La toxicité des antidépresseurs inhibiteurs de recapture de la sérotonine (IRS) dont le citalopram est le représentant le plus sélectif, est réputée faible. Or les IRS ont été rendus responsables de syndromes sérotoninergiques, de convulsions, d’anomalies électrocardiographiques, voire de troubles respiratoires et de décès. L’implication de cette classe pharmacologique au cours des intoxications médicamenteuses volontaires (IMV) apparait peu documentée par des données récentes en France. Ainsi, la morbidité des IMV impliquant un IRS aux urgences (SAU) et les symptômes les plus fréquemment observés à la suite d’une exposition toxique aux IRS sont peu décrits. De même, les mécanismes toxiques impliqués dans les décès ne sont clairs. Objectifs : Nous avons mené ces travaux dans le but de : 1- mieux connaitre l’épidémiologie des IMV dans un SAU et y préciser l’implication des IRS ; 2- explorer une éventuelle sur-morbidité liée aux IRS dans les IMV polymédicamenteuses ; 3-comprendre les mécanismes de décès induits par de fortes doses de citalopram et les moyens de les prévenir. Méthodes: Nous avons conduit une étude observationnelle des IMV admises au SAU durant 4 ans, avec appariement des patients ayant ingéré un IRS versus des patients intoxiqués non exposés à un IRS. Nous avons également mené une étude expérimentale chez le rat Sprague-Dawley pour connaitre la dose létale médiane (MLD) du citalopram et explorer la toxicité neurologique, respiratoire et systémique impliquée dans le décès consécutif à l’administration de citalopram. Des dosages de sérotonine plasmatiques et plaquettaires ont été effectués afin de caractériser le rôle de la toxicité sérotoninergique. Résultats : Les IRS étaient impliqués dans 16% des IMV au SAU, soit en 2e position après les benzodiazépines. L’attribution des symptômes observés aux effets sérotoninergiques était rarement faite (dans environ un cas sur cinq) par les médecins urgentistes en charge des patients. La survenue d’un syndrome sérotoninergique et de convulsions était plus fréquente dans le groupe de patients intoxiqués par IRS que chez les témoins appariés. Un allongement du QT a été noté chez un patient et aucune toxicité respiratoire n’a été décelée. Le recours à la ventilation mécanique était plus important du fait de troubles de la conscience, sans augmentation pour autant du nombre d’admission en réanimation en comparaison aux témoins. L’étude expérimentale nous a permis de montrer que les décès induits par le citalopram étaient toujours précédés de convulsions, et que la prévalence des convulsions étaient dose-dépendante, significativement plus fréquente pour les fortes doses de citalopram (80 et 120% de la MLD) comparativement aux autres groupes (60% de la MLD et témoins). De même, le citalopram induisait une baisse dose-dépendante de la sérotonine plaquettaire et une élévation dose-dépendante de la sérotonine plasmatique. L’incidence du syndrome sérotoninergique était, par contre, comparable. Le citalopram n’induisait ni hypoxémie, ni hypercapnie, ni hyperlactatémie ; mais il était responsable d’un allongement du temps inspiratoire et d’un « braking expiratoire » mimant un phénomène adaptatif à l’hypoxémie. Par ailleurs, le prétraitement par diazépam ou cyproheptadine des rats intoxiqués avec une dose létale de citalopram prévenait les convulsions et le décès. Conclusions : La toxicité des IRS et du citalopram en particulier, semble essentiellement neurologique, tant chez l’homme que chez l’animal. Le syndrome sérotoninergique et les convulsions devraient être rassemblés en marqueurs de la toxicité sérotoninergique. Il est nécessaire de sensibiliser les médecins urgentistes à cette toxicité, en utilisant les critères de Hunter, plus simples et probablement plus spécifique. La place des antidotes restent à définir, mais, selon notre modèle expérimental, ils pourraient être efficaces pour réduire cette toxicité spécifique. / Toxicity of the serotonin-reuptake inhibitors (SRI) including citalopram, the most selective one, is considered as relatively low. However SRI may be responsible for serotonin syndrome, seizures, electrocardiographic abnormalities, respiratory failure, and even death. Implication of SRI in deliberate drug poisonings has not been assessed by recent data in France. Morbidity of SRI-related poisonings as well as the most common resulting presentations in the emergency department (ED) remains poorly described. Moreover, mechanisms of SRI-attributed death remain unclear. Objectives: We conducted these clinical and experimental studies: 1-to better understand the