Deletion of the Na-K-2Cl cotransporter NKCC1 results in a more severe epileptic phenotype in the intrahippocampal kainate mouse model of temporal lobe epilepsy.

Increased neuronal expression of the Na-K-2Cl cotransporter NKCC1 has been implicated in the generation of seizures and epilepsy. However, conclusions from studies on the NKCC1-specific inhibitor, bumetanide, are equivocal, which is a consequence of the multiple potential cellular targets and poor brain penetration of this drug. Here, we used Nkcc1 knockout (KO) and wildtype (WT) littermate control mice to study the ictogenic and epileptogenic effects of intrahippocampal injection of kainate. Kainate (0.23 µg in 50 nl) induced limbic status epilepticus (SE) in both KO and WT mice with similar incidence, latency to SE onset, and SE duration, but the number of intermittent generalized convulsive seizures during SE was significantly higher in Nkcc1 KO mice, indicating increased SE severity. Following SE, spontaneous recurrent seizures (SRS) were recorded by continuous (24/7) video/EEG monitoring at 0-1, 4-5, and 12-13 weeks after kainate, using depth electrodes in the ipsilateral hippocampus. Latency to onset of electrographic SRS and the incidence of electrographic SRS were similar in WT and KO mice. However, the frequency of electrographic seizures was lower whereas the frequency of electroclinical seizures was higher in Nkcc1 KO mice, indicating a facilitated progression from electrographic to electroclinical seizures during chronic epilepsy, and a more severe epileptic phenotype, in the absence of NKCC1. The present findings suggest that NKCC1 is dispensable for the induction, progression and manifestation of epilepsy, and they do not support the widely held notion that inhibition of NKCC1 in the brain is a useful strategy for preventing or modifying epilepsy.

Proof-of-concept that network pharmacology is effective to modify development of acquired temporal lobe epilepsy.

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.

Disruption of the sodium-dependent citrate transporter SLC13A5 in mice causes alterations in brain citrate levels and neuronal network excitability in the hippocampus.

In addition to tissues such as liver, the plasma membrane sodium-dependent citrate transporter, NaCT (SLC13A5), is highly expressed in brain neurons, but its function is not understood. Loss-of-function mutations in the human SLC13A5 gene have been associated with severe neonatal encephalopathy and pharmacoresistant seizures. The molecular mechanisms of these neurological alterations are not clear. We performed a detailed examination of a Slc13a5 deletion mouse model including video-EEG monitoring, behavioral tests, and electrophysiologic, proteomic, and metabolomic analyses of brain and cerebrospinal fluid. The experiments revealed an increased propensity for epileptic seizures, proepileptogenic neuronal excitability changes in the hippocampus, and significant citrate alterations in the CSF and brain tissue of Slc13a5 deficient mice, which may underlie the neurological abnormalities. These data demonstrate that SLC13A5 is involved in brain citrate regulation and suggest that abnormalities in this regulation can induce seizures. The present study is the first to (i) establish the Slc13a5-knockout mouse model as a helpful tool to study the neuronal functions of NaCT and characterize the molecular mechanisms by which functional deficiency of this citrate transporter causes epilepsy and impairs neuronal function; (ii) evaluate all hypotheses that have previously been suggested on theoretical grounds to explain the neurological phenotype of SLC13A5 mutations; and (iii) indicate that alterations in brain citrate levels result in neuronal network excitability and increased seizure propensity.

Gene therapy decreases seizures in a model of Incontinentia pigmenti.

