Beneficial effects of a ketamine/atropine combination in soman-poisoned rats under a neutral thermal environment.

Exposure to organophosphorus (OP) compounds, such as pesticides and the chemical warfare agents (soman and sarin), respectively represents a major health problem and a threat for civilian and military communities. OP poisoning may induce seizures, status epilepticus and even brain lesions if untreated. We recently proved that a combination of atropine sulfate and ketamine, a glutamatergic antagonist, was effective as an anticonvulsant and neuroprotectant in mice and guinea-pigs exposed to soman. Since OP exposure may also occur in conditions of heat strain due to climate, wearing of protective gears or physical exercise, we previously demonstrated that ketamine/atropine association may be used in a hot environment without detrimental effects. In the present study, we assess soman toxicity and evaluate the effects of the ketamine/atropine combination on soman toxicity in a warm thermoneutral environment. Male Wistar rats, exposed to 31°C (easily reached under protective equipments), were intoxicated by soman and treated with an anesthetic dose of ketamine combined with atropine sulfate. Body core temperature and spontaneous locomotor activity were continuously monitored using telemetry. At the end of the warm exposure, blood chemistry and brain mRNA expression of some specific genes were measured. In soman-intoxicated animals, metabolic and genic modifications were related to convulsions rather than to soman intoxication by itself. In the warm environment, ketamine/atropine combination did not produce any side-effect on the assessed variables. Furthermore, the ketamine/atropine combination exhibited beneficial therapeutic effects on soman-intoxicated rats such as a limitation of convulsion-induced hyperthermia and of the increase in some blood chemistry markers.

Ketamine does not impair heat tolerance in rats.

Exposure to organophosphorus compounds, either pesticides or chemical warfare agents such as soman or sarin, represents a major health problem. Organophosphorus poisoning may induce seizures, status epilepticus and even brain lesions if untreated. Ketamine, an antagonist of glutamatergic receptors, was recently proved to be effective in combination with atropine sulfate as an anticonvulsant and neuroprotectant in mice and guinea pigs exposed to soman. Organophosphorus exposure may also occur in conditions of contemporary heat exposure. Since both MK-801, a more potent glutamatergic antagonist than ketamine, and atropine sulfate are detrimental for thermoregulation, we evaluated the pathophysiological consequences of ketamine/atropine combinations in a hot environment. Male wistar rats were exposed to 38°C ambient temperature and treated with atropine sulfate and/or ketamine (anesthetic and subanesthetic doses). The abdominal temperature and spontaneous locomotor activity were continuously monitored using telemetry. At the end of heat exposure, blood chemistry and the mRNA expression of some specific genes in the brain were assessed. Unlike MK-801, ketamine did not induce any deleterious effect on thermoregulation in rats. Conversely, atropine sulfate led to heatstroke and depressed the heat-induced blood corticosterone increase. Furthermore, it induced a dramatic increase in Hsp70 and c-Fos mRNA levels and a decrease in IκBα mRNA expression, both suggesting brain aggression. When combined with the anesthetic dose of ketamine, some of the atropine-induced metabolic disturbances were modified. In conclusion, ketamine can be used in hot environment and may even limit some of the biological alterations induced by atropine sulfate in these conditions.

Prediction of neuroprotective treatment efficiency using a HRMAS NMR-based statistical model of refractory status epilepticus on mouse: a metabolomic approach supported by histology.

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.

Combinations of ketamine and atropine are neuroprotective and reduce neuroinflammation after a toxic status epilepticus in mice.

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.

A new use for an old method: the Woelcke myelin stain for counting degenerating neurons in the brain of mice following status epilepticus.

The Woelcke method is classically used for myelin staining. Degenerating neurons can be revealed histologically by hemalun and phloxin (H&P) where they appear "eosinophilic". In the first 24 h following soman-induced status epilepticus, we observed that the Woelcke method also revealed condensed, dark blue/black cells (W+ cells) in the gray matter of brain regions known to be sites of seizure-related brain damage, marked by the presence of eosinophilic cells. In the present study, using adjacent brain sections alternately stained with either the Woelcke or the H&P method, we show that eosinophilic cells and W+ cells are the same degenerating cells. Moreover, we show that semi-automated quantitative evaluation of W+ cells through computerized image analysis is considerably easier and faster than that of eosinophilic cells. It is therefore concluded that the Woelcke technique could be very useful, especially for quantifying acute brain cell damage following status epilepticus.

