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Objective Cholinergic-induced status epilepticus (SE) is associated with a loss of synaptic gamma-aminobutyric acid A receptors (GABAA R) and an increase in N-methyl-D-aspartate receptors (NMDAR) and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) that may contribute to pharmacoresistance when treatment with benzodiazepine antiseizure medication is delayed. The barbiturate phenobarbital enhances inhibitory neurotransmission by binding to a specific site in the GABAA R to increase the open state of the channel, decrease neuronal excitability, and reduce glutamate-induced currents through AMPA/kainate receptors. We hypothesized that phenobarbital as an adjunct to midazolam would augment the amelioration of soman-induced SE and associated neuropathological changes and that further protection would be provided by the addition of an NMDAR antagonist. Methods We investigated the efficacy of combining antiseizure medications to include a benzodiazepine and a barbiturate allosteric GABAA R modulator (midazolam and phenobarbital, respectively) to correct loss of inhibition, and ketamine to reduce excitation caused by increased synaptic localization of NMDAR and AMPAR, which are NMDA-dependent. Rats implanted with transmitters to record electroencephalographic (EEG) activity were exposed to soman and treated with atropine sulfate and HI-6 one min after exposure and with antiseizure medication(s) 40 minutes after seizure onset. Results The triple therapy combination of phenobarbital, midazolam, and ketamine administered at 40 minutes after seizure onset effectively prevented soman-induced epileptogenesis and reduced neurodegeneration. In addition, dual therapy with phenobarbital and midazolam or ketamine was more effective than monotherapy (midazolam or phenobarbital) in reducing cholinergic-induced toxicity. Significance Benzodiazepine efficacy is drastically reduced with time after seizure onset and inversely related to seizure duration. To overcome pharmacoresistance in severe benzodiazepine-refractory cholinergic-induced SE, simultaneous drug combination to include drugs that target both the loss of inhibition (eg, midazolam, phenobarbital) and the increased excitatory response (eg, ketamine) is more effective than benzodiazepine or barbiturate monotherapy.

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.

Graphical abstract, Highlights • The median lethal dose of soman (GD) was estimated in adult male Göttingen minipigs. • GD exposure induces cholinesterase inhibition and behavioral seizure in minipigs. • GD induces neurodegeneration in visual cortex and lateral geniculate nucleus. • Prolonged seizure is associated with widespread microglial activation and cell death. • The minipig may be a useful alternative large animal model to the non-human primate., Animal models are essential for evaluating the toxicity of chemical warfare nerve agents (CWNAs) to extrapolate to human risk and are necessary to evaluate the efficacy of medical countermeasures. The Göttingen minipig is increasingly used for toxicological studies because it has anatomical and physiological characteristics that are similar to those of humans. Our objective was to determine whether the minipig would be a useful large animal model to evaluate the toxic effects of soman (GD). We determined the intramuscular (IM) median lethal dose (LD50) of GD in adult male Göttingen minipigs using an up-and-down dosing method. In addition to lethality estimates, we characterized the observable signs of toxicity, blood and tissue cholinesterase (ChE) activity and brain pathology following GD exposure. The 24 h LD50 of GD was estimated to be 4.7 μg/kg, with 95 % confidence limits of 3.6 and 6.3 μg/kg. As anticipated, GD inhibited ChE activity in blood and several tissues. Neurohistopathological analysis showed neurodegeneration and neuroinflammation in survivors exposed to 4.7 μg/kg of GD, including in the primary visual cortex and various thalamic nuclei. These findings suggest that the minipig will be a useful large animal model for assessing drugs to mitigate neuropathological effects of exposure to CWNAs.

Chemical warfare nerve agent exposure leads to status epilepticus (SE) that may progress to epileptogenesis and severe brain pathology when benzodiazepine treatment is delayed. We evaluated the dose–response effects of delayed midazolam 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 (LD(50)) of GD exposure in female ES1(−/−) mice across estrous with male mice and observed a greater LD(50) during estrus compared with proestrus or to males. Subsequently, male and female GD-exposed ES1(−/−) mice treated with a dose range of midazolam 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 midazolam 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 midazolam 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.

Objective Our objective was to evaluate the protective efficacy of the neurosteroid pregnanolone (3α‐hydroxy‐5β 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.

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.

Delayed treatment of cholinergic seizure results in benzodiazepine-refractory status epilepticus that is thought, at least in part, to result from maladaptive trafficking of N-methyl-D-aspartate (NMDA) and gamma aminobutyric acid type A (GABA(A)) 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 exposure when evaluated in a mouse model that, similar to humans, lacks plasma carboxylesterase, greatly reducing endogenous scavenging of soman. 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 soman and treated with midazolam or with midazolam and ketamine combination at 40 min after seizure onset. Ketamine and midazolam combination reduced soman-induced lethality, seizure severity and the number of mice that developed spontaneous recurrent seizure compared to midazolam monotherapy. In addition, ketamine-midazolam combination treatment reduced soman-induced neuronal degeneration and microgliosis. These results support that combination of anti-epileptic drug therapies aimed at correcting the maladaptive GABA(A) and NMDA receptor trafficking reduce the detrimental effects of soman exposure. Ketamine may be a beneficial adjunct to midazolam in reducing the epileptogenesis and neuroanatomical damage that follows nerve agent exposure and pharmacoresistant status epilepticus.

Elderly individuals compose a large percentage of the world population; however, few studies have addressed the efficacy of current medical countermeasures (MCMs) against the effects of chemical warfare nerve agent exposure in aged populations. We evaluated the efficacy of the anticonvulsant diazepam in an old adult rat model of soman (GD) poisoning and compared the toxic effects to those observed in young adult rats when anticonvulsant treatment is delayed. After determining their respective median lethal dose (LD50) of GD, we exposed young adult and old adult rats to an equitoxic 1.2 LD50 dose of GD followed by treatment with atropine sulfate and the oxime HI-6 at 1 min after exposure, and diazepam at 30 min after seizure onset. Old adult rats that presented with status epilepticus were more susceptible to developing spontaneous recurrent seizures (SRSs). Neuropathological analysis revealed that in rats of both age groups that developed SRS, there was a significant reduction in the density of mature neurons in the piriform cortex, thalamus, and amygdala, with more pronounced neuronal loss in the thalamus of old adult rats compared with young adult rats. Furthermore, old adult rats displayed a reduced density of cells expressing glutamic acid decarboxylase 67, a marker of GABAergic interneurons, in the basolateral amygdala and piriform cortex, and a reduction of astrocyte activation in the piriform cortex. Our observations demonstrate the reduced effectiveness of current MCM in an old adult animal model of GD exposure and strongly suggest the need for countermeasures that are more tailored to the vulnerabilities of an aging population.

Summary 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.