OBJECTIVE: Incontinentia pigmenti (IP) is a genetic disease leading to severe neurological symptoms, such as epileptic seizures, but no specific treatment is available. IP is caused by pathogenic variants that inactivate the Nemo gene. Replacing Nemo through gene therapy might provide therapeutic benefits. METHODS: In a mouse model of IP, we administered a single intravenous dose of the adeno-associated virus (AAV) vector, AAV-BR1-CAG-NEMO, delivering the Nemo gene to the brain endothelium. Spontaneous epileptic seizures and the integrity of the blood-brain barrier (BBB) were monitored. RESULTS: The endothelium-targeted gene therapy improved the integrity of the BBB. In parallel, it reduced the incidence of seizures and delayed their occurrence. Neonate mice intravenously injected with the AAV-BR1-CAG-NEMO vector developed no hepatocellular carcinoma or other major adverse effects 11 months after vector injection, demonstrating that the vector has a favorable safety profile. INTERPRETATION: The data show that the BBB is a target of antiepileptic treatment and, more specifically, provide evidence for the therapeutic benefit of a brain endothelial-targeted gene therapy in IP. Ann Neurol 2017;82:93-104.

Isoflurane prevents acquired epilepsy in rat models of temporal lobe epilepsy.

OBJECTIVE: Acquired epilepsy is a devastating long-term risk of various brain insults, including trauma, stroke, infections, and status epilepticus (SE). There is no preventive treatment for patients at risk. Attributable to the complex alterations involved in epileptogenesis, it is likely that multitargeted approaches are required for epilepsy prevention. We report novel preclinical findings with isoflurane, which exerts various nonanesthetic effects that may be relevant for antiepileptogenesis. METHODS: The effects of isoflurane were investigated in two rat models of SE-induced epilepsy: intrahippocampal kainate and systemic administration of paraoxon. Isoflurane was either administered during (kainate) or after (paraoxon) induction of SE. Magnetic resonance imaging was used to assess blood-brain barrier (BBB) dysfunction. Positron emission tomography was used to visualize neuroinflammation. Long-term electrocorticographic recordings were used to monitor spontaneous recurrent seizures. Neuronal damage was assessed histologically. RESULTS: In the absence of isoflurane, spontaneous recurrent seizures were common in the majority of rats in both models. When isoflurane was administered during kainate injection, duration and severity of SE were not affected, but only few rats developed spontaneous recurrent seizures. A similar antiepileptogenic effect was found when paraoxon-treated rats were exposed to isoflurane after SE. Moreover, in the latter model, isoflurane prevented BBB dysfunction and neurodegeneration, whereas isoflurane reduced neuroinflammation in the kainate model. INTERPRETATION: Given that isoflurane is a widely used volatile anesthetic, and is used for inhalational long-term sedation in critically ill patients at risk to develop epilepsy, our findings hold a promising potential to be successfully translated into the clinic. Ann Neurol 2016;80:896-908.

Various modifications of the intrahippocampal kainate model of mesial temporal lobe epilepsy in rats fail to resolve the marked rat-to-mouse differences in type and frequency of spontaneous seizures in this model.

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.

The bumetanide prodrug BUM5, but not bumetanide, potentiates the antiseizure effect of phenobarbital in adult epileptic mice.

OBJECTIVE: The loop diuretic bumetanide has been reported to potentiate the antiseizure activity of phenobarbital in rodent models of neonatal seizures, most likely as a result of inhibition of the chloride importer Na-K-Cl cotransporter isoform 1 (NKCC1) in the brain. In view of the intractability of neonatal seizures, the preclinical findings prompted a clinical trial in neonates on bumetanide as an add-on to phenobarbital, which, however, had to be terminated because of ototoxicity and lack of efficacy. We have recently shown that bumetanide penetrates only poorly into the brain, so that we developed lipophilic prodrugs such as BUM5, the N,N-dimethylaminoethylester of bumetanide, which penetrate more easily into the brain and are converted to bumetanide. METHODS: In the present study, we used a new strategy to test whether BUM5 is more potent than bumetanide in potentiating the antiseizure effect of phenobarbital. Adult mice were made epileptic by pilocarpine, and the antiseizure effects of bumetanide, BUM5, and phenobarbital alone or in combination were determined by the maximal electroshock seizure threshold test. RESULTS: In nonepileptic mice, only phenobarbital exerted seizure threshold-increasing activity, and this was not potentiated by the NKCC1 inhibitors. In contrast, a marked potentiation of phenobarbital by BUM5, but not bumetanide, was determined in epileptic mice. SIGNIFICANCE: Thus, bumetanide is not capable of potentiating phenobarbital's antiseizure effect in an adult mouse model, which, however, can be overcome by using the prodrug BUM5. These data substantiate that BUM5 is a promising tool compound for target validation and proof-of-concept studies on the role of NKCC1 in brain diseases.

