Status epilepticus (SE), a neurological emergency both in adults and in children, could lead to brain damage and even death if untreated. Generalized convulsive SE (GCSE) is the most common and severe form, an example of which is that induced by organophosphorus nerve agents. First- and second-line pharmacotherapies are relatively consensual, but if seizures are still not controlled, there is currently no definitive data to guide the optimal choice of therapy. The medical community seems largely reluctant to use ketamine, a noncompetitive antagonist of the N-methyl-d-aspartate glutamate receptor. However, a review of the literature clearly shows that ketamine possesses, in preclinical studies, antiepileptic properties and provides neuroprotection. Clinical evidences are scarcer and more difficult to analyze, owing to a use in situations of polytherapy. In absence of existing or planned randomized clinical trials, the medical community should make up its mind from well-conducted preclinical studies performed on appropriate models. Although potentially active, ketamine has no real place for the treatment of isolated seizures, better accepted drugs being used. Its best usage should be during GCSE, but not waiting for SE to become totally refractory. Concerns about possible developmental neurotoxicity might limit its pediatric use for refractory SE.

Organophosphorus nerve agents (NA), potent irreversible cholinesterase inhibitors, could induce severe seizures, status epilepticus (SE), seizure-related brain damage (SRBD) and lethality. Despite the lack of data in the case of NA, clinical evidences suggest that SE survivors could suffer from neurological/cognitive deficits and impairments such as spontaneous recurrent seizures (epilepsy) after a latent period of epileptogenesis. It is beyond doubt that an effective and quick management of the initial seizures and prevention of SRBD are critical to prevent these long-term consequences, explaining why most experimental data are focusing on the 5–40 min post-exposure time frame. However, in field conditions, treatment may be delayed and with the exception of NMDA receptor antagonists, currently no drug provides protection (against lethality, seizures, SRBD and neurological consequences) when seizures are left unabated for one hour or more. Ketamine (KET) is the only NMDA antagonist licensed as an injectable drug in different countries and remains an anesthetic of choice in some difficult field conditions. In this short review paper, after a presentation of some of the key points of the pathophysiology of NA-induced SE and a quick survey of the potential therapeutic avenues in the context of delayed treatment of NA-induced SE, we will review the recent data we obtained showing that KET, in combination with atropine sulfate (AS), with or without a benzodiazepine, considerably reduces soman-induced neuroinflammation, provides neuroprotection, histologically and functionally, and also positively modify soman-induced changes in brain metabolism. Finally, we will also mention some results from safety studies including those bringing evidence that, at difference with MK-801, KET does not impair thermoregulation and even seems to reduce AS-induced heat stress. All in all, KET, in combination, appears a good candidate for the out-of-hospital treatment of severe NA-induced SE.

Epileptic seizures and status epilepticus (SE) induced by the poisoning with organophosphorus nerve agents (OP), like soman, are accompanied by neuroinflammation whose role in seizure-related brain damage (SRBD) is not clear. Antagonists of the NMDA glutamate ionotropic receptors are currently among the few compounds able to arrest seizures and provide neuroprotection even during refractory status epilepticus (RSE). Racemic ketamine (KET), in combination with atropine sulfate (AS), was previously shown to counteract seizures and SRBD in soman-poisoned guinea-pigs. In a mouse model of severe soman-induced SE, we assessed the potentials of KET/AS combinations as a treatment for SE/RSE-induced SRBD and neuroinflammation. When starting 30min after soman challenge, a protocol involving six injections of a sub-anesthetic dose of KET (25mg/kg) was evaluated on body weight loss, brain damage, and neuroinflammation whereas during RSE, anesthetic protocols were considered (KET 100mg/kg). After confirming that during RSE, KET injection was to be repeated despite some iatrogenic deaths, we used these proof-of-concept protocols to study the changes in mRNA and related protein contents of some inflammatory cytokines, chemokines and adhesion molecules in cortex and hippocampus 48h post-challenge. In both cases, the KET/AS combinations showed important neuroprotective effects, suppressed neutrophil granulocyte infiltration and partially suppressed glial activation. KET/AS could also reduce the increase in mRNA and related pro-inflammatory proteins provoked by the poisoning. In conclusion, the present study confirms that KET/AS treatment has a strong potential for SE/RSE management following OP poisoning. The mechanisms involved in the reduction of central neuroinflammation remain to be studied.

