A biomechanical-based approach to scale blast-induced molecular changes in the brain.

Animal studies provide valuable insights on how the interaction of blast waves with the head may injure the brain. However, there is no acceptable methodology to scale the findings from animals to humans. Here, we propose an experimental/computational approach to project observed blast-induced molecular changes in the rat brain to the human brain. Using a shock tube, we exposed rats to a range of blast overpressures (BOPs) and used a high-fidelity computational model of a rat head to correlate predicted biomechanical responses with measured changes in glial fibrillary acidic protein (GFAP) in rat brain tissues. Our analyses revealed correlates between model-predicted strain rate and measured GFAP changes in three brain regions. Using these correlates and a high-fidelity computational model of a human head, we determined the equivalent BOPs in rats and in humans that induced similar strain rates across the two species. We used the equivalent BOPs to project the measured GFAP changes in the rat brain to the human. Our results suggest that, relative to the rat, the human requires an exposure to a blast wave of a higher magnitude to elicit similar brain-tissue responses. Our proposed methodology could assist in the development of safety guidelines for blast exposure.

Combination of antiseizure medications phenobarbital, ketamine, and midazolam reduces soman-induced epileptogenesis and brain pathology in rats.

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.

Investigation of the direct and indirect mechanisms of primary blast insult to the brain.

The interaction of explosion-induced blast waves with the head (i.e., a direct mechanism) or with the torso (i.e., an indirect mechanism) presumably causes traumatic brain injury. However, the understanding of the potential role of each mechanism in causing this injury is still limited. To address this knowledge gap, we characterized the changes in the brain tissue of rats resulting from the direct and indirect mechanisms at 24 h following blast exposure. To this end, we conducted separate blast-wave exposures on rats in a shock tube at an incident overpressure of 130 kPa, while using whole-body, head-only, and torso-only configurations to delineate each mechanism. Then, we performed histopathological (silver staining) and immunohistochemical (GFAP, Iba-1, and NeuN staining) analyses to evaluate brain-tissue changes resulting from each mechanism. Compared to controls, our results showed no significant changes in torso-only-exposed rats. In contrast, we observed significant changes in whole-body-exposed (GFAP and silver staining) and head-only-exposed rats (silver staining). In addition, our analyses showed that a head-only exposure causes changes similar to those observed for a whole-body exposure, provided the exposure conditions are similar. In conclusion, our results suggest that the direct mechanism is the major contributor to blast-induced changes in brain tissues.

Phosphorylated Neurofilament Heavy Chain in the Cerebrospinal Fluid Is a Suitable Biomarker of Acute and Chronic Blast-Induced Traumatic Brain Injury.

Blast-induced traumatic brain injury (bTBI) has been documented as a significant concern for both military and civilian populations in response to the increased use of improvised explosive devices. Identifying biomarkers that could aid in the proper diagnosis and assessment of both acute and chronic bTBI is in urgent need since little progress has been made towards this goal. Addressing this knowledge gap is especially important in military veterans who are receiving assessment and care often years after their last blast exposure. Neuron-specific phosphorylated neurofilament heavy chain protein (pNFH) has been successfully evaluated as a reliable biomarker of different neurological disorders, as well as brain trauma resulting from contact sports and acute stages of brain injury of different origin. In the present study, we have evaluated the utility of pNFH levels measured in the cerebrospinal fluid (CSF) as an acute and chronic biomarker of brain injury resulting from single and tightly coupled repeated blast exposures using experimental rats. The pNFH levels increased at 24 h, returned to normal levels at 1 month, but increased again at 6 months and 1 year post-blast exposures. No significant changes were observed between single and repeated blast-exposed groups. To determine whether the observed increase of pNFH in CSF corresponded with its levels in the brain, we performed fluorescence immunohistochemistry in different brain regions at the four time-points evaluated. We observed decreased pNFH levels in those brain areas at 24 h, 6 months, and 1 year. The results suggest that blast exposure causes axonal degeneration at acute and chronic stages resulting in the release of pNFH, the abundant neuronal cytoskeletal protein. Moreover, the changes in pNFH levels in the CSF negatively correlated with the neurobehavioral functions in the rats, reinforcing suggestions that CSF levels of pNFH can be a suitable biomarker of bTBI.

Blast Exposure Causes Long-Term Degeneration of Neuronal Cytoskeletal Elements in the Cochlear Nucleus: A Potential Mechanism for Chronic Auditory Dysfunctions.

