Preventing Long-Term Brain Damage by Nerve Agent-Induced Status Epilepticus in Rat Models Applicable to Infants: Significant Neuroprotection by Tezampanel Combined with Caramiphen but Not by Midazolam Treatment.

Acute exposure to nerve agents induces a peripheral cholinergic crisis and prolonged status epilepticus (SE), causing death or long-term brain damage. To provide preclinical data pertinent to the protection of infants and newborns, we compared the antiseizure and neuroprotective effects of treating soman-induced SE with midazolam (MDZ) versus tezampanel (LY293558) in combination with caramiphen (CRM) in 12- and 7-day-old rats. The anticonvulsants were administered 1 hour after soman exposure; neuropathology data were collected up to 6 months postexposure. In both ages, the total duration of SE within 24 hours after soman exposure was significantly shorter in the LY293558 plus CRM groups compared with the MDZ groups. Neuronal degeneration was substantial in the MDZ-treated groups but absent or minimal in the groups treated with LY293558 plus CRM. Loss of neurons and interneurons in the basolateral amygdala and CA1 hippocampal area was significant in the MDZ-treated groups but virtually absent in the LY293558 plus CRM groups. Atrophy of the amygdala and hippocampus occurred only in MDZ-treated groups. Neuronal/interneuronal loss and atrophy of the amygdala and hippocampus deteriorated over time. Reduction of inhibitory activity in the basolateral amygdala and increased anxiety were found only in MDZ groups. Spontaneous recurrent seizures developed in the MDZ groups, deteriorating over time; a small percentage of rats from the LY293558 plus CRM groups also developed seizures. These results suggest that brain damage can be long lasting or permanent if nerve agent-induced SE in infant victims is treated with midazolam at a delayed timepoint after SE onset, whereas antiglutamatergic treatment with tezampanel and caramiphen provides significant neuroprotection. SIGNIFICANCE STATEMENT: To protect the brain and the lives of infants in a mass exposure to nerve agents, an anticonvulsant treatment must be administered that will effectively stop seizures and prevent neuropathology, even if offered with a relative delay after seizure onset. The present study shows that midazolam, which was recently approved by the Food and Drug Administration for the treatment of nerve agent-induced status epilepticus, is not an effective neuroprotectant, whereas brain damage can be prevented by targeting glutamate receptors.

Evaluation of Midazolam-Ketamine-Allopregnanolone Combination Therapy against Cholinergic-Induced Status Epilepticus in Rats.

Status epilepticus (SE) is a life-threatening development of self-sustaining seizures that becomes resistant to benzodiazepines when treatment is delayed. Benzodiazepine pharmacoresistance is thought in part to result from internalization of synaptic GABAA receptors, which are the main target of the drug. The naturally occurring neurosteroid allopregnanolone is a therapy of interest against SE for its ability to modulate all isoforms of GABAA receptors. Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been partially effective in combination with benzodiazepines in mitigating SE-associated neurotoxicity. In this study, allopregnanolone as an adjunct to midazolam or midazolam-ketamine combination therapy was evaluated for efficacy against cholinergic-induced SE. Adult male rats implanted with electroencephalographic (EEG) telemetry devices were exposed to the organophosphorus chemical (OP) soman (GD) and treated with an admix of atropine sulfate and HI-6 at 1 minute after exposure followed by midazolam, midazolam-allopregnanolone, or midazolam-ketamine-allopregnanolone 40 minutes after seizure onset. Neurodegeneration, neuronal loss, and neuroinflammation were assessed 2 weeks after GD exposure. Seizure activity, EEG power integral, and epileptogenesis were also compared among groups. Overall, midazolam-ketamine-allopregnanolone combination therapy was effective in reducing cholinergic-induced toxic signs and neuropathology, particularly in the thalamus and hippocampus. Higher dosage of allopregnanolone administered in combination with midazolam and ketamine was also effective in reducing EEG power integral and epileptogenesis. The current study reports that there is a promising potential of neurosteroids in combination with benzodiazepine and ketamine treatments in a GD model of SE. SIGNIFICANCE STATEMENT: Allopregnanolone, a naturally occurring neurosteroid, reduced pathologies associated with soman (GD) exposure such as epileptogenesis, neurodegeneration, and neuroinflammation, and suppressed GD-induced toxic signs when used as an adjunct to midazolam and ketamine in a delayed treatment model of soman-induced status epilepticus (SE) in rats. However, protection was incomplete, suggesting that further studies are needed to identify optimal combinations of antiseizure medications and routes of administration for maximal efficacy against cholinergic-induced SE.

