Oxidative stress in the hippocampus after pilocarpine-induced status epilepticus in Wistar rats

The role of oxidative stress in pilocarpine-induced status epilepticus was investigated by measuring lipid peroxidation level, nitrite content, GSH concentration, and superoxide dismutase and catalase activities in the hippocampus of Wistar rats. The control group was subcutaneously injected with 0.9% saline. The experimental group received pilocarpine (400 mg.kg(-1), subcutaneous). Both groups were killed 24 h after treatment. After the induction of status epilepticus, there were significant increases (77% and 51%, respectively) in lipid peroxidation and nitrite concentration, but a 55% decrease in GSH content. Catalase activity was augmented 88%, but superoxide dismutase activity remained unaltered. These results show evidence of neuronal damage in the hippocampus due to a decrease in GSH concentration and an increase in lipid peroxidation and nitrite content. GSH and catalase activity are involved in mechanisms responsible for eliminating oxygen free radicals during the establishment of status epilepticus in the hippocampus. In contrast, no correlations between superoxide dismutase and catalase activities were observed. Our results suggest that GSH and catalase activity play an antioxidant role in the hippocampus during status epilepticus.     

Neuroprotective effect of pyruvate and oxaloacetate during pilocarpine induced status epilepticus in rats

Recent research data have shown that systemic administration of pyruvate and oxaloacetate causes an increased brain-to-blood glutamate efflux. Since increased release of glutamate during epileptic seizures can lead to excitotoxicity and neuronal cell death, we tested the hypothesis that glutamate scavenging mediated by pyruvate and oxaloacetate systemic administration could have a neuroprotective effect in rats subjected to status epilepticus (SE). SE was induced by a single dose of pilocarpine (350mg/kgi.p.). Thirty minutes after SE onset, a single dose of pyruvate (250mg/kgi.p.), oxaloacetate (1.4mg/kgi.p.), or both substances was administrated. Acute neuronal loss in hippocampal regions CA1 and hilus was quantitatively determined five hours after SE onset, using the optical fractionator method for stereological cell counting. Apoptotic cascade in the hippocampus was also investigated seven days after SE using caspase-1 and -3 activity assays. SE-induced neuronal loss in CA1 was completely prevented in rats treated with pyruvate plus oxaloacetate. The SE-induced caspase-1 activation was significantly reduced when rats were treated with oxaloacetate or pyruvate plus oxaloacetate. The treatment with pyruvate and oxaloacetate caused a neuroprotective effect in rats subjected to pilocarpine-induced SE.     

Pilocarpine-induced status epilepticus causes N-methyl-D-aspartate receptor-dependent inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake

Status epilepticus is associated with sustained and elevated levels of cytosolic Ca(2+). To elucidate the mechanisms associated with changes of cytosolic Ca(2+) after status epilepticus, this study was initiated to evaluate the effect of pilocarpine-induced status epilepticus on Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in microsomes isolated from rat cortex, because the Ca(2+) uptake mechanism plays a major role in regulating intracellular Ca(2+) levels. The data demonstrated that the initial rate and overall Ca(2+) uptake in microsomes from pilocarpine treated animals were significantly inhibited compared with those in microsomes from saline-treated control animals. It was also shown that the inhibition of Ca(2+) uptake caused by status epilepticus was not an artifact of increased Ca(2+) release from microsomes, selective isolation of damaged microsomes from the homogenate, or decreased Mg(2+)/Ca(2+) ATPase protein in the microsomes. Pretreatment with the NMDA antagonist dizocilpine maleate blocked status epilepticus-induced inhibition of the initial rate and overall Ca(2+) uptake. The data suggest that inhibition of microsomal Mg(2+)/Ca(2+) ATPase Ca(2+) uptake is involved in NMDA-dependent deregulation of cytosolic Ca(2+) homeostasis associated with status epilepticus.     