Prediction of soman-induced cerebral damage by distortion product otoacoustic emissions.

The organophosphorus nerve agent soman is an irreversible cholinesterase (ChE) inhibitor that can produce long-lasting seizures and seizure-related brain damage (SRBD) in which acetylcholine and glutamate are involved. Since these neurotransmitters play a key-role in the auditory function, it was hypothesized that a hearing test may be an efficient way for detecting the central effects of soman intoxication. In the present study, distortion product otoacoustic emissions (DPOAEs), a non-invasive audiometric method, were used in rats administered with soman (70 µg/kg). Four hours post-soman, DPOAE intensities were significantly decreased. They returned to baseline one day later. The amplitude of the temporary drop of the DPOAEs was well related to the severity of the intoxication. The greatest change was recorded in the rats that survived long-lasting convulsions, i.e. those that showed the highest ChE inhibition in brain and severe encephalopathy. Furthermore, the administration, immediately after soman, of a three-drug therapy composed of atropine sulfate, HI-6 and avizafone abolished the convulsions, the transient drop of DPOAEs at 4h and the occurrence of SRBD at 28 h without modifying brain ChE inhibition. This showed that DPOAE change was not directly related to soman-induced inhibition of cerebral ChE but rather to its neuropathological consequences. The present findings strongly suggest that DPOAEs represent a promising non-invasive tool to predict SRBD occurrence in nerve agent poisoning and to control the efficacy of a neuroprotective treatment.

Hypertonic mannitol in mice poisoned by a convulsive dose of soman: antilethal activity without neuroprotection.

A convulsive dose of soman induces seizure-related brain damage (SRBD), including cerebral edema (CE) and cell death. In 1993, an American study demonstrated that hypertonic mannitol (Mann) intravenously (i.v.) administered 1 min and 5h post-soman was an effective neuroprotectant in intoxicated rats. Using a similar protocol, we recently failed to reproduce this success in intoxicated mice. In the present study, also performed in mice, the persistence or the amplitude of the osmotic load was increased by reducing the time interval between two injections of Mann or by augmenting the number of injections. Mice were pre-treated with the oxime HI-6 and then intoxicated with a convulsive dose of soman (172 microg/kg). Afterward, they were administered a first i.v. bolus of Mann 20% 1 min post-challenge and a second one either 5, or 2, or 1h after. Additional animals were given either one (1 min post-soman), or two (1 min and 1h post-soman), or three (1 min, 1 and 2h post-soman) series of three injections of Mann at 5 min intervals. Non-intoxicated mice treated with Mann (same protocols as above) and intoxicated mice treated with Mann vehicle (saline) served as controls. At 24h post-intoxication, the survivors were sacrificed and their brains prepared for quantitative histological assessment of cell damage, CE, and ventricle size. Whatever the protocol, Mann had no effect on soman-induced convulsions but did provide considerable antilethal activity. Histologically, Mann did not reduce the cell damage or CE. It even showed a dose-dependent trend toward aggravation of SRBD in some regions and promoted subarachnoid hemorrhages. Conversely, in one of the treatment protocol, it reduced soman-induced enlargement of ventricle size. Although treatment with hypertonic Mann showed some benefit on mortality and ventricle size, it failed to be an effective neuroprotector in soman-intoxicated mice and even increased the detrimental impact of soman at the cerebral level. Therefore, no clear recommendation could be drawn from the present study in view of a possible clinical use of hyperosmolar treatment in the medical management of soman poisoning.

Hyperosmolar treatment of soman-induced brain lesions in mice: evaluation of the effects through diffusion-weighted magnetic resonance imaging and through histology.

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 microg/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. 1 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.

Prolonged inflammatory gene response following soman-induced seizures in mice.