Efficacy of Lacosamide and Rufinamide as Adjuncts to Midazolam-Ketamine Treatment Against Cholinergic-Induced Status Epilepticus in Rats.

Benzodiazepine pharmacoresistance develops when treatment of status epilepticus (SE) is delayed. This response may result from gamma-aminobutyric acid A receptors (GABAAR) internalization that follows prolonged SE; this receptor trafficking results in fewer GABAAR in the synapse to restore inhibition. Increase in synaptic N-methyl-D-aspartate receptors (NMDAR) also occurs in rodent models of SE. Lacosamide, a third-generation antiseizure medication (ASM), acts on the slow inactivation of voltage-gated sodium channels. Another ASM, rufinamide, similarly acts on sodium channels by extending the duration of time spent in the inactivation stage. Combination therapy of the benzodiazepine midazolam, NMDAR antagonist ketamine, and ASMs lacosamide (or rufinamide) was investigated for efficacy against soman (GD)-induced SE and neuropathology. Adult male rats implanted with telemetry transmitters for monitoring electroencephalographic (EEG) activity were exposed to a seizure-inducing dose of GD and treated with an admix of atropine sulfate and HI-6 1 minute later and with midazolam monotherapy or combination therapy 40 minutes after EEG seizure onset. Rats were monitored continuously for seizure activity for two weeks, after which brains were processed for assessment of neurodegeneration, neuronal loss, and neuroinflammatory responses. Simultaneous administration of midazolam, ketamine, and lacosamide (or rufinamide) was more protective against GD-induced SE compared with midazolam monotherapy. In general, lacosamide triple therapy had more positive outcomes on measures of epileptogenesis, EEG power integral, and the number of brain regions protected from neuropathology compared with rats treated with rufinamide triple therapy. Overall, both drugs were well tolerated in these combination models. SIGNIFICANCE STATEMENT: We currently report on improved efficacy of antiseizure medications lacosamide and rufinamide, each administered in combination with ketamine (NMDAR antagonist) and midazolam (benzodiazepine), in combatting soman (GD)-induced seizure, epileptogenesis, and brain pathology over that provided by midazolam monotherapy, or dual therapy of midazolam and lacosamide (or rufinamide) in rats. Administration of lacosamide as adjunct to midazolam and ketamine was particularly effective against GD-induced toxicity. However, protection was incomplete, suggesting the need for further study.

Mechanisms of Organophosphate Toxicity and the Role of Acetylcholinesterase Inhibition.

Organophosphorus compounds (OPs) have applications in agriculture (e.g., pesticides), industry (e.g., flame retardants), and chemical warfare (nerve agents). In high doses or chronic exposure, they can be toxic or lethal. The primary mechanism, common among all OPs, that initiates their toxic effects is the inhibition of acetylcholinesterase. In acute OP exposure, the subsequent surge of acetylcholine in cholinergic synapses causes a peripheral cholinergic crisis and status epilepticus (SE), either of which can lead to death. If death is averted without effective seizure control, long-term brain damage ensues. This review describes the mechanisms by which elevated acetylcholine can cause respiratory failure and trigger SE; the role of the amygdala in seizure initiation; the role of M1 muscarinic receptors in the early stages of SE; the neurotoxic pathways activated by SE (excitotoxicity/Ca++ overload/oxidative stress, neuroinflammation); and neurotoxic mechanisms linked to low-dose, chronic exposure (Ca++ dyshomeostasis/oxidative stress, inflammation), which do not depend on SE and do not necessarily involve acetylcholinesterase inhibition. The evidence so far indicates that brain damage from acute OP exposure is a direct result of SE, while the neurotoxic mechanisms activated by low-dose chronic exposure are independent of SE and may not be associated with acetylcholinesterase inhibition.

Treatment of cholinergic-induced status epilepticus with polytherapy targeting GABA and glutamate receptors.

