Erythropoietin preconditioning on hippocampus neuronal apoptosis following status epilepticus induced by Li-pilocarpine in rats through anti-caspase-3 expression

Selective neuronal loss following status epilepticus (SE) was first described just under 100 years ago. The acute pathology following SE was shown to be 'ischemic cell change' and was assumed to arise through hypoxia/ischemia. Recently, erythropoietin (Epo) has been shown to have potent anti-apoptosis activity in central nervous system neurons in animal models of ischaemic injury. AIMS: In this report, in order to determine Epo preconditioning on hippocampus neuronal apoptosis, we examined caspase-3 expression following SE caused by Li-pilocarpine in rats. SETTINGS AND DESIGN: Animals were classified into three groups: EP group (pilocarpine group), rhEpo-pilocarpine group and control group. Four hours after preconditioning with Epo intraperitoneally, pilocarpine hydrochloride was administered intraperitoneally and observed for behavioral manifestations of SE. The animals were sacrificed at one hour after SE onset. MATERIALS AND METHODS: At the above-mentioned time point, animals were deeply anesthetized and were perfused through the left ventricle. Detection of hippocampus neuronal apoptosis was performed with caspase-3 immunohistochemical technique on three groups. To further confirm which cell population upregulates caspase-3, brain sections were stained for NeuN (green) and caspase-3 (red). STATISTICAL ANALYSIS: ANOVA and Fisher's post hoc test was used. RESULTS: Quatification of hippocampus neurons revealed that the number of caspase-3-positive cells in the CA1/CA3 area and dentate gyrus(DG) of three groups had a significant difference. In comparison with control group, there was an increase by 74% and 534%, 42% and 272% in the CA1/CA3 area and DG of EP group and rhEpo-treated group respectively. There was a decrease by 18% and 26% in the CA1/CA3 area and DG of rhEpo-treated group compared with those in EP group. In addition, colocalization of caspase-3 with NeuN was shown. CONCLUSIONS: Systemic rhEpo therapy reduced caspase-3 expression in SE induced by Li-pilocarpine.     

Comparison of neuropathology in rats following status epilepticus induced by diisopropylfluorophosphate and soman

The increasing number of cases involving the use of nerve agents as deadly weapons has spurred investigation into the molecular mechanisms underlying nerve agent-induced pathology. The highly toxic nature of nerve agents restrict their use in academic research laboratories. Less toxic organophosphorus (OP) based agents including diisopropylfluorophosphate (DFP) are used as surrogates in academic research laboratories to mimic nerve agent poisoning. However, neuropathology resulting from DFP-induced status epilepticus (SE) has not been compared directly to neuropathology observed following nerve agent poisoning in the same study. Here, the hypothesis that neuropathology measured four days after SE is the same for rats exposed to DFP and soman was tested. Adult Sprague-Dawley rats were injected with soman or DFP to induce SE. Cortical electroencephalography (EEG) was recorded prior to and during soman-induced SE. EEG power analysis of rats administered soman revealed prolonged electrographic SE similar to that of rats that endure uninterrupted SE following injection of DFP. Rats that experienced soman-induced SE displayed less hippocampal neuroinflammation and gliosis compared to rats administered DFP. Seizure-induced weight change, blood-brain barrier (BBB) leakiness and neurodegeneration in most seizure sensitive limbic brain regions were similar for rats that endured SE following soman or DFP. The amalgamated pathology score calculated by combining pathological measures (weight loss, hippocampal neuroinflammation, gliosis, BBB integrity and neurodegeneration) was similar in rats administered the OP agents. These findings support use of the rat DFP model of SE as a suitable surrogate for investigating some, but not all delayed consequences produced by nerve agents.     

