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Epilepsy is a neurological disease affecting some 50 million people worldwide. It is characterized by recurrent seizures due to the synchronous and spontaneous overexcitation of neuronal populations in the brain. Seizures vary widely in nature, and symptoms dependon the area of the brain affected and its extent. The term ‘epileptic disorders’ is accordingly preferred. These can have many causes, including both genetic (e.g. Dravet syndrome, a rare infantile epilepsy caused in 80% of cases by the heterozygous mutation of the SCN1A gene), and environmental (e.g. after poisoning with organophosphates, compounds present in pesticides and neurotoxic warfare agents). Whether for Dravet syndrome or organophosphate poisoning, current treatments do not enable optimal control of seizures. A better understanding of the pathophysiology of these different forms of epilepsy is thus needed to find new therapeutic targets and new anticonvulsants. Microglial cells are the resident macrophages in the brain. These cells have many functions, which can vary depending on the maturity of the brain. The microglia are the guardians of cerebral homeostasis, continuously ensuring the proper functioning of neurons. They are immune cells able to modulate their activity according to the dangers they detect. In addition, microglia have a special role in synaptic plasticity and the modulation of neuronal excitability. These different roles have prompted numerous hypotheses on the involvement of these cells in the pathophysiology of epileptic disorders. In some, microglia are harmful for the excitability of neurons, through their activation and the chronic secretion of proinflammatory cytokines. Others lend them a beneficial role, with microglia buffering neuronal hyperexcitability and thus decreasing the frequency of seizures. The objective of my PhD work was to study the mechanisms of epileptogenesis involving microglial cells in order to identify new therapeutic targets. I developed two models of epilepsy in zebrafish, a genetic model of Dravet syndrome and a model of organophosphate poisoning. These enabled me to study the modifications of the central nervous system during epileptogenesis. I specifically demonstrated an excitatory/inhibitory imbalance toward excitation that could trigger epileptic seizures. Using the Dravet model, I also successfully characterized the morphological, behavioral and molecular changes of microglial cells after seizures. This work improves our understanding of the consequences of epileptic seizures in the brain and helps pave the way for the discovery of new therapeutic targets to treat different forms of epilepsy., L'épilepsie est une maladie neurologique qui touche plus de 50 millions de personnes dans le monde. Elle se caractérise par des crises récurrentes dues à la surexcitation synchrone et spontanée de populations neuronales du cerveau. Les crises sont de nature très variable et les symptômes dépendent de la zone du cerveau touchée et de son étendue. Le terme «troubles épileptiques» est par conséquent préféré. Ceux-ci peuvent avoir de nombreuses causes, soient génétiques (par exemple, le syndrome de Dravet, une épilepsie infantile rare, provoquée dans 80% des cas par la mutation hétérozygote du gène SCN1A), soient environnementales (par exemple, après un empoisonnement aux organophosphorés, des composés présents dans les pesticides et les agents de guerre neurotoxiques). Dans les deux cas, les traitements actuels ne permettent pas un contrôle optimal des crises. Une meilleure compréhension de la physiopathologie de ces différentes formes d'épilepsie est donc nécessaire pour trouver de nouvelles cibles thérapeutiques et de nouveaux anticonvulsivants. Les cellules microgliales, les macrophages résidents du cerveau ont de nombreuses fonctions qui varient en fonction de la maturité du cerveau. Les microglies sont les gardiennes de l'homéostasie cérébrale, assurant en permanence le bon fonctionnement des neurones. Ce sont des cellules immunitaires capables de moduler leur activité en fonction des dangers qu'elles détectent. De plus, elles ont un rôle particulier dans la plasticité synaptique et la modulation de l'excitabilité neuronale. Ces différents rôles ont suscité de nombreuses hypothèses sur l'implication de ces cellules dans la physiopathologie des troubles épileptiques. Pour certaines, les microglies sont nocives pour l'excitabilité des neurones, par leur activation et la sécrétion chronique de cytokines pro-inflammatoires. Pour d'autres, elles ont un rôle bénéfique, la microglie tamponnant l'hyperexcitabilité neuronale et diminuant ainsi la fréquence des crises. L'objectif de mon travail de thèse était d'étudier les mécanismes de l'épileptogenèse impliquant les cellules microgliales afin d'identifier de nouvelles cibles thérapeutiques. J'ai développé deux modèles d'épilepsie chez le poisson zèbre, un modèle génétique du syndrome de Dravet et un modèle d'empoisonnement aux organophosphorés. Ceux-ci m'ont permis d'étudier les modifications du système nerveux central au cours de l'épileptogenèse. J'ai ainsi montré un déséquilibre de la balance excitateur/inhibiteur vers l'excitation qui pourrait déclencher des crises d'épilepsie. En utilisant le modèle de Dravet, j'ai également caractérisé les changements morphologiques, comportementaux et moléculaires des cellules microgliales après des crises. Ces travaux améliorent notre compréhension des conséquences des crises d'épilepsie dans le cerveau et contribuent à ouvrir la voie à la découverte de nouvelles cibles thérapeutiques pour traiter différentes formes d'épilepsie.

