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Journal of Visualized Experiments : JoVE Jun 2018Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas...
Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.
Topics: Animals; Brain; Disease Models, Animal; GABA Antagonists; Injections, Intraperitoneal; Kindling, Neurologic; Male; Mice; Pentylenetetrazole; Seizures
PubMed: 29985308
DOI: 10.3791/56573 -
International Journal of Molecular... Dec 2021Epilepsy comorbidities and anti‑epileptic drugs (AEDs) are currently the main limitations of epilepsy treatment. Semaglutide is a glucagon like peptide‑1 analogue...
Semaglutide attenuates seizure severity and ameliorates cognitive dysfunction by blocking the NLR family pyrin domain containing 3 inflammasome in pentylenetetrazole‑kindled mice.
Epilepsy comorbidities and anti‑epileptic drugs (AEDs) are currently the main limitations of epilepsy treatment. Semaglutide is a glucagon like peptide‑1 analogue that has entered the market as a new once‑weekly drug for type II diabetes. The aim of the present study was to investigate the functions of semaglutide in epilepsy and inflammation models, in order to investigate its potential mechanism. , an inflammation model was established using lipopolysaccharide (LPS) and nigericin stimulation in BV2 cells. , chronic epilepsy model mice were generated using a pentylenetetrazole (PTZ) kindling method. BV2 cell proliferation was assessed using the Cell Counting Kit‑8. The effects of semaglutide on NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and inflammatory cytokine secretion were determined using western blotting (WB) and ELISA. A lactate dehydrogenase (LDH) assay kit was used to detect the effect of semaglutide on LDH release. Electrocorticography and the modified Racine scale were used to assess seizure severity. Cognitive function was evaluated with behavioral assessment. Morphological changes in the hippocampus were observed with Nissl staining. Double immunofluorescence staining for NeuN and Iba‑1, WB and immunofluorescence analysis of apoptosis‑related proteins were used to evaluate neuronal apoptosis. The NLRP3 inflammasome was assessed by reverse transcription‑quantitative PCR, WB and immunofluorescence staining, and inflammatory cytokine release was evaluated by WB analysis in the hippocampus of C57/BL6J model mouse. Semaglutide attenuated the LPS‑ and nigericin‑induced inflammatory response and LDH release by blocking NLRP3 inflammasome activation in BV2 cells. Moreover, semaglutide decreased seizure severity, alleviated hippocampal neuronal apoptosis, ameliorated cognitive dysfunction, blocked NLRP3 inflammasome activation and decreased inflammatory cytokine secretion in PTZ‑kindled mice. These results indicated that semaglutide reduced seizure severity, exerted neuroprotective effects and ameliorated cognitive dysfunction, possibly via inhibition of NLRP3 inflammasome activation and inflammatory cytokine secretion. Semaglutide may therefore be a novel, promising adjuvant therapeutic for epilepsy and its associated comorbidities.
Topics: Animals; Apoptosis; Cell Line; Cognitive Dysfunction; Cytokines; Disease Models, Animal; Epilepsy; Glucagon-Like Peptides; Hippocampus; Inflammasomes; Inflammation; Male; Mice; Mice, Inbred C57BL; Microglia; NLR Family, Pyrin Domain-Containing 3 Protein; Neuroprotective Agents; Pentylenetetrazole; Seizures
PubMed: 34676876
DOI: 10.3892/ijmm.2021.5052 -
Epilepsy & Behavior : E&B Jun 2019Seizure severity in experimental models of epilepsy is often evaluated by means of the Racine scale, in spite of the use of seizure induction methods that are different...
Seizure severity in experimental models of epilepsy is often evaluated by means of the Racine scale, in spite of the use of seizure induction methods that are different from those of the original paper by Racine in 1972. In such cases, the use of this scale is not always justified because some seizure behaviors are significantly different from those originally described or not present at all. Correspondingly, the pentylenetetrazole (PTZ) model, which is frequently used for antiepileptic drug research, lacked an adequate assessment tool to measure seizure severity. In 2009, an adapted intensity scale for PTZ-induced seizures was already designed for rats. Here, we evaluated electroencephalographic (EEG) and behavioral parameters after a single PTZ injection, to determine whether this scale is also suitable for use in mouse studies. We found that the scale designed for rats is quite robust and can thus be applied to score seizure severity in mice. Yet, certain convulsive behaviors and EEG characteristics were distinct between species. Therefore, a species-specific scale was designed, which included the concomitant EEG characteristic next to the behavioral expressions we observed, in order to establish a user-friendly scoring scale for PTZ-induced seizures in mice. To evaluate applicability, we utilized the scale in a seizure susceptibility study of a transgenic mouse model. We demonstrated that the maximum severity scores obtained with the newly revised Racine scale highly correlated with the administered dose. Hence, the revised scale differentiates well between different classes of seizure severity.