epidemiology of drug poisonings in one ED in Paris area and analyze SRI involvement; 2- to investigate a possible over-morbidity related to SRI in multidrug poisonings and describe the most common SRI-related complications; 3- to understand mechanisms of death induced by elevated doses of citalopram and its possible prevention. Methods: We conducted an observational study during 4 years in an ED matching patients who ingested at least one IRS with patients who did not. We also conducted an experimental study in the Sprague-Dawley rat to determine the median lethal dose (MLD) of citalopram and investigate citalopram-related neurological, respiratory, and systemic toxicity as well as mechanisms of citalopram-induced death. Platelet and plasma serotonin were measured to ensure the serotoninergic mechanism. Results: SRI were involved in 16% of the drug poisonings admitted to the ED, ranking at the second place after the benzodiazepines. Attribution of the observed signs and symptoms to the serotonin toxicity was rarely performed by the emergency physicians in charge, in only one out of five cases. Onset of serotonin syndrome and seizures were more frequent in SRI-exposed patients than in their matched controls. QT prolongation was observed in one patient while no direct respiratory toxicity was reported. Mechanical ventilation was more frequently used in SRI-exposed patients due to impaired consciousness, despite no resulting increased admission rate to the intensive care unit in comparison to the controls. Based on our rat study, citalopram-induced death always occurred after seizures which were dose-dependent, with a greater prevalence at the two highest doses of citalopram (80 and 120% of the MLD) than in the other groups (60% of control and the MLD). Citalopram-induced decrease in platelet serotonin and increase in plasma serotonin were dose-dependent. However, incidence of serotonin syndrome appears similar in all the groups. Citalopram did not induce hypoxemia, hypercapnia or hyperlactacidemia, but resulted in a slight prolongation in the inspiratory time and an "expiratory braking" that could be attributed to an adaptive phenomenon to hypoxemia. Pretreatment with diazepam and cyproheptadine prevented rats treated with lethal-doses of citalopram from seizures and death. Conclusions: SRI and citalopram in particular are mainly responsible for neurological toxicity, both in humans and rats. Serotonin syndrome and seizures should be grouped as markers of serotonin toxicity. Emergency physicians should become more aware of this specific toxicity. Using the simpler and probably more specific Hunter criteria may be useful in the ED. The exact indications of antidotes remain to be defined, but our experimental model seems to support their effectiveness to prevent IRS-related specific serotonin toxicity.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Methylmalonic acidemia is an inborn error of metabolism characterized clinically and biochemically by tissue accumulation of methylmalonic acid (MMA) and neurological dysfunction, including convulsion. Furthermore, clinical data suggest that infections conditions can precipitate metabolic crisis and cause neurological changes observed in patients of acidemia. Provided that the MMA cause neurological complications, and that the inflammation can contribute to the occurrence of convulsions and cognitive deficit in several animal models, it is possible to suggest that inflammatory mediators, such as inducible nitric oxide synthase (iNOS), facilitate MMA-induced convulsions. The iNOS is one of three isoforms of nitric oxide synthase (NOS), which generates nitric oxide (NO), a simple gaseous signaling molecule and free radical. The iNOS is induced at injury/inflammation sites, but is also constitutively expressed on some cells, such as in neurons. Studies in experimental models have already demonstrated that NO generated in the central nervous system (CNS), by endothelial and neuronal isoforms of NOS, is involved in MMA-induced convulsions. However, until the present moment are scarce the data in the literature evaluating the relationship of iNOS in experimental models of Methylmalonic Acidemia. The results published in the article has shown that iNOS knock-out C57BL/6 mice, when injected acutely with MMA (2 μmols/2 μl, intracerebroventricularly), have a shorter duration of seizures, no significant change in the mean amplitude of electroencephalographic waves (EEG); not increase the levels of nitrite and nitrate (NOx) compared to animals injected with saline, but have a partial reduction