Despite new antiseizure medications, the development of cholinergic-induced refractory status epilepticus (RSE) continues to be a therapeutic challenge as pharmacoresistance to benzodiazepines and other antiseizure medications quickly develops. Studies conducted by Epilepsia. 2005;46:142 demonstrated that the initiation and maintenance of cholinergic-induced RSE are associated with trafficking and inactivation of gamma-aminobutyric acid A receptors (GABAA R) thought to contribute to the development of benzodiazepine pharmacoresistance. In addition, Dr. Wasterlain's laboratory reported that increased N-methyl-d-aspartate receptors (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) contribute to enhanced glutamatergic excitation (Neurobiol Dis. 2013;54:225; Epilepsia. 2013;54:78). Thus, Dr. Wasterlain postulated that targeting both maladaptive responses of reduced inhibition and increased excitation that is associated with cholinergic-induced RSE should improve therapeutic outcome. We currently review studies in several animal models of cholinergic-induced RSE that demonstrate that benzodiazepine monotherapy has reduced efficacy when treatment is delayed and that polytherapy with drugs that include a benzodiazepine (eg midazolam and diazepam) to counter loss of inhibition, concurrent with an NMDA antagonist (eg ketamine) to reduce excitation provide improved efficacy. Improved efficacy with polytherapy against cholinergic-induced seizure is demonstrated by reduction in (1) seizure severity, (2) epileptogenesis, and (3) neurodegeneration compared with monotherapy. Animal models reviewed include pilocarpine-induced seizure in rats, organophosphorus nerve agent (OPNA)-induced seizure in rats, and OPNA-induced seizure in two mouse models: (1) carboxylesterase knockout (Es1-/- ) mice which, similarly to humans, lack plasma carboxylesterase and (2) human acetylcholinesterase knock-in carboxylesterase knockout (KIKO) mice. We also review studies showing that supplementing midazolam and ketamine with a third antiseizure medication (valproate or phenobarbital) that targets a nonbenzodiazepine site rapidly terminates RSE and provides further protection against cholinergic-induced SE. Finally, we review studies on the benefits of simultaneous compared with sequential drug treatments and the clinical implications that lead us to predict improved efficacy of early combination drug therapies. The data generated from seminal rodent studies of efficacious treatment of cholinergic-induced RSE conducted under Dr. Wasterlain's guidance suggest that future clinical trials should treat the inadequate inhibition and temper the excess excitation that characterize RSE and that early combination therapies may provide improved outcome over benzodiazepine monotherapy.

GABAergic circuits of the basolateral amygdala and generation of anxiety after traumatic brain injury.

Traumatic brain injury (TBI) has reached epidemic proportions around the world and is a major public health concern in the United States. Approximately 2.8 million individuals sustain a traumatic brain injury and are treated in an Emergency Department yearly in the U.S., and about 50,000 of them die. Persistent symptoms develop in 10-15% of the cases including neuropsychiatric disorders. Anxiety is the second most common neuropsychiatric disorder that develops in those with persistent neuropsychiatric symptoms after TBI. Abnormalities or atrophy in the temporal lobe has been shown in the overwhelming number of TBI cases. The basolateral amygdala (BLA), a temporal lobe structure that consolidates, stores and generates fear and anxiety-based behavioral outputs, is a critical brain region in the anxiety circuitry. In this review, we sought to capture studies that characterized the relationship between human post-traumatic anxiety and structural/functional alterations in the amygdala. We compared the human findings with results obtained with a reproducible mild TBI animal model that demonstrated a direct relationship between the alterations in the BLA and an anxiety-like phenotype. From this analysis, both preliminary insights, and gaps in knowledge, have emerged which may open new directions for the development of rational and more efficacious treatments.

Novel Genetically Modified Mouse Model to Assess Soman-Induced Toxicity and Medical Countermeasure Efficacy: Human Acetylcholinesterase Knock-in Serum Carboxylesterase Knockout Mice.

The identification of improved medical countermeasures against exposure to chemical warfare nerve agents (CWNAs), a class of organophosphorus compounds, is dependent on the choice of animal model used in preclinical studies. CWNAs bind to acetylcholinesterase and prevent the catalysis of acetylcholine, causing a plethora of peripheral and central physiologic manifestations, including seizure. Rodents are widely used to elucidate the effects of CWNA-induced seizure, albeit with a caveat: they express carboxylesterase activity in plasma. Carboxylesterase, an enzyme involved in the detoxification of some organophosphorus compounds, plays a scavenging role and decreases CWNA availability, thus exerting a protective effect. Furthermore, species-specific amino acid differences in acetylcholinesterase confound studies that use oximes or other compounds to restore its function after inhibition by CWNA. The creation of a human acetylcholinesterase knock-in/serum carboxylesterase knockout (C57BL/6-Ces1ctm1.1LocAChEtm1.1Loc/J; a.k.a KIKO) mouse may facilitate better modeling of CWNA toxicity in a small rodent species. The current studies characterize the effects of exposure to soman, a highly toxic CWNA, and evaluate the efficacy of anti-seizure drugs in this newly developed KIKO mouse model. Data demonstrate that a combination of midazolam and ketamine reduces seizure duration and severity, eliminates the development of spontaneous recurrent seizures, and protects certain brain regions from neuronal damage in a genetically modified model with human relevance to organophosphorus compound toxicity. This new animal model and the results of this study and future studies using it will enhance medical countermeasures development for both defense and homeland security purposes.