Topics: Animals; Convulsants; Disease Models, Animal; Electroencephalography; Female; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pentylenetetrazole; Random Allocation; Seizures; Severity of Illness Index
PubMed: 31026782
DOI: 10.1016/j.yebeh.2019.02.029 -
BioMed Research International 2022Epilepsy is a chronic neurological disorder affecting 1-2% of world population, and one-third of patients are refractory to pharmacological treatment. This fact has...
Epilepsy is a chronic neurological disorder affecting 1-2% of world population, and one-third of patients are refractory to pharmacological treatment. This fact has stimulated research for new antiepileptic drugs and natural products have been an important source. -Anethole (TAN) is a phenylpropanoid, component of some essential oils, extracted from plants, and its effects have been little studied. Therefore, this study is aimed at investigating the TAN effect in classic seizure models and evaluate the electroencephalographic (EEG) profile of animals treated with this substance. For this, Swiss male mice () were used, and the lethal dose was evaluated and subsequently submitted to the test maximal electroshock (MES), the pentylenetetrazole- (PTZ) induced seizure test, and the EEG profile. Initially, the LD50 for TAN was estimated in 1000 mg/kg (i.p.) dose and there was no sign of acute toxicity or death. In the MES test, TAN 300, i.p. (12.00 ± 2.9 s) and 400 mg/kg, i.p. (9.00 ± 4.4 s) doses was able to decrease tonic seizures duration induced by electric discharge (0.5 mA, 150 pulses/s, for 0.5 s). In the PTZ test (75 mg/kg, i.p.), TAN 400 mg/kg, i.p. increased the latency to myoclonic jerks (80.0 (56.0-134.0)), the latency totonic-clonic seizures (900.0 (861.0-900.0) and decrease seizure duration (0.0 (0.0-10.0)). No deaths were found in this groups compared to vehicle. EEG analysis showed an amplitude decrease of waves (ratio of baseline) in TAN 300 (1.82 ± 0.23) and 400 mg/kg (1.06 ± 0.16) groups. In this way, TAN at 400 mg/kg was able to inhibit and/or attenuate seizures by increasing the time for the onset of spasms and convulsions, as reducing the duration of seizures. The EEG profile corroborate with this results showing a reduction in the amplitude of waves compared to the PTZ group. Thus, TAN showed an anticonvulsant effect in all experimental models performed, behavioral and electroencephalographic.
Topics: Animals; Humans; Male; Mice; Allylbenzene Derivatives; Anisoles; Anticonvulsants; Disease Models, Animal; Dose-Response Relationship, Drug; Electroshock; Pentylenetetrazole; Seizures
PubMed: 35607305
DOI: 10.1155/2022/9902905 -
Biomedicine & Pharmacotherapy =... Apr 2023Tiagabine (Tia), a new-generation antiseizure drug that mimics the GABAergic signaling by inhibiting GABA transporter type-1, is the least studied molecule in chronic...
Tiagabine suppresses pentylenetetrazole-induced seizures in mice and improves behavioral and cognitive parameters by modulating BDNF/TrkB expression and neuroinflammatory markers.