Following exposure to the organophosphorus nerve agent soman, the development of long-lasting seizures and build-up of irreversible seizure-related brain damage (SRBD) still represent a therapeutic challenge. A neuro-inflammatory reaction takes place in the brain after poisoning but its characteristics and potential role in SRBD and post-status epilepticus epileptogenesis is not well understood. In the present study we have analyzed by quantitative RT-PCR the time course of changes in mRNA levels of IL-1beta, TNFalpha, IL-6, ICAM-1 and SOCS3 in hippocampus, whole cortex and cerebellum in a mouse model of severe seizures and neuropathy up to 7 days after poisoning. Mice received an injection of the oxime HI-6 (50mg/kg) 5 min prior to the administration of a convulsive dose of soman (172 microg/kg). An important and highly significant increase of the five mRNA levels was recorded in cortex and hippocampus. In the cortex, the activation was generally detected as early as 1h post-intoxication with a peak response recorded between 6 and 24h. In the hippocampus, the gene up-regulation was delayed to 6h post-soman and the peak response observed between 24 and 48 h. After peaking, the response declined (except for ICAM in the hippocampus) but remained elevated, some of them significantly, at day 7. Interestingly, in the cerebellum, some changes were also observed but were several fold smaller. In conclusion, the present study indicates a quick neuro-inflammatory gene response that does not subside over 7 days suggesting a potential role in the neurological consequences of soman-induced status epilepticus.

Distortion product otoacoustic emissions as non-invasive biomarkers and predictors of soman-induced central neurotoxicity: a preliminary study.

The organophosphorus nerve agent soman is an irreversible cholinesterase (ChE) inhibitor that can produce long-lasting seizures and brain damage in which the neurotransmitters acetylcholine and glutamate are involved. These same neurotransmitters play key-roles in the auditory function. It was then assumed that exploring the hearing function may provide markers of the central events triggered by soman intoxication. In the present study, distortion product otoacoustic emissions (DPOAEs), a non-invasive audiometric method, were used to monitor cochlear functionality in rats administered with a moderate dose of soman (45 microg/kg). DPOAEs were investigated either 4h or 24h post-challenge. In parallel, the effects of soman on whole blood and brain ChE activity and on brain histology were also studied. The first main result is that DPOAE intensities were significantly decreased 4h post-soman and returned to baseline at 24h. The amplitude changes were well related to the severity of symptoms, with the greatest change being recorded in the rats that survived long-lasting convulsions. The second main result is that baseline DPOAEs recorded 8 days before soman appear to predict the severity of symptoms produced by the intoxication. Indeed, the lowest baseline DPOAEs corresponded to the occurrence of long-lasting convulsions and brain damage and to the greatest inhibition in central ChE. These results thus suggest that DPOAEs represent a promising non-invasive tool to assess and predict the central consequences of nerve agent poisoning. Further investigations will be carried out to assess the potential applications and the limits of this non-invasive method.

Cerebral edema induced in mice by a convulsive dose of soman. Evaluation through diffusion-weighted magnetic resonance imaging and histology.

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.

Flunarizine: a possible adjuvant medication against soman poisoning?

Organophosphate (OP) nerve agents are amongst the most toxic chemicals. One of them, soman, can induce severe epileptic seizures and brain damage for which therapy is incomplete. The present study shows that pretreatment with flunarizine (Flu), a voltage-dependent calcium channel blocker, when used alone, does not produce any beneficial effect against the convulsions, neuropathology and lethality induced by soman. Flu was also tested in combination with atropine sulfate and diazepam. In this case, although only some results reach statistical significance, an encouraging general trend toward an improvement of the anticonvulsant, neuroprotective and antilethal capacities of this classical anti-OP two-drug regimen is constantly observed. In the light of these findings, it seems premature to definitely reject (or recommend) Flu as a possible adjuvant medication against soman poisoning. Further studies are required to determine its real potential interest.

Early changes in MAP2 protein in the rat hippocampus following soman intoxication.

We investigated the time course of both MAP2 (microtubule-associated protein 2) levels and its phosphorylation degree in the rat hippocampus during the first 90 min following the onset of soman-induced seizures. The quantitative immunoblot analysis of hippocampal extracts revealed that MAP2 increased significantly in response to a sustained epileptic activity (from 60 min of seizure duration). In addition, intense MAP2 dephosphorylation was also observed 60 to 90 min after the onset of seizures. The possible neuropathological consequences of these two early MAP2 changes are discussed in relation to the both excessive stimulation of glutamate receptors and subsequent dendritic spine alterations occurring in hippocampal neurons soon after soman intoxication.

Review of the value of huperzine as pretreatment of organophosphate poisoning.