Evaluation of the pentylenetetrazole seizure threshold test in epileptic mice as surrogate model for drug testing against pharmacoresistant seizures.

Resistance to antiepileptic drugs (AEDs) is a major problem in epilepsy therapy, so that development of more effective AEDs is an unmet clinical need. Several rat and mouse models of epilepsy with spontaneous difficult-to-treat seizures exist, but because testing of antiseizure drug efficacy is extremely laborious in such models, they are only rarely used in the development of novel AEDs. Recently, the use of acute seizure tests in epileptic rats or mice has been proposed as a novel strategy for evaluating novel AEDs for increased antiseizure efficacy. In the present study, we compared the effects of five AEDs (valproate, phenobarbital, diazepam, lamotrigine, levetiracetam) on the pentylenetetrazole (PTZ) seizure threshold in mice that were made epileptic by pilocarpine. Experiments were started 6 weeks after a pilocarpine-induced status epilepticus. At this time, control seizure threshold was significantly lower in epileptic than in nonepileptic animals. Unexpectedly, only one AED (valproate) was less effective to increase seizure threshold in epileptic vs. nonepileptic mice, and this difference was restricted to doses of 200 and 300 mg/kg, whereas the difference disappeared at 400mg/kg. All other AEDs exerted similar seizure threshold increases in epileptic and nonepileptic mice. Thus, induction of acute seizures with PTZ in mice pretreated with pilocarpine does not provide an effective and valuable surrogate method to screen drugs for antiseizure efficacy in a model of difficult-to-treat chronic epilepsy as previously suggested from experiments with this approach in rats.

Significant effects of sex, strain, and anesthesia in the intrahippocampal kainate mouse model of mesial temporal lobe epilepsy.

The intrahippocampal kainate mouse model of mesial temporal lobe epilepsy is increasingly being used for studies on epileptogenesis and antiepileptogenesis. Almost all previous studies used male mice for this purpose, and no study is available in this or other models of acquired epilepsy that directly compared epileptogenesis in female and male rodents. Epidemiological studies suggest that gender may affect susceptibility to epilepsy and its prognosis; therefore, one goal of this study was to investigate whether sex has an influence on latent period and epileptogenesis in the intrahippocampal kainate model in mice. Another aspect that was examined in the present study was whether mouse strain differences in epileptogenesis exist. Finally, we examined the effects of different types of anesthesia (chloral hydrate, isoflurane) on kainate-induced status epilepticus (SE) and epileptogenesis. Continuous (24/7) video-EEG monitoring was used during SE and the 2 weeks following SE as well as 4-6 weeks after SE. In male NMRI mice with chloral hydrate anesthesia during kainate injection, SE was followed by a seizure-free latent period of 10-14 days if hippocampal paroxysmal discharges (HPDs) recorded from the kainate focus were considered the onset of epilepsy. Anesthesia with isoflurane led to a more rapid onset and higher severity of SE, and not all male NMRI mice exhibited a seizure-free latent period. Female NMRI mice differed from male animals in the lack of any clear latent period, independently of anesthesia type. Furthermore, HPDs were only rarely observed. These problems were not resolved by decreasing the dose of kainate or using other strains (C57BL/6, FVB/N) of female mice. The present data are the first to demonstrate marked sex-related differences in the latent period following brain injury in a rodent model of acquired epilepsy. Furthermore, our data demonstrate that the choice of anesthestic agent during kainate administration affects SE severity and as a consequence, the latent period, which may explain some of the differences reported for this model in the literature.