Epilepsy is a neurological disease affecting some 50 million people worldwide. It is characterized by recurrent seizures due to the synchronous and spontaneous overexcitation of neuronal populations in the brain. Seizures vary widely in nature, and symptoms dependon the area of the brain affected and its extent. The term ‘epileptic disorders’ is accordingly preferred. These can have many causes, including both genetic (e.g. Dravet syndrome, a rare infantile epilepsy caused in 80% of cases by the heterozygous mutation of the SCN1A gene), and environmental (e.g. after poisoning with organophosphates, compounds present in pesticides and neurotoxic warfare agents). Whether for Dravet syndrome or organophosphate poisoning, current treatments do not enable optimal control of seizures. A better understanding of the pathophysiology of these different forms of epilepsy is thus needed to find new therapeutic targets and new anticonvulsants. Microglial cells are the resident macrophages in the brain. These cells have many functions, which can vary depending on the maturity of the brain. The microglia are the guardians of cerebral homeostasis, continuously ensuring the proper functioning of neurons. They are immune cells able to modulate their activity according to the dangers they detect. In addition, microglia have a special role in synaptic plasticity and the modulation of neuronal excitability. These different roles have prompted numerous hypotheses on the involvement of these cells in the pathophysiology of epileptic disorders. In some, microglia are harmful for the excitability of neurons, through their activation and the chronic secretion of proinflammatory cytokines. Others lend them a beneficial role, with microglia buffering neuronal hyperexcitability and thus decreasing the frequency of seizures. The objective of my PhD work was to study the mechanisms of epileptogenesis involving microglial cells in order to identify new therapeutic targets. I developed two models of epilepsy in zebrafish, a genetic model of Dravet syndrome and a model of organophosphate poisoning. These enabled me to study the modifications of the central nervous system during epileptogenesis. I specifically demonstrated an excitatory/inhibitory imbalance toward excitation that could trigger epileptic seizures. Using the Dravet model, I also successfully characterized the morphological, behavioral and molecular changes of microglial cells after seizures. This work improves our understanding of the consequences of epileptic seizures in the brain and helps pave the way for the discovery of new therapeutic targets to treat different forms of epilepsy., L'épilepsie est une maladie neurologique qui touche plus de 50 millions de personnes dans le monde. Elle se caractérise par des crises récurrentes dues à la surexcitation synchrone et spontanée de populations neuronales du cerveau. Les crises sont de nature très variable et les symptômes dépendent de la zone du cerveau touchée et de son étendue. Le terme «troubles épileptiques» est par conséquent préféré. Ceux-ci peuvent avoir de nombreuses causes, soient génétiques (par exemple, le syndrome de Dravet, une épilepsie infantile rare, provoquée dans 80% des cas par la mutation hétérozygote du gène SCN1A), soient environnementales (par exemple, après un empoisonnement aux organophosphorés, des composés présents dans les pesticides et les agents de guerre neurotoxiques). Dans les deux cas, les traitements actuels ne permettent pas un contrôle optimal des crises. Une meilleure compréhension de la physiopathologie de ces différentes formes d'épilepsie est donc nécessaire pour trouver de nouvelles cibles thérapeutiques et de nouveaux anticonvulsivants. Les cellules microgliales, les macrophages résidents du cerveau ont de nombreuses fonctions qui varient en fonction de la maturité du cerveau. Les microglies sont les gardiennes de l'homéostasie cérébrale, assurant en permanence le bon fonctionnement des neurones. Ce sont des cellules immunitaires capables de moduler leur activité en fonction des dangers qu'elles détectent. De plus, elles ont un rôle particulier dans la plasticité synaptique et la modulation de l'excitabilité neuronale. Ces différents rôles ont suscité de nombreuses hypothèses sur l'implication de ces cellules dans la physiopathologie des troubles épileptiques. Pour certaines, les microglies sont nocives pour l'excitabilité des neurones, par leur activation et la sécrétion chronique de cytokines pro-inflammatoires. Pour d'autres, elles ont un rôle bénéfique, la microglie tamponnant l'hyperexcitabilité neuronale et diminuant ainsi la fréquence des crises. L'objectif de mon travail de thèse était d'étudier les mécanismes de l'épileptogenèse impliquant les cellules microgliales afin d'identifier de nouvelles cibles thérapeutiques. J'ai développé deux modèles d'épilepsie chez le poisson zèbre, un modèle génétique du syndrome de Dravet et un modèle d'empoisonnement aux organophosphorés. Ceux-ci m'ont permis d'étudier les modifications du système nerveux central au cours de l'épileptogenèse. J'ai ainsi montré un déséquilibre de la balance excitateur/inhibiteur vers l'excitation qui pourrait déclencher des crises d'épilepsie. En utilisant le modèle de Dravet, j'ai également caractérisé les changements morphologiques, comportementaux et moléculaires des cellules microgliales après des crises. Ces travaux améliorent notre compréhension des conséquences des crises d'épilepsie dans le cerveau et contribuent à ouvrir la voie à la découverte de nouvelles cibles thérapeutiques pour traiter différentes formes d'épilepsie.