Epileptogenesis in immature rats following recurrent status epilepticus

Strong evidences link status epilepticus (SE) in childhood with the later development of epilepsy. Pilocarpine-induced SE in developing rats leads to late appearance of spontaneous epileptic seizures only when SE is induced after the 18th day of life. We examined the possibility that 3 consecutive episodes of pilocarpine-induced SE on postnatal days 7, 8 and 9 could induce behavioral, electrographic and histological epileptic changes in adult life. The animals also underwent behavioral tests (inhibitory step-down avoidance, skinner box, rota-rod, open field and elevated plus-maze). EEG recordings made at the age of 30, 60 and 90 days showed the occurrence of several episodes of spikes and/or polyspikes appearing simultaneously in hippocampus and cortex. Only three isolated spontaneous seizures were observed during the whole period of observation (120 days). The long-term effects of three consecutive episodes of SE include increased spontaneous exploratory activity, learning impairment, and reduced anxiety when tested on P60. Our findings provide evidence for EEG changes and cognitive deficits in adult life following recurrent SE on postnatal days 7–9.     

Characterization of pharmacoresistance to benzodiazepines in the rat Li-pilocarpine model of status epilepticus

Status epilepticus is usually initially treated with a benzodiazepine such as diazepam. During prolonged seizures, however, patients often lose their sensitivity to benzodiazepines, thus developing pharmacoresistant seizures. In rats, administration of LiCl followed 20–24 h later by pilocarpine induces a continuous, self-sustained, and reproducible form of status epilepticus that can be terminated with diazepam when it is administered soon after the pilocarpine injection. However, when administered after a 45 min delay, diazepam is less effective. Previous findings have suggested that the development of pharmacoresistance is related to the stage of status epilepticus. In the present study, we characterized the seizure stage-dependence of diazepam pharmacoresistance. Following administration of different doses of diazepam at varying time intervals after specific behaviorally- and electrographically-defined seizure stages, stage-, time-, and dose-dependent pharmacoresistance to diazepam developed. We also studied two other antiepileptic drugs commonly used in the treatment of status epilepticus, phenobarbital and phenytoin. Consistent with previous studies, our results indicated a similar relationship between stage, time and dose for phenobarbital, but not for phenytoin. Our data are consistent with rapid modulation of GABA_A receptors during status epilepticus that may result in pharmacoresistance to antiepileptic drugs that enhance GABA_A receptor-mediated inhibition.     

癫痫持续状态诱导发育幼鼠海马神经的发生

目的 观察发育期幼鼠癫痫持续状态(SE)后海马内神经干细胞(NSCs)的增殖、迁移与分化特点.方法 日龄7d、14 d、21 d、28 d(P7、P14、P21、P28)健康SD大鼠共320只,完全随机法分为SE组和正常对照组,戊四氮造模后各个日龄组大鼠再随机分为1、7、14、21、28 d5个时间点,每个时间点8只,通过免疫组织化学法观察发作后不同时间点的齿状回区细胞增殖、迁移情况.再选P14日龄大鼠64只随机分为SE组和正常对照组,通过荧光免疫双标记5-溴脱氧尿嘧啶核苷(Brdu)和神经元特异性核蛋白(NeuN)、Brdu和胶质纤维酸性蛋白(GFAP)观察NSCs分化情况.结果 (1)P7、P14、P21、P28幼鼠SE后1d齿状回出现nestin阳性细胞,7 d nestin阳性细胞逐渐增多,14 d达到峰值,21 d逐渐减少,28 d下降至最低.其中P7幼鼠SE后7 d nestin阳性细胞数为177.00±3.22(t=16.033)、14 d幼鼠nestin阳性细胞数为195.00±3.41(t =28.840)明显高于各自正常对照组(147.50±2.08,136.50±2.65,均P＜0.05),总体表现出先上升后下降的趋势.同时SE发作日龄越小,nestin表达强度越大,而在生理状态下幼鼠齿状回中nestin表达随着日龄的增加而逐渐减少;(2)SE后1d及7d时大部分nestin阳性细胞分布于颗粒细胞下层,14 d nestin阳性细胞逐渐向颗粒细胞层迁移,迁移时细胞形态发生改变.在P14、P21、P28 3个日龄组有少部分异位迁移入海马的门区,同时在海马CA1、CA3及顶皮质区也见到形态不同的nestin阳性细胞;(3)Brdu阳性细胞大多同时表达NeuN,有4％ ～5％共表达GFAP.结论 SE可诱导发育幼鼠海马齿状回区的神经发生,并且具有年 龄相关性特点.新生细胞大部分从齿状回颗粒细胞下层迁移到颗粒细胞层,少部分异位迁移到海马门区,多数分化为神经元,少部分分化为胶质神经细胞.     

