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The AMPA receptor antagonist NBQX exerts anti-seizure but not antiepileptogenic effects in the intrahippocampal kainate mouse model of mesial temporal lobe epilepsy
Abstract
The AMPA receptor subtype of glutamate receptors, which mediates fast synaptic excitation, is of primary importance in initiating epileptiform discharges, so that AMPA receptor antagonists exert anti-seizure activity in diverse animal models of partial and generalized seizures. Recently, the first AMPA receptor antagonist, perampanel, was approved for use as adjunctive therapy for the treatment of resistant partial seizures in patients. Interestingly, the competitive AMPA receptor antagonist NBQX has recently been reported to prevent development of spontaneous recurrent seizures (SRS) in a neonatal seizure model in rats, indicating the AMPA antagonists may exert also antiepileptogenic effects. This prompted us to evaluate competitive (NBQX) and noncompetitive (perampanel) AMPA receptor antagonists in an adult mouse model of mesial temporal lobe epilepsy. In this model, SRS develop after status epilepticus (SE) induced by intrahippocampal injection of kainate. Focal electrographic seizures in this model are resistant to several major antiepileptic drugs. In line with previous studies, phenytoin was not capable of blocking such seizures in the present experiments, while they were markedly suppressed by NBQX and perampanel. However, perampanel was less tolerable than NBQX in epileptic mice, so that only NBQX was subsequently tested for antiepileptogenic potential. When mice were treated over three days after kainate-induced SE with NBQX (20 mg/kg t.i.d.), no effect on development or frequency of seizures was found in comparison to vehicle controls. These results suggest that AMPA receptor antagonists, while being effective in suppressing resistant focal seizures, are not exerting antiepileptogenic effects in an adult mouse model of partial epilepsy.
Copyright © 2015. Published by Elsevier Ltd.
... In order to provide a vision "at a glance" of those concepts developed in the review, we built a summarizing table rat amygdala-kindling model decreases the duration of after discharge activity [62] mouse model of mesial temporal lobe epilepsy attenuates spontaneous seizures and occludes limbic seizure-induced brain damage [64] Long-Evans rats suppression of behavioral alterations and the onset of spontaneous secondary seizures [65] YM872 rat amygdala kindling model suppressed fully kindled seizures [68] YM90K DBA/2 mice potent suppressive activity against audiogenic seizure [69] rat amygdala-kindling model suppressed fully kindled seizures [70] LY293558 mouse protection versus maximal electroshock seizures and decreases in spontaneous motor activity [73] RPR117824 mouse or rat blocker of convulsions induced by supramaximal electroshock or chemoconvulsive agents [74] NS1209 mouse increased the seizure threshold for electroshock-induced tonic seizures [76] rat protects against status epilepticus induced by electrical stimulation of the amygdala [77] Negative allosteric modulators of AMPA receptors. mouse protection against seizures induced by infusion of ATPA [98] human reduces seizures when administered as an add-on treatment for drug-resistant focal epilepsy [101] Perampanel human approved for the treatment of focal epilepsy and generalized tonic-clonic epilepsy; restrains myoclonic or absence seizures [102][103][104][105][106][107][108][109] mouse protection against PTZ-induced clonic seizures and psychomotor seizures in the 6-Hz stimulation protocol [110] rat amygdala kindling model shortens after-discharge activity and prolongs latency to onset of generalized seizures [111,112] rat block of pilocarpine-induced status epilepticus [113] human good efficacy and tolerability in young children with intractable epilepsy [116] AMPA receptors in models of absence epilepsy. ...
... NBQX is a competitive AMPA receptor antagonist that showed anti-seizure but not antiepileptogenic activity in the mouse model of mesial temporal lobe epilepsy induced by intrahippocampal injection of kainate [64]. Acute treatment with NBQX soon after hypoxia-induced postnatal seizures attenuates spontaneous seizures and occludes limbic seizureinduced brain damage, as evidenced by hippocampal mossy fiber sprouting [64]. ...
... NBQX is a competitive AMPA receptor antagonist that showed anti-seizure but not antiepileptogenic activity in the mouse model of mesial temporal lobe epilepsy induced by intrahippocampal injection of kainate [64]. Acute treatment with NBQX soon after hypoxia-induced postnatal seizures attenuates spontaneous seizures and occludes limbic seizureinduced brain damage, as evidenced by hippocampal mossy fiber sprouting [64]. Occlusion of such a pathological alteration, which is reminiscent of medial temporal lobe sclerosis and correlates with secondary epileptogenesis is confirmed by data showing suppression of behavioral alterations and the onset of spontaneous secondary seizures following NBQX in adult Long-Evans rats [65]. ...
Background
A dysfunction in glutamate neurotransmission is critical for seizure. Glutamate is the major excitatory drive in the cerebral cortex, where seizures occur. Glutamate acts via (i) ionotropic (iGlu) receptors, which are ligand-gated ion channels mediating fast excitatory synaptic transmission; and (ii) G proteins coupled metabotropic (mGlu) receptors.
Objective
To overview the evidence on the role of iGlu receptors in the onset, duration, and severity of convulsive and nonconvulsive seizures to lay the groundwork for novel strategies for drug-resistant epilepsy.
Methods
We used PubMed crossed-search for “glutamate receptor and epilepsy” (sorting 3,170 reports), searched for “ionotropic glutamate receptors”, “AMPA receptors”, “NMDA receptors”, “kainate receptors”, “convulsive seizures”, “absence epilepsy”, and selected those papers focusing this Review’s scope.
Results
iGlu receptors antagonists inhibit, whereas agonists worsen experimental seizures in various animal species. Clinical development of iGlu receptor antagonists has been limited by the occurrence of adverse effects caused by inhibition of fast excitatory synaptic transmission. To date, only one drug (perampanel) selectively targeting iGlu receptors is marketed for the treatment of focal epilepsy. However, other drugs, such as topiramate and felbamate, inhibit iGlu receptors in addition to other mechanisms.
Conclusion
This review is expected to help dissecting those steps induced by iGlu receptors activation, which may be altered to provide antiepileptic efficacy without altering key physiological brain functions, thus improving safety and tolerability of iGlu-receptor directed antiepileptic agents. This effort mostly applies to drug resistant seizures, which impact the quality of life and often lead to status epilepticus, which is a medical urgency.
... Applied at 25 mg/kg and analyzed its effects in a 10-h period post-injection. This protocol was based on the phenytoin doses used in other mouse models (20-50 mg/kg; Riban et al., 2002;Klein et al., 2015;Twele et al., 2015;Bankstahl et al., 2016) and the half-life of phenytoin plasma elimination in rodents (8-16 h;Löscher, 2007;Markowitz et al., 2010). 2) Lorazepam. ...
... 3) Levetiracetam. Applied at 100 mg or 400 mg/kg, similar to the doses used in other rodent models (160-800 mg/kg; Löscher et al., 1993;Löscher and Hönack, 2000;Zhang et al., 2003;Jiqun et al., 2005;Lee et al., 2013;Shetty, 2013;Twele et al., 2015;Duveau et al., 2016). In mice submitted to intra-peritoneal injections at 200 mg/kg, the half-life of levetiracetam plasma elimination is about 1.5 h and its brain-to-blood ratio is 0.8 at 4 h post-injection (Benedetti et al., 2004;Markowitz et al., 2010). ...
... Several studies have examined phenytoin, diazepam and/or levetiracetam in a mouse model of intra-hippocampal kainate application (Riban et al., 2002;Klein et al., 2015;Twele et al., 2015;Duveau et al., 2016). In these studies, AEDs were applied by acute intra-peritoneal injections and their effects on hippocampal EEG discharges were examined in periods of 3-4 h post-injection. ...
Epilepsy is a common neurological disorder characterized by naturally-occurring spontaneous recurrent seizures and comorbidities. Kindling has long been used to model epileptogenic mechanisms and to assess antiepileptic drugs. In particular, extended kindling can induce spontaneous recurrent seizures without gross brain lesions, as seen clinically. To date, the development of spontaneous recurrent seizures following extended kindling, and the effect of the antiepileptic drugs on these seizures are not well understood. In the present study we aim to develop a mouse model of extended hippocampal kindling for the first time. Once established, we plan to evaluate the effect of three different antiepileptic drugs on the development of the extended-hippocampal-kindled-induced spontaneous recurrent seizures. Male C57 black mice were used for chronic hippocampal stimulations or handling manipulations (twice daily for up to 70 days). Subsequently, animals underwent continuous video/EEG monitoring for seizure detection. Spontaneous recurrent seizures were consistently observed in extended kindled mice but no seizures were detected in the control animals. The aforementioned seizures were generalized events characterized by hippocampal ictal discharges and concurrent motor seizures. Incidence and severity of the seizures was relatively stable while monitored over a few months after termination of the hippocampal stimulation. Three antiepileptic drugs with distinct action mechanisms were tested: phenytoin, lorazepam and levetiracetam. They were applied via intra-peritoneal injections at anticonvulsive doses and their effects on the spontaneous recurrent seizures were analyzed 10–12 h post-injection. Phenytoin (25 mg/kg) and levetiracetam (400 mg/kg) abolished the spontaneous recurrent seizures. Lorazepam (1.5 mg/kg) decreased motor seizure severity but did not reduce the incidence and duration of corresponding hippocampal discharges, implicating its inhibitory effects on seizure spread. No gross brain lesions were observed in a set of extended hippocampal kindled mice submitted to histological evaluation. All these data suggests that our model could be considered as a novel mouse model of extended hippocampal kindling. Some limitations remain to be considered.
... Before the antiepileptogenesis experiments, we evaluated the tolerability of the drug combination in a group of 6 mice that had developed epilepsy after intrahippocampal kainate. The reason for this experiment was that we had previously observed that the tolerability of NMDA and AMPA receptor antagonists in epileptic rodents is lower compared to non-epileptic rodents 8,31 . For the tolerability experiment, we chose an i.p. dose of 30 mg/kg for both NBQX and ifenprodil. ...
... Frasca et al. 18 reported that 20 mg/kg ifenprodil were neuroprotective in a rat SE model. We recently reported that 30 mg/kg NBQX exerted an anti-seizure effect in the intrahippocampal kainate mouse model 8 . Furthermore, administration of NBQX at 20 mg/kg three times daily over 3 days, starting 6 or 8 h after intrahippocampal kainate, was tolerated without any obvious adverse effects 8 . ...
... We recently reported that 30 mg/kg NBQX exerted an anti-seizure effect in the intrahippocampal kainate mouse model 8 . Furthermore, administration of NBQX at 20 mg/kg three times daily over 3 days, starting 6 or 8 h after intrahippocampal kainate, was tolerated without any obvious adverse effects 8 . The three times daily drug injection protocol chosen for the present experiments was based on the rapid elimination of NBQX and ifenprodil in rodents 33,34 . ...
Epilepsy may arise following acute brain insults, but no treatments exist that prevent epilepsy in patients at risk. Here we examined whether a combination of two glutamate receptor antagonists, NBQX and ifenprodil, acting at different receptor subtypes, exerts antiepileptogenic effects in the intrahippocampal kainate mouse model of epilepsy. These drugs were administered over 5 days following kainate. Spontaneous seizures were recorded by video/EEG at different intervals up to 3 months. Initial trials showed that drug treatment during the latent period led to higher mortality than treatment after onset of epilepsy, and further, that combined therapy with both drugs caused higher mortality at doses that appear safe when used singly. We therefore refined the combined-drug protocol, using lower doses. Two weeks after kainate, significantly less mice of the NBQX/ifenprodil group exhibited electroclinical seizures compared to vehicle controls, but this effect was lost at subsequent weeks. The disease modifying effect of the treatment was associated with a transient prevention of granule cell dispersion and less neuronal degeneration in the dentate hilus. These data substantiate the involvement of altered glutamatergic transmission in the early phase of epileptogenesis. Longer treatment with NBQX and ifenprodil may shed further light on the apparent temporal relationship between dentate gyrus reorganization and development of spontaneous seizures.
... Electroencephalographic (EEG) recordings are essential for assessing electrographic activity of neuronal populations and detecting seizures in the brain. Tethered EEG (Cavalheiro et al., 1996;Clasadonte et al., 2013;Seo et al., 2013;Leung et al., 2015;Twele et al., 2015) and telemetric EEG (Wither et al., 2012;Puttachary et al., 2015;Gross et al., 2017;Mooney et al., 2017;Scantlebury et al., 2017) have increasingly been used to examine spontaneous recurrent seizures in mouse models. The telemetric recordings offer an advantage of being free of cable-related complications thus are ideal for continuous EEG monitoring for 24 h daily, up to a few weeks. ...
... Previous studies have examined spontaneous recurrent seizures in mouse models using continuous tethered EEG and video recordings (Cavalheiro et al., 1996;Clasadonte et al., 2013;Seo et al., 2013;Leung et al., 2015;Twele et al., 2015). EEG signals were reportedly collected 24 h daily for a period of 4 days or a few months. ...
... Our present works appear to be the first to describe in detail as to how the slip ring can be used as a mouse EEG commutator. We thus anticipate that our works may add to the previously established approaches (Cavalheiro et al., 1996;Clasadonte et al., 2013;Seo et al., 2013;Leung et al., 2015;Twele et al., 2015;Bertram, 2017;Watanabe et al., 2017) and facilitate continuous EEG monitoring in mouse models. ...
We describe here a simple, cost-effective apparatus for continuous tethered electroencephalographic (EEG) monitoring of spontaneous recurrent seizures in mice. We used a small, low torque slip ring as an EEG commutator, mounted the slip ring onto a standard mouse cage and connected rotary wires of the slip ring directly to animal's implanted headset. Modifications were made in the cage to allow for a convenient installation of the slip ring and accommodation of animal ambient activity. We tested the apparatus for hippocampal EEG recordings in adult C57 black mice. Spontaneous recurrent seizures were induced using extended hippocampal kindling (≥95 daily stimulation). Control animals underwent similar hippocampal electrode implantations but no stimulations were given. Combined EEG and webcam monitoring were performed for 24 h daily for 5–9 consecutive days. During the monitoring periods, the animals moved and accessed water and food freely and showed no apparent restriction in ambient cage activities. Ictal-like hippocampal EEG discharges and concurrent convulsive behaviors that are characteristics of spontaneous recurrent seizures were reliably recorded in a majority of the monitoring experiments in extendedly kindled but not in control animals. However, 1–2 rotary wires were disconnected from the implanted headset in some animals after continuous recordings for ≥5 days. The key features and main limitations of our recording apparatus are discussed.
... The severity of SE was categorized using a score from 1 to 6 (Table S1) based on whether only nonconvulsive seizures were observed or few/many events of convulsive seizures (class 3 to 5 based on a modified Racine's scale; Table S2) were observed during the period of SE. 21 For chronic vEEG, seizures were categorized as either convulsive seizures (class 3-5) or hippocampal paroxysmal discharges (HPDs), which are events with at least twice the amplitude of the baseline recordings with a frequency of >8 Hz, as described previously in Riban et al. 22 Other pathologic evaluations included seizure-like events (SLEs), which were low-frequency high-voltage sharp waves (usually <5 Hz) that were at least 5 s in duration, as described previously by Twele et al. 23 Most HPDs and SLEs were not associated with any behavior, although, subtle events like facial clonus and freezing may be missed in the vEEG recordings. ...
... HPDs typically lasted for 20-25 s and may or may not be associated with behavioral phenotypes of nonconvulsive partial seizures (class 1-2, Racine's scale). However, the incidence of HPDs was rare (<2 HPDs/day) unlike reported previously by Riban et al. 22 This was in line with the findings from Twele et al., 23 who also reported HPDs to be rare events. Accordingly, we observed a very high incidence (average of around 15/h) of high amplitude low frequency (typically <5 Hz) spikes/sharp waves (Fig. 2D3) and defined them as SLEs as was reported in Twele et al. 23 Although comparing between the veh-SE and IL-13 MSC-SE mice, no significant differences in the convulsive seizures ( Fig. 2E; p = 0.89), HPDs ( Fig. 2F; p = 0.08), or the SLEs (Fig. 2G; p = 0.68) were observed. ...
... However, the incidence of HPDs was rare (<2 HPDs/day) unlike reported previously by Riban et al. 22 This was in line with the findings from Twele et al., 23 who also reported HPDs to be rare events. Accordingly, we observed a very high incidence (average of around 15/h) of high amplitude low frequency (typically <5 Hz) spikes/sharp waves (Fig. 2D3) and defined them as SLEs as was reported in Twele et al. 23 Although comparing between the veh-SE and IL-13 MSC-SE mice, no significant differences in the convulsive seizures ( Fig. 2E; p = 0.89), HPDs ( Fig. 2F; p = 0.08), or the SLEs (Fig. 2G; p = 0.68) were observed. Similarly, no significant differences were observed between the treatment groups for the mean duration of convulsive seizures ( Fig. 2H; p = 0.95), HPDs ( Fig. 2I; p = 0.99), or SLEs ( Fig. 2J; p = 0.97). ...
Objectives:
Neuroinflammation plays a critical role in the pathophysiology of mesial temporal lobe epilepsy. We aimed to evaluate whether intracerebral transplantation of interleukin 13-producing mesenchymal stem cells (IL-13 MSCs) induces an M2 microglia/macrophage activation phenotype in the hippocampus with an epileptogenic insult, thereby providing a neuroprotective environment with reduced epileptogenesis.
Methods:
Genetically engineered syngeneic IL-13 MSCs or vehicle was injected within the hippocampus 1 week before the intrahippocampal kainic acid-induced status epilepticus (SE) in C57BL/6J mice. Neuroinflammation was evaluated at disease onset as well as during the chronic epilepsy period (9 weeks). In addition, continuous video-electroencephalography (EEG) (vEEG) monitoring was obtained during the chronic epilepsy period (between 6 and 9 weeks after SE).
Results:
Evaluation of vEEG recordings suggested that IL-13 MSC grafts did not affect the severity and duration of SE or the seizure burden during the chronic epilepsy period, when compared to the vehicle treated SE mice. An M2-activation phenotype was induced in microglia/macrophages that infiltrated the -13 MSC graft site, as evidenced by the arginase1 expression at the graft site at both the 2-week and 9-week time-points. However, M2-activated immune cells were rarely observed outside the graft site and, accordingly, the neuroinflammatory response or cell loss related to SE induction was not altered by IL-13 MSC grafting. Moreover, an increase in the proportion of F4/80+ cells was observed in the IL-13 MSC group compared to the controls.
Significance:
Our data suggest that MSC-based IL-13 delivery to induce M2 glial activation does not provide any neuroprotective or disease-modifying effects in a mouse model of epilepsy. Moreover, use of cell grafting to deliver bioactive compounds for modulating neuroinflammation may have confounding effects in disease pathology of epilepsy due to the additional immune response generated by the grafted cells.
... We recently proposed a two-stage preclinical approach for developing such treatments [9]. In stage 1, tolerability and efficacy of potential anti-epileptogenic drugs or drug combinations is tested in the intrahippocampal kainate mouse model of TLE as recently described for the AMPA receptor antagonist NBQX [10]. The reason for choosing the mouse model for stage 1 is the high frequency of spontaneous electrographic seizures which greatly facilitates anti-epileptogenic drug testing [9,10]. ...
... In stage 1, tolerability and efficacy of potential anti-epileptogenic drugs or drug combinations is tested in the intrahippocampal kainate mouse model of TLE as recently described for the AMPA receptor antagonist NBQX [10]. The reason for choosing the mouse model for stage 1 is the high frequency of spontaneous electrographic seizures which greatly facilitates anti-epileptogenic drug testing [9,10]. In stage 2, tolerable and effective compounds or combinations of compounds resulting from stage 1 are further evaluated in the intrahippocampal kainate model of TLE in rats, thus allowing determination of whether the effect observed in mice translates to another species [9]. ...