The pilocarpine model of temporal lobe epilepsy: Marked intrastrain differences in female Sprague-Dawley rats and the effect of estrous cycle.

Rat strains such as Sprague-Dawley (SD) or Wistar are widely used in epilepsy research, including popular models of temporal lobe epilepsy in which spontaneous recurrent seizures (SRS), hippocampal damage, and behavioral alterations develop after status epilepticus (SE). Such rats are randomly outbred, and outbred strains are known to be genetically heterogeneous populations with a high intrastrain variation. Intrastrain differences may be an important reason for discrepancies between studies from different laboratories, but the extent to which such differences affect the development of seizures, neurodegeneration, and psychopathology in post-SE models of epilepsy has received relatively little attention. In the present study, we induced SE by systemic administration of pilocarpine (following pretreatment with lithium) in SD rats from different breeders (Harlan, Charles River [CRL], Taconic) as well as different breeding locations of the same breeder (Harlan-Winkelmann [HW] in Germany vs. Harlan Laboratories [HL] in the Netherlands). Some experiments were also performed in Wistar rats. Pilocarpine was administered by a ramp-up dosing protocol that allows determining interindividual differences in susceptibility to the convulsant. Marked intrastrain differences in induction of SE and its long-term consequences were found. Sprague-Dawley rats from HW were significantly more sensitive to SE induction than all other SD substrains. The majority of SD rats from different vendors developed SRS after SE except SD rats from HL. The CRL-SD rats markedly differed in basal behavior and SE-induced behavioral alterations from other SD substrains. Susceptibility to pilocarpine was hardly affected by the estrous cycle. The marked intrastrain differences provide an interesting tool to study the impact of genetic and environmental factors on seizure susceptibility, epileptogenesis, and relationship between behavior and epilepsy and vice versa.

The search for NKCC1-selective drugs for the treatment of epilepsy: Structure-function relationship of bumetanide and various bumetanide derivatives in inhibiting the human cation-chloride cotransporter NKCC1A.

The Na(+)-K(+)-Cl(-) cotransporter NKCC1 plays a major role in the regulation of intraneuronal Cl(-) concentration. Abnormal functionality of NKCC1 has been implicated in several brain disorders, including epilepsy. Bumetanide is the only available selective NKCC1 inhibitor, but also inhibits NKCC2, which can cause severe adverse effects during treatment of brain disorders. A NKCC1-selective bumetanide derivative would therefore be a desirable option. In the present study, we used the Xenopus oocyte heterologous expression system to compare the effects of bumetanide and several derivatives on the two major human splice variants of NKCCs, hNKCC1A and hNKCC2A. The derivatives were selected from a series of ~5000 3-amino-5-sulfamoylbenzoic acid derivatives, covering a wide range of structural modifications and diuretic potencies. To our knowledge, such structure-function relationships have not been performed before for NKCC1. Half maximal inhibitory concentrations (IC50s) of bumetanide were 0.68 (hNKCC1A) and 4.0µM (hNKCC2A), respectively, indicating that this drug is 6-times more potent to inhibit hNKCC1A than hNKCC2A. Side chain substitutions in the bumetanide molecule variably affected the potency to inhibit hNKCC1A. This allowed defining the minimal structural requirements necessary for ligand interaction. Unexpectedly, only a few of the bumetanide derivatives examined were more potent than bumetanide to inhibit hNKCC1A, and most of them also inhibited hNKCC2A, with a highly significant correlation between IC50s for the two NKCC isoforms. These data indicate that the structural requirements for inhibition of NKCC1 and NKCC2 are similar, which complicates development of bumetanide-related compounds with high selectivity for NKCC1.

Refinement of a model of acquired epilepsy for identification and validation of biomarkers of epileptogenesis in rats.

