Effects of L-arginine on prevention and treatment of lithium-pilocarpine-induced status epilepticus

The effects of various doses of L-arginine, a nitric oxide substrate, on lithium-pilocarpine-induced seizures were studied in rats. Rats were implanted with chronic, stainless steel screw electrodes epidurally for electrocortical recordings. A control group received 3 mEq/kg LiCl (i.p.) and 24 h later 45 mg/kg pilocarpine HCl (i.p.). Two different experimental procedures were followed: (1) L-arginine was applied in doses of 100 mg/kg, 300 mg/kg or 500 mg/kg (i.p.), 30 min before pilocarpine injection; (2) 300 mg/kg, 500 mg/kg or 1000 mg/kg (i.p.) L-arginine was injected either 5 min or 30 min after the onset of status epilepticus (SE). L-arginine (300 mg/kg) injected 30 min before pilocarpine significantly reduced the percentage of SE, but did not change the latency to SE or 24-hour survival. These parameters were not significantly affected by the 100 mg/kg or 500 mg/kg dose of L-arginine. On the other hand, no dose of L-arginine that was applied after SE had begun, had any significant influence on the seizures. We concluded that L-arginine may prevent seizure activity in some but not all doses, and does not have any effect on the ongoing seizure activity.     

Lipoic Acid Increases Hippocampal Choline Acetyltransferase and Acetylcholinesterase Activities and Improvement Memory in Epileptic Rats

In the present study we investigated the effect of seizures on rat performance in the Morris water maze task, as well as on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in rat hippocampus. Wistar rats were treated with 0.9% saline (i.p., control group), lipoic acid (20 mg/kg, i.p., LA group), pilocarpine (400 mg/kg, i.p., pilocarpine group), and the association of LA (20 mg/kg, i.p.) plus pilocarpine (400 mg/kg, i.p.), 30 min before of administration of LA (LA plus pilocarpine group). After the treatments all groups were observed for 1 h. The effect of lipoic acid administration was observed on reference and working spatial memory of seized rats. The ChAT and AChE activities were measured using spectrophotometric methods and the results compared to values obtained from saline and pilocarpine-treated animals. Its activity was also determined after behavioral task. Results showed that pretreatment with lipoic acid did not alter reference memory when compared to saline-treated animals. In the working memory task, we observed a significant day’s effect with significant differences between control and pilocarpine-induced seizures and pretreated animals with lipoic acid. In LA plus pilocarpine group was observed a significantly increased in ChAT and AChE activities, when compared to pilocarpine group. Results showed that acute administration of lipoic acid alone did not alter hippocampal ChAT and AChE activities. Our findings suggest that seizures caused cognitive dysfunction and a decrease of ChAT and AChE activities that might be related, at least in part, to the neurological problems presented by epileptic patients. Lipoic acid can reverse cognitive dysfunction observed in seized rats as well as increase the ChAT and AChE activities in hippocampus of rats prior to pilocarpine-induced seizures, suggesting that this antioxidant could be used in clinic treatment of epilepsy.     

The Effects of Alpha-Tocopherol on Hippocampal Oxidative Stress Prior to in Pilocarpine-Induced Seizures

Reactive oxygen species have been implicated in seizure-induced neurodegeneration, and there is a correlation between free radical level and scavenger enzymatic activity in the epilepsy. It has been suggested that pilocarpine-induced seizures is mediated by an increase in oxidative stress. Current research has found that antioxidant may provide, in a certain degree, neuroprotection against the neurotoxicity of seizures at the cellular level. Alpha-tocopherol has numerous nonenzymatic actions and is a powerful liposoluble antioxidant. The objective of the present study was to evaluate the neuroprotective effects of alpha-tocopherol (TP) in rats, against oxidative stress caused by pilocarpine-induced seizures. 30 min prior to behavioral observation, Wistar rats were treated with, 0.9% saline (i.p., control group), TP (200 mg/kg, i.p., TP group), pilocarpine (400 mg/kg, i.p., P400 group), or the combination of TP (200 mg/kg, i.p.) and pilocarpine (400 mg/kg, i.p.). After the treatments all groups were observed for 6 h. The enzymatic activities, lipid peroxidation and nitrite concentrations were measured using speccitrophotometric methods and these data were assayed. In P400 group mice there was a significant increase in lipid peroxidation and nitrite levels. However, no alteration was observed in superoxide dismutase (SOD) and catalase activities. In the TP and pilocarpine co-administered mice, antioxidant treatment significantly reduced the lipid peroxidation level and nitrite content, as well as increased the SOD and catalase activities in rat hippocampus after seizures. Our findings strongly support the hypothesis that oxidative stress occurs in hippocampus during pilocarpine-induced seizures, indicate that brain damage induced by the oxidative process plays a crucial role in seizures pathogenic consequences, and imply that strong protective effect could be achieved using alpha-tocopherol.     

