Acute Seizure Models Research Articles

Simultaneous lipidomic and transcriptomic profiling in mouse brain punches of acute epileptic seizure model compared to controls

In this study, we report the development of a dual extraction protocol for RNA and lipids, including phospholipids, endocannabinoids, and arachidonic acid, at high spatial resolution, e.g., brain punches obtained from whole frozen brains corresponding to four brain subregions: dorsal hippocampus, ventral hippocampus, basolateral amygdala, and hypothalamus. This extraction method combined with LC/multiple reaction monitoring for lipid quantifi-cation and quantitative PCR for RNA investigation allows lipidomic and transcriptomic profiling from submilligram amounts of tissue, thus benefiting the time and animal costs for analysis and the data reliability due to prevention of biological variability between animal batches and/or tissue heterogeneity, as compared with profiling in distinct animal batches. Moreover, the method allows a higher extraction efficiency and integrity preservation for RNA, while allowing concurrently quantitative analysis of low and high abundant lipids. The method was applied for brain punches obtained 1 h after kainic acid-induced epileptic seizures in mice (n = 10) compared with controls (n = 10), and enabled the provision of valuable new insights into the subregional lipid and RNA changes with epilepsy, highlighting its potential as a new viable tool in quantitative neurobiology.

Anticonvulsant and Antioxidant Effects of Musa sapientum Stem Extract on Acute and Chronic Experimental Models of Epilepsy.

Background:Musa sapientum (banana) plant extract has been shown to possess antioxidant activity in previous studies. Neuronal injury resulting from oxidative stress is an important factor involved in pathogenesis of epilepsy.Objective:The present study aimed to evaluate the anticonvulsant activity of M. sapientum stem extract (MSSE) in acute and chronic experimental models in mice and its effects on various markers of oxidative stress in the brain of pentylenetetrazole (PTZ)-kindled animals.Material and Methods:Maximal electroshock seizures (MES) and PTZ-induced convulsion models were used for acute studies. For the chronic study, the effect of MSSE on the development of kindling was studied. For the evaluation of the effects of MSSE on oxidative stress in brain, malondialdehyde (MDA) and reduced glutathione (GSH) levels were estimated in the brains of the kindled animals.Results:MSSE significantly increased the latency to onset of myoclonic jerks and the duration of clonic convulsions following PTZ administration. The MSSE pretreated group showed significantly reduced mean seizure score on PTZ-induced kindling. There was a significant increase in the brain MDA levels and decrease in GSH levels in response to PTZ-induced kindling. On MSSE pretreatment, there was a significant decrease in the MDA levels in the brains, though the increase in the GSH levels was not significant.Conclusion:The results from this study suggest the presence of significant anticonvulsant activity in MSSE, in both acute and chronic PTZ-induced seizure models, which could be due to its antioxidant activity, as is reflected by the change in oxidative stress markers in brain.SUMMARY Evaluation of the anticonvulsant activity of Musa sapientum and its effects on various markers of oxidative stress in the brain has not been done previously to the best of our knowledgeM. sapientum stem extract (MSSE) significantly increased the latency to onset of myoclonic jerks and the duration of clonic convulsions in the experimental modelsThe MSSE pretreated group showed significantly reduced mean seizure score on pentylenetetrazole (PTZ)-induced kindlingThere was significant increase in the brain malondialdehyde (MDA) levels and decrease in glutathione (GSH) levels in response to PTZ-induced kindlingOn MSSE pretreatment, there was a significant decrease in the MDA levels in the brain, though the increase in the GSH levels was not significant. Abbreviations Used: MSSE: Musa sapientum stem extract, PTZ: Pentylenetetrazole, MES: Maximal electroshock seizures, MDA: Malondialdehyde, GSH: Glutathione, SOD: Superoxide dismutase, THLE: Tonic hindlimb extension

Glial source of nitric oxide in epileptogenesis: A target for disease modification in epilepsy.