This work presents a model combining quantitative proton HRMAS NMR data and PLS-DA for neuropathology and neuroprotection evaluation. Metabolic data were also confronted to histopathological results obtained using the same experimental conditions. Soman, when not lethal, can induce status epilepticus (SE), brain damage, histological lesions, and profound cerebral metabolic disorders as revealed using (1)H HRMAS NMR. Our challenge was to evaluate delayed treatments, which could control refractory SE and avoid brain lesions. For this aim, we have built a statistical model of soman intoxication describing brain metabolite evolution during 7 days. We have then used this model to evaluate the efficiency of a combination of ketamine/atropine (KET/AS) administrated 1 and 2 h after SE induction, compared to the immediate anticonvulsant therapy midazolam/atropine sulfate (MDZ/AS). Furthermore, quantitation of HRMAS NMR data allowed us to follow individual evolution of 17 metabolites. N-Acetylaspartate, lactate, or taurine presented a long lasting disruption, while glutamine, alanine, glycerophosphocholine and myo-inositol showed disruptions for 3 days with a reversion at day 7. These changes were completely normalized by the administration of MDZ/AS. Interestingly, they were also almost completely reversed by KET/AS 1 h postsoman. This work suggests further the predictive interest of HRMAS and PLS-DA for neuropathology/neuroprotection studies and also confirms, on the metabolic aspects, the neuroprotective potentials of KET/AS combinations for the delayed treatment of soman-induced SE.

The mechanisms of epileptogenesis remain largely unknown and are probably diverse. The aim of this study was to investigate the role of focal cholinergic imbalance in epileptogenesis. To address this question, we monitored electroencephalogram (EEG) activity up to 12 weeks after the injection of a potent cholinesterase (ChE) inhibitor (soman) at different doses (0.53, 0.75, 1, 2, 2.8, 4 and 11 nmol) into the right dorsal hippocampus of C57BL/6 mice. Different parameters were used to choose the dose for a focal model of epileptogenesis (mainly electrographic patterns and peripheral ChE inhibition). The pattern of neuronal activation was studied by Fos immunohistochemistry (IHC). Brain damage was evaluated by hemalun-phloxin, neuronal nuclei antigen IHC and silver staining. Glial fibrillary acidic protein IHC was used to evaluate astroglial reaction. Finally, long-term behavioral consequences were characterized. At the highest dose (11 nmol), soman quickly evoked severe signs, including initial seizures and promoted epileptogenesis in the absence of tissue damage. With lower doses, late-onset seizures were evidenced, after 1-4 weeks depending on the dose, despite the absence of initial overt seizures and of brain damage. Only a weak astroglial reaction was observed. Following injection of 1 nmol, Fos changes were first evidenced in the ipsilateral hippocampus and then spread to extrahippocampal areas. A selective deficit in contextual fear conditioning was also evidenced two months after injection. Our data show that focal hypercholinergy may be a sufficient initial event to promote epilepsy and that major brain tissue changes (cellular damage, edema, neuroinflammation) are not necessary conditions.

Soman, an irreversible organophosphorus cholinesterase inhibitor, induces status epilepticus and, in sensitive brain areas, seizure-related brain damage (e.g. brain edema and neuronal loss). The brain metabolic disturbances associated with these events are ill known. In the present study, we thus evaluated these changes in a murine model of soman-induced status epilepticus up to 7 days after intoxication. Mice, protected by HI-6 and atropine methyl nitrate, were poisoned with soman (172μg/kg) and then sacrificed at set time points, from 1h to 7 days. Brain biopsies from the piriform cortex (Pir) and cerebellum (Cer) were analyzed by 1H HRMAS NMR spectroscopy. Spectra were then analyzed using both a supervised multivariate analysis and the QUEST procedure of jMRUI for the quantification of 17 metabolites. The multivariate analysis clearly showed the metabolic differences between a damaged structure (Pir) and a structure with less prominent changes (cerebellum) and helped to globally assess the time course of metabolic changes. Analysis of the individual metabolites showed that the major changes took place in the piriform cortex but that cerebellum was not change-free. The most prominent changes in the former were an early (1–4h) increase in alanine and acetate, a delayed increase in lactate, glycerophosphocholine and glutamine as well as a delayed decrease in myo-inositol and N-acetylaspartate. A week after poisoning, some metabolic disturbances were still present. Further research will be necessary to clarify what could be the involvement of these metabolites in physiological processes and how they might become useful surrogate markers of brain damage and repair.