Tiagabine (Tia), a new-generation antiseizure drug that mimics the GABAergic signaling by inhibiting GABA transporter type-1, is the least studied molecule in chronic epilepsy models with comorbid neurobehavioral and neuroinflammatory parameters. Therefore, the current study investigated the effects of Tia in a real-time manner on electroencephalographic (EEG) activity, behavioral manifestations and mRNA expression in pentylenetetrazole (PTZ)-kindled mice. Male BALB/c mice were treated with tiagabine (0.5, 1 and 2 mg/kg) for 21 days with simultaneous PTZ (40 mg/kg) injection every other day for a total of 11 injections and monitored for seizure progression with synchronized validation through EEG recordings from cortical electrodes. The post-kindling protection from anxiety and memory deficit was verified by a battery of behavioral experiments. Isolated brains were evaluated for oxidative alterations and real-time changes in mRNA expression for BDNF/TrkB, GAT-1 and GAT-3 as well as neuroinflammatory markers. Experimental results revealed that Tia at the dose of 2 mg/kg maximally inhibited the development of full bloom seizure and reduced epileptic spike discharges from the cortex. Furthermore, Tia dose-dependently exerted the anxiolytic effects and protected from PTZ-evoked cognitive impairment. Tia reduced lipid peroxidation and increased superoxide dismutase and glutathione levels in the brain via augmentation of GABAergic modulation. PTZ-induced upregulated BDNF/TrkB signaling and pro-inflammatory cytokines were mitigated by Tia with upregulation of GAT-1 and GAT-3 transporters in whole brains. In conclusion, the observed effects of Tia might have resulted from reduced oxidative stress, BDNF/TrkB modulation and mitigated neuroinflammatory markers expression leading to reduced epileptogenesis and improved epilepsy-related neuropsychiatric effects.
Topics: Animals; Male; Mice; Anticonvulsants; Brain-Derived Neurotrophic Factor; Cognition; Epilepsy; Kindling, Neurologic; Pentylenetetrazole; RNA, Messenger; Seizures; Tiagabine
PubMed: 36791567
DOI: 10.1016/j.biopha.2023.114406 -
Cells Jun 2023Zebrafish (Danio rerio) assays provide a versatile pharmacological platform to test compounds on a wide range of behaviors in a whole organism. A major challenge lies in...
Zebrafish (Danio rerio) assays provide a versatile pharmacological platform to test compounds on a wide range of behaviors in a whole organism. A major challenge lies in the lack of knowledge about the bioavailability and pharmacodynamic effects of bioactive compounds in this model organism. Here, we employed a combined methodology of LC-ESI-MS/MS analytics and targeted metabolomics with behavioral experiments to evaluate the anticonvulsant and potentially toxic effects of the angular dihydropyranocoumarin pteryxin (PTX) in comparison to the antiepileptic drug sodium valproate (VPN) in zebrafish larvae. PTX occurs in different Apiaceae plants traditionally used in Europe to treat epilepsy but has not been investigated so far. To compare potency and efficacy, the uptake of PTX and VPN into zebrafish larvae was quantified as larvae whole-body concentrations together with amino acids and neurotransmitters as proxy pharmacodynamic readout. The convulsant agent pentylenetetrazole (PTZ) acutely reduced the levels of most metabolites, including acetylcholine and serotonin. Conversely, PTX strongly reduced neutral essential amino acids in a LAT1 (SLCA5)-independent manner, but, similarly to VPN specifically increased the levels of serotonin, acetylcholine, and choline, but also ethanolamine. PTX dose and time-dependent manner inhibited PTZ-induced seizure-like movements resulting in a ~70% efficacy after 1 h at 20 µM (the equivalent of 4.28 ± 0.28 µg/g in larvae whole-body). VPN treated for 1 h with 5 mM (the equivalent of 18.17 ± 0.40 µg/g in larvae whole-body) showed a ~80% efficacy. Unexpectedly, PTX (1-20 µM) showed significantly higher bioavailability than VPN (0.1-5 mM) in immersed zebrafish larvae, possibly because VPN in the medium dissociated partially to the readily bioavailable valproic acid. The anticonvulsive effect of PTX was confirmed by local field potential (LFP) recordings. Noteworthy, both substances specifically increased and restored whole-body acetylcholine, choline, and serotonin levels in control and PTZ-treated zebrafish larvae, indicative of vagus nerve stimulation (VNS), which is an adjunctive therapeutic strategy to treat refractory epilepsy in humans. Our study demonstrates the utility of targeted metabolomics in zebrafish assays and shows that VPN and PTX pharmacologically act on the autonomous nervous system by activating parasympathetic neurotransmitters.