Epileptogenesis is the process of developing an epileptic condition and/or its progression once it is established. The molecules that initiate, promote, and propagate remarkable changes in the brain during epileptogenesis are emerging as targets for prevention/treatment of epilepsy. Epileptogenesis is a continuous process that follows immediately after status epilepticus (SE) in animal models of acquired temporal lobe epilepsy (TLE). Both SE and epileptogenesis are potential therapeutic targets for the discovery of anticonvulsants and antiepileptogenic or disease-modifying agents. For translational studies, SE targets are appropriate for screening anticonvulsive drugs prior to their advancement as therapeutic agents, while targets of epileptogenesis are relevant for identification and development of therapeutic agents that can either prevent or modify the disease or its onset. The acute seizure models do not reveal antiepileptogenic properties of anticonvulsive drugs. This review highlights the important components of epileptogenesis and the long-term impact of intervening one of these components, nitric oxide (NO), in rat and mouse kainate models of TLE. NO is a putative pleotropic gaseous neurotransmitter and an important contributor of nitro-oxidative stress that coexists with neuroinflammation and epileptogenesis. The long-term impact of inhibiting the glial source of NO during early epileptogenesis in the rat model of TLE is reviewed. The importance of sex as a biological variable in disease modification strategies in epilepsy is also briefly discussed.

Role of the Fyn-PKCδ signaling in SE-induced neuroinflammation and epileptogenesis in experimental models of temporal lobe epilepsy

Status epilepticus (SE) induces neuroinflammation and epileptogenesis, but the mechanisms are not yet fully delineated. The Fyn, a non-receptor Src family tyrosine kinase (SFK), and its immediate downstream target, PKCδ are emerging as potential mediators of neuroinflammation. In order to first determine the role of Fyn kinase signaling in SE, we tested the efficacy of a SFK inhibitor, saracatinib (25mg/kg, oral) in C57BL/6J mouse kainate model of acute seizures. Saracatinib pretreatment dampened SE severity and completely prevented mortality. We further utilized fyn−/− and fyn+/+ mice (wildtype control for the fyn−/− mice on same genetic background), and the rat kainate model, treated with saracatinib post-SE, to validate the role of Fyn/SFK in SE and epileptogenesis. We observed significant reduction in SE severity, epileptiform spikes, and electrographic non-convulsive seizures in fyn−/− mice when compared to fyn+/+ mice. Interestingly, significant reductions in phosphorylated pSrc-416 and PKCδ (pPKCδ-507) and naive PKCδ were observed in fyn−/− mice as compared to fyn+/+ mice suggesting that PKCδ signaling is a downstream mediator of Fyn in SE and epileptogenesis. Notably, fyn−/− mice also showed a reduction in key proinflammatory mediators TNF-α, IL-1β, and iNOS mRNA expression; serum IL-6 and IL-12 levels; and nitro-oxidative stress markers such as 4-HNE, gp91phox, and 3-NT in the hippocampus. Immunohistochemistry revealed a significant increase in reactive microgliosis and neurodegeneration in the hippocampus and hilus of dentate gyrus in fyn+/+ mice in contrast to fyn−/− mice. Interestingly, we did not observe upregulation of Fyn in pyramidal neurons of the hippocampus during post-SE in fyn+/+ mice, but it was upregulated in hilar neurons of the dentate gyrus when compared to naïve control. In reactive microglia, both Fyn and PKCδ were persistently upregulated during post-SE suggesting that Fyn-PKCδ may drive neuroinflammation during epileptogenesis. Since disabling the Fyn kinase prior to SE, either by treating with saracatinib or fyn gene knockout, suppressed seizures and the subsequent epileptogenic events, we further tested whether Fyn/SFK inhibition during post-SE modifies epileptogenesis. Telemetry-implanted, SE-induced, rats were treated with saracatinib and continuously monitored for a month. At 2h post-diazepam, the saracatinib (25mg/kg) or the vehicle was administered orally and repeated twice daily for first three days followed by a single dose/day for the next four days. The saracatinib post-treatment prevented epileptogenesis in >50% of the rats and significantly reduced spontaneous seizures and epileptiform spikes in the rest (one animal did not respond) when compared to the vehicle treated group, which had >24 seizures in a month. Collectively, the findings suggest that Fyn/SFK is a potential mediator of epileptogenesis and a therapeutic target to prevent/treat seizures and epileptogenesis.