Blast-induced auditory dysfunctions including tinnitus are the most prevalent disabilities in service members returning from recent combat operations. Most of the previous studies were focused on the effect of blast exposure on the peripheral auditory system and not much on the central auditory signal-processing regions in the brain. In the current study, we have exposed rats to single and tightly coupled repeated blasts and examined the degeneration of neuronal cytoskeletal elements using silver staining in the central auditory signal-processing regions in the brain at 24 h, 14 days, 1 month, 6 months, and 1 year. The brain regions evaluated include cochlear nucleus, lateral lemniscus, inferior colliculus, medial geniculate nucleus, and auditory cortex. The results obtained indicated that a significant increase in degeneration of neuronal cytoskeletal elements was observed only in the left and right cochlear nucleus. A significant increase in degeneration of neuronal cytoskeletal elements was observed in the cochlear nucleus at 24 h and persisted through 1 year, suggesting acute and chronic neuronal degeneration after blast exposure. No statistically significant differences were observed between single and repeated blasts. The localized degeneration of neuronal cytoskeletal elements in the cochlear nucleus suggests that the damage could be caused by transmission of blast shockwaves/noise through the ear canal and that use of suitable ear protection devices can protect against acute and chronic central auditory signal processing defects including tinnitus after blast exposure.

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'.

Blast Exposure Leads to Accelerated Cellular Senescence in the Rat Brain.

Blast-induced traumatic brain injury (bTBI) is one of the major causes of persistent disabilities in Service Members, and a history of bTBI has been identified as a primary risk factor for developing age-associated neurodegenerative diseases. Clinical observations of several military blast casualties have revealed a rapid age-related loss of white matter integrity in the brain. In the present study, we have tested the effect of single and tightly coupled repeated blasts on cellular senescence in the rat brain. Isoflurane-anesthetized rats were exposed to either a single or 2 closely coupled blasts in an advanced blast simulator. Rats were euthanized and brains were collected at 24 h, 1 month and 1 year post-blast to determine senescence-associated-ß-galactosidase (SA-ß-gal) activity in the cells using senescence marker stain. Single and repeated blast exposures resulted in significantly increased senescence marker staining in several neuroanatomical structures, including cortex, auditory cortex, dorsal lateral thalamic nucleus, geniculate nucleus, superior colliculus, ventral thalamic nucleus and hippocampus. In general, the increases in SA-ß-gal activity were more pronounced at 1 month than at 24 h or 1 year post-blast and were also greater after repeated than single blast exposures. Real-time quantitative RT-PCR analysis revealed decreased levels of mRNA for senescence marker protein-30 (SMP-30) and increased mRNA levels for p21 (cyclin dependent kinase inhibitor 1A, CDKN1A), two other related protein markers of cellular senescence. The increased senescence observed in some of these affected brain structures may be implicated in several long-term sequelae after exposure to blast, including memory disruptions and impairments in movement, auditory and ocular functions.

Antibodies Against Lysophosphatidic Acid Protect Against Blast-Induced Ocular Injuries.

Exposure to blast overpressure waves is implicated as the major cause of ocular injuries and resultant visual dysfunction in veterans involved in recent combat operations. No effective therapeutic strategies have been developed so far for blast-induced ocular dysfunction. Lysophosphatidic acid (LPA) is a bioactive phospholipid generated by activated platelets, astrocytes, choroidal plexus cells, and microglia and is reported to play major roles in stimulating inflammatory processes. The levels of LPA in the cerebrospinal fluid have been reported to increase acutely in patients with traumatic brain injury (TBI) as well as in a controlled cortical impact (CCI) TBI model in mice. In the present study, we have evaluated the efficacy of a single intravenous administration of a monoclonal LPA antibody (25 mg/kg) given at 1 h post-blast for protection against injuries to the retina and associated ocular dysfunctions. Our results show that a single 19 psi blast exposure significantly increased the levels of several species of LPA in blood plasma at 1 and 4 h post-blast. The anti-LPA antibody treatment significantly decreased glial cell activation and preserved neuronal cell morphology in the retina on day 8 after blast exposure. Optokinetic measurements indicated that anti-LPA antibody treatment significantly improved visual acuity in both eyes on days 2 and 6 post-blast exposure. Anti-LPA antibody treatment significantly increased rod photoreceptor and bipolar neuronal cell signaling in both eyes on day 7 post-blast exposure. These results suggest that blast exposure triggers release of LPAs, which play a major role promoting blast-induced ocular injuries, and that a single early administration of anti-LPA antibodies provides significant protection.

Rational polytherapy in the treatment of cholinergic seizures.