Transcranial photobiomodulation attenuates pentylenetetrazole‐induced status epilepticus in peripubertal rats

Convulsive status epilepticus is the most common neurological emergency in children. Transcranial photobiomodulation (tPBM) reverses elevated rodent neurotransmitters after status epilepticus (SE) yet whether tPBM can attenuate seizure behaviors remains unknown. Here, we applied near‐infrared laser at wavelength 808 nm transcranially to peripubertal Sprague‐Dawley rats prior to pentylenetetrazole (PTZ) injection. Hematoxylin‐eosin, immunofluorescence (IF) staining with anti‐parvalbumin (PV) and terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assay after IF staining was performed. Behaviorally, tPBM attenuated the mean seizure score and reduced the incidence of SE and mortality. Histochemically, tPBM reduced dark neurons in the cortex, hippocampus, thalamus and hypothalamus, lessened the apoptotic ratio of parvalbumin‐positive interneurons (PV‐INs) and alleviated the aberrant extent of PV‐positive unstained somata of PCs in the hippocampus. Conclusively, tPBM attenuated PTZ‐induced seizures, SE and mortality in peripubertal rats and reduced PTZ‐induced neuronal injury, apoptosis of PV‐INs and preserved PV positive perisomatic inhibitory network in the hippocampus.     

Exposure to Mozart music reduces cognitive impairment in pilocarpine-induced status epilepticus rats

Patients with temporal lobe epilepsy (TLE) often display cognitive deficits. However, current epilepsy therapeutic interventions mainly aim at how to reduce the frequency and degree of epileptic seizures. Recovery of cognitive impairment is not attended enough, resulting in the lack of effective approaches in this respect. In the pilocarpine-induced temporal lobe epilepsy rat model, memory impairment has been classically reported. Here we evaluated spatial cognition changes at different epileptogenesis stages in rats of this model and explored the effects of long-term Mozart music exposure on the recovery of cognitive ability. Our results showed that pilocarpine rats suffered persisting cognitive impairment during epileptogenesis. Interestingly, we found that Mozart music exposure can significantly enhance cognitive ability in epileptic rats, and music intervention may be more effective for improving cognitive function during the early stages after Status epilepticus. These findings strongly suggest that Mozart music may help to promote the recovery of cognitive damage due to seizure activities, which provides a novel intervention strategy to diminish cognitive deficits in TLE patients.     

Molecular and cellular mechanisms of excitotoxic neuronal death

Glutamate receptor-mediated excitatory neurotransmission plays a key role in neural development, differentiation and synaptic plasticity. However, excessive stimulation of glutamate receptors induces neurotoxicity, a process that has been defined as excitotoxicity. Excitotoxicity is considered to be a major mechanism of cell death in a number of central nervous system diseases including stroke, brain trauma, epilepsy and chronic neurodegenerative disorders. Unfortunately clinical trials with glutamate receptor antagonists, that would logically prevent the effects of excessive receptor activation, have been associated with untoward side effects or little clinical benefit. Therefore, uncovering molecular pathways involved in excitotoxic neuronal death is of critical importance to future development of clinical treatment of many neurodegenerative disorders where excitotoxicity has been implicated. This review discusses the current understanding of the molecular and cellular mechanisms of excitotoxicity and their roles in the pathogenesis of diseases of the central nervous system.     

Autophagy is upregulated in rats with status epilepticus and partly inhibited by Vitamin E

Autophagy, a process of bulk degradation of cellular constituents through autophagosome-lysosomal pathway, is enhanced during oxidative stress. Whether autophagy is induced during status epilepticus (SE), which induces an excess production of reactive oxygen species (ROS) and leads to oxidative stress, is not established. We also sought to determine if pretreatment with Vitamin E reduced autophagy. We used pilocarpine to elicit SE in rats. The ratio of LC3 II to LC3 I and beclin 1 were used to estimate autophagy. We found that ratio of LC3 II to LC3 I and beclin 1 increased significantly at 2, 8, 16, 24 and 72 h, peaking at 24 h after SE onset. Pretreatment with Vitamin E partially inhibited autophagy by reducing LC3 II formation and de novo synthesis of beclin 1 at 24 h after seizures. These data show that autophagy is increased in rats with pilocarpine-induced SE, and Vitamin E have a partial inhibition on autophagy.     