in the levels of 3-nitrotyrosine (3-NT) compared to wild animals that were also treated with MMA; similarly, show a partially lower inhibition of Na+,K+-ATPase, but exhibit no difference in succinate dehydrogenase (SDH) inhibition on cerebral cortex compared to wild mice which also received MMA. The results submitted in the manuscript has shown that Wistar rats, after being injected chronically with MMA (from 5th to 28th day of life, twice daily, with doses ranging from 0.76 to 1.67 mmol/g depending on the age of the animal, via subcutaneous) showed a reduced index of recognition in spatial learning/memory test, but show no anxiety at elevated plus maze test; they have a reduction in neutrophils, but an increase in the number of mononuclear leukocytes in the blood; and in addition show increased levels of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), iNOS and 3-NT in the cerebral cortex. Considering the data presented in both studies, it was concluded that the MMA can cause seizures, nitrosative stress and inhibition of Na+,K+-ATPase activity in cerebral cortex of mice by mechanisms related to NO production via iNOS; and that the MMA can also cause neurocognitive deficits, altered immune system in blood and increase of pro-inflammatory cytokines, leading to increased expression of iNOS and nitrosative stress. / A Acidemia Metilmalônica é um erro inato do metabolismo caracterizado bioquimicamente e clinicamente pelo acúmulo tecidual de ácido metilmalônico (MMA) e disfunção neurológica, incluindo convulsões e déficit cognitivo. Além disso, dados clínicos sugerem que quadros infecciosos podem precipitar crises metabólicas e causar as alterações neurológicas observadas nos pacientes com essa acidemia. Desde que o MMA causa complicações neurológicas, e que a inflamação pode contribuir para a ocorrência de convulsões e déficits cognitivos em vários modelos animais, é possível sugerir que mediadores inflamatórios, como a enzima Óxido Nítrico Sintase Induzível (iNOS), facilitem as convulsões induzidas por MMA. A iNOS é uma das três isoformas da enzima Óxido Nítrico Sintase (NOS), que gera o óxido nítrico (NO), uma molécula gasosa simples, sinalizadora e um radical livre. A iNOS é induzida em sítios de lesão/inflamação, mas também se expressa constitutivamente em algumas células, como nos neurônios. Estudos em modelos experimentais já demonstraram que o NO gerado no sistema nervos central (SNC), pelas isoformas endotelial e neuronal da NOS, tem envolvimento nas convulsões induzidas por MMA. Contudo, até o presente momento são escassos os dados na literatura avaliando a relação da iNOS em modelos experimentais da Acidemia Metilmalônica. Os resultados publicados no artigo mostraram que camundongos C57BL/6 nocaute para iNOS, ao serem injetados agudamente com MMA (2 μmols/2 μL, via intracerebroventricular), apresentam uma duração menor das convulsões, sem alteração significativa na amplitude média das ondas eletroencefalográficas (EEG); não aumentam os níveis de nitrito e nitrato (NOx) comparado aos animais injetados com solução salina, mas têm uma redução parcial nos níveis de 3-nitrotirosina (3-NT) comparado aos animais selvagens que também foram tratados com MMA; semelhantemente, mostram uma inibição parcialmente menor na atividade da enzima Na+,K+-ATPase; mas não exibem diferença na inibição da atividade da succinato desidrogenase (SDH) no córtex cerebral quando comparados aos camundongos selvagens que também receberam MMA. Os resultados apresentados no manuscrito submetido mostram que ratos Wistar, após serem injetados cronicamente com MMA (do 5º ao 28º dia de vida, duas vezes ao dia, com doses variando de 0,76 à 1,67 mmol/g em função da idade do animal, via subcutânea), apresentam um reduzido índice de reconhecimento em teste de memória/aprendizado espacial, mas não demonstram ansiedade no teste do labirinto em cruz elevado; têm uma redução no número de neutrófilos, mas um aumento no número de leucócitos mononucleares no sangue; e além disso mostram aumento nos níveis de interleucina-1beta (IL-1β), do fator de necrose tumoral-alfa (TNF-α), de iNOS e de 3-NT no córtex cerebral. Considerando os dados apresentados nos dois estudos, concluiu-se que o MMA pode causar convulsões, estresse nitrosativo e inibição da enzima Na+,K+-ATPase no córtex cerebral de camundongos por mecanismos relacionados à produção de NO via iNOS; e que o MMA também pode causar déficit neurocognitivo, alteração do sistema imunológico no sangue, e aumento de citocinas pró-inflamatórias, levando ao aumento na expressão da iNOS e estresse nitrosativo.