The initiation and maintenance phases of cholinergic status epilepticus (SE) are associated with maladaptive trafficking of synaptic GABAA and glutamate receptors. The resulting pharmacoresistance reflects a decrease in synaptic GABAA receptors and increase in NMDA and AMPA receptors, which tilt the balance between inhibition and excitation in favor of the latter. If these changes are important to the pathophysiology of SE, both should be treated, and blocking their consequences should have therapeutic potential. We used a model of benzodiazepine-refractory SE (RSE) (Tetz et al., 2006) and a model of soman-induced SE to test this hypothesis. Treatment of RSE with combinations of the GABAAR agonists midazolam or diazepam and the NMDAR antagonists MK-801 or ketamine terminated RSE unresponsive to high-dose monotherapy with benzodiazepines, ketamine or other antiepileptic drugs (AEDs). 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 sc soman-induced SE similarly showed much greater reduction of EEG power by a combination of midazolam with ketamine, compared to midazolam monotherapy. When treating late (40 min after seizure onset), there may not be enough synaptic GABAAR left to be able to restore inhibition with maximal GABAAR stimulation, and further benefit is derived from the addition of an AED which increases inhibition or reduces excitation by a non-GABAergic mechanism. The midazolam-ketamine-valproate combination is effective in terminating RSE. 3-D isobolograms demonstrate positive cooperativity between midazolam, ketamine and valproate, without any interaction between the toxicity of these drugs, so that the therapeutic index is increased by combination therapy between GABAAR agonist, NMDAR antagonist and selective AEDs. We compared this drug combination based on the receptor trafficking hypothesis to treatments based on clinical practice. The midazolam-ketamine-valproate combination is far more effective in stopping RSE than the midazolam-fosphenytoin-valproate combination inspired from clinical guidelines. Furthermore, sequential administration of midazolam, ketamine and valproate is far less effective than simultaneous treatment with the same drugs at the same dose. These data suggest that we should re-evaluate our traditional treatment of RSE, and that treatment should be based on pathophysiology. The search for a better drug has to deal with the fact that most monotherapy leaves half the problem untreated. The search for a better benzodiazepine should acknowledge the main cause of pharmacoresistance, which is loss of synaptic GABAAR. 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.

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".

Poisoning with Soman, an Organophosphorus Nerve Agent, Alters Fecal Bacterial Biota and Urine Metabolites: a Case for Novel Signatures for Asymptomatic Nerve Agent Exposure.

The experimental pathophysiology of organophosphorus (OP) chemical exposure has been extensively reported. Here, we describe an altered fecal bacterial biota and urine metabolome following intoxication with soman, a lipophilic G class chemical warfare nerve agent. Nonanesthetized Sprague-Dawley male rats were subcutaneously administered soman at 0.8 (subseizurogenic) or 1.0 (seizurogenic) of the 50% lethal dose (LD50) and evaluated for signs of toxicity. Animals were stratified based on seizing activity to evaluate effects of soman exposure on fecal bacterial biota and urine metabolites. Soman exposure reshaped fecal bacterial biota by altering Facklamia, Rhizobium, Bilophila, Enterobacter, and Morganella genera of the Firmicutes and Proteobacteria phyla, some of which are known to hydrolyze OP chemicals. However, analogous changes were not observed in the bacterial biota of the ileum, which remained the same irrespective of dose or seizing status of animals after soman intoxication. However, at 75 days after soman exposure, the bacterial biota stabilized and no differences were observed between groups. Interestingly, in considering just the seizing status of animals, we found that the urine metabolomes were markedly different. Leukotriene C4, kynurenic acid, 5-hydroxyindoleacetic acid, norepinephrine, and aldosterone were excreted at much higher rates at 72 h in seizing animals, consistent with early multiorgan involvement during soman poisoning. These findings demonstrate the feasibility of using the dysbiosis of fecal bacterial biota in combination with urine metabolome alterations as forensic evidence for presymptomatic OP exposure temporally to enable administration of neuroprotective therapies of the future.IMPORTANCE The paucity of assays to determine physiologically relevant OP exposure presents an opportunity to explore the use of fecal bacteria as sentinels in combination with urine to assess changes in the exposed host. Recent advances in sequencing technologies and computational approaches have enabled researchers to survey large community-level changes of gut bacterial biota and metabolomic changes in various biospecimens. Here, we profiled changes in fecal bacterial biota and urine metabolome following a chemical warfare nerve agent exposure. The significance of this work is a proof of concept that the fecal bacterial biota and urine metabolites are two separate biospecimens rich in surrogate indicators suitable for monitoring OP exposure. The larger value of such an approach is that assays developed on the basis of these observations can be deployed in any setting with moderate clinical chemistry and microbiology capability. This can enable estimation of the affected radius as well as screening, triage, or ruling out of suspected cases of exposures in mass casualty scenarios, transportation accidents involving hazardous materials, refugee movements, humanitarian missions, and training settings when coupled to an established and validated decision tree with clinical features.

