Modulation of benzodiazepine by lysine and pipecolic acid on pentylenetetrazol-induced seizures

L-lysine, an essential amino acid for man and animals, and its metabolite pipecolic acid (PA) have been studied for their effects on pentylenetetrazol (PTZ)-induced seizures in mice. L-Lysine or L-PA i.p. significantly increased clonic and tonic latencies in a dose-dependent manner against 90 mg/kg PTZ-induced seizures. L-Lysine but not L-PA enhanced the anticonvulsant effect of diazepam (DZ) (0.2 mg/kg). L-PA (0.1 mmol/kg) i.c.v. showed a slight decrease in clonic latency; it did not enhance the antiseizure activity of DZ; it caused seizures at 0.6 mmol/kg. D-PA (0.1 mmol/kg) i.c.v. displayed an opposite effect compared to its L-isomer. The anticonvulsant effect of L-lysine in terms of increase in seizure latency and survival was even more amplified when tested with a submaximal PTZ concentration (65 mg/kg). L-Lysine showed an enhancement of specific 3H-flunitrazepam (FZ) binding to mouse brain membranes both in vitro and in vivo. The possibility of L-lysine acting as a modulator for the GABA/benzodiazepine receptors was demonstrated. Since L-PA showed enhancement of 3H-FZ binding only in vitro but not in vivo, the anticonvulsant effect of L-PA may not be linked to the GABA/benzodiazepine receptor.     

Neuropharmacological actions of panchagavya formulation containing Emblica officinalis Gaerth and Glycyrrhiza glabra Linn in mice

A panchagavya Ayurvedic formulation containing E. officinalis, G. glabra, and cow's ghee was evaluated for its effect on pentobarbital-induced sleeping time, pentylenetetrazol-induced seizures, maximal electroshock-induced seizures, spontaneous motor activity, rota-rod performance (motor coordination) and antagonism to amphetamine in mice. The formulation (300, 500 mg/kg, po) produced a significant prolongation of pentobarbital-induced sleeping time and reduced spontaneous locomotor activity. The formulation also significantly antagonised the amphetamine induced hyper-locomotor activity (500, 750 mg/kg, po) and protected mice against tonic convulsions induced by maximal electroshock (500, 750 mg/kg, po). The formulation slightly prolonged the phases of seizure activity but did not protect mice against lethality induced by pentylenetetrazole. The formulation did not show neurotoxicity. The results suggest that the panchagavya formulation is sedative in nature.     

Effect of Withania somnifera Dunal root extract against pentylenetetrazol seizure threshold in mice: possible involvement of GABAergic system

Withania somnifera (ashwagandha) is a widely used herb in the Ayurvedic system of medicine. The objective of the present study was to elucidate the effect of W. somnifera root extract (Ws) alone or in combination with exogenous gamma-amino butyric acid (GABA), a GABA receptor agonist or with diazepam, a GABA receptor modulator against pentylenetetrazol (PTZ, iv) seizure threshold in mice. Minimal dose of PTZ (iv, mg/kg) needed to induce different phases (myoclonic jerks, generalized clonus and tonic extension) of convulsions were recorded as an index of seizure threshold. Ws (100 or 200 mg/kg, po) increased the PTZ seizure threshold for the onset of tonic extension phase whereas a lower dose (50 mg/kg, po) did not show any effect on the seizure threshold. Co-administration of a sub-effective dose of Ws (50 mg/kg, po) with a sub-protective dose of either GABA (25 mg/kg, ip) or diazepam (0.5 mg/kg, ip) increased the seizure threshold. The results suggested that the anticonvulsant effect of W. somnifera against PTZ seizure threshold paradigm involved the GABAAergic modulation.     