Treatment of acetylcholinesterase inhibitor-induced seizures with polytherapy targeting GABA and glutamate receptors.

The initiation and maintenance of cholinergic-induced status epilepticus (SE) are associated with decreased synaptic gamma-aminobutyric acid A receptors (GABAAR) and increased N-methyl-d-aspartate receptors (NMDAR) and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). We hypothesized that trafficking of synaptic GABAAR and glutamate receptors is maladaptive and contributes to the pharmacoresistance to antiseizure drugs; targeting these components should ameliorate the pathophysiological consequences of refractory SE (RSE). We review studies of rodent models of cholinergic-induced SE, in which we used a benzodiazepine allosteric GABAAR modulator to correct loss of inhibition, concurrent with the NMDA antagonist ketamine to reduce excitation caused by increased synaptic localization of NMDAR and AMPAR, which are NMDAR-dependent. Models included lithium/pilocarpine-induced SE in rats and soman-induced SE in rats and in Es1-/- mice, which similar to humans lack plasma carboxylesterase, and may better model soman toxicity. These model human soman toxicity and are refractory to benzodiazepines administered at 40 min after seizure onset, when enough synaptic GABAAR may not be available to restore inhibition. Ketamine-midazolam combination reduces seizure severity, epileptogenesis, performance deficits and neuropathology following cholinergic-induced SE. Supplementing that treatment with valproate, which targets a non-benzodiazepine site, effectively terminates RSE, providing further benefit against cholinergic-induced SE. The therapeutic index of drug combinations is also reviewed and we show the improved efficacy of simultaneous administration of midazolam, ketamine and valproate compared to sequential drug administration. These data suggest that future clinical trials should treat both the lack of sufficient inhibition and the excess excitation that characterize RSE, and include early combination drug therapies. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Cannabidiol reduces soman-induced lethality and seizure severity in female plasma carboxylesterase knockout mice treated with midazolam.

Cannabidiol, approved for treatment of pediatric refractory epilepsy, has anti-seizure effects in various animal seizure models. Chemical warfare nerve agents, including soman, are organophosphorus chemicals that can induce seizure and death if untreated or if treatment is delayed. Our objective was to evaluate whether cannabidiol would ameliorate soman-induced toxicity using a mouse model that similar to humans lacks plasma carboxylesterase. In the present study, adult female plasma carboxylesterase knockout (Es1-/-) mice were pre-treated with cannabidiol (20-150 mg/kg) or vehicle 1 h prior to exposure to a seizure-inducing dose of soman and evaluated for survival and seizure activity. The muscarinic antagonist atropine sulfate and the oxime HI-6 were administered at 1 min after exposure, and the benzodiazepine midazolam was administered at 30 min after seizure onset. Cannabidiol (150 mg/kg) pre-treatment led to a robust increase in survival rate and attenuated body weight loss in soman-exposed mice treated with medical countermeasures, compared to mice pre-treated with vehicle. In addition, mice pretreated with cannabidiol (150 mg/kg) had a modest reduction in seizure severity after midazolam treatment compared to vehicle-pretreated. These findings of improved outcome with cannabidiol administration in a severe seizure model of soman exposure provide additional pre-clinical support for the benefits of cannabidiol against exposure to seizure-inducing chemical agents and suggest cannabidiol may augment the anti-seizure effects of midazolam.

Acetylcholinesterase inhibitors (nerve agents) as weapons of mass destruction: History, mechanisms of action, and medical countermeasures.

Nerve agents are organophosphorus acetylcholinesterase inhibitors. Acute exposure to nerve agents can cause rapid death. In this review, we summarize the history of nerve agent development and use in warfare, the mechanisms by which these agents cause death or long-term brain damage, and the treatments for preventing death or long-term morbidity. The G-series nerve agents, tabun, sarin, soman, ethyl sarin, and cyclosarin, were developed by the Nazis. VX, the best-known of the V-series agents, was synthesized in the 1950's by a British scientist. Little is known about the development of the novichoks (the "A-series") by the former Soviet Union. Nerve agents were used for the first time in the battlefield by the Iraqi government in the Iran-Iraq War, in the 1980s. The Chemical Weapons Convention, in 1993, banned all chemical weapons production and use, yet, sarin was subsequently used in terrorist attacks in Japan and, recently, in the war in Syria. Pyridostigmine has been used as a prophylactic treatment, and bioscavengers are presently investigated as a better alternative. Atropine, along with an oxime, can prevent rapid death from the nerve agent-induced peripheral cholinergic crisis. Treatment with diazepam or midazolam for the cessation of nerve agent-induced status epilepticus cannot protect against brain damage, and, therefore, these benzodiazepines should be replaced by novel anticonvulsants and neuroprotectants. The AMPA/GluK1 receptor antagonist LY293558 (tezampanel) has shown superior antiseizure and neuroprotective efficacy against soman, particularly when administered in combination with caramiphen, an antagonist of muscarinic and NMDA receptors. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Ketamine as adjunct to midazolam treatment following soman-induced status epilepticus reduces seizure severity, epileptogenesis, and brain pathology in plasma carboxylesterase knockout mice.

