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.

Spontaneous recurrent seizures after status epilepticus induced by soman in Sprague-Dawley rats.

PURPOSE: Exposure to toxic levels of organophosphorus (OP) nerve agents can lead to seizures, respiratory failure, and, if untreated, death. The cholinesterase inhibitor soman belongs to the class of OP nerve agents and can cause status epilepticus (SE) and brain damage due to neuroexcitotoxicity. In the present study, electroencephalographic seizures are characterized through telemetry implants in rats exposed to soman, followed by treatment with therapeutics similar to those administered after nerve agent exposure. METHODS: Cortical electroencephalography (EEG), motor activity and body temperature were recorded continuously for 2 days preexposure and 15 days postexposure to verify the occurrence of spontaneous recurrent seizures (SRS) after soman exposure. RESULTS: Behavioral seizures were monitored and the latency to SE was 7.8 ± 4.0 min after exposure. Among the rats that showed SE, approximately 90% had prolonged seizures within the initial 3 days after soman exposure. Five percent of the rats developed stage 1 seizures, 16% stage 2, 23% stage 3, 18% stage 4, and 38% stage 5. Seventy-nine percent of the rats presented SE and epileptiform-like discharges several days after SE, and 28.9% of those with SE experienced electrographic SRS. The latency to the appearance of SRS ranged from 5-10 days. Fiber degeneration evaluated through silver staining revealed damage in cortical and subcortical areas directly correlated with SE. DISCUSSION: The presence of SRS after seizures induced by soman highlights the importance of quantifying SRS in studies where the objective is to find new therapeutics against soman-induced seizures.

Analyzing large data sets acquired through telemetry from rats exposed to organophosphorous compounds: an EEG study.

The organophosphorous compound soman is an acetylcholinesterase inhibitor that causes damage to the brain. Exposure to soman causes neuropathology as a result of prolonged and recurrent seizures. In the present study, long-term recordings of cortical EEG were used to develop an unbiased means to quantify measures of seizure activity in a large data set while excluding other signal types. Rats were implanted with telemetry transmitters and exposed to soman followed by treatment with therapeutics similar to those administered in the field after nerve agent exposure. EEG, activity and temperature were recorded continuously for a minimum of 2 days pre-exposure and 15 days post-exposure. A set of automatic MATLAB algorithms have been developed to remove artifacts and measure the characteristics of long-term EEG recordings. The algorithms use short-time Fourier transforms to compute the power spectrum of the signal for 2-s intervals. The spectrum is then divided into the delta, theta, alpha, and beta frequency bands. A linear fit to the power spectrum is used to distinguish normal EEG activity from artifacts and high amplitude spike wave activity. Changes in time spent in seizure over a prolonged period are a powerful indicator of the effects of novel therapeutics against seizures. A graphical user interface has been created that simultaneously plots the raw EEG in the time domain, the power spectrum, and the wavelet transform. Motor activity and temperature are associated with EEG changes. The accuracy of this algorithm is also verified against visual inspection of video recordings up to 3 days after exposure.

Students investigating the antiproliferative effects of synthesized drugs on mouse mammary tumor cells.

The potential for personalized cancer management has long intrigued experienced researchers as well as the naïve student intern. Personalized cancer treatments based on a tumor's genetic profile are now feasible and can reveal both the cells' susceptibility and resistance to chemotherapeutic agents. In a weeklong laboratory investigation that mirrors current cancer research, undergraduate and advanced high school students determine the efficacy of common pharmacological agents through in vitro testing. Using mouse mammary tumor cell cultures treated with "unknown" drugs historically recommended for breast cancer treatment, students are introduced to common molecular biology techniques from in vitro cell culture to fluorescence microscopy. Student understanding is assessed through laboratory reports and the successful identification of the unknown drug. The sequence of doing the experiment, applying logic, and constructing a hypothesis gives the students time to discover the rationale behind the cellular drug resistance assay. The breast cancer experiment has been field tested during the past 5 yr with more than 200 precollege/undergraduate interns through the Gains in the Education of Mathematics and Science program hosted by the Walter Reed Army Institute of Research.