... When the duration of SE was measured on the basis of these ictal EEG alterations, it ranged from 3 to 21 h without any obvious difference between rats with or without anesthesia during kainate injection (Table 3; Fig. 2D). Following these typical ictal EEG alterations during SE, a phase of EEG alterations characterized by continuous poly-spike/ waves was observed (Fig. 3D) as also reported during kainate-induced SE in mice [10]. The amplitude and frequency of these poly-spike/waves decreased over the course of SE, and normal basal EEG activity was recorded 4-54 h after SE onset without any obvious difference between rats with or without anesthesia during kainate injection (Table 3). ...
Temporal lobe epilepsy (TLE) is the most common type of acquired epilepsy in adults. TLE can develop after diverse brain insults, including traumatic brain injury, infections, stroke, or prolonged status epilepticus (SE). Post-SE rodent models of TLE are widely used to understand mechanisms of epileptogenesis and develop treatments for epilepsy prevention. In this respect, the intrahippocampal kainate model of TLE in mice is of interest, because highly frequent spontaneous electrographic seizures develop in the kainate focus, allowing evaluation of both anti-seizure and anti-epileptogenic effects of novel drugs with only short EEG recording periods, which is not possible in any other model of TLE, including the intrahippocampal kainate model in rats. In the present study, we investigated whether the marked mouse-to-rat difference in occurrence and frequency of spontaneous seizures is due to a species difference or to technical variables, such as anesthesia during kainate injection, kainate dose, or location of kainate injection and EEG electrode in the hippocampus. When, as in the mouse model, anesthesia was used during kainate injection, only few rats developed epilepsy, although severity or duration of SE was not affected by isoflurane. In contrast, most rats developed epilepsy when kainate was injected without anesthesia. However, frequent electrographic seizures as observed in mice did not occur in rats, irrespective of location of kainate injection (CA1, CA3) or EEG recording electrode (CA1, CA3, dentate gyrus) or dose of kainate injected. These data indicate marked phenotypic differences between mice and rats in this model. Further studies should explore the mechanisms underlying this species difference.
... NBQX, GYKI-52466 and MK 801 all readily penetrate into the CNS when administered peripherally (Sheardown et al., 1990; Smith et al., 1991; Vizi et al., 1996; Wong et al., 1986). Previous studies have shown that NBQX, GYKI-52466 and MK 801 block seizures in various animal seizure models (Barton et al., 2003; Chapman et al., 1991; Loscher and Honack, 1994; Twele et al., 2015). We determined the effects of these antagonists on acute seizure development in the Theiler's murine encephalomyelitis virus (TMEV)-induced seizure model, an infection-driven animal model for epilepsy [reviewed in (Libbey and Fujinami, 2011)]. ...
... †p b 0.05, chi-square test. adult mouse model (KA injection into the CA1 area of the hippocampus of FVB/N mice) of medically resistant mesial temporal lobe epilepsy (mTLE) (Twele et al., 2015). Protection following ischemia may be through both the direct action of NBQX and its induction of prolonged brain hypothermia (Lees, 2000 ). ...
... Protection following ischemia may be through both the direct action of NBQX and its induction of prolonged brain hypothermia (Lees, 2000 ). In addition, NBQX exerted antiseizure but not antiepileptogenic effects, when administered after KAinduced SE, in this mouse model of mTLE (Twele et al., 2015). Using the TMEV-induced seizure model, surprisingly we found that NBQX significantly increased the number of mice experiencing seizures compared to control (Fig. 1) and did not protect hippocampal CA1 neurons against damage (Fig. 4). ...
... The intrahippocampal kainate mouse model was used to evaluate the antiseizure effects of competitive (NBQX) and noncompetitive (perampanel) AMPA receptor antagonists in resistant focal seizures. 47,48 Whereas phenytoin did not block the spontaneous seizures that developed following kainate administration, both NBQX and perampanel suppressed the seizures, suggesting efficacy in treating resistant focal seizures. In a separate experiment, administration of NBQX for 3 days following kainate administration did not produce an antiseizure effect. ...
... In a separate experiment, administration of NBQX for 3 days following kainate administration did not produce an antiseizure effect. 48 Perampanel was not evaluated with a comparable treatment protocol. ...
This article reviews the profile of perampanel, a novel noncompetitive α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA) receptor antagonist, and its role as a potential broad‐spectrum antiepileptic drug in the treatment of epilepsy. For this narrative review, data were collected using specified search criteria. Articles reporting the evidence for perampanel's efficacy from preclinical models, phase 3 clinical studies, observational studies, and descriptive evidence were included. AMPA receptors play a key role in mediating the action of glutamate at the excitatory synapse. Preclinical research showed the AMPA receptor blockade to constitute a promising target for antiepileptic drug therapy. In animal models, perampanel proved to be protective against seizures and reduce seizure severity and duration. Four phase‐3 randomized controlled trials (3 in patients with focal seizures and one in primary generalized tonic–clonic seizures in idiopathic generalized epilepsy) have been completed. In focal (partial) onset seizures, perampanel (4, 8, and 12 mg) significantly reduced seizure frequency per 28 days (23.3%‐28.8% vs 12.8%; P < .01) and responder rates (≥50% reduction in seizures) (28.5%‐35.3% vs 19.3%; P < .05) compared with placebo. In primary generalized tonic–clonic seizures, perampanel 8 mg resulted in greater reduction in seizure frequency per 28 days (−76.5% vs −38.4%; P < .0001) and responder rate (64.2% vs 39.5%; P = .0019) than placebo. The efficacy, safety, and tolerability of perampanel have been reproduced in real‐world clinical practice, and the agent has been shown to be effective in other epilepsy syndromes. Perampanel is a potentially broad‐spectrum antiepileptic drug with a novel mechanism of action that may be a useful addition for patients with epilepsy with various seizure types. The availability of novel antiepileptic drugs for epilepsy treatment enables more individualized treatment for these patients.
... AMPA receptors regulate the fast synaptic excitation in brain regions that are related to epilepsy [7]. Specifically, AMPA receptor antagonists markedly reduce epileptiform and inhibit spread of epileptic discharges in both animal seizure models and human epilepsy [8,9]. In addition, growing evidence validate the critical role of AMPA receptors in epileptic seizures, and suggest that AMPA receptors may be a potential target for epilepsy therapy. ...
... Moreover, clinical data showed that perampanel with daily dose is effective in patients with refractory partial-onset seizures [12][13][14]. NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline-2,3-dione) is a competitive AMPA receptor antagonist and has a property that suppressed focal electrographic seizures in epileptic mice [9]. Recently, it was described that NBQX blocks the development of spontaneous recurrent seizures when treatment after neonatal seizures [15]. ...
Epilepsy is a serious brain disorder with diverse seizure types and epileptic syndromes. AMPA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline-2,3-dione (NBQX) attenuates spontaneous recurrent seizures in rats. However, the anti-epileptic effect of NBQX in chronic epilepsy model is poorly understood. Perineuronal nets (PNNs), specialized extracellular matrix structures, surround parvalbumin-positive inhibitory interneurons, and play a critical role in neuronal cell development and synaptic plasticity. Here, we focused on the potential involvement of PNNs in the treatment of epilepsy by NBQX. Rats were intraperitoneally (i.p.) injected with pentylenetetrazole (PTZ, 50 mg/kg) for 28 consecutive days to establish chronic epilepsy models. Subsequently, NBQX (20 mg/kg, i.p.) was injected for 3 days for the observation of behavioral measurements of epilepsy. The Wisteria floribundi agglutinin (WFA)-labeled PNNs were measured by immunohistochemical staining to evaluate the PNNs. The levels of three components of PNNs such as tenascin-R, aggrecan and neurocan were assayed by Western blot assay. The results showed that there are reduction of PNNs and decrease of tenascin-R, aggrecan and neurocan in the medial prefrontal cortex (mPFC) in the rats injected with PTZ. However, NBQX treatment normalized PNNs, tenascin-R, aggrecan and neurocan levels. NBQX was sufficient to decrease seizures through increasing the latency to seizures, decrease the duration of seizure onset, and reduce the scores for the severity of seizures. Furthermore, the degradation of mPFC PNNs by chondroitinase ABC (ChABC) exacerbated seizures in PTZ-treated rats. Finally, the anti-epileptic effect of NBQX was reversed by pretreatment with ChABC into mPFC. These findings revealed that PNNs degradation in mPFC is involved in the pathophysiology of epilepsy and enhancement of PNNs may be effective for the treatment of epilepsy.
... The anticonvulsant effects of LY3130481 were abolished in γ-8 −/− mice, whereas the anticonvulsant effects of GYKI52466 and perampanel remained (Fig. 4e). Perampanel (1 and 2 mg/kg) induced tremor, ataxia, running, bouncing and tonic-clonic forelimb seizures with falling in mice with kainate-induced mesial temporal lobe epilepsy, effects that have not been observed in non-epileptic mice 18 . Thus, we conducted two experiments to evaluate the effects of LY3130481 on mice with epilepsy. ...
... The MTLE mouse is induced by an initial neurotoxic event, a unilateral intrahippocampal injection of kainic acid (KA) into the dorsal hippocampus, which induces nonconvulsive s.e.m. lasting several hours. This initial event is followed by a latent phase. 2 to 3 weeks after KA injection, spontaneous recurrent hippocampal paroxysmal discharges (HPD) are recorded in the epileptic hippocampus and remain stable and stereotyped for the whole life of the animal 18,31 . These HPDs occur spontaneously about 30-60 times per hour when the animals are in a state of quiet wakefulness, generally last 15-20 s and are associated with behavioral arrest and/or mild motor automatisms. ...
Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.
... Moreover, NMDA blockers may exacerbate seizures in humans (Sveinbjornsdottir et al. 1993;Kasteleijn-Nolst Trenité et al. 2015). Pharmacological blockade of AMPA receptors has broad spectrum antiseizure activity in in vitro and animal seizure and epilepsy models (Yamaguchi et al. 1993;Twele et al. 2015). In neocortical and hippocampal tissue removed from patients with focal epilepsy, there is evidence that AMPA receptor density is increased (Mathern et al. 1998;Zilles et al. 1999;Eid et al. 2002). ...
... Adverse central nervous system effects, such as dizziness, irritability, and somnolence, are common, particularly at higher doses, emphasizing the importance of AMPA receptors in brain function. Although AMPA receptor antagonists have powerful antiseizure effects, they are not antiepileptogenic in animal models Twele et al. 2015). ...
Antiseizure drugs (ASDs), also termed antiepileptic drugs, are the main form of symptomatic treatment for people with epilepsy, but not all patients become free of seizures. The ketogenic diet is one treatment option for drug-resistant patients. Both types of therapy exert their clinical effects through interactions with one or more of a diverse set of molecular targets in the brain. ASDs act by modulation of voltage-gated ion channels, including sodium, calcium, and potassium channels; by enhancement of γ-aminobutyric acid (GABA)-mediated inhibition through effects on GABAA receptors, the GABA transporter 1 (GAT1) GABA uptake transporter, or GABA transaminase; through interactions with elements of the synaptic release machinery, including synaptic vesicle 2A (SV2A) and α2δ; or by blockade of ionotropic glutamate receptors, including α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. The ketogenic diet leads to increases in circulating ketones, which may contribute to the efficacy in treating pharmacoresistant seizures. Production in the brain of inhibitory mediators, such as adenosine, or ion channel modulators, such as polyunsaturated fatty acids, may also play a role. Metabolic effects, including diversion from glycolysis, are a further postulated mechanism. For some ASDs and the ketogenic diet, effects on multiple targets may contribute to activity. Better understanding of the ketogenic diet will inform the development of improved drug therapies to treat refractory seizures.
... The NMDA receptor antagonist ketamine was excluded from any combination because we recently found that it induces hyperlocomotion and severe running and bouncing in epileptic mice from the intrahippocampal kainate model (Twele et al., 2015). ...
... Multitargeted drug combinations that were found sufficiently tolerable by this approach will next be tested for antiepileptogenic potential in the intrahippocampal kainate mouse model of mesial TLE (Fig. 1). For this purpose, the combinations will be administered 2-3 times daily over 3-5 days, using a design that was recently described for the AMPA receptor antagonist NBQX (Twele et al., 2015). Rationally chosen drug combinations used for our approach are of course not limited to the drugs used in this study but may involve compounds with additional mechanisms to target the complex network involved in epileptogenesis . ...
Prevention of symptomatic epilepsy ("antiepileptogenesis") in patients at risk is a major unmet clinical need. Several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. Similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. In the majority of studies, drugs were given as monotherapy. However, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. We recently proposed multitargeted approaches ("network pharmacology") to interfere with epileptogenesis. One strategy, which, if effective, would allow a relatively rapid translation into the clinic, is developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis. In order to test this strategy preclinically, we developed an algorithm for testing such drug combinations, which was inspired by the established drug development phases in humans. As a first step of this algorithm, tolerability of four rationally chosen, repeatedly administered drug combinations was evaluated by a large test battery in mice: A, levetiracetam and phenobarbital; B, valproate, losartan, and memantine; C, levetiracetam and topiramate; and D, levetiracetam, parecoxib, and anakinra. As in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful translation of preclinical findings to the clinic. Except combination B, all drug cocktails were relatively well tolerated. Based on previous studies, we expected that tolerability would be lower in the latent and chronic phases following status epilepticus in mice, but, except combinations C and D, no significant differences were determined between nonepileptic and post-status epilepticus animals. As a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy.
... The anti-seizure effect of PER was short-lasting. ED50 of 0.7 mg/kg i.p [79] RTG Amygdala kindling RTG increased the threshold for induction of afterdischarges, exerting significant effects already after 0.01 mg/kg. At higher doses (2.5 --5 mg/kg i.p.; 10 --15 mg/kg p.o.), RTG exerted anticonvulsant effects on seizure severity, seizure duration, total duration of behavioral changes and afterdischarge duration [89] Hippocampal kindling RTG was able to raise ADT, reduce duration, and reduce behavioral severity and duration in rats at postnatal days 14 (2.5 mg/kg, i.p.), 21 (5 mg/kg, i.p.), and 35 (5 mg/kg, i.p.). ...
... Another recent study has investigated the effect of PER in a mouse model of MTLE induced by intrahippocampal injection of kainate. PER was tested at three doses, 0.5, 1.0 and 2.0 mg/kg i.p. showing that PER dosedependently reduced the frequency of the number of seizure like events by 39% (0.5mg/kg), 55% (1.0 mg/kg) and 96% (2.0 mg/kg) over the first 30 min after injection (Table 1) [79]. The preclinical efficacy of PER and also other non-competitive AMPA antagonists support the possibility that drugs acting through this mechanism of action including PER might be an option for the treatment of MTLE with or without HS [70,74]. ...
Introduction:
Mesial temporal lobe epilepsy associated with hippocampal sclerosis (MTLE-HS) is a syndrome that is often refractory to drug treatment. The effects on specific syndromes are not currently available from the pre-marketing clinical development of new AEDs; this does not allow the prediction of whether new drugs will be more effective in the treatment of some patients. Areas covered: We have reviewed all the existing literature relevant to the understanding of a potential effectiveness in MTLE-HS patients for the latest AEDs, namely brivaracetam, eslicarbazepine, lacosamide, perampanel and retigabine also including the most relevant clinical data and a brief description of their pharmacological profile. Records were identified using predefined search criteria using electronic databases (e.g., PubMed, Cochrane Library Database of Systematic Reviews). Primary peer-reviewed articles published up to the 15 June 2015 were included. Expert opinion: All the drugs considered have the potential to be effective in the treatment of MTLE-HS; in fact, they possess proven efficacy in animal models; currently considered valuable tools for predicting drug efficacy in TLE. Furthermore, for some of these (e.g., lacosamide and eslicarbazepine) data are already available from post-marketing studies while brivaracetam acting on SV2A like levetiracetam might have the same potential effectiveness with the possibility to be more efficacious considering its ability to inhibit voltage gated sodium channels; finally, perampanel and retigabine are very effective drugs in animal models of TLE.
... The anti-seizure effect of PER was short-lasting. ED50 of 0.7 mg/kg i.p [79] RTG Amygdala kindling RTG increased the threshold for induction of afterdischarges, exerting significant effects already after 0.01 mg/kg. At higher doses (2.5 --5 mg/kg i.p.; 10 --15 mg/kg p.o.), RTG exerted anticonvulsant effects on seizure severity, seizure duration, total duration of behavioral changes and afterdischarge duration [89] Hippocampal kindling RTG was able to raise ADT, reduce duration, and reduce behavioral severity and duration in rats at postnatal days 14 (2.5 mg/kg, i.p.), 21 (5 mg/kg, i.p.), and 35 (5 mg/kg, i.p.). ...
... Another recent study has investigated the effect of PER in a mouse model of MTLE induced by intrahippocampal injection of kainate. PER was tested at three doses, 0.5, 1.0 and 2.0 mg/kg i.p. showing that PER dosedependently reduced the frequency of the number of seizure like events by 39% (0.5mg/kg), 55% (1.0 mg/kg) and 96% (2.0 mg/kg) over the first 30 min after injection (Table 1) [79]. The preclinical efficacy of PER and also other non-competitive AMPA antagonists support the possibility that drugs acting through this mechanism of action including PER might be an option for the treatment of MTLE with or without HS [70,74]. ...
... Studies have shown that the usage of NBQX, a competitive antagonist that targets AMPARs, can prevent the development of hypoxia-induced spontaneous recurrent seizures in neonatal rats [72]. Additionally, it has been found to suppress focal electrographic seizures in mice with KA-induced epilepsy [73] and to reduce the onset of seizures induced by pentylenetetrazole (PTZ) in adult rats [74]. Similar changes in the subunit composition of AMPARs observed in animal models have been found in individuals with epilepsy. ...
Epilepsy is one of the common neurological diseases that affects not only adults but also infants and children. Because epilepsy has been studied for a long time, there are several pharmacologically effective anticonvulsants, which, however, are not suitable as therapy for all patients. The genesis of epilepsy has been extensively investigated in terms of its occurrence after injury and as a concomitant disease with various brain diseases, such as tumors, ischemic events, etc. However, in the last decades, there are multiple reports that both genetic and epigenetic factors play an important role in epileptogenesis. Therefore, there is a need for further identification of genes and loci that can be associated with higher susceptibility to epileptic seizures. Use of mouse knockout models of epileptogenesis is very informative, but it has its limitations. One of them is due to the fact that complete deletion of a gene is not, in many cases, similar to human epilepsy-associated syndromes. Another approach to generating mouse models of epilepsy is N-Ethyl-N-nitrosourea (ENU)-directed mutagenesis. Recently, using this approach, we generated a novel mouse strain, soc (socrates, formerly s8-3), with epileptiform activity. Using molecular biology methods, calcium neuroimaging, and immunocytochemistry, we were able to characterize the strain. Neurons isolated from soc mutant brains retain the ability to differentiate in vitro and form a network. However, soc mutant neurons are characterized by increased spontaneous excitation activity. They also demonstrate a high degree of Ca2+ activity compared to WT neurons. Additionally, they show increased expression of NMDA receptors, decreased expression of the Ca2+-conducting GluA2 subunit of AMPA receptors, suppressed expression of phosphoinositol 3-kinase, and BK channels of the cytoplasmic membrane involved in protection against epileptogenesis. During embryonic and postnatal development, the expression of several genes encoding ion channels is downregulated in vivo, as well. Our data indicate that soc mutation causes a disruption of the excitation–inhibition balance in the brain, and it can serve as a mouse model of epilepsy.
... Intrahippocampal kainic acid administration in mice causes dentate granule (DG) cell dispersion and CA1 cell death in the hippocampus ipsilateral to the injection site (43)(44)(45)(46)(47). To further validate the model and determine whether antagomir treatment affected IHpKa-associated morphological changes, we assessed Frontiers in Neurology 09 frontiersin.org ...
Background
Acquired epilepsies are caused by an initial brain insult that is followed by epileptogenesis and finally the development of spontaneous recurrent seizures. The mechanisms underlying epileptogenesis are not fully understood. MicroRNAs regulate mRNA translation and stability and are frequently implicated in epilepsy. For example, antagonism of a specific microRNA, miR-324-5p, before brain insult and in a model of chronic epilepsy decreases seizure susceptibility and frequency, respectively. Here, we tested whether antagonism of miR-324-5p during epileptogenesis inhibits the development of epilepsy.