Age-Related Susceptibility to Epileptogenesis and Neuronal Loss in Male Fischer Rats Exposed to Soman and Treated With Medical Countermeasures.

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.

The limitations of diazepam as a treatment for nerve agent-induced seizures and neuropathology in rats: comparison with UBP302.

Exposure to nerve agents induces prolonged status epilepticus (SE), causing brain damage or death. Diazepam (DZP) is the current US Food and Drug Administration-approved drug for the cessation of nerve agent-induced SE. Here, we compared the efficacy of DZP with that of UBP302 [(S)-3-(2-carboxybenzyl)willardiine; an antagonist of the kainate receptors that contain the GluK1 subunit] against seizures, neuropathology, and behavioral deficits induced by soman in rats. DZP, administered 1 hour or 2 hours postexposure, terminated the SE, but seizures returned; thus, the total duration of SE within 24 hours after soman exposure was similar to (DZP at 1 hour) or longer than (DZP at 2 hours) that in the soman-exposed rats that did not receive the anticonvulsant. Compared with DZP, UBP302 stopped SE with a slower time course, but dramatically reduced the total duration of SE within 24 hours. Neuropathology and behavior were assessed in the groups that received anticonvulsant treatment 1 hour after exposure. UBP302, but not DZP, reduced neuronal degeneration in a number of brain regions, as well as neuronal loss in the basolateral amygdala and the CA1 hippocampal area, and prevented interneuronal loss in the basolateral amygdala. Anxiety-like behavior was assessed in the open field and by the acoustic startle response 30 days after soman exposure. The results showed that anxiety-like behavior was increased in the DZP-treated group and in the group that did not receive anticonvulsant treatment, but not in the UBP302-treated group. The results argue against the use of DZP for the treatment of nerve agent-induced seizures and brain damage and suggest that targeting GluK1-containing receptors is a more effective approach.

The recovery of acetylcholinesterase activity and the progression of neuropathological and pathophysiological alterations in the rat basolateral amygdala after soman-induced status epilepticus: relation to anxiety-like behavior.

Organophosphorus nerve agents are powerful neurotoxins that irreversibly inhibit acetylcholinesterase (AChE) activity. One of the consequences of AChE inhibition is the generation of seizures and status epilepticus (SE), which cause brain damage, resulting in long-term neurological and behavioral deficits. Increased anxiety is the most common behavioral abnormality after nerve agent exposure. This is not surprising considering that the amygdala, and the basolateral nucleus of the amygdala (BLA) in particular, plays a central role in anxiety, and this structure suffers severe damage by nerve agent-induced seizures. In the present study, we exposed male rats to the nerve agent soman, at a dose that induce SE, and determined the time course of recovery of AChE activity, along with the progression of neuropathological and pathophysiological alterations in the BLA, during a 30-day period after exposure. Measurements were taken at 24 h, 7 days, 14 days, and 30 days after exposure, and at 14 and 30 days, anxiety-like behavior was also evaluated. We found that more than 90% of AChE is inhibited at the onset of SE, and AChE inhibition remains at this level 24 h later, in the BLA, as well as in the hippocampus, piriform cortex, and prelimbic cortex, which we analyzed for comparison. AChE activity recovered by day 7 in the BLA and day 14 in the other three regions. Significant neuronal loss and neurodegeneration were present in the BLA at 24 h and throughout the 30-day period. There was no significant loss of GABAergic interneurons in the BLA at 24 h post-exposure. However, by day 7, the number of GABAergic interneurons in the BLA was reduced, and at 14 and 30 days after soman, the ratio of GABAergic interneurons to the total number of neurons was lower compared to controls. Anxiety-like behavior in the open-field and the acoustic startle response tests was increased at 14 and 30 days post-exposure. Accompanying pathophysiological alterations in the BLA - studied in in vitro brain slices - included a reduction in the amplitude of field potentials evoked by stimulation of the external capsule, along with prolongation of their time course and an increase in the paired-pulse ratio. Long-term potentiation was impaired at 24 h, 7 days, and 14 days post-exposure. The loss of GABAergic interneurons in the BLA and the decreased interneuron to total number of neurons ratio may be the primary cause of the development of anxiety after nerve agent exposure.

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.