De nombreuses études expérimentales montrent que les canaux calciques activés par la dépolarisation membranaire, tout particulièrement les canaux calciques de type T (canaux T), jouent un rôle important dans la physiopathologie des épilepsies. Il existe trois isoformes des canaux T, Cav3.1, Cav3.2 et Cav3.3, toutes exprimées au niveau neuronal. De manière classique, c'est dans l'épilepsie absence où les canaux T ont été le plus étudiés. Les canaux T jouent également un rôle dans des modèles d'épilepsie partielle secondairement généralisée, comme le modèle pilocarpine qui mime l'épilepsie du lobe temporal (ELT). Jusqu'à présent ces canaux ne possédaient pas de pharmacologie spécifique, mais plusieurs molécules récemment synthétisées, en particulier le TTA-A2, apparaissent sélectives des canaux T. Le premier objectif de ma thèse était d'étudier l'implication des canaux T dans l'épileptogenèse. Pour cela nous avons traité des souris au TTA-A2 pendant la phase de latence du modèle pilocarpine (modèle ELT). Nos conditions expérimentales ne nous ont pas permis de conclure quant à une action protectrice du TTA-A2 dans ce modèle. Le deuxième objectif était d'étudier l'effet du TTA-A2 sur des modèles murins de convulsions généralisées : le modèle du Maximal Electroshock Seizure (MES) et le modèle pentylènetétrazole (PTZ). Deux lignées de souris inactivées pour les isoformes Cav3.1 ou Cav3.2 (KO Cav3.1 et KO Cav3.2) ont également été caractérisées dans cette étude. Nous montrons que le TTA-A2 réduit l'apparition des crises toniques dans le modèle MES et que les souris KO Cav3.1 sont également protégées, suggérant un rôle prépondérant des canaux Cav3.1 dans le développement des crises toniques. / Numerous experimental studies show that calcium channels activated by membrane depolarization, especially T-type calcium channels (T-channels), play an important role in the physiopathology of epilepsy. There are three T-channels isoforms, Cav3.1, Cav3.2 and Cav3.3, all expressed in neuronal level. Conventionally, T-channels were the most studied in absence epilepsy. T-channels are also involved in partial secondarily generalized epilepsy models, as the pilocarpine model that mimics temporal lobe epilepsy (TLE).Up to now, there was no specific pharmacology for this channels, but several molecules have recently been synthesized, particularly TTA-A2, appearing selective T-channels. The first goal of my thesis was to study the T-channels involvement in epileptogenesis. For this purpose we treated mice with TTA-A2 during the silent phase of the pilocarpine model (TLE model). Our experimental conditions do not allow us to conclude about a possible protective action of TTA-A2 on this model. The second goal was to study TTA-A2 effects on mice models of generalized seizures: the Maximal Electroshock model (MES) and the pentylenetetrazole model (PTZ). Two mice strains knock-out for Cav3.1 or Cav3.2 (KO Cav3.1 and KO Cav3.2) have also been characterized in this study. We show that the TTA-A2 reduces the appearance of tonic seizures in the MES model and the KO Cav3.1 mice are also protected, suggesting a preponderant role of Cav3.1 channels in the development of tonic seizures.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-08-24T17:29:48Z No. of bitstreams: 1 leandrovespolicampos.pdf: 1925979 bytes, checksum: a437d2a7a7488eca8d27ad667669795d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-25T11:53:35Z (GMT) No. of bitstreams: 1 leandrovespolicampos.pdf: 1925979 bytes, checksum: a437d2a7a7488eca8d27ad667669795d (MD5) / Made available in DSpace on 2017-08-25T11:53:35Z (GMT). No. of bitstreams: 1 leandrovespolicampos.pdf: 1925979 bytes, checksum: a437d2a7a7488eca8d27ad667669795d (MD5) Previous issue date: 2017-06-26 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / O Hypericum perforatum (HP) é uma espécie utilizada classicamente como um fitoterápico antidepressivo e ansiolítico. Seus diferentes compostos (hipericina e hiperforina) proporcionam muitos