Erythropoietin prevents the increase in blood-brain barrier permeability during pentylentetrazol induced seizures

Recombinant human erythropoietin (r-Hu EPO) has been shown to exert neuroprotection in ischemic, excitotoxicity, trauma, convulsions and neurodegenerative disorders. Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including neurodegenerative disorders. This study aimed to investigate the effects of r-Hu EPO on BBB integrity in pentylentetrazol (PTZ) induced seizures in rats. Seizures were observed and evaluated regard to latency and intensity for an hour. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage were observed for BBB integrity. r-Hu EPO was given intraperitoneally 24 h prior to seizure induction. Total seizure duration of 720+/-50 s after single PTZ administration (80 mg/kg i.p.) was declined to 190+/-40 s in r-Hu EPO pretreatment. A typical BBB breakdown pattern (i.e. staining in cerebellum, cerebral cortex, midbrain, hippocampus, thalamus and corpus striatum) was observed in rat brains with PTZ induced seizures; whereas, EPO pretreatment confined BBB leakage to cerebellum and cortical areas, and lessened the intensity of tonic-clonic seizures observed in PTZ seizures. The protective effect of r-Hu EPO on BBB permeability in seizures is a new and original finding. The protective action of r-Hu EPO in seizures and some of CNS pathologies warrant further investigations.     

Azolylchromans as a novel scaffold for anticonvulsant activity

A series of azolylchroman derivatives were prepared as conformationally constrained analogs of (arylalkyl)azole anticonvulsants. The anticonvulsant activities of the compounds were evaluated by determining seizure latency and protective effect against pentylenetetrazole (PTZ)-induced lethal convulsions in mice at a dose of 5mg/kg. Among these compounds, 7-chloro-3-(1H-imidazol-1-yl)chroman-4-one and 3-(1H-1,2,4-triazol-1-yl)chroman-4-one exhibited significant action in delaying seizures as well as effective protection against PTZ-induced seizures and deaths.     

Suppression of generalized seizures activity by intrathalamic 2-chloroadenosine application

In the present study, we investigated the effects of micro-injecting 2-chloroadenosine (2-CADO; an adenosine receptor agonist) into the thalamus alone and with theophylline (a nonspecific adenosine receptor antagonist) pretreatment on pentylenetetrazol (PTZ)-induced tonic-clonic seizures in male Wistar albino rats. Following intrathalamic 2-CADO injection alone or theophylline pretreatment, 50 mg kg(-1) PTZ was given ip after 1 and 24 hrs. The duration of epileptic seizure activity was recorded by cortical electroencephalogram (EEG), and seizure severity was behaviorally scored. Intrathalamic 2-CADO administration induced significant decreases in both seizure duration and seizure severity scores at 1 and 24 hrs, but the effects were more abundant on the seizures induced after 24 hrs. On the other hand, pretreatment with theophylline prevented the inhibitor effect of 2-CADO on seizure activity and increased both seizure duration and seizure scores. Present results suggest that the activation of adenosine receptors in the thalamus may represent another anticonvulsant/modulatory site of adenosine action during the course of the PTZ-induced generalized tonic-clonic seizures and provide additional data for the involvement of the adenosinergic system in the generalized seizures model.     

Antiepileptic effects of ghrelin on pentylenetetrazole-induced seizures in rats

It is well known that neuropeptide Y (NPY) and gamma-aminobutyric acid (GABA) exert antiepileptic effects in animal models. It has recently been shown that ghrelin neurons increase the activities of GABA and NPY in the brain. Therefore it can be said that ghrelin is an antiepileptic agent. In this study we aimed to investigate the antiepileptic effect of ghrelin in an acute experimental epilepsy model in pentylenetetrazole (PTZ) injected rats. Adult male Wistar albino rats were divided into a control group and four experimental groups with seven rats in each group. In order to generate epileptic seizures, PTZ (50mg/kg) was injected intraperitoneally. The experimental groups received intraperitoneal injections of ghrelin at doses of 20, 40, 60 and 80microg/kg 30min before PTZ injection. After PTZ injection, the latencies were separated into three components: first myoclonic jerk, generalized clonic seizures and tonic generalized extension. The injection of 50mg/kg PTZ-induced epileptic seizures in the control group. The onset times of the three characteristic behavioral changes were significantly delayed and the duration of tonic generalized extension was diminished by dose-dependent ghrelin administration. Our results demonstrated that ghrelin suppresses the onset time of PTZ-induced seizures. In the light of our current knowledge, it seems that ghrelin may be considered as an antiepileptic drug.     