Anticonvulsant effect of the hydroethanolic leaf extract of Psydrax subcordata (DC.) Bridson in murine models

Ethnopharmacological relevancePsydrax subcordata (DC.) Bridson is a tropical medicinal plant used traditionally for the management of epilepsy. However, there is little scientific evidence to support its use. Aim of studyThe current study investigated the anticonvulsant properties of the hydroethanolic leaf extract of Psydrax subcordata (PSE) in animal models. Materials and methodsThe anticonvulsant effects were evaluated in mouse models of acute seizures (pentylenetetrazole-, picrotoxin-, 4-aminopyridine-, strychnine- and maximal electroshock-induced seizure tests) and status epilepticus (Lithium/pilocarpine-induced SE). The role of GABAergic mechanisms in the actions of the extract was also examined by pre-treatment of animals with flumazenil in the pentylenetetrazole test. ResultsThe extract (30, 100 and 300mg/kg, p.o.) significantly delayed the onset and decreased the duration and frequency of pentylenetetrazole- and picrotoxin-convulsions. PSE also reduced the duration of tonic hind limb extensions in the maximal electroshock-induced seizure test. Furthermore, PSE pre-treatment significantly delayed the onset of seizures and improved survival in the 4-aminopyridine-induced seizure test. In the strychnine-induced seizure test, PSE treatment did not significantly affect the latency to convulsions and time until death when compared to controls. PSE exhibited anticonvulsant effects in the lithium/pilocarpine test by delaying the onset of seizures and status epilepticus as well as reducing the severity of seizures and mortality of mice. Again, the anticonvulsant effect of PSE (100mg/kg, p.o.) was blocked by pre-treatment with flumazenil in the PTZ test. ConclusionPSE has anticonvulsant activity in animal models, and this effect may be mediated, at least partly, through GABAergic mechanisms.

Methylated flavonoids as anti-seizure agents: Naringenin 4′,7-dimethyl ether attenuates epileptic seizures in zebrafish and mouse models

Epilepsy is a neurological disease that affects more than 70 million people worldwide and is characterized by the presence of spontaneous unprovoked recurrent seizures. Existing anti-seizure drugs (ASDs) have side effects and fail to control seizures in 30% of patients due to drug resistance. Hence, safer and more efficacious drugs are sorely needed. Flavonoids are polyphenolic structures naturally present in most plants and consumed daily with no adverse effects reported. These structures have shown activity in several seizure and epilepsy animal models through allosteric modulation of GABAA receptors, but also via potent anti-inflammatory action in the brain. As such, dietary flavonoids offer an interesting source for ASD and anti-epileptogenic drug (AED) discovery, but their pharmaceutical potential is often hampered by metabolic instability and low oral bioavailability. It has been argued that their drug-likeness can be improved via methylation of the free hydroxyl groups, thereby dramatically enhancing metabolic stability and membrane transport, facilitating absorption and highly increasing bioavailability. Since no scientific data is available regarding the use of methylated flavonoids in the fight against epilepsy, we studied naringenin (NRG), kaempferol (KFL), and three methylated derivatives, i.e., naringenin 7-O-methyl ether (NRG-M), naringenin 4′,7-dimethyl ether (NRG-DM), and kaempferide (4′-O-methyl kaempferol) (KFD) in the zebrafish pentylenetetrazole (PTZ) seizure model. We demonstrate that the methylated flavanones NRG-DM and NRG-M are highly effective against PTZ-induced seizures in larval zebrafish, whereas NRG and the flavonols KFL and KFD possess only a limited activity. Moreover, we show that NRG-DM is active in two standard acute mouse seizure models, i.e., the timed i.v. PTZ seizure model and the 6-Hz psychomotor seizure model. Based on these results, NRG-DM is proposed as a lead compound that is worth further investigation for the treatment of generalized seizures and drug-resistant focal seizures. Our data therefore highlights the potential of methylated flavonoids in the search for new and improved ASDs.

Chronic levetiracetam decreases hippocampal and testicular aromatase expression in normal but not kainic acid-induced experimental model of acute seizures in rats.