outros efeitos, tais como: antinociceptivo e anticonvulsivante. O objetivo desta tese foi investigar a passagem do extrato hidro-alcoólico de H. perforatum pelas barreiras placentária e hematoencefálica fetal e seus prováveis efeitos antinociceptivo, anticonvulsivante, ansiolítico e antidepressivo sobre os descendentes ao atingirem a idade adulta. Para isto, ratas Wistar receberam doses de 36, 72 e 144 mg/kg de HP ao longo de toda a gestação, por via oral. A fluorescência observada demonstrou a presença do extrato de HP em todos os tecidos analisados tanto das ratas gestantes, quanto dos fetos. Testes para avaliação da atividade antinociceptiva e anticonvulsivante do extrato de HP foram realizados em ratas F1 adultas, as quais apresentaram aumento de ambas as respostas. Testes para avaliação das atividades ansiolítica e antidepressiva do extrato de HP foram realizados com ratos F1 adultos, resultando também em aumento desses efeitos. Estes resultados sugerem que a administração de HP durante a gestação provocou mudanças no neurodesenvolvimento de regiões cerebrais relacionadas com o controle da dor, convulsão, ansiedade e depressão em seus descendentes. / Hypericum perforatum (HP) is a classically used species as an antidepressant and anxiolytic herbal remedy. Its different compounds (hypericin and hyperforin) provide many other effects, such as: antinociceptive and anticonvulsive. The objective of this thesis was to investigate the passage of the hydroalcoholic extract of H. perforatum through placental and fetal blood-brain barrier and the probable antinociceptive, anticonvulsive, anxiolytic and antidepressant effects on offspring as they reach adulthood. Wistar rats received oral doses of 36, 72 and 144 mg/kg of HP throughout gestation. The observed fluorescence indicated the presence of the extract in all tissues analyzed from both pregnant rats and fetuses. Tests for evaluation of antinociceptive and anticonvulsant activity of HP extract were performed on adult F1 rats, which showed an increase in both responses. Tests for evaluation of anxiolytic and antidepressant activities of HP extract were performed on adult F1 rats, also resulting in an increase in these effects. These results suggest that the administration of HP during gestation caused changes in the neurodevelopment of brain regions related to the control of pain, seizure, anxiety and depression in their offspring.

Glutamatergic receptors are targets for many L-glutamate structure analogues, which cause neurotoxicity. This study investigated the actions of two dicarboxylic compounds, the first had cyclic framework and rigid structure, and the other had an acyclic framework and flexible structure, on the glutamatergic neurotransmission, oxidative damage and behavior in mice. The first compound evaluated was D,L-cis-2,3-pyrrolidine dicarboxylate (D,L-cis-2,3-PDC), a new glutamate analogue. D,L-cis-2,3-PDC reduced sodium-independent [3H]-L-glutamate binding by 50% in lysed membrane preparations and had no effect on sodium-dependent glutamate binding. Intracerebroventricular administration (ICV) of D,L-cis-2,3-PDC (7.5 - 25 nmol/ 5μl) induced dose-dependent tonic-clonic convulsions. The co-administration of MK-801 (7 nmol/ 2.5 μl; ICV), a noncompetitive NMDA receptor antagonist, with D,L-cis-2,3-PDC (16.5 nmol/ 2.5 μl; ICV) fully protected the animals against D,L-cis-2,3-PDC-induced convulsions, while the co-administration of DNQX (10 nmol/ 2.5 μl; ICV), a AMPA and KA receptors antagonist, increased the latency to convulsion and did not alter the percentage of animals that had convulsions. These results suggest that D,L-cis-2,3-PDC-induced effects are mediated predominantly by NMDA receptors activation. The second compound studied was succinate, the accumulating substrate in succinate dehydrogenase (SDH) deficiencies and SDH inhibitor intoxication. Adult male mice received an ICV injection of succinate (0.7, 1.0 and 1.7 μmol/ 5 μl) or 0.9% NaCl (5 μl) and had their