The comparative effects of propofol, thiopental, and diazepam, administered intravenously, on pentylenetetrazol seizure threshold in the rabbit.

The anticonvulsant effects of propofol, thiopental, and diazepam, administered intravenously, on pentylenetetrazol (PTZ) seizure threshold were studied and compared in the rabbit. The PTZ seizure threshold determined in various rabbit groups during the control phase of conducted experiments, was found to be in the range of 10.1 +/- 2.0 to 13.5 +/- 3.7 mg/kg. Intravenous administration of comparable doses of propofol, thiopental, and diazepam resulted in marked and significant increases in PTZ seizure threshold. At all administered doses (1.25-10.0 mg/kg), propofol was found to be more effective than thiopental in increasing the PTZ threshold dose. However, the anticonvulsant effects of diazepam were more marked than those of propofol, except at a dose of 10 mg/kg where both agents exhibited equipotent activities. These data demonstrate that propofol enjoys a considerable degree of anticonvulsant activity in the rabbit. This anticonvulsant action is greater than that of thiopental at doses ranging from 2.5 to 10 mg/kg and equipotent with diazepam at the 10 mg/kg dose.     

The effect of naloxone administration on pregnancy-associated seizures

Pregnant mice are more susceptible to flurothyl-induced seizures than are non-pregnant controls. The possibility that the well-known increase in beta-endorphin concentration which accompanies pregnancy was involved in this effect was examined by testing whether naloxone administration could block the increased seizure susceptibility. Pregnant female, control female and male C3H mice were treated with 5-50 mg/kg naloxone 5 min before flurothyl seizure testing. Naloxone markedly increased clonic seizure susceptibility in all three groups at a dose of 50 mg/kg, but had little effect at lower doses. In contrast, naloxone had differential effects on myoclonic seizures in pregnant and control female mice, being anticonvulsant in the controls, but proconvulsant in the pregnant mice. A role for endogenous opiates is unlikely in mediating clonic seizures in pregnant mice, but may be involved in myoclonic seizures.     

Adrenaline potentiates antiepileptic but not sedative action of diazepam in rats

Intramuscular (i.m.) administration ofdiazepam in a dose of 10 mg/kg and adrenaline in a dose of 0.2 mg/kg prevents generalized clonic-tonic pentylenetetrazol (PTZ) seizures in 75-80 % of rats, but only in 35-40 % of rats it prevents local clonic PTZ seizures. In the above mentioned dose, diazepam causes a strong sedation, but adrenaline does not cause a sedative effects. The combined administration of diazepam and adrenaline in threshold independently ineffective doses prevents both clonic-tonic and clonic PTZ seizures in 80 % of rats without a sedation development. The basis for mechanism of potentiation of anticonvulsant action of diazepam is the stimulation of gastric mucosa afferents by adrenaline.     

Effect of calcium channel blockers on experimentally induced seizures in rats

Chemically different classes of calcium channel blockers were examined in rats for their effects on behavior, tolerability and protection against maximal electroshock seizures (MES) and pentylenetetrazol (PTZ) induced seizures. In MES test at doses (mg/kg, ip) that were devoid of side effects, felodipine, 50, afforded 100% protection, while nimodipine, 5; pimozide, 10; and thioridazine, 25, showed 50 to 66% protection. Nifedipine, 10, and diltiazem, 50, showed 30 and 66% protection respectively, but were associated with side effects. Verapamil and loperamide were ineffective against MES and PTZ induced seizures. Nimodipine, 1 mg/kg, ip, was the most potent agent and produced 100% protection against PTZ. Equieffective doses were pimozide, 25, felodipine, 50, and thioridazine, 50. The rest of the calcium channel blockers showed marginal to moderate activity against chemoshock. The data obtained suggest that some calcium channel blockers possess anticonvulsant activity and may be considered as adjuvant therapeutic agents in epileptics refractory to conventional antiepileptic medication.     