Delayed treatment of cholinergic seizure results in benzodiazepine-refractory status epilepticus (SE) that is thought, at least in part, to result from maladaptive trafficking of N-methyl-d-aspartate (NMDA) and gamma-aminobutyric acid type A (GABAA) receptors, the effects of which may be ameliorated by combination therapy with the NMDA receptor antagonist ketamine. Our objective was to establish whether ketamine and midazolam dual therapy would improve outcome over midazolam monotherapy following soman (GD) exposure when evaluated in a mouse model that, similar to humans, lacks plasma carboxylesterase, greatly reducing endogenous scavenging of GD. In the current study, continuous cortical electroencephalographic activity was evaluated in male and female plasma carboxylesterase knockout mice exposed to a seizure-inducing dose of GD and treated with midazolam or with midazolam and ketamine combination at 40 min after seizure onset. Ketamine and midazolam combination reduced GD-induced lethality, seizure severity, and the number of mice that developed spontaneous recurrent seizure (SRS) compared with midazolam monotherapy. In addition, ketamine-midazolam combination treatment reduced GD-induced neuronal degeneration and microgliosis. These results support that combination of antiepileptic drug therapies aimed at correcting the maladaptive GABAA and NMDA receptor trafficking reduces the detrimental effects of GD exposure. Ketamine may be a beneficial adjunct to midazolam in reducing the epileptogenesis and neuroanatomical damage that follows nerve agent exposure and pharmacoresistant SE.

Electroencephalographic analysis in soman-exposed 21-day-old rats and the effects of midazolam or LY293558 with caramiphen.

Acute nerve agent exposure induces status epilepticus (SE), which can cause brain damage or death. Research aiming at developing effective therapies for controlling nerve agent-induced SE is commonly performed in adult rats. The characteristics of nerve agent-induced SE in young rats are less clear; relevant knowledge is necessary for developing effective pediatric therapies. Here, we have used electroencephalographic (EEG) recordings and analysis to study seizures in postnatal day 21 rats exposed to 1.2 × LD50 of soman, and compared the antiseizure efficacy of midazolam (MDZ)-currently considered by the Food and Drug Administration to replace diazepam for treating SE in victims of nerve agent exposure-with that of LY293558, an AMPA/GluK1 receptor antagonist, administered in combination with caramiphen, an antimuscarinic with N-methyl-d-aspartate receptor antagonistic properties. Prolonged SE developed in 80% of the rats and was reflected in behavioral seizures/convulsions. Both MDZ and LY293558 + caramiphen stopped the SE induced by soman, but there was a significant recurrence of seizures within 24 h postexposure only in the MDZ-treated group, as revealed in the raw EEG data and their representation in the frequency domain using a fast Fourier transform and in spectral analysis over 24 hours. In contrast to the high efficacy of LY293558 + caramiphen, MDZ is not an effective treatment for SE induced by soman in young animals.

Delayed midazolam dose effects against soman in male and female plasma carboxylesterase knockout mice.

Chemical warfare nerve agent exposure leads to status epilepticus that may progress to epileptogenesis and severe brain pathology when benzodiazepine treatment is delayed. We evaluated the dose-response effects of delayed midazolam (MDZ) on toxicity induced by soman (GD) in the plasma carboxylesterase knockout (Es1-/- ) mouse, which, similar to humans, lacks plasma carboxylesterase. Initially, we compared the median lethal dose (LD50 ) of GD exposure in female Es1-/- mice across estrous with male mice and observed a greater LD50 during estrus compared with proestrus or with males. Subsequently, male and female GD-exposed Es1-/- mice treated with a dose range of MDZ 40 min after seizure onset were evaluated for survivability, seizure activity, and epileptogenesis. GD-induced neuronal loss and microglial activation were evaluated 2 weeks after exposure. Similar to our previous observations in rats, delayed treatment with MDZ dose-dependently increased survival and reduced seizure severity in GD-exposed mice, but was unable to prevent epileptogenesis, neuronal loss, or gliosis. These results suggest that MDZ is beneficial against GD exposure, even when treatment is delayed, but that adjunct therapies to enhance protection need to be identified. The Es1-/- mouse GD exposure model may be useful to screen for improved medical countermeasures against nerve agent exposure.

