulse excitation or inhibition in the hippocampus of rat pups tested 2-4 weeks after status epilepticus. Our results provide evidence that the immature hippocampus may be more resistant to seizure-induced changes than the mature hippocampus.     

Nitric oxide synthase immunoreactivity in the rat hippocampus after status epilepticus induced by perforant pathway stimulation

Nitric oxide has recently been implicated in mediation of neuronal excitotoxicity and damage. This study aimed at elucidating the changes in the expression of neuronal isoform of nitric oxide synthase (nNOS) in the hippocampus after status epilepticus induced by perforant pathway stimulation. nNOS-immunoreactivity (nNOS-ir) and neuronal damage, assessed by silver staining, were evaluated separately in different hippocampal subfields 2 weeks after induction of status epilepticus. Perforant pathway stimulation resulted in an increase in the number of nNOS-immunoreactive neurons in the stratum radiatum of the CA1 and CA3 subfields of the hippocampus proper, and the hilus of the dentate gyrus. The morphology and distribution of the nNOS-ir neurons resembled that of interneurons. No correlation of the number of nNOS-ir neurons to the neuronal damage score was observed. Our results suggest that status epilepticus provokes a de novo expression of nNOS protein, and the nNOS expressing neurons may be selectively resistant to epileptic brain injury.     

幼鼠早期癫痫持续状态对海马结构损伤的远期影响

目的　探讨幼鼠早期癫痫持续状态 (status epilepticus,SE)对成鼠后海马结构损伤的远期影响。方法　健康生后 1 5～ 2 0 d Wistar幼鼠 48只 ,随机分为生理盐水对照组和氯化锂 -匹罗卡品腹腔注射诱导的 SE组 ,并持续追踪至成鼠阶段 ,应用常规病理及电镜观察海马结构的形态学改变 ,同时应用 Timm组织化学染色方法进行苔藓纤维发芽研究。结果　约 2 /3 SE幼鼠发育至成鼠阶段后 ,海马结构的神经元仍可发现变性和坏死性改变 ,以 CA1区、CA3区和齿状回为重。 Timm染色见 CA3区有苔藓纤维发芽现象。约 1 /3 SE幼鼠发育至成鼠后未见海马结构损伤性改变。结论　幼鼠早期 SE造成的海马结构损伤性改变可持续至成鼠阶段 ,但存在个体差异 ,可能与个体耐受性有关。     

Synaptic reorganization in subiculum and CA3 after early-life status epilepticus in the kainic acid rat model

PURPOSE: The immature rat brain is highly susceptible to seizures, but has a resistance to pathological changes induced by seizures as compared to adult rats. However, prolonged seizures during early-life enhance cellular injury and hyperexcitability induced by convulsive insults later in adulthood. The mechanisms underlying these phenomena are not understood. In adult models, the CA1 axons reorganize their projections to subiculum. Seizure induced plasticity in this pathway has not been investigated in immature seizure models, and may contribute to the vulnerability to later seizures. METHODS: On postnatal day 15, rats experienced convulsive status epilepticus with kainic acid (KA). Seizure induced plasticity was examined with Timm histochemistry and iontophoretic injections of sodium selenite, a retrograde tracer. Cellular injury was evaluated with Fluoro-Jade B histochemistry. RESULTS: Retrograde tracing experiments determined a 67% larger dorsoventral extent of retrograde labeling in the CA1 pyramidal region after tracer injections in subiculum. The synaptic reorganization of the CA1 projection to subiculum was noted in the absence of overt neuronal injury in subiculum or CA1. In contrast, mossy fiber sprouting was detected into the stratum oriens of CA3 with limited neuronal injury to CA3 pyramidal neurons. No mossy fiber sprouting into the inner molecular layer of the dentate gyrus, or CA1 sprouting into the stratum moleculare of CA1 were noted. CONCLUSIONS: The results indicate that the developing brain has distinct mechanisms of seizure induced reorganization as compared to the adult brain. Our experiments show that the concept of "resistance of the immature brain to excitotoxicity" is considerably more complicated than generally believed. Morphological plasticity in the immature brain appears more extensive in distal, but not proximal, projections of hippocampal pathways, and across hippocampal lamellae. The abnormal connectivity between hippocampal lamellae might play a role in the increased susceptibility to injury and hyperexcitability associated with later convulsive insults.     

Hydroethidine detection of superoxide production during the lithium-pilocarpine model of status epilepticus

Hydroethidine is reported to be selectively oxidized to ethidium by superoxide. Using digital imaging and fluorescence microscopy it is possible to evaluate neuronal ethidium accumulation in specific brain regions of rats damaged in the lithium-pilocarpine model of status epilepticus. Intravenous or intraperitoneal administration of hydroethidine prior to 1 h of status epilepticus produced diffuse cytosolic distribution of ethidium fluorescence suggesting an increased neuronal production of superoxide that was not observed in control animals. A significantly increased number of neurons with the enhanced ethidium fluorescence was observed in parietal cortex, piriform cortex, perirhinal cortex, lateral amygdala, mediodorsal thalamus and laterodorsal thalamus, suggesting superoxide as a mechanism of neuronal injury in those regions. Other regions injured by lithium-pilocarpine seizures, such as the basolateral amygdala and hippocampus, did not demonstrate the enhanced neuronal ethidium fluorescence. In such regions it is possible that superoxide is not a mechanism of injury or that 1 h of status epilepticus is not sufficient to produce superoxide or other reactive oxygen species.