Reproductive disorders are more common in men with epilepsy taking anticonvulsant medications. Antiseizure/anticonvulsant drugs and seizures in medial temporal lobe structures may cause gonadal dysfunction, including infertility, decreased libido, and potency. Levels of circulating bioavailable testosterone are affected by the aromatase enzyme, which converts testosterone into estrogen and may be affected by seizure medications. However, the relationship of anticonvulsant drugs with central aromatase levels is not clear. This study investigated the possible effects of the highly efficient, new-generation antiseizure/anticonvulsant drug levetiracetam on central and gonadal aromatase expression and gonadal tissue functionality at 27 and 54 mg/kg/day doses. Epileptogenesis was generated in male Wistar rats by an intraperitoneal injection of the excitotoxic agent kainic acid. Aromatase levels were 1.5 times higher in the brain cortex of the kainic acid groups after 4 weeks and the hippocampus after 4 and 8 weeks compared with the controls. Decreased basal aromatase levels were observed after 1 week of levetiracetam treatment (27 mg/kg/day). Administration of 27 mg/kg/day levetiracetam did not alter vas deferens contractions at 1, 4, or 8 weeks compared with the controls. No histological changes were observed in the vas deferens, epididymis, or testis after 8 weeks of levetiracetam administration at both doses. Administration of 27 and 54 mg/kg/day levetiracetam downregulated testis aromatase expression at 8 weeks compared with the controls. These results suggest that levetiracetam decreases aromatase levels in the testis and increases the seizure threshold by a possible decrease in systemic estradiol levels.

Evaluation of the Anticonvulsant Effect of Brilliant Blue G, a Selective P2X7 Receptor Antagonist, in\xa0the iv PTZ-, Maximal Electroshock-, and 6\xa0Hz-Induced Seizure Tests in Mice

Epilepsy is one of the most common neurological disorders which is diagnosed in around 65 million people worldwide. Clinically available antiepileptic drugs fail to control epileptic activity in about 30% of patients and they are merely symptomatic treatments and cannot cure or prevent epilepsy. There remains a need for searching new therapeutic strategies for epileptic disorders. The P2X7 receptor has been recently investigated as a new target in epilepsy treatment. Preclinical studies revealed that P2X7 receptor antagonists have anticonvulsant properties in some models of epilepsy. We aimed to investigate whether P2X7 receptor antagonist—brilliant blue G (BBG)—is able to change seizure threshold in three acute seizure models in mice, i.e., in the intravenous pentylenetetrazole seizure threshold, maximal electroshock seizure threshold and 6 Hz psychomotor seizure threshold tests. BBG was administered acutely (50–200 mg/kg, 30 min before the tests) and sub-chronically (25–100 mg/kg, once daily for seven consecutive days). Moreover, the chimney and grip strength tests were used to estimate the influence of BBG on the motor coordination and muscular strength in mice, respectively. Our results revealed only a week anticonvulsant potential of the studied P2X7 receptor antagonist because it showed anticonvulsant action only in the 6 Hz seizure test, both after acute and sub-chronic administration. BBG did not significantly influence seizure thresholds in the remaining tests. Motor coordination and muscular strength were not affected by the studied P2X7 receptor antagonist. In summary, BBG does not possess any remarkable anticonvulsant potential in acute seizure models in mice.

Impaired hippocampal glucose metabolism during and after flurothyl-induced seizures in mice: Reduced phosphorylation coincides with reduced activity of pyruvate dehydrogenase.

To determine changes in glucose metabolism and the enzymes involved in the hippocampus ictally and postictally in the acute mouse flurothyl seizure model. [U-13 C]-Glucose was injected (i.p.) prior to, or following a 5 min flurothyl-induced seizure. Fifteen minutes later, mice were killed and the total metabolite levels and % 13 C enrichment were analyzed in the hippocampal formation using gas chromatography-mass spectrometry. Activities of key metabolic and antioxidant enzymes and the phosphorylation status of pyruvate dehydrogenase were measured, along with lipid peroxidation. During seizures, total lactate levels increased 1.7-fold; however, [M + 3] enrichment of both lactate and alanine were reduced by 30% and 43%, respectively, along with a 28% decrease in phosphofructokinase activity. Postictally the % 13 C enrichments of all measured tricarboxylic acid (TCA) cycle intermediates and the amino acids were reduced by 46-93%. At this time, pyruvate dehydrogenase (PDH) activity was 56% of that measured in controls, and there was a 1.9-fold increase in the phosphorylation of PDH at ser232. Phosphorylation of PDH is known to decrease its activity. Here, we show that the increase of lactate levels during flurothyl seizures is from a source other than [U-13 C]-glucose, such as glycogen. Surprisingly, although we saw a reduction in phosphofructokinase activity during the seizure, metabolism of [U-13 C]-glucose into the TCA cycle seemed unaffected. Similar to our recent findings in the chronic phase of the pilocarpine model, postictally the metabolism of glucose by glycolysis and the TCA cycle was impaired along with reduced PDH activity. Although this decrease in activity may be a protective mechanism to reduce oxidative stress, which is observed in the flurothyl model, ATP is critical to the recovery of ion and neurotransmitter balance and return to normal brain function. Thus we identified promising novel strategies to enhance energy metabolism and recovery from seizures.