Transcriptional profiling of mefloquine-induced disruption of calcium homeostasis in neurons in vitro.

Mefloquine is associated with adverse neurological effects that are mediated via unknown mechanisms. Recent in vitro studies have shown that mefloquine disrupts neuronal calcium homeostasis via liberation of the endoplasmic reticulum (ER) store and induction of calcium influx across the plasma membrane. In the present study, global changes in gene expression induced in neurons in response to mefloquine-induced disruption of calcium homeostasis and appropriate control agents were investigated in vitro using Affymetrix arrays. The mefloquine transcriptome was found to be enriched for important regulatory sequences of the unfolded protein response and the drug was also found to induce key ER stress proteins, albeit in a manner dissimilar to, and at higher equivalent concentrations than, known ER-tropic agents like thapsigargin. Mefloquine also down-regulated several important functional categories of genes, including transcripts encoding G proteins and ion channels. These effects may be related to intrusion of extracellular calcium since they were also observed after glutamate, but not thapsigargin, hydrogen peroxide, or low-dose mefloquine treatment. Mefloquine could be successfully differentiated from other treatments on the basis of principle component analysis of its "calcium-relevant" transcriptome. These data may aid interpretation of expression of results from future in vivo studies.

Inhibition of glutamate carboxypeptidase II (NAALADase) protects against dynorphin A-induced ischemic spinal cord injury in rats.

Glutamate carboxypeptidase (GCP) II (EC 3.4.17.21), which is also known as N-acetylated-alpha-linked acidic dipeptidase (NAALADase), hydrolyses the endogenous acidic dipeptide N-acetylaspartylglutamate (NAAG), yielding N-acetyl-aspartate and glutamate. Inhibition of this enzyme by 2-(phosphonomethyl) pentanedioic acid (2-PMPA) has been shown to protect against ischemic injury to the brain and hypoxic and metabolic injury to neuronal cells in culture, presumably by increasing and decreasing the extracellular concentrations of NAAG and glutamate, respectively. Since both NAAG and GCP II are found in especially high concentrations in the spinal cord, injuries to the spinal cord involving pathophysiological elevations in extracellular glutamate might be particularly responsive to GCP II inhibition. Lumbar subarachnoid injections of dynorphin A in rats cause ischemic spinal cord injury, elevated extracellular glutamate and a persistent hindlimb paralysis that is mediated through excitatory amino acid receptors. We therefore used this injury model to evaluate the protective effects of 2-PMPA. When coadministered with dynorphin A, 2-PMPA significantly attenuated the dynorphin A-induced elevations in cerebrospinal fluid glutamate levels and by 24 h postinjection caused significant dose-dependent improvements in motor scores that were associated with marked histopathological improvements. These results indicate that 2-PMPA provides effective protection against excitotoxic spinal cord injury.

Lipoic acid pretreatment attenuates ferric chloride-induced seizures in the rat.

Traumatic brain injury (TBI) is often complicated by the occurrence of seizures, which adversely affect clinical outcome. The risk of seizures increases to the extent that the injury is associated with sub-arachnoid hemorrhage and hematoma. A likely mechanism of seizure development post-TBI is decompartmentalization of iron from extravasated hemoglobin (Hb). It is well known that iron can catalyze formation of reactive oxygen species (ROS). Based on this proposed mechanism, a descriptive model of TBI-induced seizures, using intracortical injection of iron salts, was developed by Willmore. We have added modifications to enhance the quantifiability of seizure activity and have used the model to examine the therapeutic efficacy of lipoic acids (ROS-scavenging antioxidants). Male SD rats were pretreated with alpha-lipoic acid (ALA) and dihydrolipoic acid (DHLA) or appropriate vehicles. Under anesthesia, unilateral intracortical infusions of ferric chloride were performed stereotaxically. EEG was recorded via extradural electrodes. EEG was sampled for 10 s of every 60-s interval over a 24-h period following injection of ferric chloride. We measured the number of seconds of epileptiform discharges or seizure activity in every 10-s EEG sample during the 24 h. The EEGs of rats pretreated with ALA and DHLA exhibited 55% less seizure activity than vehicle-treated ferric chloride-injected animals, suggesting that lipoic acids may be of use in preventing or attenuating TBI-induced seizures.