Organophosphorus compounds (OP) are toxic molecules developed as insecticides and chemical warfare nerve agents (CWNAs). Most OP are neurotoxic and act as nervous system disruptors by blocking cholinergic transmission. They are therefore responsible for many poisonings worldwide. OP toxicity may result either from acute or chronic exposure, and their poisoning effect were evaluated using several animal models. These latter were also used for evaluating the efficacy of antidotes. Strategies based on enzymes that can trap (stoichiometric bioscavengers) or degrade (catalytic bioscavengers) OP, were particularly studied since they allow effective decontamination, without toxicity or environmental impact. This review summarizes the results obtained in vivo with enzymes through three levels: prophylaxis, treatment and external decontamination. The efficiency of enzymatic treatments in different animal models is presented and the relevance of these models is also discussed for a better extrapolation to humans.

Cholinesterases are the main targets of organophosphorus compounds. The two enzymes present in the blood (butyrylcholinesterase, BChE; acetylcholinesterase, AChE) are biomarkers of their systemic toxicity. Activity of the plasma BChE is very often determined as it allows a rapid diagnostic of poisoning and is a marker of the persistence of the toxicant in the blood. The activity of the red blood cell AChE gives a better picture of the synaptic inhibition in the nervous system but the assay is less commonly available in routine laboratories. Better biomarker of the exposure, it allows a diagnosis of the severity of the poisoning and helps to assess the efficacy of oxime therapy. Besides the practical aspects of blood collection and sample processing, and the interpretation of the assays, this review stresses the complementarity of both enzyme assays and recalls their crucial interest for the confirmation of poisoning with an organophosphorus in a situation of war or terrorist attack and for the monitoring of occupational exposures.

The clinical picture of organophosphate poisoning is dominated by respiratory and neurological signs. Cardiac complications are rare, serious and little known by clinicians.F.B, 17 years, was addressed intubated, at the emergencies after an organosphorus poisoning. At admission, the patient was comatose with a muscarinic syndrome. Her hemodynamic was stable. Chest radiography and ECG of admission were normal. A gastric washing and a treatment by atropine were quickly started. The toxicological analysis of gastric liquid showed presence of malathion. After 20hours, the patient presented a cardiogenic shock. Chest radiography showed signs of acute pulmonary edema and ECG: a circumferential endocardic ischaemia. In spite of vasopressors and positive inotropic treatment, the patient quickly died. The autopsies revealed multiple necrosis of the heart.The cardiac symptoms are caused by many mechanisms often associated. The cardiac attack appears primarily by many electrocardiographic modifications: arrhythmias, disturb of repolarisation, conduction's disorder... On the hemodynamic plan, a circulatory insufficiency is possible. The ventricular arrhythmias, which are not treated by lidocaine or electric cardioversion are treated effectively by injection of isoproterenol and/or ventricular stimulation, and recently by the use of magnesium salts.If the clinical picture of the IOP is dominated by neurological and respiratory signs, it is always necessary to remember the possibility of cardiac complications (early or late) whose pronostic is very often bad.