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 Seizures Produce Alterations on Choline Acetyltransferase and Acetylcholinesterase Activities and Deficit Memory in Rats

In the present study, we investigated the effect of seizures on rat performance in the Morris water maze task, as well as on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in rat hippocampus. Wistar rats were treated with 0.9% saline (i.p., control group) and pilocarpine (400 mg/kg, i.p., pilocarpine group). After the treatments all groups were observed for 1 h. The changes on reference and working spatial memory caused by pilocarpine administration were observed in seized rats. The ChAT and AChE activities were measured using spectrophotometric methods and the results compared to values obtained from saline animals. Its activities were also determined after behavioral task. Results showed that seizures alter reference memory when compared to saline-treated animals. In the working memory task, we observed a significant day’s effect with significant differences between control and pilocarpine-induced seizures. In pilocarpine group, it was observed a significant decreased in ChAT and AChE activities, when compared to control group. Our findings suggest that seizures caused cognitive dysfunction and a decrease of ChAT and AChE activities that might be related, at least in part, to the neurological problems presented by seizures induced by pilocarpine.     

Dynamics of hippocampal acetylcholine release during lithium‐pilocarpine‐induced status epilepticus in rats

The lithium‐pilocarpine model is a rat model of epilepsy that mimics status epilepticus in humans. Here, we report changes of acetylcholine (ACh) release in the hippocampus before, during and after status epilepticus as monitored by microdialysis in unanesthetized rats. Administration of pilocarpine (30 mg/kg s.c.) to rats pretreated with lithium chloride (127 mg/kg i.p.) caused a massive, six‐fold increase of hippocampal ACh release, paralleling the development of tonic seizures. When seizures were stopped by administration of diazepam (10 mg/kg i.p.) or ketamine (75 mg/kg i.p.), ACh levels returned to normal. Extracellular concentrations of glutamate remained unchanged during this procedure. Administration of atropine (1 mg/kg i.p.) 2 h after pilocarpine caused a further increase of ACh but did not affect seizures, whereas injection of mecamylamine (5 mg/kg i.p.) reduced ACh levels and seizures in a delayed fashion. Local infusion of tetrodotoxin, 1 μM locally) or hemicholinium (10 μM locally) strongly reduced ACh release and had delayed effects on seizures. Administration of glucose or inositol (250 mg/kg each i.p.) had no visible consequences. In parallel experiments, lithium‐pilocarpine‐induced status epilepticus also enhanced striatal ACh release, and hippocampal ACh levels equally increased when status epilepticus was induced by kainate (30 mg/kg i.p.). Taken together, our results demonstrate that seizure development in status epilepticus models is accompanied by massive increases of extracellular ACh, but not glutamate, levels. Treatments that reduce seizure activity also reliably reduce extracellular ACh levels.