Methods
We used the intrahippocampal kainic acid (IHpKa) model to initiate epileptogenesis in male wild type C57BL/6 J mice aged 6–8 weeks. Twenty-four hours after IHpKa, we administered a miR-324-5p or scrambled control antagomir intracerebroventricularly and implanted cortical surface electrodes for EEG monitoring. EEG data was collected for 28 days and analyzed for seizure frequency and duration, interictal spike activity, and EEG power. Brains were collected for histological analysis.
Results
Histological analysis of brain tissue showed that IHpKa caused characteristic hippocampal damage in most mice regardless of treatment. Antagomir treatment did not affect latency to, frequency, or duration of spontaneous recurrent seizures or interictal spike activity but did alter the temporal development of frequency band-specific EEG power.
Conclusion
These results suggest that miR-324-5p inhibition during epileptogenesis induced by status epilepticus does not convey anti-epileptogenic effects despite having subtle effects on EEG frequency bands. Our results highlight the importance of timing of intervention across epilepsy development and suggest that miR-324-5p may act primarily as a proconvulsant rather than a pro-epileptogenic regulator.
... Of them, antioxidants, antagonists of ionotropic excitatory amino acid, NMDA, AMPA and kainate receptors and inhibitors of various cell death signaling pathways have been most extensively studied in animal models of epileptogenesis. It was found that glutamate receptor antagonists suppressed seizures but not epileptogenesis in animal models [23]. Substances which act through more than one molecular mechanism could be more efficient in halting the development of the pathological cascade, which leads to seizures. ...
The pharmacological treatment of epilepsy is purely symptomatic. Despite many decades of intensive research, causal treatment of this common neurologic disorder is still unavailable. Nevertheless, it is expected that advances in modern neuroscience and molecular biology tools, as well as improved animal models may accelerate designing antiepileptogenic and epilepsy-modifying drugs. Epileptogenesis triggers a vast array of genomic, epigenomic and transcriptomic changes, which ultimately lead to morphological and functional transformation of specific neuronal circuits resulting in the occurrence of spontaneous convulsive or nonconvulsive seizures. Recent decades unraveled molecular processes and biochemical signaling pathways involved in the proepileptic transformation of brain circuits including oxidative stress, apoptosis, neuroinflammatory and neurotrophic factors. The “omics” data derived from both human and animal epileptic tissues, as well as electrophysiological, imaging and neurochemical analysis identified a plethora of possible molecular targets for drugs, which could interfere with various stages of epileptogenetic cascade, including inflammatory processes and neuroplastic changes. In this narrative review, we briefly present contemporary views on the neurobiological background of epileptogenesis and discuss the advantages and disadvantages of some more promising molecular targets for antiepileptogenic pharmacotherapy.
... Among them, NBQX was the first compound to exhibit a high selectivity for AMPAR versus NMDAR (Table 1) and has been used as the referential antagonist of choice in numerous in vitro and in vivo models. The neuroprotective, anticonvulsant, anxiolytic, and antinociceptive effects of NBQX have been widely investigated and reported [69,[73][74][75][76][77][78][79][80][81]. Neuroprotective properties of NBQX in ischemic stroke were confirmed in clinical studies; however, due to the low aqueous solubility and associated nephrotoxicity at therapeutic doses, it was rejected in the second phase of clinical studies [82]. ...
Since the 1990s, ionotropic glutamate receptors have served as an outstanding target for drug discovery research aimed at the discovery of new neurotherapeutic agents. With the recent approval of perampanel, the first marketed non-competitive antagonist of AMPA receptors, particular interest has been directed toward ‘non-NMDA’ (AMPA and kainate) receptor inhibitors. Although the role of AMPA receptors in the development of neurological or psychiatric disorders has been well recognized and characterized, progress in understanding the function of kainate receptors (KARs) has been hampered, mainly due to the lack of specific and selective pharmacological tools. The latest findings in the biology of KA receptors indicate that they are involved in neurophysiological activity and play an important role in both health and disease, including conditions such as anxiety, schizophrenia, epilepsy, neuropathic pain, and migraine. Therefore, we reviewed recent advances in the field of competitive and non-competitive kainate receptor antagonists and their potential therapeutic applications. Due to the high level of structural divergence among the compounds described here, we decided to divide them into seven groups according to their overall structure, presenting a total of 72 active compounds.
... Some ASMs that are thought to act predominantly at one target are shown for comparison. Data are from Gladding et al. (1985); Löscher (1980), Löscher et al. (1986); Löscher and Nolting (1991), Löscher and Hönack (1993); Dalby and Nielsen (1997); Otsuki et al. (1998), Barton et al. (2001), Riban et al. (2002); Stöhr et al. (2007); Hanada et al. (2011, Twele et al. (2015); Duveau et al. (2016); Klitgaard et al. (2016); Wu et al. (2019), Leclercq et al. (2020), Löscher et al. (2021) 2014). Furthermore, specific genetic animal models for pediatric genetic epilepsies, such as Lennox-Gastaut syndrome, infantile spasms (West syndrome), Dravet syndrome, and tuberous sclerosis complex (TSC) can be used to discover novel ASMs for the difficult-to-treat seizures in these syndromes (Demarest and Brooks-Kayal, 2018). ...
Rationally designed multi-target drugs (also termed multimodal drugs, network therapeutics, or designed multiple ligands) have emerged as an attractive drug discovery paradigm in the last 10–20 years, as potential therapeutic solutions for diseases of complex etiology and diseases with significant drug-resistance problems. Such agents that modulate multiple targets simultaneously are developed with the aim of enhancing efficacy or improving safety relative to drugs that address only a single target or to combinations of single-target drugs. Although this strategy has been proposed for epilepsy therapy >25 years ago, to my knowledge, only one antiseizure medication (ASM), padsevonil, has been intentionally developed as a single molecular entity that could target two different mechanisms. This novel drug exhibited promising effects in numerous preclinical models of difficult-to-treat seizures. However, in a recent randomized placebo-controlled phase IIb add-on trial in treatment-resistant focal epilepsy patients, padsevonil did not separate from placebo in its primary endpoints. At about the same time, a novel ASM, cenobamate, exhibited efficacy in several randomized controlled trials in such patients that far surpassed the efficacy of any other of the newer ASMs. Yet, cenobamate was discovered purely by phenotype-based screening and its presumed dual mechanism of action was only described recently. In this review, I will survey the efficacy of single-target vs. multi-target drugs vs. combinations of drugs with multiple targets in the treatment and prevention of epilepsy. Most clinically approved ASMs already act at multiple targets, but it will be important to identify and validate new target combinations that are more effective in drug-resistant epilepsy and eventually may prevent the development or progression of epilepsy.
... mg,p.o.); it is effective against audiogenic seizures in DBA/2 mice; and it has activity in the maximal electroshock (MES) and 6 Hz tests, as well as in kindled seizures (Hanada et al. 2011). Perampanel also reduces the frequency of focal electrographic seizures in mice that exhibit spontaneous recurrent seizures following intrahippocampal injection of kainic acid (Twele et al. 2015). Finally, perampanel suppresses behavioral and electrographic seizures in the rat lithium-pilocarpine (Hanada et al. 2014) and diisopropylfluorophosphate (Dhir et al. 2020) models of status epilepticus. ...
Tetramethylenedisulfotetramine (TETS), a noncompetitive GABA A receptor antagonist, is a potent, highly lethal convulsant that is considered to be a chemical threat agent. Here, we assessed the ability of the AMPA receptor antagonist perampanel to protect against TETS-induced seizures and lethality in mice when administered before or after treatment with the toxicant. For comparison, we conducted parallel testing with diazepam, which is a first-line treatment for chemically induced seizures in humans. Pre-treatment of mice with either perampanel (1–4 mg/kg, i.p.) or diazepam (1–5 mg/kg, i.p.) conferred protection in a dose-dependent fashion against tonic seizures and lethality following a dose of TETS (0.2 mg/kg, i.p.) that rapidly induces seizures and death. The ED 50 values for protection against mortality were 1.6 mg/kg for perampanel and 2.1 mg/kg for diazepam. Clonic seizures were unaffected by perampanel and only prevented in a minority of animals by high-dose diazepam. Neither treatment prevented myoclonic body twitches. Perampanel and diazepam also conferred protection against tonic seizures and lethality when administered 15 min following a 0.14 mg/kg, i.p., dose of TETS and 5 min following a 0.2 mg/kg, i.p., dose of TETS. Both posttreatments were highly potent at reducing tonic seizures and lethality in animals exposed to the lower dose of TETS whereas greater doses of both treatments were required in animals exposed to the larger dose of TETS. Neither treatment was as effective suppressing clonic seizures. In an experiment where 0.4 mg/kg TETS was administered by oral gavage and the treatment drugs were administered 5 min later, perampanel only partially protected against lethality whereas diazepam produced nearly complete protection. We conclude that perampanel and diazepam protect against TETS-induced tonic seizures and lethality but have less impact on clonic seizures. Both drugs could have utility in the treatment of TETS intoxication but neither eliminates all seizure activity.
... Several previous studies indicated that epilepsy is difficult to prevent or modify in the intrahippocampal kainate model, including studies with glutamate receptor antagonists (Twele et al., 2015;Schidlitzki et al., 2017), mTOR antagonists (Shima et al., 2015;Gericke et al., 2020), an inhibitor of adenosine kinase (Sandau et al., 2019), genetically engineered cells (Ali et al., 2017), and genetic manipulation of urokinasetype plasminogen activator receptor (Ndode-Ekane and Pitkänen, 2013) or BDNF-mediated TrkB signaling . This may be related to the double-hit insult produced in this model by the traumatic insult caused by surgical implantation of the EEG electrode into the hippocampus and the intrahippocampal injection of the excitotoxic kainate (Brackhan et al., 2018). ...
Epileptogenesis, the gradual process that leads to epilepsy after brain injury or genetic mutations, is a complex network phenomenon, involving a variety of morphological, biochemical and functional brain alterations. Although risk factors for developing epilepsy are known, there is currently no treatment available to prevent epilepsy. We recently proposed a multitargeted, network-based approach to prevent epileptogenesis by rationally combining clinically available drugs and provided first proof-of-concept that this strategy is effective. Here we evaluated eight novel rationally chosen combinations of 14 drugs with mechanisms that target different epileptogenic processes. The combinations consisted of 2–4 different drugs per combination and were administered systemically over 5 days during the latent epileptogenic period in the intrahippocampal kainate mouse model of acquired temporal lobe epilepsy, starting 6 h after kainate. Doses and dosing intervals were based on previous pharmacokinetic and tolerability studies in mice. The incidence and frequency of spontaneous electrographic and electroclinical seizures were recorded by continuous (24/7) video linked EEG monitoring done for seven days at 4 and 12 weeks post-kainate, i.e., long after termination of drug treatment. Compared to vehicle controls, the most effective drug combination consisted of low doses of levetiracetam, atorvastatin and ceftriaxone, which markedly reduced the incidence of electrographic seizures (by 60%; p < 0.05) and electroclinical seizures (by 100%; p < 0.05) recorded at 12 weeks after kainate. This effect was lost when higher doses of the three drugs were administered, indicating a synergistic drug-drug interaction at the low doses. The potential mechanisms underlying this interaction are discussed. We have discovered a promising novel multitargeted combination treatment for modifying the development of acquired epilepsy.
... Perampanel is an anticonvulsant with a unique pharmacological profile, namely, it is a non-competitive antagonist of AMPA receptors (Hanada et al., 2011;French et al., 2012;Krauss et al., 2012;Potschka & Trinka, 2019). Perampanel has been demonstrated to inhibit the AMPA receptor-mediated current in single neurons (Barygin, 2016;Ceolin et al., 2012;Chen, Matt, Hell, & Rogawski, 2014), reduce in vitro epileptiform discharges with extracellular recording in human neocortical slices (Augustin et al., 2018) and decrease seizure behaviours in experimental models (Citraro et al., 2017;Hanada et al., 2011;Russmann, Salvamoser, Rettenbeck, Komori, & Potschka, 2016;Twele, Bankstahl, Klein, Römermann, & Löscher, 2015;Wu, Nagaya, & Hanada, 2014). Although the molecular action of perampanel on AMPA receptors and the ameliorating effect of perampanel on seizure discharges or behaviours have been separately documented and epileptiform discharges can be inhibited by the other AMPA antagonists (Avoli et al., 1996;Gean, 1990), it remains elusive how the anti-epileptic effect of perampanel is achieved by inhibition of AMPAergic transmission at the neural circuit level. ...
Background and purpose:
Perampanel is a newly-approved anticonvulsant uniquely targeting AMPA receptors, the most abundant excitatory synapses in the brain. However, the network mechanisms underlying the antiepileptic effect of the AMPAergic antagonist remain to be explored.
Experimental approach:
The mechanisms of perampanel action were studied with the basolateral amygdalar (BLA) network containing pyramidal-inhibitory neuronal (PN-IN) resonators in seizure models of 4-aminopyridine (4-AP) and electrical kindling.
Key results:
Application of either 4-AP or electrical kindling to BLA readily induces AMPAergic transmission-dependent reverberating activities between PN-IN resonators, which are chiefly characterized by burst discharges in INs and corresponding recurrent inhibitory postsynaptic potentials in PNs. Perampanel reduces post-kindling "paroxysmal depolarization shift" especially in PNs and, counterintuitively, eliminates burst activities in inhibitory neurons and inhibitory synaptic inputs onto excitatory PNs to result in prevention of epileptiform discharges and seizure behaviors. Intriguingly, similar effects can be obtained with not only the AMPAergic antagonist CNQX but also the GABAergic antagonist bicuculline which is in general considered as a proconvulsant.
Conclusion and implications:
Ictogenesis depends on AMPAergic recruitment of coordinated PN-IN network oscillations tuned by dynamic glutamatergic and GABAergic transmission. The anticonvulsant effect of perampanel stems from disruption of coordinating network activities rather than simply decreased neuronal excitability or excitatory transmission. Positive or negative modulation of epileptic reverberations may be pro- or anti-ictogenic, respectively, constituting a more applicable rationale for the therapy against seizures.
... By contrast, CLB, CLZ, PER, and GBP have not been previously evaluated for their effects against post-SE spontaneous seizures, although a comparable dose of PER was effective in reducing electrographic seizures in a mouse model of TLE. 36 LEV has previously been shown to reduce spontaneous seizures following pilocarpine-induced 37 or electrically induced 38 SE, but this effect can decline quickly after treatment is initiated. 38 TPM has previously been shown to dose-dependently inhibit spontaneous seizures. ...
Objective
Approximately 30% of patients with epilepsy do not experience full seizure control on their antiseizure drug (ASD) regimen. Historically, screening for novel ASDs has relied on evaluating efficacy following a single administration of a test compound in either acute electrical or chemical seizure induction. However, the use of animal models of spontaneous seizures and repeated administration of test compounds may better differentiate novel compounds. Therefore, this approach has been instituted as part of the National Institute of Neurological Disorders and Stroke Epilepsy Therapy Screening Program screening paradigm for pharmacoresistant epilepsy.
Methods
Rats were treated with intraperitoneal kainic acid to induce status epilepticus and subsequent spontaneous recurrent seizures. After 12 weeks, rats were enrolled in drug screening studies. Using a 2‐week crossover design, selected ASDs were evaluated for their ability to protect against spontaneous seizures, using a video‐electroencephalographic monitoring system and automated seizure detection. Sixteen clinically available compounds were administered at maximally tolerated doses in this model. Dose intervals (1‐3 treatments/d) were selected based on known half‐lives for each compound.
Results
Carbamazepine (90 mg/kg/d), phenobarbital (30 mg/kg/d), and ezogabine (15 mg/kg/d) significantly reduced seizure burden at the doses evaluated. In addition, a dose‐response study of topiramate (20‐600 mg/kg/d) demonstrated that this compound reduced seizure burden at both therapeutic and supratherapeutic doses. However, none of the 16 ASDs conferred complete seizure freedom during the testing period at the doses tested.
Significance
Despite reductions in seizure burden, the lack of full seizure freedom for any ASD tested suggests that this screening paradigm may be useful for testing novel compounds with potential utility in pharmacoresistant epilepsy.
... 134,135 Perampanel has shown to reduce neuronal cell death in the hippocampus and the piriform cortex in the lithium-pilocarpine post-SE model, 136 but does not have anti-epileptogenic properties. 137 Similar neuroprotective effects have been described with other AMPA antagonists. 138 ...
There is a growing body of clinical and experimental evidence that neurodegenerative diseases and epileptogenesis after an acquired brain insult, may share common etiological mechanisms. Acquired epilepsy commonly develops as a comorbid condition in patients with neurodegenerative diseases such as Alzheimer's Disease, although it is likely much under diagnosed in practice. Progressive neurodegeneration has also been described after traumatic brain injury, stroke and other forms of brain insults. Moreover, recent evidence has shown that acquired epilepsy is often a progressive disorder that is associated with the development of drug resistance, cognitive decline and worsening of other neuropsychiatric comorbidities. Therefore, new pharmacological therapies that target neurobiological pathways which underpin neurodegenerative diseases have potential to have both an antiepileptogenic or disease modifying effect on the seizures in patients with acquired epilepsy, and also mitigate the progressive neurocognitive and neuropsychiatric comorbidities. Here, we review the neurodegenerative pathways that are plausible targets for the development of novel therapies that could prevent the development or modify the progression of acquired epilepsy, and the supporting published experimental and clinical evidence.
... 5,20 Thus, onset of pharmacological treatment several hours after IHK injection, as typically done in studies evaluating drugs for antiepileptogenic efficacy, may be too late to prevent hippocampal damage and epileptogenesis. This problem is illustrated by our recent experiments with N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists, which did not prevent neuronal damage and epileptogenesis in the IHK mouse model when treatment was initiated 6-8 hours after kainate, 37,38 whereas NMDA and AMPA receptor antagonists prevent kainateinduced excitotoxic cell death when administered before or shortly after the convulsant. 34,39 With respect to testing ASDs, the present study indicates that the IAK model is an interesting approach to evaluating the response of electroclinical seizures to ASDs, but such experiments are laborious, because prolonged continuous (24/7) video-EEG recording is needed, which is a disadvantage compared to models with more frequent focal SRSs, such as the IHK model (Figure 4), or models with induced seizures, such as the 6-Hz mouse model of partial seizures. ...
Objective:
Intracranial (intrahippocampal or intra-amygdala) administration of kainate in rodents leads to spatially restricted brain injury and development of focal epilepsy with characteristics that resemble mesial temporal lobe epilepsy. Such rodent models are used both in the search for more effective antiseizure drugs (ASDs) and in the development of antiepileptogenic strategies. However, it is not clear which of the models is best suited for testing different types of epilepsy therapies.
Methods:
In the present study, we performed a face-to-face comparison of the intra-amygdala kainate (IAK) and intrahippocampal kainate (IHK) mouse models using the same mouse inbred strain (C57BL/6). For comparison, some experiments were performed in mouse outbred strains.
Results:
Intra-amygdala kainate injection led to more severe status epilepticus and higher mortality than intrahippocampal injection. In male C57BL/6 mice, the latent period to spontaneous recurrent seizures (SRSs) was short or absent in both models, whereas a significantly longer latent period was determined in NMRI and CD-1 outbred mice. When SRSs were recorded from the ipsilateral hippocampus, relatively frequent electroclinical seizures were determined in the IAK model, whereas only infrequent electroclinical seizures but extremely frequent focal electrographic seizures were determined in the IHK model. As a consequence of the differences in SRS frequency, prolonged video-electroencephalographic monitoring and drug administration were needed for testing efficacy of the benchmark ASD carbamazepine in the IAK model, whereas acute drug testing was possible in the IHK model. In both models, carbamazepine was only effective at high doses, indicating ASD resistance to this benchmark drug.
Significance:
We found a variety of significant differences between the IAK and IHK models, which are important when deciding which of these models is best suited for studies on novel epilepsy therapies. The IAK model appears particularly interesting for studies on disease-modifying treatments, whereas the IHK model is well suited for studying the antiseizure activity of novel ASDs against difficult-to-treated focal seizures.