Effect of Anacyclus pyrethrum on Pentylenetetrazole-Induced Kindling, Spatial Memory, Oxidative Stress and Rho-Kinase II Expression in Mice

Anacyclus pyrethrum (A. pyrethrum) has been reported to exhibit anticonvulsant activity. In the present study, the effect of hydro-alcoholic extract of A. pyrethrum root (HEAP) on pentylenetetrazole (PTZ) induced kindling, spatial memory, oxidative stress and rho kinase (ROCK II) was assessed. Male albino mice (25–30 g) were used in the study. PTZ (35 mg/kg, i.p. on alternate days) was injected to induce kindling and PTZ (70 mg/kg, i.p) challenge was given 7 days post-kindling. HEAP was administered orally daily in the doses of 100, 250 and 500 mg/kg along with PTZ injections during the kindling process and continued till PTZ challenge post kindling. Spatial memory was assessed using Morris water maze test. Oxidative stress parameters [malondialdehyde (MDA) and reduced glutathione (GSH)] and ROCK II expression were estimated in whole brain at the end of the study. Pre-treatment with HEAP (250 and 500 mg/kg) showed significant increase in the myoclonic jerk latency and delay in the development of kindling. A significant decrease in mortality was observed at higher doses of HEAP (250 and 500 mg/kg). Pre-treatment with HEAP significantly increased the number of platform crossings and decreased the escape latency, as opposed to the PTZ group, thus showing protection against memory deficit. HEAP pre-treatment also attenuated the oxidative stress induced by PTZ kindling. PTZ induced kindling increased the ROCK II expression whereas, HEAP pre-treatment attenuated the increase in ROCK II expression. To conclude, HEAP pre-treatment showed antiepileptic effect and also showed protection against cognitive impairment by decreasing oxidative stress and ROCK II expression in PTZ kindled mice.     

Synthesis,in-vitro cholinesterase inhibition,in-vivo anticonvulsant activity and in-silico exploration of N-(4-methylpyridin-2-yl)thiophene-2-carboxamide analogs

In our current research, a diverse effect of acetylcholinesterase inhibitors was studied on BALB-C mice by using pentylenetetrazole (PTZ) seizure model. A series of carboxamide analogs (4a–4i) have been synthesized via Suzuki coupling reaction in moderate to good yields (35–84%). To study the efficacy of the synthesized compounds against AD, in-vitro inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was performed. A number of compounds showed inhibition in low micromolar range. Subsequently, these compounds were evaluated for anticonvulsive effects in BALB-C mice by using pentylenetetrazole (PTZ) seizure model. The compound 4e displayed potential anticonvulsive effect and displayed 50% and 80% protection from mortality at the dose of 10 mg/kg, and 30 mg/kg respectively. The compound 4h showed some protection (33%) from mortality at 10 mg/kg and was not further explored based on non-significant delay in onset of myoclonic seizures. While, other compounds from the series did not show any anticonvulsive activity. To rationalize the observed biological activity, we performed docking studies against AChE and BChE targets. To explore the rationale of the mechanism of in-vivo anticonvulsant activity, docking studies were performed on GABAergic receptors. Moreover, in order to establish a relationship between physiochemical data of the synthesized compounds and their in-vivo performance, we employed in-silico pharmacokinetic predictions. Our in-silico predictions suggest that the plasma protein binding, low to moderate blood brain barrier penetration and low solubility are the main attributes of low in-vivo performance.     

Protective effect of safranal on pentylenetetrazol-induced seizures in the rat: Involvement of GABAergic and opioids systems