     

Lipoic Acid Alters δ-Aminolevulinic Dehydratase,Glutathione Peroxidase and Na+,K+-ATPase Activities and Glutathione-Reduced Levels in Rat Hippocampus After Pilocarpine-Induced Seizures

In the present study, we investigated the effects of lipoic acid (LA) in the brain oxidative stress caused by pilocarpine-induced seizures in adult rats. Wistar rats were treated with 0.9% saline (i.p., control group), lipoic acid (10 mg/kg, i.p., LA group), pilocarpine (400 mg/kg, i.p., pilocarpine group), and the association of LA (10 mg/kg, i.p.) plus pilocarpine (400 mg/kg, i.p.), 30 min before the administration of LA (LA plus pilocarpine group). After the treatments, all groups were observed for 1 h. The enzyme activities [δ-aminolevulinic dehydratase (δ-ALA-D), glutathione peroxidase (GPx), glutathione reductase (GR), and Na+,K+-ATPase] as well as the glutathione-reduced (GSH) and ascorbic acid (AA) concentrations were measured using spectrophotometric methods, and the results were compared to values obtained from saline and pilocarpine-treated animals. Protective effects of LA were also evaluated on the same parameters. In pilocarpine group, no changes were observed in GPx and GR activities and AA content. Moreover, in the same group, decrease in GSH levels as well as a reduction in δ-ALA-D and Na+,K+-ATPase activities after seizures was observed. In turn, in LA plus pilocarpine group, the appearance of seizures was abolished, and the decreases in δ-ALA-D and Na+,K+-ATPase activities produced by seizures as well as increases in GSH levels and GPx activity were reversed, when compared to the pilocarpine seizing group. The results of the present study demonstrated that preadministration of LA abolished seizure episodes induced by pilocarpine in rat, probably by reducing oxidative stress in rat hippocampus caused by seizures.     

NaK ATPase activity in the rat hippocampus: A study in the pilocarpine model of epilepsy

Biochemical abnormalities have been implicated in possible mechanisms underlying the epileptic phenomena. Some of these alterations include changes in the activity of several enzymes present in epileptic tissues. Systemic administration of pilocarpine in rats induces electrographic and behavioral limbic seizures and status epilepticus, that is followed by a transient seizure-free period (silent period). Finally a chronic phase ensues, characterized by spontaneous and recurrent seizures (chronic period), that last for the rest of the animal's life. The present work aimed to study the activity of the enzyme Na⁺ K⁺ ATPase, in rat hippocampus, during the three phases of this epilepsy model. The enzyme activity was determined at different time points from pilocarpine administration (1 and 24 h of status epilepticus, during the silent and chronic period) using a spectrophotometric assay previously described by Mishra and Delivoria-Papadopoulos [Neurochem. Res. (1988) 13, 765–770]. The results showed decreased enzyme activities during the acute and silent periods and increased Na⁺K⁺ ATPase activity during the chronic phase. These data show that changes in Na⁺K⁺ ATPase activity could be involved in the appearance of spontaneous and recurrent seizures following brain damage induced by pilocarpine injection.     

Anticonvulsant and Antioxidant Effects of Cyano-carvone and Its Action on Acetylcholinesterase Activity in Mice Hippocampus

The anticonvulsant effect of cyano-carvone, a monoterpene monocyclic, was investigated in epilepsy model induced by pilocarpine. Cyano-carvone at doses of 25, 50 or 75 mg/kg promoted a reduction of 16.7, 33 and 66.7%, respectively, against pilocarpine-induced seizures, and it was efficacious in increasing both the latency to first seizures and the survival percentage, resulting in 33.3, 67 and 91.7% of protection against death induced by seizures, respectively (P < 0.05). The reference drug atropine (25 mg/kg) also produced a significant protection (100%). Its monoterpene, at 25, 50 and 75 mg/kg, was also capable to increase the latency for installation of status epilepticus induced by pilocarpine, and presented a significant protection against lipid peroxidation and nitrite formation in mice hippocampus (P < 0.05). In addition, it was observed that the cyano-carvone pretreatment increased the acetylcholinesterase activity in mice hippocampus after pilocarpine-induced seizures. The present results clearly indicate the anticonvulsant ability of cyano-carvone, which can be, at least in part, explained by the increased activity of the acetylcholinesterase enzyme. Our data suggest that the action mechanism can also be due to a direct activation of the antioxidant enzymes that could be associated with a reduction observed in oxidative stress in mice hippocampus, probably involving an inhibition of free radical production.     