Pentoxifylline ameliorates lithium-pilocarpine induced status epilepticus in young rats

The neuroprotective effects of pentoxifylline (PTX) against lithium-pilocarpine (Li-Pc)-induced status epilepticus (SE) in young rats are described. Animals treated with PTX (0, 20, 40, and 60 mg/kg) before induction of SE were examined for latency to and frequency of SE, behavioral changes, oxidative stress, neurochemical alterations in the hippocampus and striatum, and histological abnormalities in the hippocampus. Treatment with PTX significantly ameliorated the frequency and severity of epileptic seizures in a dose-dependent manner. Our behavioral studies using the elevated plus-maze, rotarod, and water maze tests suggested a significant reduction in anxiety, enhanced motor performance, and improved learning and memory in PTX-treated rats. Li-Pc-induced neuronal cell loss and sprouting of mossy fibers in the hippocampus were also attenuated by PTX. The neuroprotective activity of PTX was accompanied by reduction in oxidative stress and reversal of SE-induced depletion of dopamine and 5-hydroxytryptamine in hippocampus and striatum. The results of this study provide a good rationale to explore the prophylactic/therapeutic potential of PTX in SE.     

Human neural stem cell transplantation reduces spontaneous recurrent seizures following pilocarpine-induced status epilepticus in adult rats

Transplantation of neural stem cells (NSCs) can replace lost neurons and improve the functional deficits. Cell transplantation strategies have been tried in the epileptic disorder, but the effect of exogenous NSCs is unknown. In this study, we attempted to test the anti-epileptogenic effect of NSCs in adult rats with status epilepticus. Experimental status epilepticus was induced by lithium-pilocarpine injection, and beta galactosidase-encoded human NSCs were transplanted intravenously on the next day of status epilepticus. Spontaneous recurrent seizures were monitored with Racine's seizure severity scale. Immunohistochemistry with anti-beta gal, Tuj-1, NeuN, GFAP, CNPase, GluR2, parvalbumin, and GABA were performed and extracellular field excitatory postsynaptic potentials (fEPSP) were recorded. Human NSCs suppressed spontaneous recurrent seizure formation and transplanted NSCs were differentiated into GABA-immunoreactive interneurons in the damaged hippocampus. Amplitude of fEPSP in the hippocampal CA1 was reduced, which was reversed by picrotoxin. These findings suggest that NSCs could be differentiated into inhibitory interneurons and decrease neuronal excitability, which could prevent spontaneous recurrent seizure formation in adult rats with pilocarpine-induced status epilepticus.     