Myeloid differentiation factor 88 is up-regulated in epileptic brain and contributes to experimental seizures in rats

Accumulating evidence supports that activation of inflammatory pathways is a crucial factor contributing to the pathogenesis of seizures. In particular, the activation of interleukin-1 beta (IL-1β) system exerts proconvulsant effects in a large variety of seizure models. Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein in the signaling cascade elicited by IL-1β. The present study aimed to investigate the expression pattern of MyD88 in rat models of seizures and in patients with refractory temporal lobe epilepsy (TLE), and to study the role of MyD88 in epileptic seizures. Our results revealed that MyD88 was up-regulated in the hippocampus of rats in the lithium-pilocarpine model of acute seizures. Importantly, MyD88 overexpression was also significantly present in the brain from chronic epileptic rats and the temporal neocortex specimens from drug-resistant TLE patients. In the acute seizure model, both the behavioral and electrographic seizure activities were record and analyzed in rats for 90min, starting immediately after pilocarpine injection. ST2825, a synthetic MyD88 inhibitor, was administered intracerebroventricularly (2.5–5.0–10μg in 2μl) 20min before pilocarpine injection. We found that ST2825 at doses of 5μg and 10μg significantly inhibited the pilocarpine-induced behavioral and electrographic seizures. Moreover, 10μg ST2825 prevented the proconvulsant actions of IL-1β. As previous evidence suggested that IL-1β proconvulsant effects was mediated by enhancing the phosphorylation level of the NR2B subunit of N-methyl-d-aspartate (NMDA) receptor, we then probed whether this molecular was involved in the effect of the pharmacological inhibition. Our results revealed that 10μg ST2825 markedly reversed the increased Tyr1472-phosphorylation of the NR2B subunit of NMDA receptor observed in the proconvulsant conditions of IL-1β and in seizures induced by pilocarpine alone. These findings indicate that altered expression of MyD88 might contribute to the pathogenesis of seizures and targeting of this adaptor protein might represent a novel therapeutic strategy to suppress seizure activities.

Photolysis of Caged-GABA Rapidly Terminates Seizures In Vivo: Concentration and Light Intensity Dependence.

The therapy of focal epilepsy remains unsatisfactory for as many as 25% of patients. The photolysis of caged-γ-aminobutyric acid (caged-GABA) represents a novel and alternative option for the treatment of intractable epilepsy. Our previous experimental results have demonstrated that the use of blue light produced by light-emitting diode to uncage ruthenium-bipyridine-triphenylphosphine-c-GABA (RuBi-GABA) can rapidly terminate paroxysmal seizure activity both in vitro and in vivo. However, the optimal concentration of RuBi-GABA, and the intensity of illumination to abort seizures, remains unknown. The aim of this study was to explore the optimal anti-seizure effects of RuBi-GABA by using implantable fibers to introduce blue light into the neocortex of a 4-aminopyridine-induced acute seizure model in rats. We then investigated the effects of different combinations of RuBi-GABA concentrations and light intensity upon seizure. Our results show that the anti-seizure effect of RuBi-GABA has obvious concentration and light intensity dependence. This is the first example of using an implantable device for the photolysis of RuBi-GABA in the therapy of neocortical seizure, and an optimal combination of RuBi-GABA concentration and light intensity was explored. These results provide important experimental data for future clinical translational studies.

DV21 decreases excitability of cortical pyramidal neurons and acts in epilepsy

Epilepsy is one of the most common neurological disorders and the administration of antiepileptic drugs (AEDs) is the most common treatment. Although there are more than 15 AEDs available, a third of epilepsy patients remain refractory to available drugs, so novel effective drugs are needed. Here, we found that DV21, which is a natural triterpenoid compound extracted from plants of the Asclepiadaceae family, significantly decreased the incidence and stages of seizures in three classical drug-induced acute seizure models in C57BL/6 mice. Furthermore, we also found that the antiepileptic effect of DV21 might be partly mediated through reducing the excitability of cortical pyramidal neurons by increasing M current, which are low-threshold non-inactivating voltage-gated potassium currents. Moreover, the application of XE991, an inhibitor of M current, could block most the antiepileptic effect of DV21. Taken together, our results indicated that DV21 might be a novel leading compound for the treatment of epilepsy.