Nerve agents could be potentially used as during a conflict or a terrorist attack. If an emergency treatment is not provided after poisoning, irreversible brain damages and behavioural sequels are expected to appear. In vivo cell therapy seems to be a promising approach for delayed treatment to contribute to brain repair. A mobilization of endogenous neural progenitors would be the basis of such an approach. After migrating, these progenitors would engraft in damaged brain regions and subsequently differentiate into functional neurons. In this review, after a few reminders regarding nerve agent poisoning and the emergency treatment of such an intoxication, progresses in terms of cell therapy and its potential application to nerve agent-induced brain lesions are summarized.

Catalytic bioscavengers show promise for the prevention and treatment of organophosphate poisoning. Catalytic bioscavengers are enzymes capable of binding and hydrolyzing organophosphorus compounds (OPs). These enzymes could be used to degrade OPs before they reach their biological targets. As such, they could be administered as antidotes before or after exposure, and be used for skin protection and decontamination. Protein engineering can yield enzymes with improved catalytic properties, that are stable during storage and in the bloodstream, and that are immunologically compatible. Large-scale production must be feasible and reasonably inexpensive.

Acute organophosphate (OP) intoxications, accidental or voluntary, are responsible for a high mortality. They cause extensive muscular paralysis by acetyl cholinesterase activity inhibition at the neuromuscular junction level.To underline the rarity and the characteristic electrophysiological pattern during cholinergic crisis.A 28-year-old woman was admitted to the medical intensive care unit for Malathion acute intoxication with signs of glandular hypersecretion, complicated tetraparesis, and respiratory distress. The cholinesterase activity was 17%. The electroneuromyography showed multiple motor responses to the same stimulation, which is characteristic of the cholinergic crisis. Other electrophysiological parameters, in particular low-frequency repetitive stimulations, were normal. The evolution was favourable after symptomatic treatment and respiratory assistance.Organophosphate intoxications evolve in three phases: acute cholinergic crisis, intermediate syndrome, and delayed neuropathy. While the electrophysiological aspects of delayed neuropathy are best characterized, those of crisis and intermediate syndrome remain very little studied. The persistence of acetylcholine in the synaptic slit would explain the multiple motor responses to single stimulation during the crisis.

Amongst organophosphate compounds, both pesticides and warfare neurotoxics are probably the most representative. These compounds are irreversible acetylcholinesterase inhibitors. Usual clinical signs observed after acute poisoning are mainly respiratory distress, convulsions and seizures. Following acute poisoning, an emergency treatment must be provided as soon as possible (maximum delay of 1 hour post-poisoning), to prevent irreversible brain damage and patient death. At the present time, there is no efficient delayed treatment which could be provided if this 1 hour latency is overpassed. However, neurogenesis by stem cell engraftment, eventually complemented by gene therapy strategy, could be a potential therapeutic approach to repair organophosphate-induced brain damage. Main stem cell engraftement strategies successfully used for brain damage of various origins are reviewed in this Article.

Suicides attempts with an organophosphate pesticide still occur in Brittany, an agricultural French area. The acute poisoning due to this nerve agent, responsible for the inhibition of the cholinesterases enzymes, is severe and can be fatal. The diagnosis is based on the decrease of cholinesterases activity, an assay which has been performed because of the patient context. The measurement of butyrylcholinesterases activity in emergency guarantees an appropriate and rapid management of the patient in order to rapidly regenerate cholinesterases. Even if the decrease level of butyrylcholinesterases activity does not give a significant prognostic value, its follow-up with time indicates the efficacy of the treatment and the gradual remission. In routine the measurement of acetylcholinesterase activity is more useful for the biological follow-up of subjects exposed to organophosphate phytosanitary compounds.

Acute intoxication by organophosphate pesticides is frequent in Morocco. We report two cases of malathion poisoning complicated by intermediate syndrome. The purpose of this work is to describe distinctive features of this syndrome, it arises 48-96 h after the cholinergic crisis and it is characterized by respiratory paresis with difficulties of weaning from the assisted respiratory, deficit of proximal limbs, neck flexors, and cranial nerves. This syndrome coincides with the prolonged inhibition of the acetylcholinesterase, and is not due to the necrosis of muscular fiber's necrosis. Both clinical and electromyographic features are explained by a combined pre- and postsynaptic dysfunction of the neuromuscular transmission. The difficulty of this syndrome lies in its rarity and also its severity, because of the respiratory failure, which justifies medical supervision in intensive care unit, for at least 96 h, in expectation for the respiratory distress, all the more cholinergic syndrome is intense.