... In the present study, treatment with either drug alone, starting 6 h after SE, did not significantly modify epileptogenesis in the intrahippocampal kainate mouse model. Indeed, several previous studies indicated that epilepsy is difficult to prevent or modify in this model, including studies with glutamate receptor antagonists (Twele et al., 2015;Schidlitzki et al., 2017), the mTOR antagonist rapamycin (Shima et al., 2015), genetically engineered cells (Ali et al., 2017), and genetic manipulation of urokinase-type plasminogen activator receptor (Ndode-Ekane and or BDNF-mediated TrkB signaling . This may be related to the double-hit insult produced in this model by the traumatic insult caused by surgical implantation of the EEG electrode into the hippocampus and the intrahippocampal injection of the excitotoxic kainate (Brackhan et al., 2018). ...
Epilepsy is a complex network phenomenon that, as yet, cannot be prevented or cured. We recently proposed network-based approaches to prevent epileptogenesis. For proof of concept we combined two drugs (levetiracetam and topiramate) for which in silico analysis of drug-protein interaction networks indicated a synergistic effect on a large functional network of epilepsy-relevant proteins. Using the intrahippocampal kainate mouse model of temporal lobe epilepsy, the drug combination was administered during the latent period before onset of spontaneous recurrent seizures (SRS). When SRS were periodically recorded by video-EEG monitoring after termination of treatment, a significant decrease in incidence and frequency of SRS was determined, indicating antiepileptogenic efficacy. Such efficacy was not observed following single drug treatment. Furthermore, a combination of levetiracetam and phenobarbital, for which in silico analysis of drug-protein interaction networks did not indicate any significant drug-drug interaction, was not effective to modify development of epilepsy. Surprisingly, the promising antiepileptogenic effect of the levetiracetam/topiramate combination was obtained in the absence of any significant neuroprotective or anti-inflammatory effects as indicated by multimodal brain imaging and histopathology. High throughput RNA-sequencing (RNA-seq) of the ipsilateral hippocampus of mice treated with the levetiracetam/topiramate combination showed that several genes that have been linked previously to epileptogenesis, were significantly differentially expressed, providing interesting entry points for future mechanistic studies. Overall, we have discovered a novel combination treatment with promise for prevention of epilepsy.
... [6] Animal models and preclinical evaluation of PER and other noncompetitive AMPA receptor antagonists have suggested a promising effect against MTLE. [7] Krestel et al showed increasing levels of calcium-permeable AMPA receptors in the hippocampus rat models of seizure, thus, leading to circuit hyperexcitability and increased seizure susceptibility. [8] In patients with treatment-resistant TLE, AMPA receptor was found to be upregulated. ...
Mesial temporal lobe epilepsy (MTLE) is a common epilepsy syndrome often refractory to antiepileptic drug (AED) treatment. The purpose of this study was to evaluate the effectiveness and tolerability of perampanel (PER) as add-on treatment for patients of MTLE.
We pooled retrospective data from adult patients with MTLE, from a tertiary center in Taiwan, who were prescribed PER between March 2016 and December 2016. The retention, responder, and seizure-free rate as well as the treatment emergent adverse events were assessed after 6 months of PER adjunctive treatment in this single-center postmarketing study.
Review of medical records revealed that adequate data were available for 44 patients who were being administered PER (mean age: 42.0 ± 13.3 years, 24 females; baseline mean seizure frequency: 5.4 per 28 days). Twelve patients exhibited hippocampal sclerosis (HS). Open-label PER was added to ongoing medications. Twelve patients withdrew because of ineffectiveness (n = 6) or adverse effects (n = 6). The retention rate was 72.7% at 6 months. On final evaluation, with a mean PER dose of 5.7 mg/day for 6 months, a ≥50% reduction in seizure frequency was observed in 46.9% of the patients, and 5 patients became seizure-free. The effectiveness was similar for patients with or without HS. Twenty-three patients (52.3%) experienced adverse effects. The most common adverse effects were dizziness, ataxia, and irritability.
Our results suggest that PER, at doses of 2 to 12 mg/day, reduces seizure frequency effectively with acceptable safety profiles for adults with MTLE.
... ** P<0.001, significantly different from control (saline) group. by electrical stimulation (25) or by various chemical substances, such as NMDA (26), pilocarpine (27), lidocaine (28), 4-aminopyridine (22), bicuculline (29), pentylenetetrazole (30), and kainate (31). The doses used in the present study for ketamine, 5 and 10 mg/ kg, were suggested to represent optimal doses for anticonvulsant activity. ...
Objectives:
Fasted rodents treated with antimuscarinics develop convulsions after refeeding. Food deprivation for 48 hr produces changes in [3H]glutamate binding suggesting glutamatergic contribution to the underlying mechanism of the seizures that are somewhat unresponsive to antiepileptics. Studies in animals and epileptic patients yielded considerable information regarding the anticonvulsant effect of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine. Thus, this study evaluated the efficacy of ketamine and its combinations with valproate and carbamazepine on convulsions in fasted animals.
Materials and methods:
Following 24 hr of fasting, mice were given saline, 5 or 10 mg/kg ketamine, 250 mg/kg sodium valproate, 24 mg/kg carbamazepine, 5 mg/kg ketamine+sodium valproate, or 5 mg/kg ketamine+carbamazepine and then were treated with saline or 2.4 mg/kg atropine (5-9 mice per group). The animals were observed for the occurrence of convulsions after being allowed to eat ad libitum.
Results:
Ketamine, valproate and carbamazepine pretreatments were ineffective in preventing the convulsions developed after atropine treatment and food intake in fasted animals. The incidence of convulsions was significantly higher in 5 and 10 mg/kg ketamine, carbamazepine, and carbamazepine+ketamine groups, but not in the valproate and valproate+ketamine treated animals.
Conclusion:
In contrast to previous findings obtained with the NMDA antagonist dizocilpine (MK-801), ketamine lacks activity against convulsions developed after fasting. The drug does not enhance the efficacy of valproate and carbamazepine either. Using different doses of ketamine or other NMDA antagonists, further studies may better clarify the anticonvulsant effect of ketamine and/or role of glutamate in these seizures.
... Most studies addressing the effects of seizures on the hippocampal NSC pool have used cSZ triggered by systemically administered chemoconvulsants like KA or pilocarpine (Parent et al., 1997;Bouilleret et al., 1999;Parent et al., 2006;Jessberger et al., 2007;Sibbe et al., 2012;Miltiadous et al., 2013;Cho et al., 2015;Twele et al., 2015). This may represent a limitation in the translational power of these studies, since not all epilepsy patients experience cSZ (Labovitz et al., 2001;Korff and Nordli, 2007;Rosenow et al., 2007). ...
Convulsive seizures promote adult hippocampal neurogenesis (AHN) through a transient activation of neural stem/progenitor cells (NSPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG). However, in a significant population of epilepsy patients, non-convulsive seizures (ncSZ) are observed. The response of NSPCs to non-convulsive seizure induction has not been characterized before. We here studied first the short-term effects of controlled seizure induction on NSPCs fate and identity. We induced seizures of controlled intensity by intrahippocampally injecting increasing doses of the chemoconvulsant kainic acid (KA) and analyzed their effect on subdural EEG recordings, hippocampal structure, NSPC proliferation and the number and location of immature neurons shortly after seizure onset. After establishing a KA dose that elicits ncSZ, we then analyzed the effects of ncSZ on NSPC proliferation and NSC identity in the hippocampus. ncSZ specifically triggered neuroblast proliferation, but did not induce proliferation of NSPCs in the SGZ, 3 days post seizure onset. However, ncSZ induced significant changes in NSPC composition in the hippocampus, including the generation of reactive NSCs. Interestingly, intrahippocampal injection of a combination of two anti microRNA oligonucleotides targeting microRNA-124 and -137 normalized neuroblast proliferation and prevented NSC loss in the DG upon ncSZ. Our results show for the first time that ncSZ induce significant changes in neuroblast proliferation and NSC composition. Simultaneous antagonism of both microRNA-124 and -137 rescued seizure-induced alterations in NSPC, supporting their coordinated action in the regulation of NSC fate and proliferation and their potential for future seizure therapies.
... AMPA receptors, as primary mediators of glutamatergic transmission, are central to hyperexcitability, but they may also be directly involved in excitotoxicity by allowing Ca 21 influx (Kwak and Weiss, 2006;Yuan and Bellone, 2013). Their importance as targets for antiepileptic drugs has been emphasized previously (Rogawski, 2013;Serdyuk et al., 2014;Twele et al., 2015); however, the selectivity of LY293558 for the kainate receptors containing the GluK1 subunit is probably at least equally important as selective GluK1 receptor antagonists with minimal antagonistic activity for AMPA receptors can alone block pilocarpine-induced seizures (Smolders et al., 2002), as well soman-induced SE and neuropathology . The mechanisms underlying the role of GluK1 kainate receptors in hyperexcitability and neuronal damage (Jane et al., 2009) involve not only postsynaptic mediation of glutamatergic transmission in principal neurons but also suppression of GABA release (Braga et al., 2003), facilitation of glutamate release (Aroniadou-Anderjaska et al., 2012), and permeability to Ca 21 Joseph et al., 2011). ...
The currently FDA-approved anticonvulsant for the treatment of status epilepticus (SE) induced by nerve agents is the benzodiazepine diazepam. However, diazepam does not appear to offer neuroprotective benefits. This is particularly concerning with respect to the protection of children, because in the developing brain, synaptic transmission mediated via GABAA receptors, the target of diazepam, is still weak. In the present study, we exposed 21-day-old male rats to 1.2xLD50 soman, and compared the antiseizure, anti-lethality, and neuroprotective efficacy of diazepam (10 mg/kg), LY293558 (an AMPA/GluK1 receptor antagonist; 15 mg/kg), caramiphen (an antimuscarinic with NMDA receptor-antagonistic properties; 50 mg/kg), and LY293558 (15 mg/kg)+caramiphen (50 mg/kg), administered 1 h post-exposure. Diazepam, LY293558, and LY293558+caramiphen, but not caramiphen alone, terminated SE, with the LY293558+caramiphen treatment acting significantly faster, and produced survival rate greater than 85%. Thirty days after soman exposure, neurodegeneration in limbic regions was most severe in the caramiphen-treated group, minimal to severe--depending on the region--in the diazepam group, absent to moderate in the LY293558-treated group, and totally absent in the LY293558+caramiphen group. Amygdala and hippocampal atrophy, severe reduction of spontaneous inhibitory activity in the basolateral amygdala, and increased anxiety-like behavior in the open field and acoustic startle response tests were present in the diazepam and caramiphen groups, while the LY293558 and LY293558+caramiphen groups did not differ from controls. The combined administration of LY293558 and caramiphen, by blocking mainly AMPA, GluK1, and NMDA receptors, is a very effective anticonvulsant and neuroprotective therapy against soman in young rats.
... ligands effectively block seizure activity [169,170]. Recent data further suggest, however, that AMPA receptor blockade by NBQX does not exert antiepileptogenic but anticonvulsive effects in the kainic acid SE model [171]. However, the Ca 2+ -related changes in epileptic tissue are non-equivocal. ...
In many patients who suffer from epilepsies, recurrent epileptic seizures do not start at birth but develop later in life. This holds particularly true for epilepsies with a focal seizure origin including focal cortical dysplasias (FCDs) and temporal lobe epilepsy (TLE). TLE most frequently has its seizure onset in the hippocampal formation. Hippocampal biopsies of pharmacoresistant TLE patients undergoing epilepsy surgery for seizure control most frequently reveal the damage pattern of hippocampal sclerosis, i.e. segmental neuronal cell loss and concomitant astrogliosis. Many TLE patients report on transient brain insults early in life, which is followed by a ‚latency’ period lacking seizure activity of months or even years before chronic recurrent seizures start. The plethora of structural and cellular mechanisms that convert the hippocampal formation to become chronically hyperexcitable after a transient insult to the brain are summarized under the term epileptogenesis. In contrast to the obstacles arising for experimental studies of epileptogenesis aspects in human surgical hippocampal tissue, recent animal model approaches allow insights into mechanisms of epileptogenesis.
... For this study we chose male mice of the NMRI outbred strain, a general purpose strain in many fields of biology as well as in pharmacology and toxicology, 13 which was previously used for this model by our and other groups. 3,8,[14][15][16][17] We expected that our study adds to the current discussion about how to best define a "seizure" in experimental models of acquired epilepsy and how careful study of control animals can clarify this issue. 11,[18][19][20][21][22] The few previous studies on the intrahippocampal kainate model in which sham controls were included used only few animals for this purpose, 2,23,24 whereas we chose a relatively large group size for our study, thus allowing statistical comparisons with kainate-injected mice. ...
Objective
There is an ongoing debate about definition of seizures in experimental models of acquired epilepsy and how important adequate sham controls are in this respect. For instance, several mouse and rat strains exhibit high‐voltage rhythmic spike or spike‐wave discharges in the cortical electroencephalogram (EEG), which has to be considered when using such strains for induction of epilepsy by status epilepticus, traumatic brain injury, or other means. Mice developing spontaneous recurrent nonconvulsive and convulsive seizures after intrahippocampal injection of kainate are increasingly being used as a model of mesial temporal lobe epilepsy. We performed a prospective study in which EEG alterations occurring in this model were compared with the EEGs in appropriate sham controls, using hippocampal electrodes and video‐EEG monitoring.
Methods
Experiments with intrahippocampal kainate (or saline) injections started when mice were about 8 weeks of age. Continuous video‐EEG recording via hippocampal electrodes was performed 6 weeks after surgery in kainate‐injected mice and sham controls, that is, at an age of about 14 weeks. Three days of continuous video‐EEG monitoring were compared between kainate‐injected mice and experimental controls.
Results
As reported previously, kainate‐injected mice exhibited two types of highly frequent electrographic seizures: high‐voltage sharp waves, which were often monomorphic, and polymorphic hippocampal paroxysmal discharges. In addition, generalized convulsive clinical seizures were infrequently observed. None of these electrographic or electroclinical seizures were observed in sham controls. The only infrequently observed EEG abnormalities in sham controls were isolated spikes or spike clusters, which were also recorded in epileptic mice.
Significance
This study rigorously demonstrates, by explicit comparison with the EEGs of sham controls, that the nonconvulsive paroxysmal events observed in this model are consequences of the induced epilepsy and not features of the EEG expected to be seen in some experimental control mice or unintentionally induced by surgical procedures.
... Hence, seizure-induced conversion of AMPARs to CPAMPARs may also be relevant to epilepsy in the adult as well as the immature brain. Finally, post-seizure rescue of published outcome measures by NBQX treatment appears to be specific to early-life seizures, as a recent study found that NBQX does not have antiepileptogenic effects in adults ( Twele et al., 2015). Therefore, the mechanism described in this report appears to be particularly sensitive in the P10 time window (developmentally similar to humans at term), providing an essential piece of preclinical information to guide the development of specific treatment options. ...
Calcium (Ca(2+))-mediated(4) signaling pathways are critical to synaptic plasticity. In adults, the NMDA glutamate receptor (NMDAR) represents a major route for activity-dependent synaptic Ca(2+) entry. However, during neonatal development, when synaptic plasticity is high, many AMPA glutamate receptors (AMPARs) are also permeable to Ca(2+) (CP-AMPAR) due to low GluA2 subunit expression, providing an additional route for activity- and glutamate-dependent Ca(2+) influx and subsequent signaling. Therefore, altered hippocampal Ca(2+) signaling may represent an age-specific pathogenic mechanism. We thus aimed to assess Ca(2+) responses 48h after hypoxia-induced neonatal seizures (HS) in postnatal day (P)10 rats, a post-seizure time point at which we previously reported LTP attenuation. We found that Ca(2+) responses were higher in brain slices from post-HS rats than in controls and this increase was CP-AMPAR-dependent. To determine whether synaptic CP-AMPAR expression was also altered post-HS, we assessed the expression of GluA2 at hippocampal synapses and the expression of long-term depression (LTD), which has been linked to the presence of synaptic GluA2. Here we report a decrease 48h after HS in synaptic GluA2 expression at synapses and LTD in hippocampal CA1. Given the potentially critical role of AMPAR trafficking in disease progression, we aimed to establish whether post-seizure in vivo AMPAR antagonist treatment prevented the enhanced Ca(2+) responses, changes in GluA2 synaptic expression, and diminished LTD. We found that NBQX treatment prevents all three of these post-seizure consequences, further supporting a critical role for AMPARs as an age-specific therapeutic target.
... The afferent conduction block of ketamine observed in the cell body of the 1 st -order neurons is a time-and concentration-dependent, which is attributed at least to the inhibition of voltage-gated Na + channels [42,43], whereas, the presynaptic transmission block-mediated by AMPA and NMDA receptors is a central mechanism of ketamine at afferent terminals of the 1 st -order neurons [4,44,45]. Voltage-gated Na + channels (Nav1.7 and Nav1.8) are critical for afferent conduction, and the current observation showed that both Nav1.7 (TTX-sensitive, TTX-S) and Nav1.8 (TTX-resistant, TTX-R) are both involved in conduction in Ah-type afferents, which could be blocked by ketamine leading to the conduction failure of vagal and solitary track neurotransmission revealed by the present (Figure 1A and Figure 4B) and previous observation [4]. However, several questions remain to be answered. ...
Background:
Ketamine enhances autonomic activity, and unmyelinated C-type baroreceptor afferents are more susceptible to be blocked by ketamine than myelinated A-types. However, the presynaptic transmission block in low-threshold and sex-specific myelinated Ah-type baroreceptor neurons (BRNs) is not elucidated.
Methods:
Action potentials (APs) and excitatory post-synaptic currents (EPSCs) were investigated in BRNs/barosensitive neurons identified by conduction velocity (CV), capsaicin-conjugated with Iberiotoxin-sensitivity and fluorescent dye using intact nodose slice and brainstem slice in adult female rats. The expression of mRNA and targeted protein for NMDAR1 was also evaluated.
Results:
Ketamine time-dependently blocked afferent CV in Ah-types in nodose slice with significant changes in AP discharge. The concentration-dependent inhibition of ketamine on AP discharge profiles were also assessed and observed using isolated Ah-type BRNs with dramatic reduction in neuroexcitability. In brainstem slice, the 2nd-order capsaicin-resistant EPSCs were identified and ~50% of them were blocked by ketamine concentration-dependently with IC50 estimated at 84.4 µM compared with the rest (708.2 µM). Interestingly, the peak, decay time constant, and area under curve of EPSCs were significantly enhanced by 100 nM iberiotoxin in ketamine-more sensitive myelinated NTS neurons (most likely Ah-types), rather than ketamine-less sensitive ones (A-types).
Conclusions:
These data have demonstrated, for the first time, that low-threshold and sex-specific myelinated Ah-type BRNs in nodose and Ah-type barosensitive neurons in NTS are more susceptible to ketamine and may play crucial roles in not only mean blood pressure regulation but also buffering dynamic changes in pressure, as well as the ketamine-mediated cardiovascular dysfunction through sexual-dimorphic baroreflex afferent pathway.
Introduction:
Epilepsy is characterized by having two or more unprovoked seizures. Understanding the pathogenesis of epilepsy, requires deep investigation into the molecular mechanisms. This helps develop diagnostic techniques, treatments, and pharmacotherapy. It also enhances precision medicine and individualized treatment processes. This article reviews all the molecular mechanisms predisposing to epileptogenesis, presents the current diagnostic techniques and drug therapy, and suggests future perspectives in treating Epilepsy in a more comprehensive and holistic approach.
Methodology:
Four authors searched keywords concerning epilepsy at a molecular level, Epilepsy diagnostic techniques and technologies, and antiepileptic drug therapy and precision medicine. Separate search strategies were conducted for each concern and retrieved articles were reviewed for relevant results.
Results:
The traditional diagnostic techniques for Epilepsy and its pathogenesis are insufficient in highlighting dynamic brain changes. For this, emerging technologies including genetic sequencing and profiling, and functional neuroimaging techniques are prevailing. Concerning treatment, the current approach focuses on managing symptoms and stopping seizures using antiseizure medications. However, their usage is limited by developing resistance to such drugs. Some therapies show promise, although most antiseizure drugs do not prevent epilepsy.