exploratory behavior assessed in an open field for 10 min. Succinate (0.7 and 1.0 μmol/ 5 μl) decreased locomotor activity behavior and increased thiobarbituric acid reactive substances (TBARS) and protein carbonylation in the forebrain. Conversely, 1.7 μmol of succinate did not alter locomotor activity or oxidative damage parameters. The involvement of NMDA receptors in the succinate-induced increase of total protein carbonylation content and exploratory behavior inhibition was assessed by co-administrating MK-801 (7 nmol/ 2.5 μl, ICV) with succinate (1 μmol/ 2.5 μl, ICV). The co-administration of MK-801 protected against succinate-induced increase of total protein carbonylation and decrease of locomotor activity. These results suggest the involvement of NMDA receptors in these effects of succinate, which may of particular relevance for succinate-accumulating conditions, such as SDH inhibitors intoxication and inherited SDH deficiencies. / Os receptores glutamatérgicos são alvos da ação de muitas neurotoxinas análogas ao L-glutamato. Neste estudo foram investigadas as ações de dois compostos dicarboxílicos, um de cadeia cíclica e estrutura rígida e o outro de cadeia acíclica e estrutura flexível, sobre a neurotransmissão glutamatérgica, dano oxidativo e comportamento em roedores. No primeiro trabalho foi investigado se o D,L-cis-2,3-dicarboxilato de pirrolidina (D,L-cis-2,3-PDC) altera a ligação de [3H]-L-glutamato em membranas plasmáticas de córtex de ratos adultos e se os receptores N-metil-D-aspartato (NMDA) estão envolvidos nas convulsões induzidas por este composto. O D,L-cis-2,3-PDC reduziu a ligação de [3H]-L-glutamato Na+-independente em 50% nas preparações de membranas rompidas e não apresentou efeito sobre a ligação de [3H]-L-glutamato Na+-dependente. A administração intracerebroventricular (ICV) de D,L-cis-2,3-PDC (7,5; 25 nmol/ 5 μl) induziu convulsões generalizadas do tipo tônico-clônica nos camundongos, de uma maneira dose-dependente. A co-administração de MK-801 (7 nmol/ 2,5 μl; ICV), um antagonista não-competitivo dos receptores NMDA, com D,L-cis-2,3-PDC (16,5 nmol/ 2,5 μl; ICV), protegeu totalmente os animais das convulsões induzidas por D,L-cis-2,3-PDC, enquanto que a co-administração de DNQX (10 nmol/ 2,5 μl; ICV), um antagonista dos receptores AMPA e KA, aumentou a latência das convulsões, mas não alterou a percentagem de animais que tiveram convulsões. Estes resultados sugerem que os efeitos induzidos por D,L-cis-2,3-PDC são mediados principalmente pela ativação dos receptores NMDA. No segundo estudo, foi investigado se o sucinato, substrato que se acumula nas deficiências da enzima sucinato desidrogenase (SDH) e nas intoxicações por inibidores da SDH, causa lipoperoxidação e carbonilação protéica, e se os receptores NMDA estão envolvidos no dano oxidativo induzido por sucinato. Camundongos machos adultos receberam uma injeção ICV de sucinato (0,7; 1,0; 1,7 μmol/ 5 μl) ou 0,9 % de NaCl (5 μl) e seu comportamento foi analisado em um campo aberto por 10 minutos. Sucinato (0,7; 1,0 μmol/ 5 μl) diminuiu a atividade locomotora e aumentou as substâncias que reagem ao ácido tiobarbitúrico (TBARS) e carbonilação protéica no cérebro. Por outro lado, 1,7 μmol de sucinato não alterou a atividade locomotora ou os parâmetros de dano oxidativo. O envolvimento dos receptores NMDA no aumento induzido por sucinato do conteúdo de carbonilação protéica e da inibição do comportamento exploratório foi avaliado pela co-administração de MK-801 (7nmol/ 2,5 μl, ICV) com sucinato (1 μmol/ 2,5 μl, ICV). A co-administração de MK801 protegeu contra o aumento induzido por sucinato da carbonilação protéica e na diminuição da atividade locomotora. Esses resultados sugerem o envolvimento dos receptores NMDA nesses efeitos do sucinato, os quais são de grande relevância nas condições em que acumula sucinato, tais como as intoxicações com inibidores da SDH e deficiências dessa enzima causadas por erros inatos do metabolismo.

No description available.