Evaluation of Cannabidiol in Animal Seizure Models by the Epilepsy Therapy Screening Program (ETSP).

Cannabidiol (CBD) is a cannabinoid component of marijuana that has no significant activity at cannabinoid receptors or psychoactive effects. There is considerable interest in CBD as a therapy for epilepsy. Almost a third of epilepsy patients are not adequately controlled by clinically available anti-seizure drugs (ASDs). Initial studies appear to demonstrate that CBD preparations may be a useful treatment for pharmacoresistant epilepsy. The National Institute of Neurological Disorders and Stroke (NINDS) funded Epilepsy Therapy Screening Program (ETSP) investigated CBD in a battery of seizure models using a refocused screening protocol aimed at identifying pharmacotherapies to address the unmet need in pharmacoresistant epilepsy. Applying this new screening workflow, CBD was investigated in mouse 6Hz 44mA, maximal electroshock (MES), corneal kindling models and rat MES and lamotrigine-resistant amygdala kindling models. Following intraperitoneal (i.p.) pretreatment, CBD produced dose-dependent protection in the acute seizure models; mouse 6Hz 44mA (ED50 164mg/kg), mouse MES (ED50 83.5mg/kg) and rat MES (ED50 88.9mg/kg). In chronic models, CBD produced dose-dependent protection in the corneal kindled mouse (ED50 119mg/kg) but CBD (up to 300mg/kg) was not protective in the lamotrigine-resistant amygdala kindled rat. Motor impairment assessed in conjunction with the acute seizure models showed that CBD exerted seizure protection at non-impairing doses. The ETSP investigation demonstrates that CBD exhibits anti-seizure properties in acute seizure models and the corneal kindled mouse. However, further preclinical and clinical studies are needed to determine the potential for CBD to address the unmet needs in pharmacoresistant epilepsy.

Therapeutic effects of anti-HMGB1 monoclonal antibody on pilocarpine-induced status epilepticus in mice

Inflammatory processes in brain tissue have been described in human epilepsy of various etiologies and in experimental models of seizures. High mobility group box-1 (HMGB1) is now recognized as representative of damage-associated molecular patterns (DAMPs). In the present study, we focused on whether anti-HMGB1 antibody treatment could relieve status epilepticus- triggered BBB breakdown and inflammation response in addition to the seizure behavior itself. Pilocarpine and methyl-scopolamine were used to establish the acute seizure model. Anti-HMGB1 mAb showed inhibitory effects on leakage of the BBB, and on the HMGB1 translocation induced by pilocarpine. The expression of inflammation-related factors, such as MCP-1, CXCL-1, TLR-4, and IL-6 in hippocampus and cerebral cortex were down-regulated by anti-HMGB1 mAb associated with the number of activated astrocytes, microglial cells as well as the expression of IL-1β. Both hematoxylin & eosin and TUNEL staining showed that the apoptotic cells could be reduced after anti-HMGB1 mAb treatment. The onset and latency of Racine stage five were significantly prolonged in the anti-HMGB1 mAb group. These results suggested that anti-HMGB1 mAb prevented the BBB permeability, reduced HMGB1 translocation while inhibiting the expression of inflammation-related factors, protected against neural cell apoptosis and prolonged Racine stage 5 seizure onset and latency.

Animal Models of Seizures and Epilepsy: Past, Present, and Future Role for the Discovery of Antiseizure Drugs.