Fate of chemicals in skin after dermal application: does the in vitro skin reservoir affect the estimate of systemic absorption?

Recent international guidelines for the conduct of in vitro skin absorption studies put forward different approaches for addressing the status of chemicals remaining in the stratum corneum and epidermis/dermis at the end of a study. The present study investigated the fate of three chemicals [dihydroxyacetone (DHA), 7-(2H-naphtho[1,2-d]triazol-2-yl)-3-phenylcoumarin (7NTPC), and disperse blue 1 (DB1)] in an in vitro absorption study. In these studies, human and fuzzy rat skin penetration and absorption were determined over 24 or 72 h in flow-through diffusion cells. Skin penetration of these chemicals resulted in relatively low receptor fluid levels but high skin levels. For DHA, penetration studies found approximately 22% of the applied dose remaining in the skin (in both the stratum corneum and viable tissue) as a reservoir after 24 h. Little of the DHA that penetrates into skin is actually available to become systemically absorbed. 7NTPC remaining in the skin after 24 h was approximately 14.7% of the applied dose absorbed. Confocal laser cytometry studies with 7NTPC showed that it is present across skin in mainly the epidermis and dermis with intense fluorescence around hair. For DB1, penetration studies found approximately 10% (ethanol vehicle) and 3% (formulation vehicle) of the applied dose localized in mainly the stratum corneum after 24 h. An extended absorption study (72 h) revealed that little additional DB1 was absorbed into the receptor fluid. Skin levels should not be considered as absorbed material for DHA or DB1, while 7NTPC requires further investigation. These studies illustrate the importance of determining the fate of chemicals remaining in skin, which could significantly affect the estimates of systemically available material to be used in exposure estimates. We recommend that a more conclusive means to determine the fate of skin levels is to perform an extended study as conducted for DB1.

N-acetylaspartylglutamate and beta-NAAG protect against injury induced by NMDA and hypoxia in primary spinal cord cultures.

The acidic dipeptide N-acetylaspartylglutamate (NAAG) is the most prevalent peptide in the central nervous system. NAAG is a low potency agonist at the NMDA receptor, and hydrolysis of NAAG yields the more potent excitatory amino acid neurotransmitter glutamate. beta-NAAG is a competitive inhibitor of the NAAG hydrolyzing enzyme N-acetylated alpha-linked acidic dipeptidase (NAAG peptidase activity) or glutamate carboxypeptidase II, and may also act as a NAAG-mimetic at some of the sites of NAAG pharmacological activity. Since NAAG has been shown to have neuroprotective characteristics in a number of experimental preparations, it is the purpose of the present study to specifically evaluate the possible efficacy of NAAG and beta-NAAG against NMDA- and hypoxia-induced injury to spinal cord mixed neuronal and glial cell cultures. NAAG (500-1000 microM) protected against NMDA- or hypoxia-induced injuries to spinal cord cultures, and the nonhydrolyzable analog beta-NAAG (250-1000 microM) completely eliminated the loss of viability caused by either insult. Both peptides also attenuated NMDA-induced increases in intraneuronal Ca(2+). Nonspecific mGluR antagonists, pertussis toxin, a stable cAMP analog, and manipulation of NAAG peptidase activity did not by themselves alter cell damage and did not influence the neuroprotective effects of NAAG. NAAG was not protective against kainate- or AMPA-induced cellular injury, while beta-NAAG was partially neuroprotective against both insults. At 2 mM, NAAG and beta-NAAG reduced neuronal survival and increased intraneuronal Ca(2+); these effects were only marginally attenuated by dizocilpine and APV. The results indicate that NAAG and beta-NAAG protect against excitotoxic and hypoxic injury to spinal cord neurons, and do so predominantly by interactions with NMDA and not mGluR receptors.