Topics: Humans; Animals; Pentylenetetrazole; Valproic Acid; Zebrafish; Vagus Nerve Stimulation; Serotonin; Acetylcholine; Tandem Mass Spectrometry; Seizures; Anticonvulsants; Choline
PubMed: 37296660
DOI: 10.3390/cells12111540 -
Journal of Traditional Chinese Medicine... Dec 2019To evaluate the effects of Rongchang capsule and Xifeng capsule on pentylenetetrazole- induced epilepsy in zebrafish larvae and to explore the possible mechanisms behind...
OBJECTIVE
To evaluate the effects of Rongchang capsule and Xifeng capsule on pentylenetetrazole- induced epilepsy in zebrafish larvae and to explore the possible mechanisms behind their actions.
METHODS
We utilized a trajectory tracking system to monitor seizures in zebrafish larva to confirm that certain concentrations of Rongchang capsule and Xifeng capsule produce antiepileptic effects. c-fos expression was assessed by quantitative reverse transcription-polymerase chain reaction to validate the efficacy of the capsules. Rest/wake behavior and correlation analysis predicted the targets of Rongchang capsule and Xifeng capsule.
RESULTS
Larval movement times and total distances traveled by zebrafish larvae experiencing pentylenetetrazole (PTZ)-induced seizures were decreased by valproate treatment. Rongchang (500 μg/mL) and Xifeng (200 μg/mL) rescued the epileptic behaviors and down-regulated c-fos expression in the brains of larvae, which indicated antiepileptic effects. The rest/wake behavioral profiles showed that Rongchang and Xifeng differentially decreased rest time at night and increased larval locomotor activities during the day. Based on correlation between the actions of the two capsules and known compounds, we predicted that they might change rest/wake behaviors by affecting serotonin, GABAergic and histamine signaling pathways.
CONCLUSION
The efficacy of Rongchang capsule and Xifeng capsule in alleviating epilepsy-like behaviors and molecular responses was confirmed. Our study provides insight into the capsules' effect on epilepsy.
Topics: Animals; Anticonvulsants; Drugs, Chinese Herbal; Larva; Male; Pentylenetetrazole; Rest; Seizures; Wakefulness; Zebrafish
PubMed: 32186159
DOI: No ID Found -
BMC Medicine Dec 2023Exposure to general anesthesia influences neuronal functions during brain development. Recently, interneurons were found to be involved in developmental neurotoxicity by...
BACKGROUND
Exposure to general anesthesia influences neuronal functions during brain development. Recently, interneurons were found to be involved in developmental neurotoxicity by anesthetic exposure. But the underlying mechanism and long-term consequences remain elusive.
METHODS
Pregnant mice received 2.5% sevoflurane for 6-h on gestational day 14.5. Pentylenetetrazole (PTZ)-induced seizure, anxiety- and depression-like behavior tests were performed in 30- and 60-day-old male offspring. Cortical interneurons were labeled using Rosa26-EYFP/-; Nkx2.1-Cre mice. Immunofluorescence and electrophysiology were performed to determine the cortical interneuron properties. Q-PCR and in situ hybridization (ISH) were performed for the potential mechanism, and the finding was further validated by in utero electroporation (IUE).
RESULTS
In this study, we found that maternal sevoflurane exposure increased epilepsy susceptibility by using pentylenetetrazole (PTZ) induced-kindling models and enhanced anxiety- and depression-like behaviors in adolescent offspring. After sevoflurane exposure, the highly ordered cortical interneuron migration was disrupted in the fetal cortex. In addition, the resting membrane potentials of fast-spiking interneurons in the sevoflurane-treated group were more hyperpolarized in adolescence accompanied by an increase in inhibitory synapses. Both q-PCR and ISH indicated that CXCL12/CXCR4 signaling pathway downregulation might be a potential mechanism under sevoflurane developmental neurotoxicity which was further confirmed by IUE and behavioral tests. Although the above effects were obvious in adolescence, they did not persist into adulthood.
CONCLUSIONS
Our findings demonstrate that maternal anesthesia impairs interneuron migration through the CXCL12/CXCR4 signaling pathway, and influences the interneuron properties, leading to the increased epilepsy susceptibility in adolescent offspring. Our study provides a novel perspective on the developmental neurotoxicity of the mechanistic link between maternal use of general anesthesia and increased susceptibility to epilepsy.