Discussion:
Understanding epileptogenesis at a molecular and genetic level aids in developing new antiepileptic pharmacotherapy. The aim is to develop therapies that could prevent seizures or modify disease course, decreasing the severity and avoiding drug resistance. Gene therapy and precision medicine are promising but applications are limited due to the heterogeneity in studying the Epileptic brain, dynamically. The dynamic investigation of the epileptic brain with its comorbidities works hand‐in‐hand with precision medicine, in developing personalized treatment plans.
Background
This study was performed to test the hypothesis that systemic leukocyte gene expression has prognostic value differentiating low from high seizure frequency refractory temporal lobe epilepsy (TLE).
Methods
A consecutive series of patients with refractory temporal lobe epilepsy was studied. Based on a median baseline seizure frequency of 2.0 seizures per month, low versus high seizure frequency was defined as ≤ 2 seizures/month and > 2 seizures/month, respectively. Systemic leukocyte gene expression was analyzed for prognostic value for TLE seizure frequency. All differentially expressed genes were analyzed, with Ingenuity® Pathway Analysis (IPA®) and Reactome, to identify leukocyte gene expression and biological pathways with prognostic value for seizure frequency.
Results
There were ten males and six females with a mean age of 39.4 years (range: 16 to 62 years, standard error of mean: 3.6 years). There were five patients in the high and eleven patients in the low seizure frequency cohorts, respectively. Based on a threshold of twofold change (p < 0.001, FC > 2.0, FDR < 0.05) and expression within at least two pathways from both Reactome and Ingenuity® Pathway Analysis (IPA®), 13 differentially expressed leukocyte genes were identified which were all over-expressed in the low when compared to the high seizure frequency groups, including NCF2, HMOX1, RHOB, FCGR2A, PRKCD, RAC2, TLR1, CHP1, TNFRSF1A, IFNGR1, LYN, MYD88, and CASP1. Similar analysis identified four differentially expressed genes which were all over-expressed in the high when compared to the low seizure frequency groups, including AK1, F2R, GNB5, and TYMS.
Conclusions
Low and high seizure frequency TLE are predicted by the respective upregulation and downregulation of specific leukocyte genes involved in canonical pathways of neuroinflammation, oxidative stress and lipid peroxidation, GABA (γ-aminobutyric acid) inhibition, and AMPA and NMDA receptor signaling. Furthermore, high seizure frequency-TLE is distinguished prognostically from low seizure frequency-TLE by differentially increased specific leukocyte gene expression involved in GABA inhibition and NMDA receptor signaling. High and low seizure frequency patients appear to represent two mechanistically different forms of temporal lobe epilepsy based on leukocyte gene expression.
Ionotropic glutamate receptors (iGluRs) mediate the majority of excitatory neurotransmission and are implicated in various neurological disorders. In this review, we discuss the role of the two fastest iGluRs subtypes, namely, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors, in the pathogenesis and treatment of Parkinson’s disease, epilepsy, and amyotrophic lateral sclerosis. Although both AMPA and kainate receptors represent promising therapeutic targets for the treatment of these diseases, many of their antagonists show adverse side effects. Further studies of factors affecting the selective subunit expression and trafficking of AMPA and kainate receptors, and a reasonable approach to their regulation by the recently identified novel compounds remain promising directions for pharmacological research.
Lipocalin-type prostaglandin D synthase (L-PGDS) is a secretory lipid-transporter protein that was shown to bind a wide variety of hydrophobic ligands in vitro. Exploiting this function, we previously examined the feasibility of using L-PGDS as a novel delivery vehicle for poorly water-soluble drugs. However, the mechanism by which human L-PGDS binds to poorly water-soluble drugs is unclear. In this study, we determined the solution structure of human L-PGDS and investigated the mechanism of L-PGDS binding to 6-nitro-7-sulfamoyl-benzo[f]quinoxalin-2,3-dione (NBQX), an α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor antagonist. NMR experiments showed that human L-PGDS has an eight-stranded antiparallel β-barrel structure that forms a central cavity, a short 310 -helix, and two α-helices. Titration with NBQX was monitored using 1 H-15 N HSQC spectroscopy. At higher NBQX concentrations, some cross-peaks of the protein exhibited fast-exchanging shifts with a curvature, indicating at least two binding sites. These residues were located in the upper portion of the cavity. Singular value decomposition analysis revealed that human L-PGDS has two NBQX binding sites. Large chemical shift changes were observed in the H2-helix and A-, B-, C-, D-, H-, and I-strands and H2-helix upon NBQX binding. Calorimetric experiments revealed that human L-PGDS binds two NBQX molecules with dissociation constants of 46.7 μM for primary binding and 185.0 μM for secondary binding. Molecular docking simulations indicated that these NBQX binding sites are located within the β-barrel. These results provide new insights into the interaction between poorly water-soluble drugs and human L-PGDS as a drug carrier.
Objective:
Intrahippocampal injection of kainate (KA) is a reliable model of temporal lobe epilepsy (TLE) that replicates spontaneous recurrent seizures. Both electrographic seizures and electroclinical seizure (most generalized seizure) can be detected in KA model. Electrographic seizures such as high-voltage sharp waves (HVSWs) and hippocampal paroxysmal discharges (HPDs) are far more common and attracting much attention. A comprehensive study on the anticonvulsant effects of classic and novel antiseizure medications (ASMs) on spontaneous electroclinical seizures, especially during long-term treatment, is still lacking. Here, we evaluated the effects of six ASMs in this model on electroclinical seizures over eight weeks.
Methods:
Using 24-hour continuous electroencephalographical (EEG) monitoring in free-moving mice, we tested the effectiveness of six ASMs (valproic acid, VPA; carbamazepine, CBZ; lamotrigine, LTG; perampanel, PER; brivaracetam, BRV; and everolimus, EVL) on the electroclinical seizures over eight weeks in the intrahippocampal kainate mouse model.
Results:
VPA, CBZ, LTG, PER and BRV significantly suppressed electroclinical seizures in the early stages of treatment, but the mice gradually developed resistance to these drugs. Overall, the mean frequency of electroclinical seizures was not significantly lower during the 8-week treatment than that at baseline in any ASM-treated group. The individual responses to ASMs varied widely.
Conclusion:
Long-term treatment with VPA, LTG, CBZ, PER, BRV and EVL did not relieve electroclinical seizures in this TLE model. Additionally, the window for screening new ASMs in this model should be set to at least 3 weeks to account for drug resistance.
Introduction: This study was performed to test the hypothesis that systemic leukocyte gene expression has prognostic value differentiating low from high seizure frequency refractory temporal lobe epilepsy (TLE).
Methods: A consecutive series of sixteen patients with refractory temporal lobe epilepsy was studied. Based on a median baseline seizure frequency of 2.0 seizures per month, low versus high seizure frequency was defined as < 2 seizures/month and > 2 seizures/month,
respectively. Systemic leukocyte gene expression was analyzed for prognostic value for TLE seizure frequency. All differentially expressed genes were analyzed, with Ingenuity® Pathway Analysis (IPA®) and Reactome, to identify leukocyte gene expression and biological pathways with prognostic value for seizure frequency.
Results: There were ten males and six females with a mean age of 39.4 years (range: 16 to 62 years, standard error of mean: 3.6 years). There were five patients in the high and eleven patients in the low seizure frequency cohorts, respectively. Based on a threshold of 2-fold change (p < 0.001, FC > 2.0, FDR < 0.05) and expression within at least two pathways from both Reactome and Ingenuity® Pathway Analysis (IPA®), 13 differentially expressed leukocyte genes were identified which were all over-expressed in the low when compared to the high seizure frequency groups, including NCF2, HMOX1, RHOB, FCGR2A, PRKCD, RAC2, TLR1, CHP1, TNFRSF1A, IFNGR1, LYN, MYD88, and CASP1. Similar analysis identified four differentially expressed genes which were all over-expressed in the high when compared to the low seizure frequency groups, including AK1, F2R, GNB5, and TYMS.
Conclusions: Low and high seizure frequency TLE are predicted by the respective upregulation and downregulation of specific leukocyte genes involved in canonical pathways of neuroinflammation, oxidative stress and lipid peroxidation, GABA (γ-aminobutyric acid) inhibition, and AMPA and NMDA receptor signaling. Furthermore, high seizure frequency-TLE is distinguished prognostically from low seizure frequency-TLE by differentially increased specific leukocyte gene expression involved in GABA inhibition and NMDA receptor signaling. High and low seizure frequency patients appear to represent two mechanistically different forms of temporal lobe epilepsy based on leukocyte gene expression.
This chapter describes current and novel therapeutic medical countermeasures (MCMs) for organophosphate (OP) pesticide and chemical nerve agent induced neurotoxicity. Therapeutic windows are explained along with the standard-of-care MCMs used for victims of OP exposure. The standard-of-care MCMs for post-exposure, and in some cases therapeutic, timepoints include benzodiazepines which do not effectively prevent or mitigate all symptoms of nerve agent intoxication. Mechanistic studies of benzodiazepine-resistant seizures are described along with the development of novel MCMs to address this unmet medical need. There are also two drug class categories with active drug development ongoing and each are discussed in detail. Neurosteroids are proposed as next-generation anticonvulsants which are superior to the benzodiazepines for the treatment of OP intoxication. They offer robust neuroprotection by reducing neuronal injury and neuroinflammation. Glutamate receptor antagonists are being developed as anticonvulsants and also may be effective in attenuating long-term neuropsychiatric deficits caused by OP exposure.
Epilepsy is caused when rhythmic neuronal network activity escapes normal control mechanisms, resulting in seizures. There is an extensive and growing body of evidence that the onset and maintenance of epilepsy involves alterations in the trafficking, synaptic surface expression and signalling of kainate and AMPA receptors (KARs and AMPARs). The KAR subunit GluK2 and AMPAR subunit GluA2 are key determinants of the properties of their respective assembled receptors. Both subunits are subject to extensive protein interactions, RNA editing and post-translational modifications. In this review we focus on the cell biology of GluK2-containing KARs and GluA2-containing AMPARs and outline how their regulation and dysregulation is implicated in, and affected by, seizure activity. Further, we discuss role of KARs in regulating AMPAR surface expression and plasticity, and the relevance of this to epilepsy.
This article is part of the special issue on ‘Glutamate Receptors - Kainate receptors’.
Experimental models of epilepsy are useful to identify potential mechanisms of epileptogenesis, seizure genesis, comorbidities, and treatment efficacy. The kainic acid (KA) model is one of the most commonly used. Several modes of administration of KA exist, each producing different effects in a strain, species, gender, and age-dependent manner. In this review, we discuss the advantages and limitations of the various forms of KA administration (systemic, intrahippocampal, and intranasal), as well as the histological, electrophysiological, and behavioral outcomes in different strains and species. We attempt a personal perspective and discuss areas where work is needed. The diversity of KA models and their outcomes offers researchers a rich palette of phenotypes, which may be relevant to specific traits found in patients with temporal lobe epilepsy.Significance statementThis review aims to help researchers use a knowledge-based approach to study specific aspects of human epilepsy phenotypes. We focus on the Kainic Acid model of temporal lobe epilepsy in rodents, presenting it as a set of sub-models, describing the various administration routes, and the differences in outcome between species, strain, age, and sex. We have reviewed more than 200 research articles, summarizing the data with a ready-to-use structure.
Background
Temporal Lobectomy (TL) is an accepted surgical procedure for refractory seizures.
Aims
To assess the effects of Perampanel (PER) therapy in patients with refractory temporal lobe epilepsy.
Methodology
Perampanel therapy was administered in two cases with refractory seizures post temporal lobectomy and in two others with refractory seizures who were eligible for temporal lobectomy.
Results
There was significant control in seizures in all the four cases with Perampanel as a single add on to ongoing refractory state.
Background
Perampanel (PER) is a novel antiepileptic drug approved as an add-on therapy for focal onset seizures with or without generalization and primary generalized tonic-clonic seizures. Aim of this study was to evaluate PER efficacy and tolerability as add-on therapy in patients with drug-resistant focal onset seizures and especially temporal lobe epilepsy (TLE).
Methods
An observational, prospective, multicentre study on adult with drug-resistant focal epilepsy consecutively recruited from six Italian tertiary epilepsy centres. All patients received add-on PER according to indication and clinical judgement. Seizure frequency and adverse events (AEs) were recorded at 6 and 12 months after PER introduction.
Results
Study sample comprised 246 patients, 77 of which with TLE. Seventy-five (35.9%) out of 209 and 66 (38.8%) out of 170 patients still taking PER resulted to be responders (i.e. ≥50% of seizure frequency or seizure free) after six and 12 months, respectively. In the TLE group, 39 (57.3%) out of 68 subjects on PER after 6 months and 32 (60.4%) out of 53 subjects taking PER after 12 months were responders. Overall reported incidence of AEs was 26.1%. In 28 cases (11.3%) AEs lead/contributed to PER discontinuation. The most frequently reported AE were dizziness (14/84) and somnolence (14/84). Regarding TLE patients, 25.9% of them experienced at least one AE and discontinuation for AEs occurred in eight (10.4%).
Conclusions
This study confirmed the good efficacy and safety of PER for drug-resistant focal epilepsy in real-life conditions and, above all, for the first time provide its effectiveness in patients with TLE.
Animal models are still indispensable in experimental epilepsy research in order to overcome restrictions in clinical research. Models of mesial temporal lobe epilepsy (mTLE) aim to replicate phenotypic and pathophysiological criteria of the disease. The mTLE models following chemoconvulsive or electrically induced status epilepticus in rodents are used most frequently. Knowledge of validity criteria and limitations of these and other models are the basis for the classification of animal experimental findings and are presented in this overview.
Endophilin A1 is a member of the endophilin A family and is primarily expressed in the central nervous system. Endophilin A1 can mediate neuronal excitability by regulating neuronal synaptic plasticity, which indicates that the protein may be involved in epilepsy. However, to date, its role in epilepsy remains unclear. To explore the role of endophilin A1 in epilepsy, we aimed to investigate the expression patterns of endophilin A1 in patients with temporal lobe epilepsy (TLE) and in a pentylenetetrazole (PTZ)-kindled epileptic mouse model and to conduct behavioral and electrophysiological analyses after lentivirus-mediated knockdown of endophilin A1 in the hippocampus of epileptic mice. This study found that the expression of endophilin A1 was significantly up-regulated in the temporal neocortex of TLE patients and in the hippocampus and adjacent temporal cortex of the PTZ-kindled epileptic mouse model. Behavioral analyses indicated that knockdown of endophilin A1 in the mouse hippocampus increased the latency of the first seizure and reduced the frequency and duration of seizure activity. Whole-cell patch-clamp recordings of pyramidal neurons in the hippocampal CA3 area indicated that knockdown of endophilin A1 in the mouse hippocampus resulted in a reduced frequency of action potentials and decreased amplitudes of miniature excitatory postsynaptic currents (mEPSCs) and evoked AMPA-dependent EPSCs. Moreover, western blotting analysis showed that the surface expression of the AMPAR GluR2 subunit was also decreased after endophilin A1 knockdown, and co-immunoprecipitation indicated an association between endophilin A1 and AMPAR GluR2 in the mouse hippocampus. Further, when AMPARs were activated by CX546, the antiepileptic function of endophilin A1 knockdown was decreased. Based on these results, endophilin A1 plays a critical role in epilepsy, and its suppression in the mouse hippocampus can restrain neuronal excitability and seizure activity via regulating AMPARs.
This paper presents a physiological account of seizure activity and its evolution over time using a rat model of induced epilepsy. We analyse spectral activity recorded in the hippocampi of three rats who received kainic acid injections in the right hippocampus. We use dynamic causal modelling of seizure activity and Bayesian model reduction to identify the key synaptic and connectivity parameters that underlie seizure onset. Using recent advances in hierarchical modelling (parametric empirical Bayes), we characterise seizure onset in terms of slow fluctuations in synaptic excitability of specific neuronal populations. Our results suggest differences in the pathophysiology – of seizure activity in the lesioned versus the non-lesioned hippocampus – with pronounced changes in excitation-inhibition balance and temporal summation on the lesioned side. In particular, our analyses suggest that marked reductions in the synaptic time constant of the deep pyramidal cells and the self-inhibition of inhibitory interneurons (in the lesioned hippocampus) are sufficient to explain changes in spectral activity. Although these synaptic changes are consistent over rats, the resulting electrophysiological phenotype can be quite diverse.
Mesiotemporal lobe Epilepsy (MTLE), the most frequent form of focal epilepsy, is often drug-resistant. Enriching the epileptic focus with GABA-releasing engineered cells has been proposed as a strategy to prevent seizures. However, ex vivo data from animal models and MTLE patients suggest that, due to changes in chloride homeostasis, GABAA receptor activation is depolarizing and partly responsible for focal interictal discharges and seizure initiation. To understand how these two contradictory aspects of GABAergic neurotransmission coexist in MTLE, we used an established mouse model of MTLE presenting hippocampal sclerosis and recurrent hippocampal paroxysmal discharges (HPDs) 30-40days after a unilateral injection of kainate in the dorsal hippocampus. We first showed that injections of GABAA receptor agonists either systemically or directly into hippocampus suppressed HPDs. Western-blotting and immunostaining revealed that levels of α1, α3 and γ2 GABAA receptor subunits were increased in epileptic mice, compared to saline controls, while levels of R1 and R2 GABAB receptor subunits but also NR1, NR2A and NR2B NMDA receptor subunits and GluR1 and GluR2 AMPA receptor subunits were decreased. In addition, we showed that the expression of the transporter NKCC1, which load neurons with chloride, was increased, whereas KCC2, a chloride extruder, was decreased and that HPDs were suppressed by injection of blockers of NKCC1. These different changes were integrated in a numerical model, and in silico simulations supported the notion that chloride imbalance impair local inhibitory control of pyramidal neurons' activity in this model of MTLE. However, our numerical model also suggested that lasting activation of these receptors restore physiological intracellular chloride concentrations and suppress HPDs. Overall, our study suggests that activation of GABAA receptor remains an effective antiepileptic strategy to suppress focal seizures in MTLE, and demonstrates that modeling and simulation studies provide new insights about the cellular and synaptic mechanisms of this disease.
Despite the introduction of over 15 third-generation anti-epileptic drugs, current medications fail to control seizures in 20-30% of patients. However, our understanding of the mechanisms mediating the development of epilepsy and the causes of drug resistance has grown substantially over the past decade, providing opportunities for the discovery and development of more efficacious anti-epileptic and anti-epileptogenic drugs. In this Review we discuss how previous preclinical models and clinical trial designs may have hampered the discovery of better treatments. We propose that future anti-epileptic drug development may be improved through a new joint endeavour between academia and the industry, through the identification and application of tools for new target-driven approaches, and through comparative preclinical proof-of-concept studies and innovative clinical trials designs.
Three recent phase III trials have shown that adjunctive treatment with perampanel—a first-in-class, noncompetitive AMPA antagonist—decreases seizure frequency in patients with refractory focal epilepsy. Although the introduction of perampanel offers more treatment choice for epilepsy, whether it brings urgently needed clinical benefit over existing drugs remains to be addressed.
In order to understand the physiopathology of epilepsies and develop antiepileptic drugs, animal models have been developed. These models appear to be valuable predictors of treatment efficacy; however, several of the currently used models remain questionable and probably inappropriate for the search for new treatments, in particular for epilepsies that cannot be treated by current antiepileptic drugs. In the present review, we report the results of a recent survey conducted by neurologists in charge of an epilepsy programme based at different hospitals in France. The 36 experts were questioned, via the internet, on the most critical features of four prototypic forms of epilepsy (idiopathic generalised epilepsies with convulsive seizures, absence epilepsy, focal epilepsy associated with dysplasia, and focal epilepsy associated with hippocampal sclerosis) that should be taken into account with regards to the relevance of animal models of epilepsy. Their answers suggest that most current models for focal epilepsies associated with either dysplasia or hippocampal sclerosis do not address the most relevant features. The models currently used in mice and rats are discussed in light of the data obtained in our survey.