The aim of the present study was to evaluate the effects of safranal, an active constituent of Crocus sativus L. stigmas, on seizures induced by pentylenetetrazol. Intracerebroventricular (i.c.v.) microinjection of safranal (4.84, 9.68 and 24.2 micromol) had no effects on tonic and clonic phases as well as mortality upon seizures induced by PTZ (90mg/kg body wt., i.p.). Peripheral administration of safranal (72.75, 145.5 and 291 mg/kg body wt., i.p.), however, induced a dose-dependent decrease in the incidence of both minimal clonic seizures (MCS) (145.5 mg/kg body wt., p<0.01) and generalized tonic-clonic seizures (GTCS) (145.5 mg/kg body wt., p<0.001) following PTZ administration. Safranal also increased MCS and GTCS latency, significantly. Percent of protection against GTCS was 30%, 100% and 100% and mortality protection percent was 40%, 100% and 100% for the mentioned doses, respectively. Pretreatment with flumazenil (5 nmol, i.c.v.) and naloxone (5.5 nmol, i.c.v. and 2 mg/kg body wt., i.p.), 15 min prior to safranal administration (145.5 mg/kg body wt., i.p.), abolished the protective effect of safranal on MCS. Flumazenil also decreased the effect of safranal on incidence as well as latency of GTCS, significantly. These effects were not, however, significant for naloxone (5.5 nmol, i.c.v. and 2mg/kg body wt., i.p.). Results of this study demonstrated that safranal could exert anticonvulsant activity in the PTZ model and this effect may be mediated, at least partly, through GABA(A)-benzodiazepine receptor complex.     

Differential effects of perinatal hypoxia and anoxia on long term seizure susceptibility in the rat.

We have previously demonstrated that hypoxia is acutely epileptogenic in the immature rat but not in the adult. The window during which hypoxia induces seizures in the rat ranges from postnatal day (P) 5-17, with the most severe seizures occurring at P10-12. Perinatal hypoxia resulted in significantly more acute seizure activity than perinatal anoxia. The present study evaluates the long term effects of perinatal hypoxia versus anoxia. Animals were exposed to hypoxia (3%O2) or anoxia (0%O2) at P10 and challenged later in adulthood (P55-60) with administration of pentylenetetrazol (PTZ) (45 mg/kg subcutaneously). Compared to normal littermate controls, the animals which had been exposed to perinatal hypoxia had a significantly higher frequency of generalized convulsions (GC) and a significantly shorter latency to the first myoclonic jerk (MJ) after PTZ. In contrast, perinatal anoxia did not alter long term seizure susceptibility. These results are discussed in context of previous studies which have shown variable long term effects using different models of perinatal hypoxia and/or ischemia.     

Serotonin mediation of the protective effect of clonidine against pentylenetetrazol-induced seizures in rats

An intraperitoneal injection of 0.5 mg/kg clonidine significantly increased the latency to the first convulsion and reduced tonic seizures and mortality caused by pentylenetetrazol (PTZ), 90 mg/kg, administered subcutaneously to rats. 1 mg/kg clonidine produced similar effects except that tonic seizures were not significantly affected. No effect was observed with 0.01 or 0.1 mg/kg clonidine. Metergoline (1 mg/kg) and methysergide (10 mg/kg), administered intraperitoneally, completely prevented the effect of 0.5 mg/kg clonidine on PTZ-induced seizures. An intraperitoneal injection of 5 mg/kg of d-fenfluramine, a releaser of 5HT from nerve terminals, significantly reduced tonic seizures and completely blocked mortality caused by PTZ but did not significantly modify the latency to the first convulsion. The results suggest that serotonin plays an important role in the protective effect of 0.5 mg/kg clonidine against PTZ-induced seizures. Possible reasons for the different effects of clonidine on different experimental seizures are discussed.     

Quinolinic Acid-induced Seizures Stimulate Glutamate Uptake into Synaptic Vesicles from Rat Brain: Effects Prevented by Guanine-based Purines