Purpose: A convulsive dose of soman induces seizure-related brain damage (SRBD), including cerebral edema (CE) and neuronal loss. In the present Study on soman-intoxicated mice, we applied diffusion-weighted magnetic resonance imaging (DW-MRI) and quantitative histology, and we measured brain water content to investigate the antiedematous and neuroprotective efficacies of two hyperosmolar treatments: mannitol (Mann) and hypertonic saline (HTS).Methods: Mice intoxicated with soman (172 mu g/kg after a protective pretreatment) were administered 1 min and 5-h post-challenge an i.v. bolus of saline, of Mann or of HTS. I day later, mice were examined with DW-MRI and then sacrificed for brain histology. Additional animals were intoxicated and treated similarly for the measurement of the brain water content (dry/wet weight method).Results: In intoxicated controls, a significant decrease of the apparent diffusion coefficient (ADC). numerous damaged (eosinophilic) cells, high edema scores, and a significant increase in brain water content were detected 24-h post-challenge in sensitive brain Structures. These soman-induced changes were not significantly modified by treatment with Mann or HTS.Conclusions: Treatment with hyperosmolar solutions did not reduce the effects of soman on ADC, on cell damage and on CE. Therefore, despite similar treatment protocols, the prominent protection by Mann that was previously demonstrated by others in poisoned rats, was not reproduced in our murine model. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Quantitation of High Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) signals enables establishing reference metabolite profiles of ex vivo tissues. Signals are often contaminated by a background signal originating mainly from macromolecules and lipids and by residual water which hampers proper quantitation. We show that automatic quantitation of HRMAS signals, even in the presence of a background, can be achieved by the semi‐parametric algorithm QUEST based on prior knowledge of a metabolite basis‐set. The latter was quantum‐mechanically simulated with NMR‐SCOPE and requires accurate spin parameters. The region of interest of spectra is a small part of the full spectral bandwidth. Reducing the computation time inherent to the large number of data‐points is possible by using ER‐Filter in a preprocessing step. Through Monte‐Carlo studies, we analyze the performances of quantitation without and with ER‐Filtering.

Among organophosphorus nerve agents, soman is one of the most studied, being one of the most dangerous and the most difficult to counteract. When it does not kill rapidly, soman produces epileptic seizures and related brain damage which, if not treated immediately, may resist current therapies. The present review will address four points: (i) the medical issues raised by the central effects of soman poisoning; (ii) the mechanism of soman-induced brain damage; (iii) the use of antagonists of ionotropic glutamate receptors as anticonvulsants and neuroprotectants; and (iv) the search for new non-invasive methods to assess the progression of soman-induced brain damage.

The LS electromagnetic band (1-4 GHz) is widely used both in domestic and industrial domains. Several studies suggested that the biological systems would exhibit a specific sensitivity to the 2.45 GHz microwaves (water resonance frequency). Potential human health hazards and especially a disruption of the cholinergic system have been reported, due to exposure to microwaves even at low power density. This work presents a multiparametric study of freely moving rat where neurophysiology was investigated during 70 hours using neurochemical (microdialysis technique), electrophysiological, behavioral (vigilance stages quantification) and thermophysiological approaches. The rats were exposed 24 hours to a 2.45 GHz pulsed electromagnetic field at low power density. In this exposure conditions, no significant effect have been reported.

The LS electromagnetic hand (1-4 GHz) is widely used both in domestic and industrial domains. Several studies suggested that the biological systems would exhibit a specific sensitivity to the 2.45 GHz microwaves (water resonance frequency). Potential human health hazards and especially a disruption of the cholinergic system have been reported, due to exposure to microwaves even at low power density. This work presents a multiparametric study of freely moving rat where neurophysiology was investigated during 70 hours using neurochemical (microdialysis technique), electrophysiological, behavioral (vigilance stages quantification) and thermophysiological approaches. The rats were exposed 24 hours to a 2.45 GHz pulsed electromagnetic field at low power density. In this exposure conditions, no significant effect have been reported. (C) 2007 Elsevier Masson SAS. Tous droits reserves.