In rodent models in which status epilepticus (SE) is used to induce epilepsy, typically most animals develop spontaneous recurrent seizures (SRS). The SE duration for induction of epileptogenesis depends on the type of SE induction. In models with electrical SE induction, the minimum duration of SE to induce epileptogenesis in >90% of animals ranges from 3-4h. A high incidence of epilepsy is an advantage in the search of antiepileptogenic treatments, whereas it is a disadvantage in the search for biomarkers of epileptogenesis, because it does not allow a comparison of potential biomarkers in animals that either develop or do not develop epilepsy. The aim of this project was the refinement of an established SE rat model so that only ~50% of the animals develop epilepsy. For this purpose, we used an electrical model of SE induction, in which a self-sustained SE develops after prolonged stimulation of the basolateral amygdala. Previous experiments had shown that the majority of rats develop SRS after 4-h SE in this model so that the SE reduced duration to 2.5h by administering diazepam. This resulted in epilepsy development in only 50% of rats, thus reaching the goal of the project. The latent period to onset of SRS wa s >2weeks in most rats. Development of epilepsy could be predicted in most rats by behavioral hyperexcitability, whereas seizure threshold did not differentiate rats that did and did not develop SRS. The refined SE model may offer a platform to identify and validate biomarkers of epileptogenesis.

Effective termination of status epilepticus by rational polypharmacy in the lithium-pilocarpine model in rats: Window of opportunity to prevent epilepsy and prediction of epilepsy by biomarkers.

The pilocarpine rat model, in which status epilepticus (SE) leads to epilepsy with spontaneous recurrent seizures (SRS), is widely used to study the mechanisms of epileptogenesis and develop strategies for epilepsy prevention. SE is commonly interrupted after 30-90min by high-dose diazepam or other anticonvulsants to reduce mortality. It is widely believed that SE duration of 30-60min is sufficient to induce hippocampal damage and epilepsy. However, resistance to diazepam develops during SE, so that an SE that is longer than 30min is difficult to terminate, and SE typically recurs several hours after diazepam, thus forming a bias for studies on epileptogenesis or antiepileptogenesis. We developed a drug cocktail, consisting of diazepam, phenobarbital, and scopolamine that allows complete and persistent SE termination in the lithium-pilocarpine model. A number of novel findings were obtained with this cocktail. (a) In contrast to previous reports with incomplete SE suppression, a SE of 60min duration did not induce epilepsy, whereas epilepsy with SRS developed after 90 or 120min SE; (b) by comparing groups of rats with 60 and 90min of SE, development of epilepsy could be predicted by behavioral hyperexcitability and decrease in seizure threshold, indicating that these read-outs are suited as biomarkers of epileptogenesis; (c) CA1 damage was prevented by the cocktail, but rats exhibited cell loss in the dentate hilus, which was related to development of epilepsy. These data demonstrate that the duration of SE needed for induction of epileptogenesis in this model is longer than previously thought.

A novel prodrug-based strategy to increase effects of bumetanide in epilepsy.

OBJECTIVE: There is considerable interest in using bumetanide, a chloride importer Na-K-Cl cotransporter antagonist, for treatment of neurological diseases, such as epilepsy or ischemic and traumatic brain injury, that may involve deranged cellular chloride homeostasis. However, bumetanide is heavily bound to plasma proteins (~98%) and highly ionized at physiological pH, so that it only poorly penetrates into the brain, and chronic treatment with bumetanide is compromised by its potent diuretic effect. METHODS: To overcome these problems, we designed lipophilic and uncharged prodrugs of bumetanide that should penetrate the blood-brain barrier more easily than the parent drug and are converted into bumetanide in the brain. The feasibility of this strategy was evaluated in mice and rats. RESULTS: Analysis of bumetanide levels in plasma and brain showed that administration of 2 ester prodrugs of bumetanide, the pivaloyloxymethyl (BUM1) and N,N-dimethylaminoethylester (BUM5), resulted in significantly higher brain levels of bumetanide than administration of the parent drug. BUM5, but not BUM1, was less diuretic than bumetanide, so that BUM5 was further evaluated in chronic models of epilepsy in mice and rats. In the pilocarpine model in mice, BUM5, but not bumetanide, counteracted the alteration in seizure threshold during the latent period. In the kindling model in rats, BUM5 was more efficacious than bumetanide in potentiating the anticonvulsant effect of phenobarbital. INTERPRETATION: Our data demonstrate that the goal of designing bumetanide prodrugs that specifically target the brain is feasible and that such drugs may resolve the problems associated with using bumetanide for treatment of neurological disorders.