The identification of potential therapeutic agents for the treatment of epilepsy requires the use of seizure models. Except for some early treatments, including bromides and phenobarbital, the antiseizure activity of all clinically used drugs was, for the most part, defined by acute seizure models in rodents using the maximal electroshock and subcutaneous pentylenetetrazole seizure tests and the electrically kindled rat. Unfortunately, the clinical evidence to date would suggest that none of these models, albeit useful, are likely to identify those therapeutics that will effectively manage patients with drug resistant seizures. Over the last 30 years, a number of animal models have been developed that display varying degrees of pharmacoresistance, such as the phenytoin- or lamotrigine-resistant kindled rat, the 6-Hz mouse model of partial seizures, the intrahippocampal kainate model in mice, or rats in which spontaneous recurrent seizures develops after inducing status epilepticus by chemical or electrical stimulation. As such, these models can be used to study mechanisms of drug resistance and may provide a unique opportunity for identifying a truly novel antiseizure drug (ASD), but thus far clinical evidence for this hope is lacking. Although animal models of drug resistant seizures are now included in ASD discovery approaches such as the ETSP (epilepsy therapy screening program), it is important to note that no single model has been validated for use to identify potential compounds for as yet drug resistant seizures, but rather a battery of such models should be employed, thus enhancing the sensitivity to discover novel, highly effective ASDs. The present review describes the previous and current approaches used in the search for new ASDs and offers some insight into future directions incorporating new and emerging animal models of therapy resistance.

Synergistic anticonvulsant effects of pregabalin and amlodipine on acute seizure model of epilepsy in mice.

Status epilepticus is a life threatening neurological medical emergency. It may cause serious damage to the brain and even death in many cases if not treated properly. There is limited choice of drugs for the short term and long term management of status epilepticus and the dugs recommended for status epilepticus possess various side effects. The present study was designed to investigate synergistic anticonvulsant effects of pregabalin with amlodipine on acute seizure model of epilepsy in mice. Pentylenetetrazole was used to induce acute seizures which mimic status epilepticus. Pregabalin and amlodipine were used in combination to evaluate synergistic anti-seizure effects on acute seizure model of epilepsy in mice. Diazepam and valproate were used as reference dugs. The acute anti-convulsive activity of pregabalin with amlodipine was evaluated in vivo by the chemical induced seizures and their anti-seizure effects were compared with pentylenetetrazole, reference drugs and to their individual effects. The anti-seizure effects of tested drugs were recorded in seconds on seizure characteristics such as latency of onset of threshold seizures, rearing and fallings and Hind limbs tonic extensions. The seizure protection and mortality to the animals exhibited by the drugs were recorded in percentage. Combination regimen of pregabalin with amlodipine exhibited dose dependent significant synergistic anticonvulsant effects on acute seizures which were superior to their individual effects and equivalent to reference drugs.

Therapeutic effects of cannabinoids in animal models of seizures, epilepsy, epileptogenesis, and epilepsy-related neuroprotection.

The isolation and identification of the discrete plant cannabinoids in marijuana revived interest in analyzing historical therapeutic claims made for cannabis in clinical case studies and anecdotes. In particular, sources as old as the 11th and 15th centuries claimed efficacy for crude marijuana extracts in the treatment of convulsive disorders, prompting a particularly active area of preclinical research into the therapeutic potential of plant cannabinoids in epilepsy. Since that time, a large body of literature has accumulated describing the effects of several of the >100 individual plant cannabinoids in preclinical models of seizures, epilepsy, epileptogenesis, and epilepsy-related neuroprotection. We surveyed the literature for relevant reports of such plant cannabinoid effects and critically reviewed their findings. We found that acute CB1R agonism in simple models of acute seizures in rodents typically produces anti-convulsant effects whereas CB1R antagonists exert converse effects in the same models. However, when the effects of such ligands are examined in more complex models of epilepsy, epileptogenesis and neuroprotection, a less simplistic narrative emerges. Here, the complex interactions between (i) brain regions involved in a given model, (ii) relative contributions of endocannabinoid signaling to modulation of synaptic transmission in such areas, (iii) multi-target effects, (iv) cannabinoid type 1 and type 2 receptor signaling interactions and, (v) timing, (vi) duration and (vii) localization of ligand administration suggest that there is both anti-epileptic therapeutic potential and a pro-epileptic risk in up- and down-regulation of endocannabinoid signaling in the central nervous system. Factors such receptor desensitization and specific pharmacology of ligands used (e.g. full vs partial agonists and neutral antagonists vs inverse agonists) also appear to play an important role in the effects reported. Furthermore, the effects of several plant cannabinoids, most notably cannabidiol (CBD) and cannabidavarin (CBDV), in models of seizures, epilepsy, epileptogenesis, and neuroprotection are less ambiguous, and consistent with reports of therapeutically beneficial effects of these compounds in clinical studies. However, continued paucity of firm information regarding the therapeutic molecular mechanism of CBD/CBDV highlights the continued need for research in this area in order to identify as yet under-exploited targets for drug development and raise our understanding of treatment-resistant epilepsies. The recent reporting of positive results for cannabidiol treatment in two Phase III clinical trials in treatment-resistant epilepsies provides pivotal evidence of clinical efficacy for one plant cannabinoid in epilepsy. Moreover, risks and/or benefits associated with the use of unlicensed Δ9-THC containing marijuana extracts in pediatric epilepsies remain poorly understood. Therefore, in light of these paradigm-changing clinical events, the present review's findings aim to drive future drug development for newly-identified targets and indications, identify important limitations of animal models in the investigation of plant cannabinoid effects in the epilepsies, and focuses future research in this area on specific, unanswered questions regarding the complexities of endocannabinoid signaling in epilepsy. This article is part of a Special Issue titled Cannabinoids and Epilepsy.