Depression and/or potentiation of cortical responses after status epilepticus in immature rats

Lithium-pilocarpine status epilepticus (SE) resulted in delayed changes of single cortical interhemisperic (transcallosal) responses in immature rats. Low-frequency stimulation inducing depression and/or potentiation was studied to analyze possible dynamic changes in cortical responses. Status was elicited in 12-day-old (SE12) or 25-day-old (SE25) rats. Control siblings received saline instead of pilocarpine. Interhemispheric responses were elicited by stimulation of the sensorimotor region of the cerebral cortex 3, 6, 9, 13, or 26 days after status. A series of 5 biphasic pulses with intensity equal to twofold threshold were used for stimulation. The interval between pulses was 100, 125, 160, 200 or 300 ms, eight responses were always averaged. Peak amplitude of the first positive, first negative and second positive waves was measured and responses to the second, third, fourth and fifth pulse were compared with the first one. Animals after status epilepticus as well as lithium-paraldehyde controls exhibit a frequency depression at nearly all the intervals studied. An outlined increase of responses in SE rats in comparison with the controls three days after SE stayed just below the level of statistical significance. In addition, animals in the SE12 group exhibited potentiation of responses at this interval after SE. With longer intervals after SE, the relation between SE and control animals changed twice resulting in a tendency to lower amplitude of responses in SE than in control rats 26 days after SE. Rats in the SE25 group exhibited higher responses than controls 13 days after status, but this difference was not present at the longest interval after SE. Low-frequency stimulation did not reveal increased cortical excitability as a long-lasting consequence of status epilepticus induced in immature rats. In addition, the outlined differences between SE and control rats changed with the time after SE.     

+]-Huperzine A treatment protects against N-methyl-D-aspartate-induced seizure/status epilepticus in rats

The toxicity of organophosphorous (OP) nerve agents is attributed to their irreversible inhibition of acetylcholinesterase (AChE), which leads to excessive accumulation of acetylcholine (ACh) and is followed by the release of excitatory amino acids (EAA). EAAs sustain seizure activity and induce neuropathology due to over-stimulation of N-methyl-d-aspartate (NMDA) receptors. Huperzine A (Hup A), a blood-brain barrier permeable selective reversible inhibitor of AChE, has been shown to reduce EAA-induced cell death by interfering with glutamate receptor-gated ion channels in primary neuronal cultures. Although [-]-Hup A, the natural isomer, inhibits AChE approximately 38-fold more potently than [+]-Hup A, both [-]- and [+]-Hup A block the NMDA channel similarly. Here, we evaluated the protective efficacy of [+]-Hup A for NMDA-induced seizure in a rat model. Rats implanted with radiotelemetry probes to record electroencephalography (EEG), electrocardiography (ECG), body temperature, and physical activity were administered various doses of [+]-Hup A (intramuscularly) and treated with 20mug/kg NMDA (intracerebroventricular) 20-30min later. For post-exposure, rats were treated with [+]-Hup A (3mg/kg, intramuscularly) 1min after NMDA (20mug/kg). Our data showed that pre- and post-exposure, [+]-Hup A (3mg/kg) protects animals against NMDA-induced seizures. Also, NMDA-administered animals showed increased survival following [+]-Hup A treatment. [+]-Hup A has no visible effect on EEG, heart-rate, body temperature, or physical activity, indicating a reduced risk of side effects, toxicity, or associated pathology. Our results suggest that [+]-Hup A protects against seizure and status epilepticus (SE) by blocking NMDA-induced excitotoxicity in vivo. We propose that [+]-Hup A, or a unique combination of [+]- and [-]-Hup A, may prove to be effective for pre- and post-exposure treatment of lethal doses of OP-induced neurotoxicity.     