Experimental evidence and clinical observations indicate that brain inflammation is an important factor in epilepsy. In particular, induction of interleukin-converting enzyme (ICE)/caspase-1 and activation of interleukin (IL)-1β/IL-1 receptor type 1 axis both occur in human epilepsy, and contribute to experimentally induced acute seizures. In this study, the anticonvulsant activity of VX-765 (a selective ICE/caspase-1 inhibitor) was examined in a mouse model of chronic epilepsy with spontaneous recurrent epileptic activity refractory to some common anticonvulsant drugs. Moreover, the effects of this drug were studied in one acute model of seizures in mice, previously shown to involve activation of ICE/caspase-1. Quantitative analysis of electroencephalogram activity was done in mice exposed to acute seizures or those developing chronic epileptic activity after status epilepticus to assess the anticonvulsant effects of systemic administration of VX-765. Histological and immunohistochemical analysis of brain tissue was carried out at the end of pharmacological experiments in epileptic mice to evaluate neuropathology, glia activation and IL-1β expression, and the effect of treatment. Repeated systemic administration of VX-765 significantly reduced chronic epileptic activity in mice in a dose-dependent fashion (12.5-200 mg/kg). This effect was observed at doses ≥ 50 mg/kg, and was reversible with discontinuation of the drug. Maximal drug effect was associated with inhibition of IL-1β synthesis in activated astrocytes. The same dose regimen of VX-765 also reduced acute seizures in mice and delayed their onset time. These results support a new target system for anticonvulsant pharmacological intervention to control epileptic activity that does not respond to some common anticonvulsant drugs.
The highest incidence of seizures during lifetime is found in the neonatal period and neonatal seizures lead to a propensity for epilepsy and long-term cognitive deficits. Here, we identify potential mechanisms that elucidate a critical role for AMPA receptors (AMPARs) in epileptogenesis during this critical period in the developing brain. In a rodent model of neonatal seizures, we have shown previously that administration of antagonists of the AMPARs during the 48 h after seizures prevents long-term increases in seizure susceptibility and seizure-induced neuronal injury. Hypoxia-induced seizures in postnatal day 10 rats induce rapid and reversible alterations in AMPAR signaling resembling changes implicated previously in models of synaptic potentiation in vitro. Hippocampal slices removed after hypoxic seizures exhibited potentiation of AMPAR-mediated synaptic currents, including an increase in the amplitude and frequency of spontaneous and miniature EPSCs as well as increased synaptic potency. This increased excitability was temporally associated with a rapid increase in phosphorylation at GluR1 S845/S831 and GluR2 S880 sites and increased activity of the protein kinases CaMKII (calcium/calmodulin-dependent protein kinase II), PKA, and PKC, which mediate the phosphorylation of these AMPAR subunits. Postseizure administration of AMPAR antagonists NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline), topiramate, or GYKI-53773 [(1)-1-(4-aminophenyl)-3-acetyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine] attenuated the AMPAR potentiation, phosphorylation, and kinase activation and prevented the concurrent increase in in vivo seizure susceptibility. Thus, the potentiation of AMPAR-containing synapses is a reversible, early step in epileptogenesis that offers a novel therapeutic target in the highly seizure-prone developing brain.
This study compared the ability of three N-methyl-D-aspartate (NMDA) receptor antagonists to prevent neuronal degeneration in an animal model of global cerebral ischemia. The model employed is characterized by damage to the striatum, hippocampus, and neocortex. Antagonists were administered to gerbils either before or after a 5-min bilateral carotid occlusion. The intraischemic rectal temperature was either maintained at 36-37 degrees C or allowed to fall passively to 28-32 degrees C. Antagonists and doses tested were 1 and 10 mg/kg of MK-801 (pre- or postischemia), 30 mg/kg of CGS 19755 preischemia, four 25 mg/kg doses of CGS 19755 administered between 0.5 and 6.5 h postischemia, and 40 mg/kg of MDL 27,266 (pre- or postischemia). All three NMDA receptor antagonists exhibited some degree of neuroprotective activity when the carotid occlusion was performed under normothermic conditions. Most of the treatments with antagonist markedly reduced striatal damage. CA1 hippocampal and neocortical pyramidal cells were spared by only three of the treatments, however, and the extent of neuroprotection varied widely from case to case. Toxic doses of antagonist were required to protect CA1 pyramidal cells from ischemic damage. Ischemic damage to hippocampal areas CA2-CA3a and CA4 appeared to be resistant to all of these treatments. Most CA1 pyramidal cells that were protected from degeneration by an NMDA receptor antagonist were histologically abnormal. The neuroprotective effects of MK-801 and intraischemic hypothermia appeared to be additive. MK-801 (10 mg/kg) consistently reduced the postischemic brain temperature, but only the magnitude of hypothermia produced soon after reperfusion correlated with its neuroprotective action. These results suggest that NMDA receptor antagonists are relatively poor neuroprotective agents against a moderately severe ischemic insult.
Several laboratories have reported a significant reduction of ischemia-induced injury to hippocampal neurons in rodents treated with competitive and noncompetitive N-methyl-D-aspartate (NMDA) receptor-channel antagonists. This study examined the effects of the noncompetitive antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) in Mongolian gerbils subjected to 5 min of bilateral carotid artery occlusion. In adult female gerbils, single doses of MK-801 injected 1 hr prior to ischemia significantly (p less than 0.01) reduced damage to CA1 hippocampal neurons. However, the drug rendered the postischemic animals comatose and hypothermic for several hours compared with the saline-treated animals. In subsequent experiments, animals pretreated with MK-801 and maintained normothermic during and after forebrain ischemia demonstrated no amelioration of hippocampal damage. Gerbils not treated with MK-801, but kept hypothermic in the postischemic period to approximately the same degree (34.5 degrees C) and duration (8 hr) as was induced by MK-801 therapy showed significant (p less than 0.01) protection of CA1 neurons against ischemia. The neuroprotective activity of MK-801 against transient global ischemia appears to be largely a consequence of postischemic hypothermia rather than a direct action on NMDA receptor-channels.
Despite more than 20 clinically approved antiepileptic drugs (AEDs), there remains a substantial unmet clinical need for patients with refractory (AED-resistant) epilepsy. Animal models of refractory epilepsy are needed for at least two goals; (1) better understanding of the mechanisms underlying resistance to AEDs, and (2) development of more efficacious AEDs for patients with refractory seizures. It is only incompletely understood why two patients with seemingly identical types of epilepsy and seizures may respond differently to the same AED. Prompted by this well-known clinical phenomenon, we previously evaluated whether epileptic rats respond differently to AEDs and discovered AED responsive and resistant animals in the same models. In the present study, we used the same approach for the widely used intrahippocampal kainate mouse model of mesial temporal lobe epilepsy. In a first step, we examined anti-seizure effects of 6 AEDs on spontaneous recurrent focal electrographic seizures and secondarily generalized convulsive seizures in epileptic mice, showing that the focal nonconvulsive seizures were resistant to carbamazepine and phenytoin, whereas valproate and levetiracetam exerted moderate and phenobarbital and diazepam marked anti-seizure effects. All AEDs seemed to suppress generalized convulsive seizures. Next we investigated the inter-individual variation in the anti-seizure effects of these AEDs and, in case of focal seizures, found responders and nonresponders to all AEDs except carbamazepine. Most nonresponders were resistant to more than one AED. Our data further validate the intrahippocampal kainate mouse model as a model of difficult-to-treat focal seizures that can be used to investigate the determinants of AED efficacy.
Copyright © 2014. Published by Elsevier Ltd.
Ethanol is commonly used as a solvent in injectable formulations of poorly watersoluble drugs. The concentrations of ethanol in such formulations are generally considered reasonably safe. It is long known that ethanol can potentiate central effects of sedatives and tranquillizers, particularly the benzodiazepines, most likely as a result of a synergistic interaction at the GABA(A) receptor. However, whether this occurs at the low systemic doses of ethanol resulting from its use as solvent in parenteral formulations of benzodiazepines is not known. In the present study we evaluated whether a commercial ethanol-containing aqueous solution of diazepam exerts more potent anti-seizure effects than an aqueous solution of diazepam hydrochloride or an aqueous emulsion of this drug in the intrahippocampal kainate model of temporal lobe epilepsy in mice. Spontaneous epileptic seizures in this model are known to be resistant to major antiepileptic drugs. Administration of the ethanol-containing formulation of diazepam caused an almost complete suppression of seizures. This was not seen when the same dose (5 mg/kg) of diazepam was administered as aqueous solution or emulsion, although all three diazepam formulations resulted in similar drug and metabolite concentrations in plasma. Our data demonstrate that ethanol-containing solutions of diazepam are superior to block difficult-to-treat seizures to other formulations of diazepam. To our knowledge, this has not been demonstrated before and, if this finding can be translated to humans, may have important consequences for emergency treatment of acute seizures, series of seizures, and initial treatment of status epilepticus in patients.
Epileptic seizures occur as a result of episodic abnormal synchronous discharges in cerebral neuronal networks. Although a variety of non-conventional mechanisms may play a role in epileptic synchronization, cascading excitation within networks of synaptically connected excitatory glutamatergic neurons is a classical mechanism. As is the case throughout the central nervous system, fast synaptic excitation within and between brain regions relevant to epilepsy is mediated predominantly by AMPA receptors. By inhibiting glutamate-mediated excitation, AMPA receptor antagonists markedly reduce or abolish epileptiform activity in in vitro preparations and confer seizure protection in a broad range of animal seizure models. NMDA receptors may also contribute to epileptiform activity, but NMDA receptor blockade is not sufficient to eliminate epileptiform discharges. AMPA receptors move into and out of the synapse in a dynamic fashion in forms of synaptic plasticity, underlying learning and memory. Often, the trigger for these dynamic movements is the activation of NMDA receptors. While NMDA receptor antagonists inhibit these forms of synaptic plasticity, AMPA receptor antagonists do not impair synaptic plasticity and do not inhibit memory formation or retrieval. The demonstrated clinical efficacy of perampanel, a high-potency, orally active non-competitive AMPA receptor antagonist, supports the concept that AMPA receptors are critical to epileptic synchronization and the generation and spread of epileptic discharges in human epilepsy.
Perampanel [2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile; E2007] is a potent, selective, orally active non-competitive AMPA receptor antagonist developed for the treatment of epilepsy. Perampanel has a 2,3′-bipyridin-6′-one core structure, distinguishing it chemically from other AMPA receptor antagonist classes. Studies in various physiological systems indicate that perampanel selectively inhibits AMPA receptor-mediated synaptic excitation without affecting NMDA receptor responses. Blocking of AMPA receptors occurs at an allosteric site that is distinct from the glutamate recognition site. Radioligand-binding studies suggest that the blocking site coincides with that of the non-competitive antagonist GYKI 52466, believed to be on linker peptide segments of AMPA receptor subunits that transduce agonist binding into channel opening. As is typical for AMPA receptor antagonists, perampanel exhibits broad-spectrum antiseizure activity in diverse animal seizure models. Perampanel has high oral bioavailability, dose-proportional kinetics, and undergoes oxidative metabolism, primarily via CYP3A4, followed by glucuronidation. The terminal half-life (t½) in humans is 105 h; however, in the presence of a strong CYP3A4 inducer (such as carbamazepine), the t½ can be reduced. In sum, perampanel is a selective, centrally acting, negative allosteric modulator of AMPA receptors with good oral bioavailability and favorable pharmacokinetic properties.
Introduction:
Perampanel is a novel AMPA receptor antagonist, approved in over 35 countries as an adjunctive therapy for the treatment of partial-onset seizures with or without secondarily generalized seizures in patients with epilepsy aged 12 years and older (18 years and older in Canada). These countries include the members of the European Union, the USA, Canada and Switzerland. The AMPA receptor antagonist, perampanel, is the first approved antiepileptic drug to inhibit excitation of postsynaptic membranes through the selective inhibition of glutamate receptors.
Areas covered:
This drug discovery case history focuses on the discovery and profiling of perampanel. It analyzes the pharmacological, behavioral and molecular mechanisms of perampanel and how they contribute to the therapeutic benefits of the drug. The article is based on the data reported in published preclinical and clinical studies, product labels and poster presentations.
Expert opinion:
Preclinical studies of perampanel have identified its broad-spectrum antiseizure effects in acute seizure models, with a narrow therapeutic index in the rotarod test similar to other AMPA receptor antagonists. This narrow therapeutic index is a potential problem for AMPA receptor antagonists. However, the discovery that perampanel has a very long half-life in humans, with gradual accumulation in plasma, could contribute to the development of tolerance. This, coupled with the identification of an optimal dosing strategy for individual patients, may help to maximize the utility of perampanel in the treatment of epilepsy.
Quality of life is directly related to the number and severity of adverse effects, and a successful antiepileptic medication must demonstrate a good balance between efficacy and tolerability. Perampanel is a newly licensed antiepileptic medication for the adjunctive treatment of patients (age 12 and older) with partial epilepsy with or without secondary generalization. Safety endpoints in the three phase III trials (304, 305, and 306) included treatment-emergent adverse events (TEAEs), vital signs, clinical laboratory parameters, and electrocardiography studies (ECGs). The most common adverse drug reactions in patients receiving perampanel were dizziness, somnolence, fatigue, irritability, nausea, and falls. Of particular concern to patients are cognitive and psychiatric side effects. Overall, depression and aggression were reported more frequently in patients taking perampanel, particularly at higher doses, than in patients taking placebo. TEAEs necessitated the withdrawal of perampanel in 99 patients (9.5%) and placebo in 21 patients (4.8%). Typically this was due to dizziness, convulsion, and somnolence. There were no clinically important changes or treatment group differences in vital signs, ECG measures, or biochemical or hematologic parameters. Weight increase of greater than 7% was seen in 14.6% of perampanel-treated patients versus 7.1% of placebo-treated patients. Overall, perampanel appears to be associated with a relatively low incidence of serious adverse effects, particularly at low doses, and the majority of TEAEs were mild or moderate in intensity. The incidence of predictable side effects, such as somnolence and dizziness, is seen more frequently at higher doses. Of importance is the greater rate of psychiatric side effects in patients treated with perampanel, principally, irritability and aggression, than with placebo. However, the rate of serious psychiatric TEAEs was low.
The diuretic bumetanide, which acts by blocking the Na-K-Cl cotransporter (NKCC), is widely used to inhibit neuronal NKCC1, particularly when NKCC1 expression is abnormally increased in brain diseases such as epilepsy. However, bumetanide poorly penetrates into the brain and, in rodents, is rapidly eliminated because of extensive oxidation of its N-butyl sidechain, reducing the translational value of rodent experiments. Inhibition of oxidation by piperonyl butoxide (PBO) has previously been reported to increase the half-life and diuretic activity of bumetanide in rats. Here we studied whether inhibition of bumetanide metabolism by PBO also increases brain levels of bumetanide in rats, and whether this alters pharmacodynamic effects in the kindling model of epilepsy. Furthermore, we studied the effects of PBO in mice. Mice eliminated bumetanide less rapidly than rats (elimination half-life 47 min vs. 13 min). Pretreatment with PBO increased the half-life in mice to average values (70 min) previously determined in humans, and markedly elevated brain levels of bumetanide. In rats, the increase in plasma and brain levels of bumetanide by PBO was less marked than in mice. PBO significantly increased the diuretic activity of bumetanide in rats and, less effectively, in mice. In epileptic mice, bumetanide (with PBO) did not suppress spontaneous seizures. In the rat kindling model, bumetanide (with or without PBO) did not exert anticonvulsant effects on fully kindled seizures, but dose-dependently altered kindling development. These data indicate that PBO offers a simple means to enhance the translational properties of rodent experiments with bumetanide, particularly when using mice.
To determine whether AMPA receptor (AMPAR) antagonist NBQX can prevent early mammalian target of rapamycin (mTOR) pathway activation and long-term sequelae following neonatal seizures in rats, including later-life spontaneous recurrent seizures, CA3 mossy fiber sprouting, and autistic-like social deficits.
Long-Evans rats experienced hypoxia-induced neonatal seizures (HS) at postnatal day (P)10. NBQX (20 mg/kg) was administered immediately following HS (every 12 h × 4 doses). Twelve hours post-HS, we assessed mTOR activation marker phosphorylated p70-S6 kinase (p-p70S6K) in hippocampus and cortex of vehicle (HS + V) or NBQX-treated post-HS rats (HS + N) versus littermate controls (C + V). Spontaneous seizure activity was compared between groups by epidural cortical electroencephalography (EEG) at P70-100. Aberrant mossy fiber sprouting was measured using Timm staining. Finally, we assessed behavior between P30 and P38.
Postseizure NBQX treatment significantly attenuated seizure-induced increases in p-p70S6K in the hippocampus (p < 0.01) and cortex (p < 0.001). Although spontaneous recurrent seizures increased in adulthood in HS + V rats compared to controls (3.22 ± 1 seizures/h; p = 0.03), NBQX significantly attenuated later-life seizures (0.14 ± 0.1 seizures/h; p = 0.046). HS + N rats showed less aberrant mossy fiber sprouting (115 ± 8.0%) than vehicle-treated post-HS rats (174 ± 10%, p = 0.004), compared to controls (normalized to 100%). Finally, NBQX treatment prevented alterations in later-life social behavior; post-HS rats showed significantly decreased preference for a novel over a familiar rat (71.0 ± 12 s) compared to controls (99.0 ± 15.6 s; p < 0.01), whereas HS + N rats showed social novelty preference similar to controls (114.3 ± 14.1 s).
Brief NBQX administration during the 48 h postseizure in P10 Long-Evans rats suppresses transient mTOR pathway activation and attenuates spontaneous recurrent seizures, social preference deficits, and mossy fiber sprouting observed in vehicle-treated adult rats after early life seizures. These results suggest that acute AMPAR antagonist treatment during the latent period immediately following neonatal HS can modify seizure-induced activation of mTOR, reduce the frequency of later-life seizures, and protect against CA3 mossy fiber sprouting and autistic-like social deficits.
The association between hippocampal sclerosis (HS) and epilepsy has been known for almost two centuries. For many years, HS was studied in postmortem series; however, since the mid-20th century, surgical specimens from temporal lobe resections have provided important new knowledge. HS is the most common pathology underlying drug-resistant mesial temporal lobe epilepsy (MTLE), a syndrome with a characteristic history and seizure semiology. In the early 1990s, it was recognized that magnetic resonance imaging (MRI) could detect HS. The standard MRI protocol for temporal lobe abnormalities uses coronal slices perpendicular to the long axis of the hippocampus. The MRI features of HS include reduced hippocampal volume, increased signal intensity on T(2) -weighted imaging, and disturbed internal architecture. The histopathologic diagnosis of HS is usually straightforward, with neuronal loss and chronic fibrillary gliosis centered on the pyramidal cell layer. There are several patterns or subtypes of HS recognized from surgical series based on qualitative or quantified assessments of regional neuronal loss. The pathologic changes of HS include granule cell dispersion, mossy fiber sprouting, and alterations to interneurons. There may also be more extensive sclerosis of adjacent structures in the medial temporal lobe, including the amygdala and parahippocampal gyrus. Subtle cortical neuropathologies may accompany HS. The revised classification of dysplasias in epilepsy denotes these as focal cortical dysplasias type IIIa. Sometimes, HS occurs with a second lesion, either in the temporal lobe or extratemporal, most often ipsilateral to the HS. HS on preoperative MRI strongly predicts good seizure outcome following temporal lobe resection (TLR). If adequate MRI shows no structural correlate in patients with MTLE, functional imaging studies are valuable, especially if they are in agreement with ictal electroencephalography (EEG) findings. Focal hypometabolism on 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) ipsilateral to the symptomatic temporal lobe predicts a good surgical outcome; the added value of (11) C-Flumazenil-PET (FMZ-PET) and proton magnetic resonance spectroscopy (MRS) is less clear. Surgical methods have evolved, particularly resecting less tissue, aiming to preserve function without compromising seizure outcome. Around two thirds of patients operated for MTLE with HS obtain seizure freedom. However, the best surgical approach to optimize seizure outcome remains controversial.