Therapeutic potential of an anti-high mobility group box-1 monoclonal antibody in epilepsy

Brain inflammation is a major factor in epilepsy, and the high mobility group box-1 (HMGB1) protein is known to contribute significantly to the generation of seizures. Here, we investigated the therapeutic potential of an anti-HMGB1 monoclonal antibody (mAb) in epilepsy. anti-HMGB1 mAb attenuated both acute seizure models (maximal electroshock seizure, pentylenetetrazole-induced and kindling-induced), and chronic epilepsy model (kainic acid-induced) in a dose-dependent manner. Meanwhile, the anti-HMGB1 mAb also attenuated seizure activities of human brain slices obtained from surgical resection from drug-resistant epilepsy patients. The mAb showed an anti-seizure effect with a long-term manner and appeared to be minimal side effects at even very high dose (no disrupted physical EEG rhythm and no impaired basic physical functions, such as body growth rate and thermoregulation). This anti-seizure effect of mAb results from its inhibition of translocated HMGB1 from nuclei following seizures, and the anti-seizure effect was absent in toll-like receptor 4 knockout (TLR4−/−) mice. Interestingly, the anti-HMGB1 mAb also showed a disease-modifying anti-epileptogenetic effect on epileptogenesis after status epileptics, which is indicated by reducing seizure frequency and improving the impaired cognitive function. These results indicate that the anti-HMGB1 mAb should be viewed as a very promising approach for the development of novel therapies to treat refractory epilepsy.

Chronic metformin treatment facilitates seizure termination

The AMP-activated protein kinase (AMPK) is a key energy sensor. Its activator metformin could suppress epileptogenesis in the pentylenetetrazol (PTZ) kindling model. However, the effect of metformin on the acute and chronic seizures has not been studied. We first detected the expression of AMPK in the brain tissue of human and mice with chronic seizures, as well as in mice with acute seizures. Second, using behavioral assay and local filed potentials (LFPs) recording, we investigated the effect of chronic metformin treatment on seizures in a acute seizure model and a chronic seizure model. Our results showed that AMPK was expressed in neurons in the epileptic brain. The expression level was decreased in the brain tissue that experienced chronic and acute seizures. In PTZ-induced acute seizures model, behavioral assay showed that chronic metformin treatment decreased the mortality, and LFPs recording showed that chronic metformin treatment shortened the duration of generalized tonic-clonic seizures and prolonged the duration of postictal depression. Moreover, in kainic acid-induced chronic seizures model, LFPs recording showed that chronic metformin treatment shortened the duration of epileptic activity. Our study suggests that chronic metformin treatment could facilitate seizure termination.

Noninvasive Targeted Transcranial Neuromodulation via Focused Ultrasound Gated Drug Release from Nanoemulsions.

Targeted, noninvasive neuromodulation of the brain of an otherwise awake subject could revolutionize both basic and clinical neuroscience. Toward this goal, we have developed nanoparticles that allow noninvasive uncaging of a neuromodulatory drug, in this case the small molecule anesthetic propofol, upon the application of focused ultrasound. These nanoparticles are composed of biodegradable and biocompatible constituents and are activated using sonication parameters that are readily achievable by current clinical transcranial focused ultrasound systems. These particles are potent enough that their activation can silence seizures in an acute rat seizure model. Notably, there is no evidence of brain parenchymal damage or blood-brain barrier opening with their use. Further development of these particles promises noninvasive, focal, and image-guided clinical neuromodulation along a variety of pharmacological axes.