Human plasma paraoxonase (PON1) is a calcium-dependent organophosphate-hydrolase. In plasma, PON1 is bound to high-density lipoproteins (HDL). PON1 prevents the oxidation of LDL and scavenges oxidized phospholipids, thus protecting from atherogenesis. Improving the prophylaxis and treatment of organophosphate (OP) poisoning is a public health concern that also interests the civilian safety and the military. In this context, engineering and formulation of enzymes able to scavenge or hydrolyze OPs, such as PON1, are widely studied. Determination of the PON1 three-dimensional structure is a key step toward improvement of the enzyme functional properties.

Chemical methods remain a credible threat in 1999. The doctrine for their use not only includes the battlefield but also domestic terrorism as was disclosed during the Tokyo metro attempt in 1995. International Treaties have not yet proven their efficacy. The arsenal of chemical weapons has changed little since the second World War but is now dispersed into many high-risk zones throughout the world. There has also been little change in antidotes: therapeutic prevention with pyridostigmine against organo-phosphorus compounds, protective treatment for seizure-induced brain lesions using anticonvulsants in association with oxime for acetylcholinesterase reactivation, and atropine are combined in a three-compartment syringe. Preventive measures against vesicants and other suffocating or toxic intracellular substances (CN, AsH(3), fluorocarbons.) can only be achieved with protective skin covering or protective breathing devices. There is no specific treatment and we often have to use symptomatic medications. Future perspectives include: phosphotriesterases as organo-phosphorus scavengers, huperzine as pretreatment and gacyclidine (GCK 11) which would effectively complete emergency multiple drug therapy against nerve agents. A new two-compartment syringe is now prepared with atropine, avisafone and HI6 or pralidoxine. A gel made of cyclodextrines for external and eventually internal use is under study.

Classical emergency treatment of organophosphate poisoning includes the combined administration of a cholinesterase reactivator (an oxime), a muscarinic cholinergic receptor antagonist (atropine) and a benzodiazepine anticonvulsant (diazepam). In subjects taking pyridostigmine and trained to autoadminister at least one autoinjector at the first signs of poisoning, classical emergency treatment ensures survival but only an antiglutamatergic compound like gacyclidine appears to be able to ensure optimal management of nerve agent poisoning in terms of rapid normalization of EEG activity, clinical recovery and total neuroprotection. All of this reinforces the therapeutical value of gacyclidine, which is in the process of approval for human use in France for treatment of head injury, as a central nervous system protective agent for the treatment of OP poisoning.

Pyridostigmine which is widely used as pretreatment of organophosphate poisoning protects cholinesterases of peripheral nervous system. Other molecules able to also protect the central nervous system are under study and, among them, huperzine A. This paper gives an overview of the current investigations about the efficacy and the innocuity of this molecule (study of the mechanisms of action, biological targets, behavioural manifestations) and brings out its potential interests.

Among the insecticides, organophosphorous compounds are the agents most involved in deliberate and involuntary intoxications. The toxic effect is mainly due to the inhibition of cholinesterase at the cholinergic synapses. Two cases of severe involuntary intoxication are described which were characterized by the development of ARDS.

Lymphocytic NTE activity was measured after intoxication by organo phosphorus compounds and in chronic alcoholics at the beginning of alcohol withdrawal. The results showed a rapid fall in lymphocytic NTE activity; neuropathy developed when the inhibition was 75 p. 100 or more. In chronic alcoholics, the fall in NTE activity is a new concept which must be taken into account when considering the causal factors of neurotoxicity in exposed professions and in patients taking potentially neurotoxic drugs. The NTE may be a useful parameter to demonstrate the toxic origin of chronic neuropathies, especially those caused by drugs. The capacity for lymphocytic NTE activity to return to normal seems to be different in acute, compared with chronic, intoxications.

Poisoning by Neurotoxic organo phosphorus products is frequent problem in Iran. Reanimation of these patients depends on the following: 1 - To begin reanimation rapidly. 2- Usage of atropine and special anticholisterasics antidotes. In case of need for surgical intervention, the use of succinylcholin is prohibited and other muscle relexants should be used with most care.