Purpose: To study the possible relation between spontaneous recurrent seizures (SRS) and the derangement of cognitive memory.
Methods: Status epilepticus (SE) was induced in adult Long-Evans rats by pilocarpine (320 mg/kg, i.p.) and interrupted after 2 h by clonazepam (CZPs mg/kg, i.p.). In addition to the animals that were given pilocarpine and CZP (group P), two groups received ketamine (100 mg/kg, i.p.): the first group 15 minutes after SE onset (group K15), and the second immediately after the CZP (group K120). Control groups were formed from animals not treated with pilocarpine as well as animals that received pilocarpine but did not develop motor seizures. Spatial cognitive memory was tested in the Morris water maze.
Results: Testing was impossible for more than 6 days after SE in group P. Ketamine shortened this period for the two groups that received it. During the silent period, deteriorated cognitive memory progressively improved, but the performance of group P started to worsen before the appearance of SRS. Group K120 only expressed a tendency toward declining performance, whereas group K15 never developed SRS, and the behavior of these animals did not differ from that of the controls after the postseizure period was over. Histologically, massive hippocampal cell loss was seen in group P. Ketamine protected hippocampal cells in a time-dependent manner; group K15 did not exhibit any obvious necrosis in the hippocampus.
Conclusions: There is no close relation between cognitive functions and the appearance of SRS, because ketamine, administered 120 min after the beginning of SE, prevented the derangment of cognitive functions but not the appearance of SRSs.
Patients with temporal lobe epilepsy are frequently afflicted with psychiatric comorbidity and deficits in spatial and other forms of declarative memory. The relationship between epilepsy and psychopathology is poorly understood, so that systematic research in this area is important. In the present study, we characterized various behaviors and learning and memory in a mouse model in which major aspects of mesial temporal lobe epilepsy can be reproduced. In this model, a single unilateral injection of kainate into the dorsal hippocampus induces a nonconvulsive status epilepticus, followed by development of spontaneous recurrent seizures and ipsilateral lesions of CA1, CA3c and dentate hilus neurons. Unexpectedly, the epileptic mice exhibited only few alterations in a behavioral test battery used to investigate locomotor activity and function, emotionality, depression-related behavior and learning and memory. In contrast to recent experiments with the same test battery in epileptic mice generated by systemic administration of pilocarpine, mice with focal kainate administration did not exhibit reduced explorative behavior or increases of anxiety-related behavior. However, similar to pilocarpine-treated mice, a decrease in depression-like behavior was observed in the forced swimming test. In the Morris water maze test, kainate-treated animals exhibited retarded acquisition and impaired retention of visual-spatial information. Our data suggest that the focal kainate model of mesial temporal lobe epilepsy may contribute to understanding the neurobiological mechanisms underlying the association between epilepsy and behavioral or cognitive alterations.
Basal ganglia are engaged in seizure propagation, control of seizures, and in epilepsy-induced neuroplasticity. Here, we tested the hypothesis that previously observed histological and neurochemical changes in the striatum of amygdala-kindled rats as a model of temporal lobe epilepsy are reflected in alterations of spontaneous striatal firing rates and patterns. Because experimental histological and clinical imaging studies indicated a bilateral involvement of the striatum in epilepsy-induced neuroplasticity, in vivo single-unit recordings were done bilaterally 1 day after a kindled seizure in rats kindled via the right amygdala.Compared to control animals, we observed (1) an increased irregularity of firing of neurons classified as striatal projection neurons and located in the anterior striatum ipsilateral to the kindling side and (2) an increased spontaneous activity of neurons classified as striatal projection neurons and located in the anterior striatum contralateral to the kindling side. These hyperactive neurons were located within the dorsolateral (sensorimotor) subregion of the striatum.The present study represents the first evidence of kindling-induced bilateral changes in electrophysiological properties of striatal neurons and demonstrates that the striatum is strongly affected by the functional reorganization of neurocircuits associated with kindling.The changes are probably caused by a combination of several factors including disturbed bilateral limbic and neocortical input as well as disturbed intrastriatal GABAergic function. The changes reflect a pathophysiological state predisposing the brain to epileptic discharge propagation or else (contralateral striatum) could represent a compensatory network of inhibitory circuits activated to prevent the propagation of seizure activity. The findings are relevant for a better understanding of kindling-induced network changes and might provide new targets for therapeutic manipulations in epilepsies.
Epileptogenesis, i.e., the process leading to epilepsy, is a presumed consequence of brain insults including head trauma, stroke, infections, tumors, status epilepticus (SE), and complex febrile seizures. Typically, brain insults produce morphological and functional alterations in the hippocampal formation, including neurodegeneration in CA1, CA3, and, most consistently, the dentate hilus. Most of these alterations develop gradually, over several days, after the insult, providing a therapeutic window of opportunity for neuroprotective agents in the immediate post-injury period. We have previously reported that prolonged (four weeks) treatment with the antiepileptic drug valproate (VPA) after SE prevents hippocampal damage and most of the behavioral alterations that occur after brain insult, but not the development of spontaneously occurring seizures. These data indicated that VPA, although not preventing epilepsy, might be an effective disease-modifying treatment following brain insult. The present study was designed to (1) determine the therapeutic window for the neuroprotective effect of VPA after SE; (2) compare the efficacy of different intermittent i.p. versus continuous i.v. VPA treatment protocols; and (3) compare VPA with the glutamate (AMPA) receptor antagonist NS1209. As in our previous study with VPA, SE was induced by sustained electrical stimulation of the basolateral amygdala in rats and terminated after 4 h by diazepam. In vehicle controls, >90% of the animals developed significant neurodegeneration in the dentate hilus, whereas damage in CA1 and CA3 was more variable. Hilar parvalbumin-expressing interneurons were more sensitive to the effects of seizures than somatostatin-stained hilar interneurons or hilar mossy cells. Among the various VPA treatment protocols, continuous infusion of VPA for 24 immediately following the SE was the most effective neuroprotective treatment, preventing most of the neuronal damage. Infusion with NS1209 for 24 h exhibited similar neuroprotective efficacy. These data demonstrate that short treatment after SE with either VPA or NS1209 is powerfully neuroprotective, and may be disease-modifying treatments following brain insult.
Prevention of epileptogenesis is an unmet need in medicine. During the last 3 years, however, several preclinical studies have demonstrated remarkable favorable effects of novel treatments on genetic and acquired epileptogenesis. These include the use of immunosuppressants and treatments that modify cellular adhesion, proliferation, and/or plasticity. In addition, the use of antiepileptic drugs in rats with genetic epilepsy or proconvulsants in acquired epilepsy models has provided somewhat unexpected favorable effects. This review summarizes these studies, and introduces some caveats when interpreting the data. In particular, the effect of genetic background, the severity of epileptogenic insult, the method and duration of seizure monitoring, and size of animal population are discussed. Furthermore, a novel scheme for defining epileptogenesis-related terms is presented.
Daily electrical stimulations of the amygdala and hippocampus at intensities sufficient to evoke after-discharges (ADs) resulted in the development of motor seizures, which could not initially be evoked by these stimulations. The triggering of ADs was critical for this development, as well as for the development of permanent changes in the characteristics of the AD. The wave form of the AD "spikes" became more complex. The frequency of these spikes and the duration of AD increased. The amplitude of the AD spikes increased in the structure stimulated as well as in secondary structures to which the AD was "projected". This increase in amplitude of "projected" spikes often correlated with the appearance of motor seizures. Other electrographic developments are discussed including the appearance of spontaneous "inter-ictal" spiking in the amygdala. It was found that the development of motor seizures by stimulation of the amygdala resulted in an increased ability of the contralateral amygdala, and the septal area, but not of the hippocampus, to drive motor seizures when stimulated ("transfer"). Motor seizure development in the hippocampus transferred to the contralateral hippocampus. These developments were shown, by means of control subjects, with lesions in the primary focus to involve changes outside the primary focus. The implications of these developments with respect to seizure development are discussed.
To assess the pharmacology of perampanel and its antiseizure activity in preclinical models. Perampanel [2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl) benzonitrile] is a novel, orally active, prospective antiepileptic agent currently in development for refractory partial-onset seizures.
Perampanel pharmacology was assessed by examining changes in intracellular free Ca(2+) ion concentration ([Ca(2+) ](i) ) in primary rat cortical neurones, and [(3) H]perampanel binding to rat forebrain membranes. Antiseizure activity of orally administered perampanel was examined in amygdala-kindled rats and in mice exhibiting audiogenic, maximal electroshock (MES)-induced, pentylenetetrazole (PTZ) -induced, or 6 Hz-induced seizures.
In cultured rat cortical neurones, perampanel inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced increases in [Ca(2+) ](i) (IC(50) 93 nm vs. 2 μm AMPA). Perampanel had a minimal effect on N-methyl-d-aspartate (NMDA)-induced increases in [Ca(2+) ](i) , and only at a high concentration (30 μm). [(3) H]Perampanel binding to rat forebrain membranes was not significantly displaced by glutamate or AMPA but was displaced by the noncompetitive AMPA receptor antagonists CP465022 (K(i) 11.2 ± 0.8 nm) and GYKI52466 (K(i) 12.4 ± 1 μm). In mice, perampanel showed protective effects against audiogenic, MES-induced, and PTZ-induced seizures (ED(50) s 0.47, 1.6, and 0.94 mg/kg, respectively). Perampanel also inhibited 6 Hz electroshock-induced seizures when administered alone or in combination with other antiepileptic drugs (AEDs). In amygdala-kindled rats, perampanel significantly increased afterdischarge threshold (p<0.05 vs. vehicle), and significantly reduced motor seizure duration, afterdischarge duration, and seizure severity recorded at 50% higher intensity than afterdischarge threshold current (p<0.05 for all measures vs. vehicle). Perampanel caused dose-dependent motor impairment in both mice (TD(50) 1.8 mg/kg) and rats (TD(50) 9.14 mg/kg), as determined by rotarod tests. In mice, the protective index (TD(50) in rotarod test/ED(50) in seizure test) was 1.1, 3.8, and 1.9 for MES-induced, audiogenic, and PTZ-induced seizures, respectively. In rat, dog, and monkey, perampanel had a half-life of 1.67, 5.34, and 7.55 h and bioavailability of 46.1%, 53.5%, and 74.5%, respectively.
These data suggest that perampanel is an orally active, noncompetitive, selective AMPA receptor antagonist with potential as a broad spectrum antiepileptic agent.
Prevention of epileptogenesis after brain trauma is an unmet medical challenge. Recent molecular profiling studies have provided an insight into molecular changes that contribute to formation of ictogenic neuronal networks, including genes regulating synaptic or neuronal plasticity, cell death, proliferation, and inflammatory or immune responses. These mechanisms have been targeted to prevent epileptogenesis in animal models. Favourable effects have been obtained using immunosuppressants, antibodies blocking adhesion of leucocytes to endothelial cells, gene therapy driving expression of neurotrophic factors, pharmacological neurostimulation, or even with conventional antiepileptic drugs by administering them before the appearance of genetic epilepsy. Further studies are needed to clarify the optimum time window and aetiological specificity of treatments. Questions related to adverse events also need further consideration. Encouragingly, the recent experimental studies emphasise that the complicated process of epileptogenesis can be favourably modified, and that antiepileptogenesis as a treatment indication might not be an impossible mission.
Mesio-temporal lobe epilepsy (MTLE), the most common drug-resistant epilepsy syndrome, is characterized by the recurrence of spontaneous focal seizures after a latent period that follows, in most patients, an initial insult during early childhood. Many of the mechanisms that have been associated with the pathophysiology of MTLE are known to be regulated by brain-derived neurotrophic factor (BDNF) in the healthy brain and an excess of this neurotrophin could therefore play a critical role in MTLE development. However, such a function remains controversial as other studies revealed that BDNF could, on the contrary, exert protective effects regarding epilepsy development. In the present study, we further addressed the role of increased BDNF/TrkB signaling on the progressive development of hippocampal seizures in the mouse model of MTLE obtained by intrahippocampal injection of kainate. We show that hippocampal seizures progressively developed in the injected hippocampus during the first two weeks following kainate treatment, within the same time-frame as a long-lasting and significant increase of BDNF expression in dentate granule cells. To determine whether such a BDNF increase could influence hippocampal epileptogenesis via its TrkB receptors, we examined the consequences of (i) increased or (ii) decreased TrkB signaling on epileptogenesis, in transgenic mice overexpressing the (i) TrkB full-length or (ii) truncated TrkB-T1 receptors of BDNF. Epileptogenesis was significantly facilitated in mice with increased TrkB signaling but delayed in mutants with reduced TrkB signaling. In contrast, TrkB signaling did not influence granule cell dispersion, an important feature of this mouse model which is also observed in most MTLE patients. These results suggest that an increase in TrkB signaling, mediated by a long-lasting BDNF overexpression in the hippocampus, promotes epileptogenesis in MTLE.
Diverse brain insults, including traumatic brain injury, stroke, infections, tumors, neurodegenerative diseases, and prolonged acute symptomatic seizures, such as complex febrile seizures or status epilepticus (SE), can induce "epileptogenesis," a process by which normal brain tissue is transformed into tissue capable of generating spontaneous recurrent seizures. Furthermore, epileptogenesis operates in cryptogenic causes of epilepsy. In view of the accumulating information about cellular and molecular mechanisms of epileptogenesis, it should be possible to intervene in this process before the onset of seizures and thereby either prevent the development of epilepsy in patients at risk or increase the potential for better long-term outcome, which constitutes a major clinical need. For identifying pharmacological interventions that prevent, interrupt or reverse the epileptogenic process in people at risk, two groups of animal models, kindling and SE-induced recurrent seizures, have been recommended as potentially useful tools. Furthermore, genetic rodent models of epileptogenesis are increasingly used in assessing antiepileptogenic treatments. Two approaches have been used in these different model categories: screening of clinically established antiepileptic drugs (AEDs) for antiepileptogenic or disease-modifying potential, and targeting the key causal mechanisms that underlie epileptogenesis. The first approach indicated that among various AEDs, topiramate, levetiracetam, carisbamate, and valproate may be the most promising. On the basis of these experimental findings, two ongoing clinical trials will address the antiepileptogenic potential of topiramate and levetiracetam in patients with traumatic brain injury, hopefully translating laboratory discoveries into successful therapies. The second approach has highlighted neurodegeneration, inflammation and up-regulation of immune responses, and neuronal hyperexcitability as potential targets for antiepileptogenesis or disease modification. This article reviews these areas of progress and discusses the challenges associated with discovery of antiepileptogenic therapies.
A variety of acute brain insults bear the risk of subsequent development of chronic epilepsy. Enhanced understanding of the brain alterations underlying this process may ultimately lead to interventions that prevent, interrupt or reverse epileptogenesis in people at risk. Various interventions have been evaluated in rat models of symptomatic epilepsy, in which epileptogenesis was induced by status epilepticus (SE) or traumatic brain injury (TBI). Paradoxically, recent data indicated that administration of proconvulsant drugs after TBI or SE exerts antiepileptogenic or disease-modifying effects, although epilepsy is often considered to represent a decrease in seizure threshold. Surprisingly, to our knowledge, it is not known whether alterations in seizure threshold occur during the latent period following SE. This prompted us to study seizure threshold during and after the latent period following SE induced by lithium/pilocarpine in rats. Timed intravenous infusion of the GABA(A) receptor antagonist pentylenetetrazole (PTZ) was used for this purpose. The duration of the latent period was determined by continuous video/EEG monitoring. Compared to control seizure threshold determined before SE, threshold significantly decreased two days after SE, but returned to pre-SE control thereafter. Moreover, the duration of PTZ-induced seizures was significantly increased throughout the latent period, which ranged from 6 to 10 days after SE. This increased susceptibility to PTZ likely reflects the complex alterations in GABA-mediated transmission that occur during the latent period following SE. The data will allow developing dosing regimens for evaluation of whether treatment with subconvulsant doses of PTZ during the latent period affects the development of epilepsy.
Cell damage and spatial localization deficits are often reported as long-term consequences of pilocarpine-induced status epilepticus. In this study, we investigated the neuroprotective effects of repeated drug administration after long-lasting status epilepticus. Groups of six to eight Wistar rats received microinjections of pilocarpine (2.4 mg/microl, 1 microl) in the right dorsal hippocampus to induce a status epilepticus, which was attenuated by thiopental injection (35 mg/kg, i.p.) 3 hrs after onset. Treatments consisted of i.p. administration of diazepam, ketamine, carbamazepine, or phenytoin at 4, 28, 52, and 76 hr after the onset of status epilepticus. Two days after the treatments, rats were tested in the Morris water maze and 1 week after the cognitive tests, their brains were submitted to histology to perform haematoxylin and eosin staining and glial fibrillary acidic protein (GFAP) immunofluorescence detection. Post-status epilepticus rats exhibited extensive gliosis and cell loss in the hippocampal CA1, CA3 (70% cell loss for both areas) and dentate gyrus (60%). Administration of all drugs reduced cell loss in the hippocampus, with best effects observed in brains slices of diazepam-treated animals, which showed less than 30% of loss in the three areas and decreased GFAP immunolabelling. Treatments improved spatial navigation during training trials and probe trial, with exception of ketamine. Interestingly, in the probe trial, only diazepam-treated animals showed preference for the goal quadrant. Our data point to significant neuroprotective effects of repeated administration of diazepam against status epilepticus-induced cell damage and cognitive disturbances.
Epileptogenesis refers to a process in which an initial brain-damaging insult triggers a cascade of molecular and cellular changes that eventually lead to the occurrence of spontaneous seizures. Cellular alterations include neurodegeneration, neurogenesis, axonal sprouting, axonal injury, dendritic remodeling, gliosis, invasion of inflammatory cells, angiogenesis, alterations in extracellular matrix, and acquired channelopathies. Large-scale molecular profiling of epileptogenic tissue has provided information about the molecular pathways that can initiate and maintain cellular alterations. Currently we are learning how these pathways contribute to postinjury epileptogenesis and recovery process and whether they could be used as treatment targets.
RÉSUMÉ
Malgré les besoins indiscutables en matiére de thérapies nouvelles, l'évaluation de la valeur commerciale d'un nouveau medicament anti‐epileptique risque d'etre defavorisee aujourd'hui si elle est entreprise par une firme pharmaceutique dépourvue de moyens suffisants. Au cours des 10 dernieres annees, I'Epilepsy Branch (du programme des maladies neurologiques des Instituts Nationaux de la Sante aux U.S.A.) a essays de contribuer aux expertises scientifiques et aux subventions financieres concernant cette evaluation. Le programme pour le developpement des medicaments antiepileptiques de I'Epilepsy Branch a, non seulement aide a com‐mercialiser les trois medicaments antiepileptiques les plus nouveaux, mais il a provoque un renouvellement d'intérêt sur l''épilepsie et les medicaments qui la traitent.
RESUMEN
A pesar de la indudable necesidad de terapeiiticas nuevas, es muy probable que la estimación del valor comercial de una nueva droga antiepileptica no fuera favorable si su desarrollo se llevara a cabo por firmas farmaceuticas que operan sin un soporte comple‐mentario. Durante los liltimos 10 anos, la Epilepsy Branch ha intentado contribuir con los necesarios soporte econdmico y experiencia cientifica, para alterar la afirmación previa. El Programa de Desarrolo de Drogas Antiepildpticas ha servido no solo para presentar en el mercado las tres drogas antiepilépticas mas recientes sino tambidn para renovar el interds en la epilepsia y en las drogas que la tratan.