Today, organophosphate (OP) nerve agents are still considered as potential threats in both military or terrorism situations. OP agents are potent irreversible inhibitors of central and peripheral acetylcholinesterases. Pretreatment of OP poisoning relies on the subchronic administration of the reversible acetylcholinesterase (AChE) inhibitor pyridostigmine (PYR). Since PYR does not penetrate into the brain, it does not afford protection against seizures and subsequent neuropathology induced by an OP agent such as soman. Comparatively, huperzine (HUP) is a reversible AChE inhibitor that crosses the blood-brain barrier. HUP is presently approved for human use or is in course of clinical trials for the treatment of Alzheimer's disease or myasthenia gravis. HUP is also used as supplementary drug in the USA for correction of memory impairment. Besides, HUP has also been successfully tested for pretreatment of OP poisoning. This review summarizes the therapeutical value of HUP in this field. Moreover, the modes of action of HUP underlying its efficacy against OP agents are described. Efficacy appears mainly related to both the selectivity of HUP for red cell AChE which preserves scavenger capacity of plasma butyrylcholinesterases for OP agents and to the protection conferred by HUP on cerebral AChE. Finally, recent data, showing that HUP seems to be devoid of deleterious effects in healthy subjects, are also presented. Globally, this review reinforces the therapeutical value of HUP for the optimal pretreatment of OP poisoning.

Subchronic administration of pyridostigmine or huperzine to primates: compared efficacy against soman toxicity.

Organophosphonate (OP) nerve agents, such as soman, are potent irreversible inhibitors of central and peripheral acetylcholinesterases (AChEs). Pre-treatment of OP poisoning relies on the subchronic administration of a reversible AChE inhibitor. In the present limited study, the protective effects against soman toxicity of such compounds, i.e., the current pre-treatment pyridostigmine and huperzine, a proposed pre-treatment, are compared in primates. This is the first time primates are used to study the potential of pre-treatment with hyperzine. Indeed, previous studies with huperzine used nonprimate models which are not the most appropriate for pre-treatment in humans. Each medication is given via a subcutaneous mini-osmotic pump for 6 days at a delivery rate providing about 20% inhibition of red cell AChE activity. In this trial with only four primates, huperzine selectively inhibits red cell AChE activity whereas pyridostigmine also inhibits plasma butyrylcholinesterase (BuChE). This latter may act as endogenous scavenger of OP compounds helping to confer additional protection against OPs. During intoxication, the cumulative dose of soman needed to produce convulsions and epileptic activity is 1.55-fold higher in the animals pre-treated with huperzine compared to those pre-treated with pyridostigmine. Thus, replacing PYR by HUP for a subchronic pre-treatment of primates gives them better tolerance to the epileptic effects of soman.

Inhibition of crotoxin phospholipase A(2) activity by manoalide associated with inactivation of crotoxin toxicity and dissociation of the heterodimeric neurotoxic complex.

Crotoxin (CACB complex) is a convulsant heterodimeric neurotoxic phospholipase A(2) (PLA(2)). The role of phospholipid hydrolysis in its epileptogenic properties remains unresolved. We, thus, studied the effect of manoalide (MLD), a PLA(2) inhibitor, on the toxin catalytic activity and its central and peripheral toxicity. Incubation of crotoxin with MLD fully and irreversibly inactivated its enzymatic activity. Interestingly, crotoxin also lost its central neurotoxicity after intracerebroventricular injection and peripheral toxicity after intravenous administration. MLD-treated crotoxin prevented the high affinity binding of [125I]-radiolabeled crotoxin on rat cortex synaptic plasma membranes. Further analysis of MLD-treated crotoxin by non-denaturing PAGE and surface plasmon resonance indicated that the crotoxin complex was dissociated after MLD treatment. Although the loss of MLD-treated crotoxin peripheral neurotoxicity could not be attributed to this dissociation, the presence of free CA subunit might explain the observed competition in binding experiments. In conclusion, the dissociation of the crotoxin complex by MLD, as demonstrated in this study, did not permit to specify the role of the enzymatic activity in crotoxin epileptogenic properties. Other approaches would be required to resolve this question.

Effects of chronic administration of huperzine A on memory in guinea pigs.

Effects of subchronic administration of huperzine A, a cholinesterase inhibitor, on spatial memory were studied in guinea pig. Spatial memory was appreciated by the Morris water maze test. At a dose of 0.25 microgram/h, inhibiting 36% of blood AChE and 14-20% of central AChE, no effect on spatial learning was found. At a dose of 1 microgram/h, inhibiting 20% of blood AChE and 14-20% of central AChE, no memory impairment was found, on the other hand, a memory enhancing effect, limited to the first day was shown. It thus appears that subchronic administration of huperzine A did not induce deleterious effects on spatial memory.