The role of muscarinic acetylcholine receptor-mediated activation of extracellular signal-regulated kinase 1/2 in pilocarpine-induced seizures

Pilocarpine-induced seizures are mediated by the M(1) subtype of muscarinic acetylcholine receptor (mAChR), but little is known about the signaling mechanisms linking the receptor to seizures. The extracellular signal-regulated kinase (ERK) signaling cascade is activated by M(1) mAChR and is elevated during status epilepticus. Yet, the role of ERK activation prior to seizure has not been evaluated. Here, we examine the role of pilocarpine-induced ERK activation in the induction of seizures in mice by pharmacological and behavioral approaches. We show that pilocarpine induces ERK activation prior to the induction of seizures by both western blot and immunocytochemistry with an antibody to phosphorylated ERK. In addition, we show that the ERK pathway inhibitor SL327 effectively blocks the pilocarpine-induced ERK activation. However, SL327 pretreatment has no effect on the initiation of seizures. In fact, animals treated with SL327 had higher seizure-related mortality than vehicle-treated animals, suggesting activated ERK may serve a protective role during seizures. In addition, ERK inhibition had no effect on the development of the long-term sequelae of status epilepticus (SE), including mossy fiber sprouting, neuronal death and spontaneous recurrent seizures.     

Attenuating effects of melatonin on pilocarpine-induced seizures in rats

Daily melatonin (10-50 mg/kg, i.p.) treatment at 08.30 h or 17.00 h for 1 week of female rats (2-months-old) increased the latency to the appearance of the first convulsion in the pilocarpine-induced seizure model. Other behavior parameters remained unaltered. The anticonvulsant effect of melatonin seemed to be more intense at the light-dark transition. Moreover, the effect of repeated melatonin treatment was also age-related, since it showed a lower threshold in 2-month-old than in 21-day-old rats, and the acute treatment was not efficient. [3H]N-methylscopolamine binding was unaltered in the hippocampus and striatum of adult rats after the association of melatonin and pilocarpine. While muscarinic binding was unaltered in adult rats, it increased in the hippocampus of young rats in the presence of melatonin (50 mg/kg) and pilocarpine, and did not change in the striatum. Melatonin partially recovered [3H]GABA binding in the hippocampus in the presence of pilocarpine-induced seizures, and intensified pilocarpine effects in the striatum of adult rats.     

Gabapentin Administration Reduces Reactive Gliosis and Neurodegeneration after Pilocarpine-Induced Status Epilepticus

The lithium-pilocarpine model of epilepsy reproduces in rodents several features of human temporal lobe epilepsy, by inducing an acute status epilepticus (SE) followed by a latency period. It has been proposed that the neuronal network reorganization that occurs during latency determines the subsequent appearance of spontaneous recurrent seizures. The aim of this study was to evaluate neuronal and glial responses during the latency period that follows SE. Given the potential role of astrocytes in the post-SE network reorganization, through the secretion of synaptogenic molecules such as thrombospondins, we also studied the effect of treatment with the α2δ1 thrombospondin receptor antagonist gabapentin. Adult male Wistar rats received 3 mEq/kg LiCl, and 20 h later 30 mg/kg pilocarpine. Once SE was achieved, seizures were stopped with 20 mg/kg diazepam. Animals then received 400 mg/kg/day gabapentin or saline for either 4 or 14 days. In vitro experiments were performed in dissociated mixed hippocampal cell culture exposed to glutamate, and subsequently treated with gabapentin or vehicle. During the latency period, the hippocampus and pyriform cortex of SE-animals presented a profuse reactive astrogliosis, with increased GFAP and nestin expression. Gliosis intensity was dependent on the Racine stage attained by the animals and peaked 15 days after SE. Microglia was also reactive after SE, and followed the same pattern. Neuronal degeneration was present in SE-animals, and also depended on the Racine stage and the SE duration. Polysialic-acid NCAM (PSA-NCAM) expression was increased in hippocampal CA-1 and dentate gyrus of SE-animals. Gabapentin treatment was able to reduce reactive gliosis, decrease neuronal loss and normalize PSA-NCAM staining in hippocampal CA-1. In vitro, gabapentin treatment partially prevented the dendritic loss and reactive gliosis caused by glutamate excitotoxicity. Our results show that gabapentin treatment during the latency period after SE protects neurons and normalizes PSA-NCAM probably by direct interaction with neurons and glia.     