ZUSAMMENFASSUNG
Trotz der eigentlich unbezweifelten Notwendigkeit, neue Therapiemöglichkeiten zu finden, wirkt sich die Feststellung des kommerziellen Wertes neuer Antiepileptica heutzutage wahrscheinlich dann ungunstig aus, wenn die Entwicklung von einer pharmazeutischen Firma alleine ohne Unterstiitzung vorgenommen wird. Wahrend der letzten 10 Jahre hat die Sektion Epilepsie versucht, wissenschaftliche Gutachten und finanzielle Unterstiitzung beizusteuern, um eine Anderung dieser Sachlage herbeizufiihren. Das Programm zur Entwicklung antiepileptischer Medikamente hat nicht nur dazu beigetragen, die 3 neuesten Antiepileptica auf den Markt zu bringen sondern es hat auch zu einem neuerlichen Interesse an der Epilepsie und an den Medikamenten gefiihrt, die zu ihrer Behandlung notwendig sind.
Outside the nervous system myelomonocytic cells are known to play an important role in the inflammatory response and tissue repair after injury. In this study we have examined the myelomonocytic response to neuronal destruction following unilateral injection of the excitotoxin kainic acid into the mouse hippocampus. Intrahippocampal injection of kainate induces rapid, synchronous neuronal death. There is no neutrophil recruitment and a delay of at least 48 h before macrophage-microglial cell numbers increase. The microglial reaction in the injected hippocampus consists of altered morphology, a 6-9-fold increase in mononuclear phagocyte cell numbers and enhanced expression of the macrophage-specific plasma membrane antigen, F4/80, assessed immunohistochemically and by Western blotting. Microglia also respond at distant sites related to the projection pathway and terminals of killed pyramidal cells but the reaction varies in cell numbers, kinetics and morphology. The absence of neutrophil recruitment and the delay in an increase in macrophage or microglial cells shows that the CNS differs from other sites in the body with regard to the kinetics and nature of the myelomonocytic cell inflammatory response. The role of mononuclear phagocytes in tissue repair in the CNS remains to be defined.
Although seizure models using electrical stimulation for the induction of generalized tonic-clonic seizures in rodents are widely employed to identify potential anticonvulsants, the important role of various technical, biological and pharmacological factors in the interpretation of results obtained with these models is often not recognized. The aim of this study was to delineate factors other than sex, age, diet, climate and circadian rhythms, which are generally known. For this purpose, experiments with 8 clinically established antiepileptic drugs were undertaken in the following electroshock seizure models: (1) the maximal (tonic extensor) electroshock seizure threshold (MEST) in mice; (2) the traditional maximal electroshock seizure (MES) test with supra-threshold stimulation in mice; and (3) the MES test with suprathreshold stimulation in rats. When drugs were dissolved in vehicles which did not themselves exert effects on seizure susceptibility, the most important factors which influenced drug potencies were (1) marked differences between drugs and species in terms of peak drug effect, duration of action and the formation of active metabolites; (2) differences in drug potencies calculated on the basis of administered doses compared to potency calculations based on active drug concentrations; (3) the equipment used for seizure induction; (4) marked effects of current strength on results obtained in electroshock seizure models; (5) site of application of the electrical stimulus (transcorneal vs. transauricular). In order to reduce the variability among estimates of anticonvulsant activity, the various factors delineated in this study should be rigidly controlled in experimental situations involving assay of anticonvulsant agents.
The ability of three derivatives of folic acid, N-5-methyltetrahydrofolic acid (MTHF), tetrahydrofolic acid (THF) and dihydrofolic acid (DHF) to mimic the actions of kainic acid (KA) in a number of in vitro and in vivo systems known to be sensitive to KA was examined. None of the three folate derivatives at 100 microM concentration significantly inhibited the specific binding of [3H]-KA to striatal membranes although 2 microM L-glutamate produced a 40% inhibition. None of the three folate derivatives stimulated the formation of cyclic GMP in cerebellar slices incubated in vitro although KA (0.5 mM) increased cyclic GMP levels by 2.5-fold. Whereas intrahippocampal injection of 2.3 nmoles of KA produces prolonged abnormalities of the EEG, limbic-type seizures and a characteristic pattern of neuronal degeneration in the hippocampal formation and related structures, intrahippocampal injection of a 100-fold greater dose of THF caused only minor and transient EEG abnormalities, no overt seizures and a highly restricted lesion. Whereas intrastriatal injection of 5.6 nmoles of KA caused a profound reduction in the specific activities of choline acetyltransferase and glutamate decarboxylase, markers for striatal intrinsic cholinergic and GABAergic neurons, 50-fold greater doses of MTHF did not affect either enzyme although this high dose of THF did cause a significant 33% reduction in choline acetyltransferase activity. These findings support the suggestion that THF may have weak neurotoxic effects in brain but indicate that the actions of this compound and the related MTHF and DHF are not mediated through KA-specific receptors.
Intraperitoneal or intrahippocampal injections of kainate induce both hippocampal cell death and axonal remodeling of the dentate gyrus granular neurons. We report here that injection of kainate into the dorsal hippocampus of adult mice may also trigger a conspicuous and long-lasting global trophic response of granule cells. Morphological changes include somatic and dendritic growth and increased nuclear volume with ultrastructural features characteristic of neuronal development. The trophic response is correlated with a specific overexpression of brain-derived neurotrophic factor that is maintained for at least six months. This shows that plasticity in adult neurons can, in addition to axonal remodeling, extend to generalized cell growth. Our results further suggest that brain-derived neurotrophic factor could be involved in the activation and/or maintenance of this phenomenon.
Excitatory amino acid transmitters are involved in the initiation of seizures and their propagation. Most attention has been directed to synapses using N-methyl-D-aspartate (NMDA) receptors, although more recent evidence indicates potential roles for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors as well. In the present experiments in amygdala-kindled rats, i.e. a model of partial epilepsy, competitive and uncompetitive NMDA antagonists exerted only weak anticonvulsant effects, whereas the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX) potently increased focal seizure threshold and inhibited seizure spread from the focus. These effects of NBQX were dramatically increased by pretreatment with low doses of NMDA antagonists, whereas adverse effects of NBQX were not potentiated. These data suggest that both non-NMDA and NMDA receptors are critically involved in the kindled state, and that combinations of AMPA and NMDA receptor antagonists provide a new strategy for treatment of epileptic seizures.
NBQX [6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione] has proven effective in protecting against cerebral ischemic insult in rodents. The preclinical development included pharmacokinetic and toxicological investigations in mice, rats, and dogs. For these purposes, NBQX was given as an intravenous bolus dose (in mice, rats, and dogs) or as a constant infusion for up to 4 weeks in rats and dogs. In NMRI mice t1/2, CL, and V2 were 1-4 hr, 0.6-1 liter/kg/hr, and 1-4 liters/kg following 3, 10, or 30 mg/kg. In Wistar and Sprague-Dawley rats, the mean +/- SD values of t1/2, CL, and Vz were 0.8 +/- 0.35 hr, 3.2 +/- 1.0 liters/kg/hr, and 4.0 +/- 1.1 liters/kg, respectively. About 33 +/- 5.2% of the dose was excreted unchanged in urine. The CLR was 0.90 +/- 0.20 liter/kg/hr. The pH of the urine samples ranged from pH 6.2 to 8.8, with a mean of 7.9 +/- 0.72. The plasma concentrations were proportional to the dose rate in the dose range 0.3-10 mg/kg/hr, independent of sex, and did not change during 4 weeks of infusion. CL and CLR were decreased to half their value when NBQX was administered in combination with probenecid. In beagle dogs, t1/2 and Vz were 1-3 hr and 1-3 liters/kg, respectively. The CL was determined to be 1.5 +/- 0.4 liters/kg/hr (N = 18) following 2 days of infusion (0.2-1 mg/kg/hr), but after 1 month CL had decreased significantly (p < 0.0001) to 1.0 +/- 0.1 liter/kg/hr.(ABSTRACT TRUNCATED AT 250 WORDS)
We investigated the anticonvulsant and adverse effects of various dose combinations of the AMPA receptor antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline) and the low-affinity, rapidly channel blocking NMDA receptor antagonist memantine in the kindling model of epilepsy. While memantine was ineffective when given alone, co-administration with NBQX markedly potentiated the increase in focal seizure threshold induced by NBQX alone. This synergistic (i.e. over-additive) interaction was seen at doses of both drugs which did not induce behavioural adverse effects. The data substantiate that combinations of AMPA and NMDA receptor antagonists provide a new strategy for the treatment of epileptic seizures.
Glutamate is the principal excitatory neurotransmitter in the brain and, as such, it inevitably plays a role in the initiation and spread of seizure activity. It also plays a critical role in epileptogenesis. The process of "kindling" limbic seizures in rodents by repeated electrical stimulation is dependent on activation of N-methyl-D-aspartate (NMDA) receptors. The function of these receptors is enhanced in the hippocampus of kindled rats and in the cerebral cortex of patients with focal epilepsy. Microdialysis studies show an increase in the extracellular concentration of glutamate and aspartate before or during seizure onset, suggesting that either enhanced amino acid release or impaired uptake contributes to seizure initiation. Glutamate antagonists selective for NMDA or non-NMDA receptors are potent anticonvulsants when given systemically in a wide variety of animal models of epilepsy. They are of limited efficacy against kindled seizures in rats and (on the basis of preliminary evidence) in patients with drug-refractory complex partial seizures. Cognitive side effects appear to be a significant problem with competitive, as well as noncompetitive, NMDA antagonists. Glutamate receptor antagonists provide significant protection against brain damage following global or focal cerebral ischemia or acute traumatic injury in rodent models. Anticonvulsant compounds of the lamotrigine type, which act on sodium channels and reduce ischemia-induced glutamate release, are cerebroprotective in rodent ischemia models and are free from the cognitive side effects of NMDA-receptor antagonists.
A competitive (NBQX) and a non-competitive (GYKI 52466) AMPA antagonist, and a competitive NMDA antagonist (D-CPPene) were tested against the development of kindling and against fully kindled seizures in amygdala-kindled rats. GYKI 52466, 10 mg/kg given i.p. 5 min prior to electrical stimulation in fully kindled animals, reduces both the cortical after-discharge duration and the behavioural seizure score. GYKI 52466, 20 mg/kg, reduces seizure score and after-discharge duration significantly (after 5-30 min) but the animals show severe motor side effects and an irregular cortical and hippocampal EEG. Administration of GYKI 52466, 10 mg/kg, prior to kindling stimulation on days 3-8, does not slow the development of kindling. NBQX, 20 mg/kg or 40 mg/kg i.p., 30 min prior to stimulation, significantly reduces the seizure score in fully kindled animals. NBQX 20 mg/kg i.p. has no effect on the development of kindling. D-CPPene, 8 mg/kg or 12 mg/kg, 120 min prior to stimulation reduces the behavioural seizure score in fully kindled animals. D-CPPene, 8 mg/kg on days 3-8, delays the development of kindling. NMDA receptors play a key role in the kindling process. Expression of kindled seizures involves non-NMDA and NMDA receptors.
To investigate the role of non-NMDA receptors in epileptic seizures, we examined the antiepileptogenic and anticonvulsant effects of NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline), a potent and selective AMPA receptor antagonist, in the rat kindling model. Systemic administration of 10-40 mg/kg NBQX significantly and dose dependently suppressed previously kindled seizures from the amygdala (AM), assessed in terms of the motor seizure stage and afterdischarge (AD) duration. The maximal effects were observed at 0.5-1 h after drug injection. When the intensity of electrical stimulation was increased to twice the generalized seizure-triggering threshold (GST), the anticonvulsant effects of NBQX on AM-kindled seizures were not reversed, suggesting that the effects were not due to non-specific elevation of the GST. In contrast to AM-kindled seizures, 20-40 mg/kg NBQX significantly suppressed only the motor seizure stage without reducing the AD duration of previously hippocampal-kindled seizures. Daily administration of 15 or 30 mg/kg NBQX prior to each electrical stimulation of the AM markedly and significantly suppressed the development of kindling. During drug sessions, the growth of the AD duration was blocked almost completely, while the waveform of ADs became more complex. These results indicate that NBQX has potent antiepileptogenic and anticonvulsant actions on kindling, at least from the AM and that non-NMDA receptors have an important role in seizure propagation.
It is widely accepted that excitatory amino acid transmitters such as glutamate are involved in the initiation of seizures and their propagation. Most attention has been directed to synapses using NMDA receptors, but more recent evidence indicates potential roles for ionotropic non-NMDA (AMPA/kainate) and metabotropic glutamate receptors as well. Based on the role of glutamate in the development and expression of seizures, antagonism of glutamate receptors has long been thought to provide a rational strategy in the search for new, effective anticonvulsant drugs. Furthermore, because glutamate receptor antagonists, particularly those acting on NMDA receptors, protect effectively in the induction of kindling, it was suggested that they may have utility in epilepsy prophylaxis, for example, after head trauma. However, first clinical trials with competitive and uncompetitive NMDA receptor antagonists in patients with partial (focal) seizures, showed that these drugs lack convincing anticonvulsant activity but induce severe neurotoxic adverse effects in doses which were well tolerated in healthy volunteers. Interestingly, the only animal model which predicted the unfavorable clinical activity of competitive NMDA antagonists in patients with chronic epilepsy was the kindling model of temporal lobe epilepsy, indicating that this model should be used in the search for more effective and less toxic glutamate receptor antagonists. In this review, results from a large series of experiments on different categories of glutamate receptor antagonists in fully kindled rats are summarized and discussed. NMDA antagonists, irrespective whether they are competitive, high- or low-affinity uncompetitive, glycine site or polyamine site antagonists, do not counteract focal seizure activity and only weakly, if at all, attenuate propagation to secondarily generalized seizures in this model, indicating that once kindling is established, NMDA receptors are not critical for the expression of fully kindled seizures. In contrast, ionotropic non-NMDA receptor antagonists exert potent anticonvulsant effects on both initiation and propagation of kindled seizures. This effect can be markedly potentiated by combination with low doses of NMDA antagonists, suggesting that an optimal treatment of focal and secondarily generalized seizures may require combined use of both non-NMDA and NMDA antagonists. Given the promising results obtained with novel AMPA/kainate antagonists and glycine/NMDA partial agonists in the kindling model, the hope for soon having potentially useful glutamate antagonists for use in epileptic patients is increasing.
Although the paradoxical ability of antiepileptic drugs (AEDs) to increase seizure activity has been recognized for decades, the underlying mechanisms are poorly understood and few systematic studies have addressed this problem. This article is intended to provide a critical review of available literature on this topic.
Information was collected by means of computerized literature searches, screening of journals and textbooks, and consultation with colleagues. Mechanisms which potentially might precipitate underlying drug-induced exacerbation of seizures were considered based on available pharmacologic and clinical knowledge.
The reviewed information suggests that a paradoxical increase in seizure frequency may occur as a result of at least two separate mechanisms. The first appears to involve a nonspecific manifestation of drug intoxication; seizure-worsening in this context is usually reversible by dosage reduction or elimination of unnecessary polypharmacy. Conversely, the other mechanism may involve a distinct adverse primary action of the drug in specific seizure types or in syndromic forms. Carabamazepine, in particular, has been reported to precipitate or exacerbate a variety of seizures, most notably absence, atonic, or myoclonic seizures in patients with generalized epilepsies characterized by bursts of diffuse and bilaterally synchronous spike-and-wave EEG activity. Phenytoin and vigabatrin also have been implicated in worsening of seizures, particularly generalized seizures, whereas gabapentin has been associated repeatedly with precipitation of myoclonic jerks. Benzodiazepines occasionally have been reported to precipitate tonic seizures, especially when given intravenously to control other seizure types in patients with Lennox-Gastaut syndrome. Seizure deterioration has been reported also with other drugs; though in most cases evidence is still insufficient for meaningful conclusions to be drawn.
Drug-induced exacerbation of seizures is a serious and common clinical problem that is often unrecognized or overlooked by the treating physician. Its occurrence appears to be related to three possible causes: an incorrect diagnosis of seizure type or syndromic form, lack of knowledge about certain drugs that are contraindicated in specific types of epilepsies, or to prescription of excessive drug dosages and drug combinations. Further studies are required to evaluate the prevalence of this phenomenon of drug-induced exacerbation of seizures, to investigate its mechanisms in greater detail and to characterize additional prognostic factors that may be used for early identification of patients at risk.
Human mesial temporal lobe epilepsy is characterized by hippocampal seizures associated with pyramidal cell loss in the hippocampus and dispersion of dentate gyrus granule cells. A similar histological pattern was recently described in a model of extensive neuroplasticity in adult mice after injection of kainate into the dorsal hippocampus [Suzuki et al. (1995) Neuroscience 64, 665–674]. The aim of the present study was to determine whether (i) recurrent seizures develop in mice after intrahippocampal injection of kainate, and (ii) the electroencephalographic, histopathological and behavioural changes in such mice are similar to those in human mesial temporal lobe epilepsy. Adult mice receiving a unilateral injection of kainate (0.2 μg; 50 nl) or saline into the dorsal hippocampus displayed recurrent paroxysmal discharges on the electroencephalographic recordings associated with immobility, staring and, occasionally, clonic components. These seizures started immediately after kainate injection and recurred for up to eight months. Epileptiform activities occurred most often during sleep but occasionally while awake. The pattern of seizures did not change over time nor did they secondarily generalize. Glucose metabolic changes assessed by [¹⁴C]2-deoxyglucose autoradiography were restricted to the ipsilateral hippocampus for 30 days, but had spread to the thalamus by 120 days after kainate. Ipsilateral cell loss was prominent in hippocampal pyramidal cells and hilar neurons. An unusual pattern of progressive enlargement of the dentate gyrus was observed with a marked radial dispersion of the granule cells associated with reactive astrocytes. Mossy fibre sprouting occurred both in the supragranular molecular layer and infrapyramidal stratum oriens layer of CA3. The expression of the embryonic form of the neural cell adhesion molecule coincided over time with granule cell dispersion.
To investigate the role of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) type glutamate receptors in epileptic seizures, we examined the antiepileptogenic and anticonvulsant effects of YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3-(1H,4H)-quinoxalinedione hydrochloride], a potent and selective new AMPA receptor antagonist, in the rat amygdala-kindling model of epilepsy. Pretreatment with YM90K (7.5-30 mg/kg i.p.) markedly retarded the evolution of kindling. Once kindling was established, administration of YM90K (7.5-30 mg/kg i.p.) significantly and dose-dependently suppressed fully kindled seizures. The maximal effects were observed 15-30 min after injection. When the intensity of electrical stimulation was increased to twice the generalized seizure-triggering threshold, the anticonvulsant effects of YM90K were reversed, suggesting that they were due to elevation of the generalized seizure-triggering threshold. Furthermore, an anticonvulsant dose (15 mg/kg) of YM90K affected neither field potentials nor long-term potentiation in the hippocampus in vivo. These results indicate that AMPA receptors play an important role in the seizure expression mechanism and the development of kindling-induced epileptogenesis, and suggest the possible clinical usefulness of AMPA receptor antagonists as antiepileptic drugs.
To study the possible relation between spontaneous recurrent seizures (SRS) and the derangement of cognitive memory.
Status epilepticus (SE) was induced in adult Long-Evans rats by pilocarpine (320 mg/kg, i.p.) and interrupted after 2 h by clonazepam (CZPs mg/kg, i.p.). In addition to the animals that were given pilocarpine and CZP (group P), two groups received ketamine (100 mg/kg, i.p.): the first group 15 minutes after SE onset (group K15), and the second immediately after the CZP (group K120). Control groups were formed from animals not treated with pilocarpine as well as animals that received pilocarpine but did not develop motor seizures. Spatial cognitive memory was tested in the Morris water maze.
Testing was impossible for more than 6 days after SE in group P. Ketamine shortened this period for the two groups that received it. During the silent period, deteriorated cognitive memory progressively improved, but the performance of group P started to worsen before the appearance of SRS. Group K120 only expressed a tendency toward declining performance, whereas group K15 never developed SRS, and the behavior of these animals did not differ from that of the controls after the postseizure period was over. Histologically, massive hippocampal cell loss was seen in group P. Ketamine protected hippocampal cells in a time-dependent manner; group K15 did not exhibit any obvious necrosis in the hippocampus.
There is no close relation between cognitive functions and the appearance of SRS, because ketamine, administered 120 min after the beginning of SE, prevented the derangment of cognitive functions but not the appearance of SRSs.