Consequences of inhibition of bumetanide metabolism in rodents on brain penetration and effects of bumetanide in chronic models of epilepsy.

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.

The novel antiepileptic drug imepitoin compares favourably to other GABA-mimetic drugs in a seizure threshold model in mice and dogs.

Recently, the imidazolinone derivative imepitoin has been approved for treatment of canine epilepsy. Imepitoin acts as a low-affinity partial agonist at the benzodiazepine (BZD) site of the GABAA receptor and is the first compound with such mechanism that has been developed as an antiepileptic drug (AED). This mechanism offers several advantages compared to full agonists, including less severe adverse effects and a lack of tolerance and dependence liability, which has been demonstrated in rodents, dogs, and nonhuman primates. In clinical trials in epileptic dogs, imepitoin was shown to be an effective and safe AED. Recently, seizures in dogs have been proposed as a translational platform for human therapeutic trials on new epilepsy treatments. In the present study, we compared the anticonvulsant efficacy of imepitoin, phenobarbital and the high-affinity partial BZD agonist abecarnil in the timed i.v. pentylenetetrazole (PTZ) seizure threshold test in dogs and, for comparison, in mice. Furthermore, adverse effects of treatments were compared in both species. All drugs dose-dependently increased the PTZ threshold in both species, but anticonvulsant efficacy was higher in dogs than mice. At the doses selected for this study, imepitoin was slightly less potent than phenobarbital in increasing seizure threshold, but markedly more tolerable in both species. Effective doses of imepitoin in the PTZ seizure model were in the same range as those suppressing spontaneous recurrent seizures in epileptic dogs. The study demonstrates that low-affinity partial agonists at the benzodiazepine site of the GABAA receptor, such as imepitoin, offer advantages as a new category of AEDs.

The anticonvulsant response to valproate in kindled rats is correlated with its effect on neuronal firing in the substantia nigra pars reticulata: a new mechanism of pharmacoresistance.

Resistance to antiepileptic drugs (AEDs) is a major problem in epilepsy treatment. However, mechanisms of resistance are only incompletely understood. We have recently shown that repeated administration of the AED phenytoin allows selecting resistant and responsive rats from the amygdala kindling model of epilepsy, providing a tool to study mechanisms of AED resistance. We now tested whether individual amygdala-kindled rats also differ in their anticonvulsant response to the major AED valproate (VPA) and which mechanism may underlie the different response to VPA. VPA has been proposed to act, at least in part, by reducing spontaneous activity in the substantia nigra pars reticulata (SNr), a main basal ganglia output structure involved in seizure propagation, seizure control, and epilepsy-induced neuroplasticity. Thus, we evaluated whether poor anticonvulsant response to VPA is correlated with low efficacy of VPA on SNr firing rate and pattern in kindled rats. We found (1) that good and poor VPA responders can be selected in kindled rats by repeatedly determining the effect of VPA on the electrographic seizure threshold, and (2) a significant correlation between the anticonvulsant response to VPA in kindled rats and its effect on SNr firing rate and pattern. The less VPA was able to raise seizure threshold, the lower was the VPA-induced reduction of SNr firing rate and the VPA-induced regularity of SNr firing. The data demonstrate for the first time an involvement of the SNr in pharmacoresistant experimental epilepsy and emphasize the relevance of the basal ganglia as target structures for new treatment options.