Molecular mechanisms underlying specificity of excitotoxic signaling in neurons

The central role of glutamate receptors in mediating excitotoxic neuronal death in stroke, epilepsy and trauma has been well established. Glutamate is the major excitatory amino acid transmitter within the CNS and it's signaling is mediated by a number of postsynaptic ionotropic and metabotropic receptors. Although calcium ions are considered key regulators of excitotoxicity, new evidence suggests that specific second messenger pathways rather than total Ca(2+) load, are responsible for mediating neuronal degeneration. Glutamate receptors are found localized at the synapse within electron dense structures known as the postsynaptic density (PSD). Localization at the PSD is mediated by binding of glutamate receptors to submembrane proteins such as actin and PDZ containing proteins. PDZ domains are conserved motifs that mediate protein-protein interactions and self-association. In addition to glutamate receptors PDZ-containing proteins bind a multitude of intracellular signal molecules including nitric oxide synthase. In this way PDZ proteins provide a mechanism for clustering glutamate receptors at the synapse together with their corresponding signal transduction proteins. PSD organization may thus facilitate the individual neurotoxic signal mechanisms downstream of receptors during glutamate overactivity. Evidence exists showing that inhibiting signals downstream of glutamate receptors, such as nitric oxide and PARP-1 can reduce excitotoxic insult. Furthermore we have shown that uncoupling the interaction between specific glutamate receptors from their PDZ proteins protects neurons against glutamate-mediated excitotoxicity. These findings have significant implications for the treatment of neurodegenerative diseases using therapeutics that specifically target intracellular protein-protein interactions.     

Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus

Long non‐coding RNAs (lncRNAs) have been implicated in the regulation of gene expression at various levels. However, to date, the expression profile of lncRNAs in status epilepticus (SE) was unclear. In our study, the expression profile of lncRNAs was investigated by high‐throughput sequencing based on a lithium/pilocarpine‐induced SE model in immature rats. Furthermore, weighted correlation network analysis (WGCNA), gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to construct co‐expression networks and establish functions of the identified hub lncRNAs in SE. The functional role of a hub lncRNA (NONRATT010788.2) in SE was investigated in an in vitro model. Our results indicated that 7082 lncRNAs (3522 up‐regulated and 3560 down‐regulated), which are involved in cell proliferation, inflammatory responses, angiogenesis and autophagy, were dysregulated in the hippocampus of immature rats with SE. Additionally, WGCNA identified 667 up‐regulated hub lncRNAs in turquoise module that were involved in apoptosis, inflammatory responses and angiogenesis via regulation of HIF‐1, p53 and chemokine signalling pathways and via inflammatory mediator regulation of TRP channels. Knockdown of an identified hub lncRNA (NONRATT010788.2) inhibited neuronal apoptosis in vitro. Taken together, our study is the first to demonstrate the expression profile and potential function of lncRNAs in the hippocampus of immature rats with SE. The defined hub lncRNAs may participate in the pathogenesis of SE via lncRNA‐miRNA‐mRNA network.     

Treatment of status epilepticus in children.

Status epilepticus is a medical emergency requiring immediate treatment. General treatment is aimed at restoring physiologic homeostasis, and drug therapy is aimed at arresting the convulsive activity and preventing its recurrence. Concurrent administration of diazepam and either phenytoin or phenobarbital will be effective in most cases. After the status epilepticus has been treated the underlying cause should be investigated and, if possible, treated.     

Treatment of status epilepticus in adults.

Status epilepticus is a medical emergency requiring immediate treatment. General treatment is aimed at restoring physiologic homeostasis, and drug therapy is aimed at arresting the convulsive activity and preventing its recurrence. Concurrent administration of diazepam and either phenytoin or phenobarbital will be effective in most cases. After the status epilepticus has been treated the underlying cause should be investigated and, if possible, treated.     

Chlormethiazole in treatment of status epilepticus.

Chlormethiazole (Heminevrin) was successful in controlling fits in seven out of nine episodes of intractable status epilepticus. It was administered as a constant intravenous injection at rates of up to 0.7g/h. No serious side effects were encountered, and the drug deserves wider recognition as a useful therapeutic agent in the management of status epilepticus.     