Brain-derived neurotrophic factor superinduction parallels anti-epileptic--neuroprotective treatment in the pilocarpine epilepsy model

Antiepileptic drugs provide neuroprotection in several animal models of brain damage, including those induced by status epilepticus (SE). The mechanisms involved in this action are unknown, but neurotrophic factors such as brain-derived neurotrophic factor (BDNF) may play a role. In this study we investigated the changes in BDNF levels in rats in which SE had been induced by pilocarpine injection (400 mg/kg i.p.) and continued for several hours (unprotected group). In other animals (protected groups), SE was suppressed after 30 min by intraperitoneal injection of either diazepam (10 mg/kg) + pentobarbital (30 mg/kg) or paraldehyde (0.3 mg/kg). In diazepam + pentobarbital-treated rats the hippocampal damage caused by SE was significantly lower (p < 0.05) than in unprotected animals. In addition, 2 and 24 h after pilocarpine injection, the levels of BDNF mRNA were moderately increased in the unprotected group, but 'superinduced' in protected animals, especially in the neocortex and hippocampus. A time-dependent increase in BDNF immunoreactivity was also found by western blot analysis in rats treated with diazepam + pentobarbital. In contrast, a decrease of BDNF immunoreactivity occurred in the unprotected group. In conclusion, these results show that neuroprotection induced by anti-epileptic drugs in pilocarpine-treated rats is accompanied by strong potentiation of BDNF synthesis in brain regions involved in SE.     

Alpha melanocyte stimulating hormone (α-MSH) does not modify pentylenetetrazol- and pilocarpine-induced seizures

Aims

Alpha-melanocyte stimulating hormone (α-MSH) is a pro-opiomelanocortin (POMC)-derived peptide involved in different neurological functions that also exerts anti-inflammatory effects, including in the central nervous system (CNS). Although inflammation has been implicated in seizures and epilepsy, no study has systematically investigated whether α-MSH modifies seizures. Therefore, in the current study we determined whether α-MSH alters pentylenetetrazol (PTZ)- and pilocarpine-induced seizures.

Main methods

Adult male Swiss mice were injected with α-MSH (1.66, 5 or 15 μg/3 μL, intracerebroventricular (i.c.v.)) or systemic (0.1, 0.3 or 1 mg/kg, intraperitoneally (i.p.)). Five to sixty minutes after the injection of the peptide, animals were injected with PTZ (60 mg/kg, i.p.) or pilocarpine (370 mg/kg, i.p.). Latency to myoclonic jerks and tonic–clonic seizures, number of seizure episodes, total time spent seizing and seizure intensity, assessed by the Racine and Meurs scales were recorded. Interleukin 1 beta (IL-1β) levels in the hippocampus were measured by a commercial enzyme-linked immunoabsorbent assay (ELISA).

Key findings

Neither intracerebroventricular (1.66, 5 or 15 μg/3 μL, i.c.v.) nor systemic (0.1, 0.3 or 1 mg/kg, i.p.) administration of α-MSH altered PTZ- and pilocarpine-induced seizures. IL-1β levels in the hippocampi were not altered by α-MSH, PTZ or pilocarpine.

Significance

Although inflammation has been implicated in seizures and epilepsy and α-MSH is a potent anti-inflammatory peptide, our results do not support a role for α-MSH in seizure control.     