Characterization of status epilepticus induced by two organophosphates in rats

Organophosphates (OPs) inhibit the enzyme cholinesterase and cause accumulation of acetylcholine, and are known to cause seizures and status epilepticus (SE) in humans. The animal models of SE caused by organophosphate analogs of insecticides are not well characterized. SE caused by OPs paraoxon and diisopropyl fluorophosphate (DFP) in rats was characterized by electroencephalogram (EEG), behavioral observations and response to treatment with the benzodiazepine diazepam administered at various stages of SE. A method for SE induction using intrahippocampal infusion of paraoxon was also tested. Infusion of 200nmol paraoxon into the hippocampus caused electrographic seizures in 43/52 (82.7%) animals tested; and of these animals, 14/43 (30%) had self-sustaining seizures that lasted 4-18h after the end of paraoxon infusion. SE was also induced by peripheral subcutaneous injection of diisopropyl fluorophosphate (DFP, 1.25mg/kg) or paraoxon (1.00mg/kg) to rats pretreated with atropine (2mg/kg) and 2-pralidoxime (2-PAM, 50mg/kg) 30min prior to OP injection. SE occurred in 78% paraoxon-treated animals and in 79% of DFP-treated animals. Diazepam (10mg/kg) was administered 10min and 30min after the onset of continuous EEG seizures induced by paraoxon and it terminated SE in a majority of animals at both time points. DFP-induced SE was terminated in 60% animals when diazepam was administered 10min after the onset of continuous EEG seizure activity but diazepam did not terminate SE in any animal when it was administered 30min after the onset of continuous seizures. These studies demonstrate that both paraoxon and DFP can induce SE in rats but refractoriness to diazepam is a feature of DFP induced SE.     

大鼠癫痫持续状态后水通道蛋白-4的表达

目的 探讨癫痫持续状态（SE）后大鼠水通道蛋白-4（AQP4）的表达变化与脑水肿形成之间的关系。方法 54只SD大鼠随机分为对照组（n=6），SE后6h，12h，24h，48h，72h，96h，120h，168h组（n=6）。腹腔注射锂-匹罗卡品建立大鼠SE模型，免疫组织化学染色和逆转录PCR方法检测AQP4蛋白和基因在SE形成后的表达。结果 SE后AQV4蛋白和mRNA24h表达水平明显增加，48h达到高峰，持续72h后下降，168h时仍有表达。SE后AQP4表达变化和脑水肿形成过程在时间上呈明显的正相关（r=0．73，氏0．05）。结论 SE后AOP4表达明显增强，在时间上与脑水肿形成呈正相关，提示AQP4在SE后脑水肿形成过程中起着重要作用。     

Beta adrenergic blockade prevents cardiac dysfunction following status epilepticus in rats

Status epilepticus (SE) can result in temporary cardiac dysfunction in patients, characterized by reduced ejection fraction, decreased ventricular contractility, and alterations in electrical activity of the heart. Although reversible, the cardiac effects of seizures are acutely life threatening, and may contribute to the delayed mortality following SE. The precise mechanisms mediating acute cardiac dysfunctions are not known. These studies evaluated effects of self-sustaining limbic SE in rats on cardiac performance 24h following seizures, and determined if sympathetic nervous system activation during seizures contributed to cardiac dysfunction. Rats subjected to SE received either vehicle (saline) or the B1 adrenergic antagonist atenolol (AT) prior to and during 90 min of seizure activity. Control rats were similarly treated, except they did not undergo seizures. Twenty-four hours after SE, animals were anesthetized and catheterized for measurement of cardiac performance variables. Animals undergoing SE demonstrated significantly reduced cardiac output, decreased ventricular contractility and relaxation, increased blood pressure, and prolonged QT interval. However, heart rate was not altered. Treatment with AT prevented all changes in cardiac performance due to SE, and attenuated the increase in QT interval. These data demonstrate that SE in the rat results in cardiac dysfunction 24h following seizures, mediated by the sympathetic nervous system.     

Development of focal chronic epilepsy following focal status epilepticus in adult patients.

In several experimental models, status epilepticus (SE) leads to secondary brain hyperexcitability and epileptogenesis. In humans, such phenomena have been rarely demonstrated, particularly in cases of SE involving the neocortical structures. We report a 36 year old woman that presented partial SE in May 1991 involving the right cerebral hemisphere. The patient was then treated in the intensive care unit with artificial ventilation and anesthesia by pentobarbital and clometiazole. MRI showed transient right parietal and temporal posterior cortical hyperintensity. The cause of SE was not determined. Three months later, the patient developed partial complex seizures with aura characterized by vertigo, nausea and auditory hallucination. Ictal video/EEG recording showed a clear right temporal posterior onset of the discharges. We speculate that status epilepticus created the lesions which subsequently caused the focal chronic epilepsy.     