Glutamate uptake into synaptic vesicles is a vital step for glutamatergic neurotransmission. Quinolinic acid (QA) is an endogenous glutamate analog that may be involved in the etiology of epilepsy and is related to disturbances on glutamate release and uptake. Guanine-based purines (GBPs) guanosine 5′-monophosphate (GMP and guanosine) have been shown to exert anticonvulsant effects against QA-induced seizures. The aims of this study were to investigate the effects of in vivo administration of several convulsant agents on glutamate uptake into synaptic vesicles and investigate the role of MK-801, guanosine or GMP (anticonvulsants) on glutamate uptake into synaptic vesicles from rats presenting QA-induced seizures. Animals were treated with vehicle (saline 0.9%), QA 239.2 nmoles, kainate 30 mg/kg, picrotoxin 6 mg/kg, PTZ (pentylenetetrazole) 60 mg/kg, caffeine 150 mg/kg or MES (maximal transcorneal electroshock) 80 mA. All convulsant agents induced seizures in 80–100% of animals, but only QA stimulated glutamate uptake into synaptic vesicle. Guanosine or GMP prevented seizures induced by QA (up to 52% of protection), an effect similar to the NMDA antagonist MK-801 (60% of protection). Both GBPs and MK-801 prevented QA-induced glutamate uptake stimulation. This study provided additional evidence on the role of QA and GBPs on glutamatergic system in rat brain, and point to new perspectives on seizures treatment.     

Anticonvulsant profile of nimodipine and nitrendipine against pentylenetetrazole induced seizures in rats

The activity of nimodipine and nitrendipine against pentylenetetrazole (PTZ) induced seizures in Albino rats was studied alone and in combination with valproate. The median effective dose [ED50] of valproate, nimodipine and nitrendipine were initially determined. All the 3 drugs were injected i.p. 30 min before the induction of seizures. Seizures were induced by PTZ 85 mg/kg i.p., and subsequently the effect of combining ED50 doses of nimodipine and nitrendipine with ED50 dose of valproate was evaluated. ED50 of valproate and nitrendipine were 129 and 2.5 mg/kg respectively. ED50 of nimodipine could not be established since a dose-response relationship was not obtained. Hence, for the purpose of combination studies, 4 mg/kg of nimodipine was used. Both nimodipine (4 mg/kg) and nitrendipine (2.5 mg/kg) decreased the ED50 of valproate from 129 to 40 mg/kg. Both nimodipine and nitrendipine potentiate the activity of valproate against PTZ induced seizures and can be considered as potential adjuvant anticonvulsants which merit further study.     

Differences between the tolerance characteristics of two anticonvulsant benzodiazepines in the amygdaloid-kindled rat

The characteristics of the development of tolerance to the anticonvulsant effects of chronic treatment by dipotassium clorazepate and diazepam using amygdaloid-kindled rats were investigated. Dipotassium clorazepate (5 mg/kg) or diazepam (5 mg/kg) were intraperitoneally administered for 10 consecutive days. Tolerance to the anticonvulsant effect of dipotassium clorazepate developed in seizure stage on day 6, after-discharge duration on day 7 and seizure latency on day 4. In contrast, tolerance to the effects of diazepam developed more rapidly in seizure stage on day 4, after-discharge duration on day 4 and seizure latency on day 3. Thus tolerance to the anticonvulsive effect of dipotassium clorazepate developed relatively slower than that to diazepam. All rats had stage 5 convulsions 24 hr after cessation of the administration of dipotassium clorazepate and diazepam. Concomitant determinations of plasma concentrations of the main metabolite of dipotassium clorazepate and diazepam, desmethyldiazepam, showed no statistical difference during treatment, suggesting that the developed tolerance was not metabolic but functional.     

Antiepileptic effects of nifedipine]

In experiments on freely moving male Wistar rats it was shown that nifedipine in a dose 10 mg/kg (i.p.) suppressed the penicillin-induced focal epileptic activity in cerebral cortex. A similar suppressing effect of nifedipine was shown on acute generalized tonic-clonic pentylenetetrazol (PTZ) seizures (75 mg/kg, i.p.). Nifedipine in the same dose was not effective on chronic PTZ administration (PTZ-kindling, 30 mg/kg i.p. during 28 days): when injected 30 min before each PTZ administration it didn't delay the development of kindling induced seizure susceptibility and had no effect on the severity of seizures. The administration of nifedipine in a dose of 10 or 30 mg/kg to control kindled animals which had not been treated with nifedipine had no influence on the severity of seizures provoked by a testing dose of PTZ (30 mg/kg i.p.): its intensity was similar to that of caused by PTZ injection along.