Deleterious effects on healthcare and particularly disruption of the cholinergic system have been reported after exposure to radiofrequency field at low power density. This work presents a 72 hours multiparametric study, where cholinergic system was investigated using a neurochemical, electrophysiological and physiological approaches. Free moving rats were exposed 24 hours to RF GSM signal at 1.8 GHz at low power density (1.2 and 9 W/m(2)). Acetylcholine (ACh) release in the hippocampus was simultaneously monitored using the microdialysis technique, electroencephalogram (EEG), electromyogram (EMG) and subcutaneous temperature. A spectral analysis of EEG was also performed and sleep stages were determined. After experimental time, the animals were sacrificed and a NMR study was performed on lipid brain extract. No significant parameters modification was observed under RF exposure. The only significant difference was the lack of increase in time spent in REM sleep, the third day, for the 1.2 W/m(2) group. This observation appeared difficult to explain and could not be reasonably related with RF exposure. Similarly, the NMR study also failed to show any effect of RF.

Soman poisoning is known to induce full-blown tonic–clonic seizures, status epilepticus (SE), seizure-related brain damage (SRBD) and lethality. Previous studies in guinea-pigs have shown that racemic ketamine (KET), with atropine sulfate (AS), is very effective in preventing death, stopping seizures and protecting sensitive brain areas when given up to 1h after a supra-lethal challenge of soman. The active ketamine isomer, S(+) ketamine (S-KET), is more potent than the racemic mixture and it also induces less side-effects. To confirm the efficacy of KET and to evaluate the potential of S-KET for delayed medical treatment of soman-induced SE, we studied different S-KET dose regimens using the same paradigm used with KET. Guinea-pigs received pyridostigmine (26μg/kg, IM) 30min before soman (62μg/kg, 2 LD50, IM), followed by therapy consisting of atropine methyl nitrate (AMN) (4mg/kg, IM) 1min following soman exposure. S-KET, with AS (10mg/kg), was then administered IM at different times after the onset of seizures, starting at 1h post-soman exposure. The protective efficacy of S-KET proved to be comparable to KET against lethality and SRBD, but at doses two to three times lower. As with KET, delaying treatment by 2h post-poisoning greatly reduced efficacy. Conditions that may have led to an increased S-KET brain concentration (increased doses or number of injections, adjunct treatment with the oxime HI-6) did not prove to be beneficial. In summary, these observations confirm that ketamine, either racemic or S-KET, in association with AS and possibly other drugs, could be highly effective in the delayed treatment of severe soman intoxication.

PURPOSE:In the present study, diffusion-weighted magnetic resonance imaging (DW-MRI) and histology were used to assess cerebral edema and lesions in mice intoxicated by a convulsive dose of soman, an organophosphate compound acting as an irreversible cholinesterase inhibitor. METHODS:Three hours and 24 h after the intoxication with soman (172 microg/kg), the mice were anesthetized with an isoflurane/N(2)O mixture and their brain examined with DW-MRI. After the imaging sessions, the mice were sacrificed for histological analysis of their brain. RESULTS:A decrease in the apparent diffusion coefficient (ADC) was detected as soon as 3 h after the intoxication and was found strongly enhanced at 24 h. A correlation was obtained between the ADC change and the severity of the overall brain damage (edema and cellular degeneration): the more severe the damage, the stronger the ADC drop. Anesthesia was shown to interrupt soman-induced seizures and to attenuate edema and cell change in certain sensitive brain areas. Finally, brain water content was assessed using the traditional dry/wet weight method. A significant increase of brain water was observed following the intoxication. CONCLUSIONS:The ADC decrease observed in the present study suggests that brain edema in soman poisoning is mainly intracellular and cytotoxic. Since entry of water into the brain was also evidenced, this type of edema is certainly mixed with others (vasogenic, hydrostatic, osmotic). The present study confirms the potential of DW-MRI as a non-invasive tool for monitoring the acute neuropathological consequences (edema and neurodegeneration) of soman-induced seizures.

We present a neurochemical, electrophysiological and physiological study on freely moving rats. During 3 days, we have simultaneously monitored acetylcholine (ACh) release in the hippocampus using the microdialysis technique, electroencephalogram (EEG), electromyogram (EMG) and subcutaneous temperature. A spectral analysis of EEG was performed and sleep stages were determined. Energy ratio in the delta (0-4 Hz), slow theta (4-6.5 Hz) and fast theta (6.5-9 Hz) band was calculated. Sleep stages were quantified using an automatic staging method.The circadian cycle of these parameters was observed. Waking, body temperature and ACh release presented synchronized cycles with close acrophases.The relationship between the central cholinergic system and the other parameters is discussed. The influence of handling on the measured parameters, as well as possible artifacts linked to the use of neostigmine in the microdialysis method are considered. Attention was focused on the cholinergic control of EEG theta rhythms. (c) 2005 Elsevier B.V. All rights reserved.