Kindling as a model of temporal lobe epilepsy induces bilateral changes in spontaneous striatal activity.

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.

Network pharmacology for antiepileptogenesis: Tolerability and neuroprotective effects of novel multitargeted combination treatments in nonepileptic vs. post-status epilepticus mice.

Network-based approaches in drug discovery comprise both development of novel drugs interacting with multiple targets and repositioning of drugs with known targets to form novel drug combinations that interact with cellular or molecular networks whose function is disturbed in a disease. Epilepsy is a complex network phenomenon that, as yet, cannot be prevented or cured. We recently proposed multitargeted, network-based approaches to prevent epileptogenesis by combinations of clinically available drugs chosen to impact diverse epileptogenic processes. In order to test this strategy preclinically, we developed a multiphase sequential study design for evaluating such drug combinations in rodents, derived from human clinical drug development phases. Because pharmacokinetics of such drugs are known, only the tolerability of novel drug combinations needs to be evaluated in Phase I in öhealthy" controls. In Phase IIa, tolerability is assessed following an epileptogenic brain insult, followed by antiepileptogenic efficacy testing in Phase IIb. Here, we report Phase I and Phase IIa evaluation of 7 new drug combinations in mice, using 10 drugs (levetiracetam, topiramate, gabapentin, deferoxamine, fingolimod, ceftriaxone, α-tocopherol, melatonin, celecoxib, atorvastatin) with diverse mechanisms thought to be important in epileptogenesis. Six of the 7 drug combinations were well tolerated in mice during prolonged treatment at the selected doses in both controls and during the latent phase following status epilepticus induced by intrahippocampal kainate. However, none of the combinations prevented hippocampal damage in response to kainate, most likely because treatment started only 16-18 h after kainate. This suggests that antiepileptogenic or disease-modifying treatment may need to start earlier after the brain insult. The present data provide a rich collection of tolerable, network-based combinatorial therapies as a basis for antiepileptogenic or disease-modifying efficacy testing.

Cerebral influx of Na+ and Cl- as the osmotherapy-mediated rebound response in rats.

BACKGROUND: Cerebral edema can cause life-threatening increase in intracranial pressure. Besides surgical craniectomy performed in severe cases, osmotherapy may be employed to lower the intracranial pressure by osmotic extraction of cerebral fluid upon intravenous infusion of mannitol or NaCl. A so-called rebound effect can, however, hinder continuous reduction in cerebral fluid by yet unresolved mechanisms. METHODS: We determined the brain water and electrolyte content in healthy rats treated with osmotherapy. Osmotherapy (elevated plasma osmolarity) was mediated by intraperitoneal injection of NaCl or mannitol with inclusion of pharmacological inhibitors of selected ion-transporters present at the capillary lumen or choroidal membranes. Brain barrier integrity was determined by fluorescence detection following intravenous delivery of Na+-fluorescein. RESULTS: NaCl was slightly more efficient than mannitol as an osmotic agent. The brain water loss was only ~ 60% of that predicted from ideal osmotic behavior, which could be accounted for by cerebral Na+ and Cl- accumulation. This electrolyte accumulation represented the majority of the rebound response, which was unaffected by the employed pharmacological agents. The brain barriers remained intact during the elevated plasma osmolarity. CONCLUSIONS: A brain volume regulatory response occurs during osmotherapy, leading to the rebound response. This response involves brain accumulation of Na+ and Cl- and takes place by unresolved molecular mechanisms that do not include the common ion-transporting mechanisms located in the capillary endothelium at the blood-brain barrier and in the choroid plexus epithelium at the blood-CSF barrier. Future identification of these ion-transporting routes could provide a pharmacological target to prevent the rebound effect associated with the widely used osmotherapy.