Changes in phosphorylation of the NMDA receptor in the rat hippocampus induced by status epilepticus

Systemic administration of pilocarpine preceded by lithium induces status epilepticus (SE) that results in neurodegeneration and may lead to the development of spontaneous recurrent seizures. We investigated the effect of Li/pilocarpine-induced SE on phosphorylation of the NMDA receptor in rat hippocampus. Phosphorylation of NR1 by PKC on Ser890 was decreased to 45% of control values immediately following 1 h of SE. During the first 3 h following the termination of SE, phosphorylation of Ser890 increased 4-fold before declining to control values by 24 h. Phosphorylation of NR1 by PKA was also depressed relative to controls immediately following SE and transiently increased above control values upon the termination of SE. SE was accompanied by a general increase in tyrosine phosphorylation of hippocampal proteins that lasted for several hours following the termination of seizures. Tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDAR increased 3-4-fold over control values during SE, continued to increase during the first hour following SE and then declined to control levels by 24 h. SE resulted in the activation of Src and Pyk2 associated with the postsynaptic apparatus, suggesting a role for these enzymes in the SE-induced increase in tyrosine phosphorylation. Changes in phosphorylation of the NMDA receptor may play a role in the pathophysiological consequences of SE.     

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.     

Neuroprotective Effects of Idebenone Against Pilocarpine-Induced Seizures: Modulation of Antioxidant Status, DNA Damage and Na+, K+-ATPase Activity in Rat Hippocampus

The current study investigated the neuroprotective activity of idebenone against pilocarpine-induced seizures and hippocampal injury in rats. Idebenone is a ubiquinone analog with antioxidant, and ATP replenishment effects. It is well tolerated and has low toxicity. Previous studies reported the protective effects of idebenone against neurodegenerative diseases such as Friedreich’s ataxia and Alzheimer’s disease. So far, the efficacy of idebenone in experimental models of seizures has not been tested. To achieve this aim, rats were randomly distributed into six groups. Two groups were treated with either normal saline (0.9 %, i.p., control group) or idebenone (200 mg/kg, i.p., Ideb200 group) for three successive days. Rats of the other four groups (P400, Ideb50 + P400, Ideb100 + P400, and Ideb200 + P400) received either saline or idebenone (50, 100, 200 mg/kg, i.p.) for 3 days, respectively followed by a single dose of pilocarpine (400 mg/kg, i.p.). All rats were observed for 6 h post pilocarpine injection. Latency to the first seizure, and percentages of seizures and survival were recorded. Surviving animals were sacrificed, and the hippocampal tissues were separated and used for the measurement of lipid peroxides, total nitrate/nitrite, glutathione and DNA fragmentation levels, in addition to catalase and Na⁺, K⁺-ATPase activities. Results revealed that in a dose-dependent manner, idebenone (100, 200 mg/kg) prolonged the latency to the first seizure, elevated the percentage of survival and diminished the percentage of pilocapine-induced seizures in rats. Significant increases in lipid peroxides, total nitrate/nitrite, DNA fragmentation levels and catalase activity, in addition to a significant reduction in glutathione level and Na⁺, K⁺-ATPase activity were observed in pilocarpine group. Pre-administration of idebenone (100, 200 mg/kg, i.p.) to pilocarpine-treated rats, significantly reduced lipid peroxides, total nitrate/nitrite, DNA fragmentation levels, and normalized catalase activity. Moreover, idebenone prevented pilocarpine-induced detrimental effects on brain hippocampal glutathione level, and Na⁺, K⁺-ATPase enzyme activity in rats. Data obtained from the current investigation emphasized the critical role of oxidative stress in induction of seizures by pilocarpine and elucidated the prominent neuroprotective and antioxidant activities of idebenone in this model.     

Variations in elemental compositions of rat hippocampal formation between acute and latent phases of pilocarpine-induced epilepsy: an X-ray fluorescence microscopy study