Dataset of EEG power integral, spontaneous recurrent seizure and behavioral responses following combination drug therapy in soman-exposed rats.

This article investigated the efficacy of the combination of antiepileptic drug therapy in protecting against soman-induced seizure severity, epileptogenesis and performance deficits. Adult male rats with implanted telemetry transmitters for continuous recording of electroencephalographic (EEG) activity were exposed to soman and treated with atropine sulfate and the oxime HI-6 one minute after soman exposure and with midazolam, ketamine and/or valproic acid 40 min after seizure onset. Rats exposed to soman and treated with medical countermeasures were evaluated for survival, seizure severity, the development of spontaneous recurrent seizure and performance deficits; combination anti-epileptic drug therapy was compared with midazolam monotherapy. Telemetry transmitters were used to record EEG activity, and a customized MATLAB algorithm was used to analyze the telemetry data. Survival data, EEG power integral data, spontaneous recurrent seizure data and behavioral data are illustrated in figures and included as raw data. In addition, edf files of one month telemetry recordings from soman-exposed rats treated with delayed midazolam are provided as supplementary materials. Data presented in this article are related to research articles "Rational Polytherapy in the Treatment of Cholinergic Seizures" [1] and "Early polytherapy for benzodiazepine-refractory status epilepticus [4].

Early polytherapy for benzodiazepine-refractory status epilepticus.

The transition from single seizures to status epilepticus (SE) is associated with malaptive trafficking of synaptic gamma-aminobutyric acid (GABAA) and glutamate receptors. The receptor trafficking hypothesis proposes that these changes are key events in the development of pharmacoresistance to antiepileptic drugs (AEDs) during SE, and that blocking their expression will help control drug-refractory SE (RSE). We tested this hypothesis in a model of SE induced by very high-dose lithium and pilocarpine (RSE), and in a model of SE induced by sc soman. Both models are refractory to benzodiazepines when treated 40 min after seizure onset. Our treatments aimed to correct the loss of inhibition because of SE-associated internalization of synaptic GABAA receptors (GABAAR), using an allosteric GABAAR modulator, sometimes supplemented by an AED acting at a nonbenzodiazepine site. At the same time, we reduced excitation because of increased synaptic localization of NMDA and AMPA (?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate) receptors (NMDAR, AMPAR (?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, N-methyl-D-aspartate receptors)) with an NMDAR channel blocker, since AMPAR changes are NMDAR-dependent. Treatment of RSE with combinations of the GABAAR allosteric modulators midazolam or diazepam and the NMDAR antagonists dizocilpine or ketamine terminated RSE unresponsive to high-dose monotherapy. It also reduced RSE-associated neuronal injury, spatial memory deficits, and the occurrence of spontaneous recurrent seizures (SRS), tested several weeks after SE. Treatment of soman-induced SE also reduced seizures, behavioral deficits, and epileptogenesis. Addition of an AED further improved seizure outcome in both models. Three-dimensional isobolograms demonstrated positive cooperativity between midazolam, ketamine, and valproate, without any interaction between the toxicity of these drugs, so that the therapeutic index was increased by combination therapy. The midazolam-ketamine-valproate combination based on the receptor trafficking hypothesis was far more effective in stopping RSE than the midazolam-fosphenytoin-valproate combination inspired from clinical guidelines for the treatment of SE. Furthermore, sequential administration of midazolam, ketamine, and valproate was far less effective than simultaneous treatment with the same drugs at the same dose. These data suggest that treatment of RSE should be based at least in part on its pathophysiology. The search for a better treatment should focus on the cause of pharmacoresistance, which is loss of synaptic GABAAR and gain of synaptic glutamate receptors. Both need to be treated. Monotherapy addresses only half the problem. Improved pharmacokinetics will not help pharmacoresistance because of loss of receptors. Waiting for one drug to fail before giving the second drugs gives pharmacoresistance time to develop. Future clinical trials should consider treating both the failure of inhibition and the runaway excitation which characterize RSE, and should include an early polytherapy arm. This article is part of the Special Issue "Proceedings of the 7th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures".

Neurosteroid and benzodiazepine combination therapy reduces status epilepticus and long-term effects of whole-body sarin exposure in rats.