Motor and electrographic response of refractory experimental status epilepticus in rats and effect of calcium channel blockers

Effects of various calcium channel inhibitors have been studied in lithium-pilocarpine model of status epilepticus. Status epilepticus was induced by administration of lithium chloride (3 meq/kg) followed 21 hr later by pilocarpine (30 mg/kg). Diltiazem (5 and 10 mg/kg) was not effective in delaying onset of convulsions. Verapamil (20 mg/kg) showed protection against lithium-pilocarpine-induced convulsions. The dihydropyridine nifidepine (2.5 and 5 mg/kg) did not show any protection in this model. Amlodipine (5 and 10 mg/kg) as partially protective. Flunarizine (10 and 20 mg/kg) delayed the onset of forelimb clonus and rearing and only 60% of the rats underwent status in the 20 mg/kg group. Pre-treatment of MK-801 led to a potentiation of the antiseizure activity of calcium channel inhibitors. The percent increase in amplitude at various time points with amlodipine pretreatment was significant only at the 30th min recording, and at the rest of the time frames was practically similar as the controls. It can be concluded that the anticonvulsant action of MK-801 can be enhanced by centrally acting calcium channel inhibitors.     

Prevention of sudden cardiac death by the atypical neuroleptic acepromazine following status epilepticus in rats

Normal male rats in which status epilepticus has been induced by injecting 30 mg/kg of pilocarpine after a single systemic administration of lithium (sufficient to produce blood levels of 0.2 mEq/L) invariably die within 24 hr. Real-time monitoring indicated sudden cardiac death; it was preceded by progressive intensification of arrhythmia. A single systemic injection (25 mg/kg) of the atypical phenothiazine acepromazine prevented the mortality and virtually eliminated the cardiac instability.     

Hippocampal desynchronization of functional connectivity prior to the onset of status epilepticus in pilocarpine-treated rats

Status epilepticus (SE), a pro-epileptogenic brain insult in rodent models of temporal lobe epilepsy, is successfully induced by pilocarpine in some, but not all, rats. This study aimed to identify characteristic alterations within the hippocampal neural network prior to the onset of SE. Sixteen microwire electrodes were implanted into the left hippocampus of male Sprague-Dawley rats. After a 7-day recovery period, animal behavior, hippocampal neuronal ensemble activities, and local field potentials (LFP) were recorded before and after an intra-peritoneal injection of pilocarpine (350 mg/kg). The single-neuron firing, population neuronal correlation, and coincident firing between neurons were compared between SE (n?=?9) and nonSE rats (n?=?12). A significant decrease in the strength of functional connectivity prior to the onset of SE, as measured by changes in coincident spike timing between pairs of hippocampal neurons, was exclusively found in SE rats. However, single-neuron firing and LFP profiles did not show a significant difference between SE and nonSE rats. These results suggest that desynchronization in the functional circuitry of the hippocampus, likely associated with a change in synaptic strength, may serve as an electrophysiological marker prior to SE in pilocarpine-treated rats.     

Cerebral amino acids and lipids in drug-induced status epilepticus

—Dogs were given repeated doses of pentylenetetrazol to maintain a condition of status epilepticus for periods of 30–40 min. Analyses of cerebral tissue frozen in situ showed significant increases in alanine, arginine, γ-aminobutyric acid, glycine, histidine, leucine, lysine, phenylalanine, serine, tyrosine, and valine. Decreases occurred in the glutamic and aspartic acid levels. Other measured amino compounds were unchanged. Ammonia was increased, but not more than occurs in seizures of brief duration. A large decrease was noted in the ganglioside fraction, and a decrease also in a fraction containing the lecithins and sphingomyelins. The cephalin, cerebroside–sulphatide, and cholesterol fractions were not affected. Similar repetitive seizures induced by bemegride and continuing for shorter periods (5–9 min) brought about less extensive changes. Glutamic acid was increased, in contrast to the pentylenetetrazol effect, and the apparent decrease in aspartic acid was not statistically significant. Increases were noted in alanine, γ-aminobutyric acid, lysine, and ammonia. The ganglioside and lecithin-sphingomyelin fractions were decreased.