Sleep structure following status epilepticus

OBJECTIVES: To determine the effect of status epilepticus (SE) on sleep. BACKGROUND: SE has a high incidence of morbidity and mortality. The study of sleep structure following SE may have implications for recovery in these patients. METHODS: Twenty-four hour polysomnography was recorded in a 52-year-old patient following generalized convulsive SE not complicated by other medical or neurologic conditions. Another patient with no known history of seizures was recorded under similar conditions. RESULTS: The first day following SE was associated with markedly abnormal sleep structure, consisting largely of stage 1 with minimal slow wave or REM sleep. Over 4 days, slow wave and REM returned to normal values and no rebound was seen. The control patient demonstrated normal sleep parameters for their age, demonstrating that sleep disruption was not due to recording conditions alone. CONCLUSIONS: This case demonstrates that sleep structure is markedly abnormal following generalized convulsive SE. As sleep may serve a restorative function, improving sleep postictally may hasten or improve recovery. Larger studies will be required to determine whether this is a common finding in such patients, and whether outcome is associated with improved sleep quality.     

Early-life status epilepticus induces ectopic granule cells in adult mice dentate gyrus

A large number of aberrant hilar granule cells (GCs) are found in the patients and animal models of adult temporal lobe epilepsy (TLE), and these “ectopic” GCs have synchronous epileptiform bursting with other hippocampal neurons. In this study, we investigated whether early-life status epilepticus (SE) induces hilar ectopic GCs that remain in the adulthood because TLE patients frequently experience seizures in the early childhood when a large number of postnatally born GCs migrate in the hilus. To label newborn GCs, bromodeoxyuridine (BrdU) was injected daily for three consecutive days to C57BL/6J mice at different postnatal days starting at postnatal-0-day-old (P0) (Group1), P7 (Group2), or P35 (Group3). Mice in each group underwent pilocarpine-induced SE at P14. Six months later, to determine whether SE induces ectopic GCs, we plotted the distribution of postnatally born GCs which were immunohistochemically defined as BrdU- and the GC marker Prox1-colabeled cells. We also examined whether SE causes the granule cell layer (GCL) dispersion and/or the mossy fiber (MF) sprouting, other representative pathologies of TLE hippocampus. Only SE-experiencing mice in Group1 had significantly more neonatally born ectopic GCs compared with control mice. Neither control nor SE mice had dispersed GCL. All mice that underwent SE had sprouted MFs in CA3. We conclude that early-life SE disrupts a normal incorporation of GCs born pre-SE but not post-SE, inducing ectopic GCs in the adult hilus. Interestingly, the results also indicate that developmentally earlier born GCs are more responsive to early-life SE in terms of the emergence of ectopic GCs.     

Brain amino acid concentration changes during status epilepticus induced by lithium and pilocarpine

Amino acid concentrations were measured in specific structures from the brains of rats decapitated before and during the course of status epilepticus induced by lithium and pilocarpine, with the stages of status defined by the electroencephalographic (EEG) pattern displayed. Early status was marked by discrete seizures on EEG, mid status by continuous spiking, and late status by periodic epileptiform discharges. Aspartate levels were lower than control levels in most regions prior to the onset of status. The decline continued and reached statistical significance in different regions at times from early to late status. Glutamate concentrations were typically higher than control just prior to status onset and then decreased in a manner similar to aspartate, but with less percentage change. gamma-Aminobutyric acid increased during status, with the earliest statistically significant differences observed in mid status. These changes were observed in most forebrain structures studied, but the largest percentage changes in excitatory amino acid concentration were found in substantia nigra, where they fell to less than half of control.     

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.