OBJECTIVE: Our objective was to evaluate the protective efficacy of the neurosteroid pregnanolone (3α-hydroxy-5ß pregnan-20-one), a GABAA receptor-positive allosteric modulator, as an adjunct to benzodiazepine therapy against the chemical warfare nerve agent (CWNA) sarin (GB), using whole-body exposure, an operationally relevant route of exposure to volatile GB. METHODS: Rats implanted with telemetry transmitters for the continuous measurement of cortical electroencephalographic (EEG) activity were exposed for 60 minutes to 3.0 LCt50 of GB via whole-body exposure. At the onset of toxic signs, rats were administered an intramuscular injection of atropine sulfate (2 mg/kg) and the oxime HI-6 (93.6 mg/kg) to increase survival rate and, 30 minutes after seizure onset, treated subcutaneously with diazepam (10 mg/kg) and intravenously with pregnanolone (4 mg/kg) or vehicle. Animals were evaluated for GB-induced status epilepticus (SE), spontaneous recurrent seizures (SRS), impairment in spatial memory acquisition, and brain pathology, and treatment groups were compared. RESULTS: Delayed dual therapy with pregnanolone and diazepam reduced time in SE in GB-exposed rats compared to those treated with delayed diazepam monotherapy. The combination therapy of pregnanolone with diazepam also prevented impairment in the Morris water maze and reduced the neuronal loss and neuronal degeneration, evaluated at one and three months after exposure. SIGNIFICANCE: Neurosteroid administration as an adjunct to benzodiazepine therapy offers an effective means to treat benzodiazepine-refractory SE, such as occurs following delayed treatment of GB exposure. This study is the first to present data on the efficacy of delayed pregnanolone and diazepam dual therapy in reducing seizure activity, performance deficits and brain pathology following an operationally relevant route of exposure to GB and supports the use of a neurosteroid as an adjunct to standard anticonvulsant therapy for the treatment of CWNA-induced SE.

Soman-induced status epilepticus, epileptogenesis, and neuropathology in carboxylesterase knockout mice treated with midazolam.

OBJECTIVE: Exposure to chemical warfare nerve agents (CWNAs), such as soman (GD), can induce status epilepticus (SE) that becomes refractory to benzodiazepines when treatment is delayed, leading to increased risk of epileptogenesis, severe neuropathology, and long-term behavioral and cognitive deficits. Rodent models, widely used to evaluate novel medical countermeasures (MCMs) against CWNA exposure, normally express plasma carboxylesterase, an enzyme involved in the metabolism of certain organophosphorus compounds. To better predict the efficacy of novel MCMs against CWNA exposure in human casualties, it is crucial to use appropriate animal models that mirror the human condition. We present a comprehensive characterization of the seizurogenic, epileptogenic, and neuropathologic effects of GD exposure with delayed anticonvulsant treatment in the plasma carboxylesterase knockout (ES1-/-) mouse. METHODS: Electroencephalography (EEG) electrode-implanted ES1-/- and wild-type (C57BL/6) mice were exposed to various seizure-inducing doses of GD, treated with atropine sulfate and the oxime HI-6 at 1 minute after exposure, and administered midazolam at 15-30 minutes following the onset of seizure activity. The latency of acute seizure onset and spontaneous recurrent seizures (SRS) was assessed, as were changes in EEG power spectra. At 2 weeks after GD exposure, neurodegeneration and neuroinflammation were assessed. RESULTS: GD-exposed ES1-/- mice displayed a dose-dependent response in seizure severity. Only ES1-/- mice exposed to the highest tested dose of GD developed SE, subchronic alterations in EEG power spectra, and SRS. Degree of neuronal cell loss and neuroinflammation were dose-dependent; no significant neuropathology was observed in C57BL/6 mice or ES1-/- mice exposed to lower GD doses. SIGNIFICANCE: The US Food and Drug Administration (FDA) animal rule requires the use of relevant animal models for the advancement of MCMs against CWNAs. We present evidence that argues for the use of the ES1-/- mouse model to screen anticonvulsant, antiepileptic, and/or neuroprotective drugs against GD-induced toxicity, as well as to identify mechanisms of GD-induced epileptogenesis.

Full Protection Against Soman-Induced Seizures and Brain Damage by LY293558 and Caramiphen Combination Treatment in Adult Rats.