Behavioral and EEG effects of GABAergic manipulation of the nigro-tectal pathway in the Wistar audiogenic rat (WAR) strain II: an EEG wavelet analysis and retrograde neuronal tracer approach.

The role of the substantia nigra pars reticulata (SNPr) and superior colliculus (SC) network in rat strains susceptible to audiogenic seizures still remain underexplored in epileptology. In a previous study from our laboratory, the GABAergic drugs bicuculline (BIC) and muscimol (MUS) were microinjected into the deep layers of either the anterior SC (aSC) or the posterior SC (pSC) in animals of the Wistar audiogenic rat (WAR) strain submitted to acoustic stimulation, in which simultaneous electroencephalographic (EEG) recording of the aSC, pSC, SNPr and striatum was performed. Only MUS microinjected into the pSC blocked audiogenic seizures. In the present study, we expanded upon these previous results using the retrograde tracer Fluorogold (FG) microinjected into the aSC and pSC in conjunction with quantitative EEG analysis (wavelet transform), in the search for mechanisms associated with the susceptibility of this inbred strain to acoustic stimulation. Our hypothesis was that the WAR strain would have different connectivity between specific subareas of the superior colliculus and the SNPr when compared with resistant Wistar animals and that these connections would lead to altered behavior of this network during audiogenic seizures. Wavelet analysis showed that the only treatment with an anticonvulsant effect was MUS microinjected into the pSC region, and this treatment induced a sustained oscillation in the theta band only in the SNPr and in the pSC. These data suggest that in WAR animals, there are at least two subcortical loops and that the one involved in audiogenic seizure susceptibility appears to be the pSC-SNPr circuit. We also found that WARs presented an increase in the number of FG+ projections from the posterior SNPr to both the aSC and pSC (primarily to the pSC), with both acting as proconvulsant nuclei when compared with Wistar rats. We concluded that these two different subcortical loops within the basal ganglia are probably a consequence of the WAR genetic background.

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.

Behavioral and EEG effects of GABAergic manipulation of the nigrotectal pathway in the Wistar audiogenic rat strain.

The superior colliculus (SC), substantia nigra pars reticulata (SNPr), and striatum have been characterized as important structures involved in the modulation of seizure activity. In the current study, bicuculline (GABA(A) antagonist) and muscimol (GABA(A) agonist) were microinjected into the deep layers of either the anterior SC (aSC) or posterior SC (pSC) of genetically developed Wistar audiogenic rats. Behavior and EEG activity were studied simultaneously. Only muscimol microinjected into the pSC had behavioral and EEG anticonvulsant effects in Wistar audiogenic rats, eliciting EEG oscillation changes in both SNPr and pSC, primarily during tonic seizures. The SC of Wistar audiogenic rats thus comprises two functionally different subregions, pSC and aSC, defined by distinct behavioral and EEG features. The pSC has proconvulsant audiogenic seizure activity in Wistar audiogenic rats. Our data suggest that this phenomenon may be a consequence of the genetic selection of the Wistar audiogenic rat strain.

Role of the superior colliculus in the expression of acute and kindled audiogenic seizures in Wistar audiogenic rats.

PURPOSE: The role of the superior colliculus (SC) in seizure expression is controversial and appears to be dependent upon the epilepsy model. This study shows the effect of disconnection between SC deep layers and adjacent tissues in the expression of acute and kindling seizures. METHODS: Subcollicular transections, ablation of SC superficial and deep layers, and ablation of only the cerebral cortex were evaluated in the Wistar audiogenic rat (WAR) strain during acute and kindled audiogenic seizures. The audiogenic seizure kindling protocol started 4 days after surgeries, with two acoustic stimuli per day for 10 days. Acute audiogenic seizures were evaluated by a categorized seizure severity midbrain index (cSI) and kindled seizures by a severity limbic index (LI). RESULTS: All subcollicular transections reaching the deep layers of the SC abolished audiogenic seizures or significantly decreased cSI. In the unlesioned kindled group, a reciprocal relationship between limbic and brainstem pattern of seizures was seen. The increased number of stimuli provoked an audiogenic kindling phenomenon. Ablation of the entire SC (ablation group) or of the cerebral cortex only (ctx-operated group) hampered the acquisition of limbic behaviors. There was no difference in cSI and LI between the ctx-operated and ablation groups, but there was a difference between ctx-operated and the unlesioned kindled group. There was also no difference in cSI between SC deep layer transection and ablation groups. Results of histologic analyses were similar for acute and kindled audiogenic seizure groups. CONCLUSIONS: SC deep layers are involved in the expression of acute and kindled audiogenic seizure, and the cerebral cortex is essential for audiogenic kindling development.