There is growing experimental evidence that tracing the elements involved in brain hyperexcitability, excitotoxicity, and/or subsequent neurodegeneration could be a valuable source of data on the molecular mechanisms triggering or promoting further development of epilepsy. The most frequently used experimental model of the temporal lobe epilepsy observed in clinical practice is the one based on pilocarpine-induced seizures. In the frame of this study, the elemental anomalies occurring for the rat hippocampal tissue in acute and silent periods after injection of pilocarpine in rats were compared. X-ray fluorescence microscopy was applied for the topographic and quantitative elemental analysis. The differences in the levels of elements such as P, S, K, Ca, Fe, Cu, and Zn between the rats 3 days (SE72) and 6 h (SE6) after pilocarpine injection as well as naive controls were examined. Comparison of SE72 and control groups showed, for specific areas of the hippocampal formation, lower levels of P, K, Cu, and Zn, and an increase in Ca accumulation. These results as well as further analysis of the differences between the SE72 and SE6 groups confirmed that seizure-induced excitotoxicity as well as mossy fiber sprouting are the mechanisms involved in the neurodegenerative processes which may finally lead to spontaneous seizures in the chronic period of the pilocarpine model. Moreover, in the light of the results obtained, Cu seems to play a very important role in the pathogenesis of epilepsy in this animal model. For all areas analyzed, the levels of this element recorded in the latent period were not only lower than those for controls but were even lower than the levels found in the acute period. The decreased hippocampal accumulation of Cu in the phase of behavior and EEG stabilization, a possible inhibitory effect of this element on excitatory amino acid receptors, and enhanced seizure susceptibility in Menkes disease (an inherited Cu transport disorder leading to Cu deficiency in the brain) suggest a neuroprotective role rather than neurodegenerative and proconvulsive roles of Cu in pilocarpine-induced epilepsy.     

锂-匹罗卡品颞叶癫模型的建立及苔藓纤维出芽的动态变化

目的研究锂-匹罗卡品颞叶癫模型大鼠致后性发作的行为学特点及海马结构病理改变的动态变化。方法将所有Wistar大鼠随机分为对照组和实验组,实验组大鼠腹腔依次注射氯化锂、匹罗卡品诱发癫持续状态（SE）后,观察其自发性癫发作（SRS）,分别于SE后1周至10周5个不同时间点取材,Nissl染色和Timm染色分别观察海马神经元损伤及苔藓纤维出芽（MFS）的变化。结果注射匹罗卡品后84%的大鼠可诱发出SE,经过10～20d的缄默期后,可观察到Ⅰ～Ⅲ级的反复SRS,病理学检查可见海马神经元的损伤及齿状回内分子层MFS。结论锂-匹罗卡品颞叶癫模型与人类颞叶癫有类似发作特点及病理改变,是一种理想的颞叶癫动物模型。     

Differential patterns of synaptotagmin7 mRNA expression in rats with kainate- and pilocarpine-induced seizures

Previous studies in rat models of neurodegenerative disorders have shown disregulation of striatal synaptotagmin7 mRNA. Here we explored the expression of synaptotagmin7 mRNA in the brains of rats with seizures triggered by the glutamatergic agonist kainate (10 mg/kg) or by the muscarinic agonist pilocarpine (30 mg/kg) in LiCl (3 mEq/kg) pre-treated (24 h) rats, in a time-course experiment (30 min-1 day). After kainate-induced seizures, synaptotagmin7 mRNA levels were transiently and uniformly increased throughout the dorsal and ventral striatum (accumbens) at 8 and 12 h, but not at 24 h, followed at 24 h by somewhat variable upregulation within different parts of the cerebral cortex, amigdala and thalamic nuclei, the hippocampus and the lateral septum. By contrast, after LiCl/pilocarpine-induced seizures, there was a more prolonged increase of striatal Synaptotagmin7 mRNA levels (at 8, 12 and 24 h), but only in the ventromedial striatum, while in some other of the aforementioned brain regions there was a decline to below the basal levels. After systemic post-treatment with muscarinic antagonist scopolamine in a dose of 2 mg/kg the seizures were either extinguished or attenuated. In scopolamine post-treated animals with extinguished seizures the striatal synaptotagmin7 mRNA levels (at 12 h after the onset of seizures) were not different from the levels in control animals without seizures, while in rats with attenuated seizures, the upregulation closely resembled kainate seizures-like pattern of striatal upregulation. In the dose of 1 mg/kg, scopolamine did not significantly affect the progression of pilocarpine-induced seizures or pilocarpine seizures-like pattern of striatal upregulation of synaptotagmin7 mRNA. In control experiments, equivalent doses of scopolamine per se did not affect the expression of synaptotagmin7 mRNA. We conclude that here described differential time course and pattern of synaptotagmin7 mRNA expression imply regional differences of pathophysiological brain activation and plasticity in these two models of seizures.