Acute exposure to nerve agents induces status epilepticus (SE), which causes brain damage or death. LY293558, an antagonist of AMPA and GluK1 kainate receptors is a very effective anticonvulsant and neuroprotectant against soman; however, some neuronal damage is still present after treatment of soman-exposed rats with LY293558. Here, we have tested whether combining LY293558 with an NMDA receptor antagonist can eliminate the residual damage. For this purpose, we chose caramiphen (CRM), an antimuscarinic compound with NMDA receptor antagonistic properties. Adult male rats were exposed to 1.2 × LD50 soman, and at 20 min after soman exposure, were injected with atropine + HI-6, or atropine + HI-6 + LY293558 (15 mg/kg), or atropine + HI-6 + LY293558 + CRM (50 mg/kg). We found that (1) the LY293558 + CRM treatment terminated SE significantly faster than LY293558 alone; (2) after cessation of the initial SE, seizures did not return in the LY293558 + CRM-treated group, during 72 h of monitoring; (3) power spectrum analysis of continuous EEG recordings for 7 days post-exposure showed increased delta and decreased gamma power that lasted beyond 24 h post-exposure only in the rats who did not receive anticonvulsant treatment; (4) spontaneous recurrent seizures appeared on day 7 only in the group that did not receive anticonvulsant treatment; (5) significant neuroprotection was achieved by LY293558 administration, while the rats who received LY293558 + CRM displayed no neurodegeneration; (6) body weight loss and recovery in the LY293558 + CRM-treated rats did not differ from those in control rats who were not exposed to soman. The data show that treatment with LY293558 + CRM provides full antiseizure and neuroprotective efficacy against soman.

Exposure to nerve agents: from status epilepticus to neuroinflammation, brain damage, neurogenesis and epilepsy.

Epilepsy is a common neurological disorder characterized by an initial injury due to stroke, traumatic brain injury, brain infection, or febrile seizures causing status epilepticus (SE). This phenomenon precedes recurrent (secondary) seizures, the latent period (period without seizures) and downstream appearance of spontaneous recurrent seizures (SRS). Epilepsy inducers include the organophosphorous (OP) compounds modified as chemical warfare nerve agents, such as soman. SE induced by soman is a result of cholinergic system hyperactivity caused by the irreversible inhibition of acetylcholinesterase, and the subsequent increase in the amount of the neurotransmitter acetylcholine at central and peripheral sites. SE leads to profound, permanent, complex and widespread brain damage and associated cognitive and behavioral deficits, accompanied by impaired neurogenesis. Several anticonvulsant and neuroprotective strategies have been studied in order to avoid the epileptogenesis which occurs after SE caused by soman exposure. In recent studies, we showed that SRS occur post-soman exposure and neuropathology can be reduced with diazepam (DZP) and valproic acid (VPA) when administered in combination treatment. These effects are accompanied by neurogenesis seen 15 days post-exposure in the hippocampal dentate gyrus (DG). This review discusses several findings about epilepsy induced by soman exposure such as behavioral changes, EEG anomalies, neuropathology, neuroinflammation, neurogenesis, possible circuitry changes and current strategies for treatment. The soman seizure model is an important model of temporal lobe epilepsy (TLE) and comparable in certain respects with well studied models in the literature such as pilocarpine and kainic acid. All these models together allow for a greater understanding of the different mechanisms of seizure induction, propagation and options for treatment. These studies are very necessary for current military and civilian treatment regimens, against OP nerve agent exposure, which fail to prevent SE resulting in severe neuropathology and epilepsy.

Combined diazepam and HDAC inhibitor treatment protects against seizures and neuronal damage caused by soman exposure.

The occurrence of status epilepticus (SE) is considered the main cause of brain lesions and morphological alterations, such as hippocampal neuron loss, that result in chronic epilepsy. Previous work demonstrated the convulsive and widespread neuropathological effects of soman, an organophosphorus compound that causes SE and severe recurrent seizures as a result of exposure. Seizures begin rapidly after exposure, can continue for hours, and contribute to prolonged physical incapacitation of the victim. This study attempts to identify anticonvulsive and neuroprotective drugs against soman exposure. Male Sprague-Dawley rats were exposed to 1.0 LD(50) soman. EEGraphical and neuropathological (Fluoro-Jade B staining) effects were analyzed at 72 h post-exposure to soman and subsequent treatments with diazepam (DZP) alone or in combination with histone deacetylase inhibitors, suberoylanilide hydroxamic acid (SAHA) or valproic acid (VPA). The extent of brain damage was dependent on the length of SE and not on the number of recurrent seizures. DZP treatment alone decreased SE time and damage in hippocampus, amygdala, thalamus and cortex, but not in piriform nuclei. The combination of DZP and VPA 100 mg/kg showed more anticonvulsive effects, decreased SE time, and afforded more neuroprotection in the hippocampus, mainly the ventral portion. The combination DZP and SAHA 25 mg/kg was more neuroprotective, but not more anticonvulsant than DZP alone. The DZP combination with VPA HDAC inhibitor proved to be a good treatment for SE and neuronal damage caused by soman exposure.