Topics: Humans; Pregnancy; Female; Mice; Animals; Male; Sevoflurane; Pentylenetetrazole; Maternal Exposure; Interneurons; Epilepsy
PubMed: 38129829
DOI: 10.1186/s12916-023-03210-0 -
Frontiers in Endocrinology 2020Still circa 25% to 30% of patients with epilepsy cannot be efficiently controlled with available antiepileptic drugs so newer pharmacological treatment options have been...
Still circa 25% to 30% of patients with epilepsy cannot be efficiently controlled with available antiepileptic drugs so newer pharmacological treatment options have been continuously searched for. In this context, a group of endogenous or exogenous neurosteroids allosterically positively modulating GABA-A receptors may offer a promising approach. Among endogenous neurosteroids synthesized in the brain, allopregnanolone or allotetrahydrodeoxycorticosterone have been documented to exert anticonvulsant activity in a number of experimental models of seizures-pentylenetetrazol-, bicuculline- pilocarpine-, or 6 Hz-induced convulsions in rodents. Neurosteroids can also inhibit fully kindled seizures and some of them have been reported to counteract maximal electroshock-induced convulsions. An exogenous neurosteroid, alphaxalone, significantly elevated the threshold for maximal electroconvulsions in mice but it did not potentiate the anticonvulsive action of a number of conventional antiepileptic drugs against maximal electroshock-induced seizures. Androsterone not only elevated the threshold but significantly enhanced the protective action of carbamazepine, gabapentin and phenobarbital against maximal electroshock in mice, as well. Ganaxolone (a 3beta-methylated analog of allopregnanolone) needs special consideration for two reasons. First, it performed better than conventional antiepileptic drugs, diazepam or valproate, in suppressing convulsive and lethal effects of pentylenetetrazol in pentylenetetrazol-kindled mice. Second, ganaxolone has been evaluated in the randomized, double-blind, placebo-controlled phase 2 trial in patients with intractable partial seizures, taking maximally 3 antiepileptic drugs. The initial results indicate that add-on therapy with ganaxolone resulted in reduced seizure frequency with adverse effect being mainly mild to moderate. Possibly, ganaxolone may be also considered against catamenial seizures. Some positive effects of ganaxolone as an adjuvant were also observed in children with refractory seizures and its use may also prove efficient for the management of neonatal seizures associated with hypoxic injury. Neurosteroids positively modulating GABA-A receptor complex exert anticonvulsive activity in many experimental models of seizures. Their interactions with antiepileptic drugs seem ambiguous in mice. Initial clinical data indicate that ganaxolone may provide a better seizure control in patients with drug-resistant epilepsy.
Topics: Allosteric Regulation; Animals; Anticonvulsants; Epilepsy; GABA-A Receptor Agonists; Humans; Neurosteroids; Seizures; Treatment Outcome
PubMed: 33117274
DOI: 10.3389/fendo.2020.541802 -
Disease Models & Mechanisms Jul 2021Danio rerio (zebrafish) are a powerful experimental model for genetic and developmental studies. Adaptation of zebrafish to study seizures was initially established...
Danio rerio (zebrafish) are a powerful experimental model for genetic and developmental studies. Adaptation of zebrafish to study seizures was initially established using the common convulsant agent pentylenetetrazole (PTZ). Larval PTZ-exposed zebrafish exhibit clear behavioral convulsions and abnormal electrographic activity, reminiscent of interictal and ictal epileptiform discharge. By using this model, our laboratory developed simple locomotion-based and electrophysiological assays to monitor and quantify seizures in larval zebrafish. Zebrafish also offer multiple advantages for rapid genetic manipulation and high-throughput phenotype-based drug screening. Combining these seizure assays with genetically modified zebrafish that represent Dravet syndrome, a rare genetic epilepsy, ultimately contributed to a phenotype-based screen of over 3500 drugs. Several drugs identified in these zebrafish screens are currently in clinical or compassionate-use trials. The emergence of this 'aquarium-to-bedside' approach suggests that broader efforts to adapt and improve upon this zebrafish-centric strategy can drive a variety of exciting new discoveries.
Topics: Animals; Anticonvulsants; Epilepsy; Pentylenetetrazole; Seizures; Zebrafish
PubMed: 34231838
DOI: 10.1242/dmm.049080