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.

Monosialoganglioside (GM1) is a component of most cell membranes and is thought to play a role in development, recognition and cellular differentiation. Furthermore, GM1 is a neuroprotective agent that has been reported to scavenge free radicals generated during reperfusion and to protect receptors and enzymes from oxidative damage. In the present study we investigate the effect of GM1 on the catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, on the spontaneous chemiluminescence and total radical-trapping potential (TRAP) in cortex of rats ex vivo and in vitro. Systemic GM1 administration (50 mg/kg, i.p.; twice) reduced spontaneous chemiluminescence and increased CAT activity ex vivo. On the other hand, GM1 (103-104 nM) reduced CAT activity in vitro. The other parameters were not affected by GM1 administration. These findings agree with the view that the antioxidant action of GM1 is not due to an intrinsic antioxidant activity of this glycolipid, but due to a secondary decrease of reactive species generation and/or increase of antioxidant defenses. Moreover, we evaluated whether GM1 could have a neuroprotective action on the experimental model of glutaric acidemia, an inherited metabolic disorder characterized by glutaric acid (GA) accumulation and neurological dysfunction, as striatal degeneration and convulsion. The systemic GM1 administration (50 mg/kg, i.p. twice) protected against the convulsions, oxidative damage markers increase (total protein carbonylation and thiobarbituric acid-reactive substances - TBARS) production and Na+,K+-ATPase activity inhibition induced by GA (4 mol/ 2 l) in striatum of rats. Furthermore, convulsive episodes induced by GA strongly correlated with Na+,K+-ATPase activity inhibition in the injected striatum, but not with oxidative stress marker measures. In addition, GM1 (50-200 M) protected against Na+,K+-ATPase inhibition induced by GA (6 mM), but not against oxidative damage in vitro. Intrastriatal administration of muscimol (46 pmol/striatum), a GABAA receptor agonist, but not glutamatergic receptor antagonists MK-801 (3 nmol/striatum) and DNQX (8 nmol/striatum), prevented GA-induced convulsions and inhibition of Na+,K+-ATPase activity. The protection of GM1 and muscimol against GA-induced seizures strongly correlated with Na+,K+-ATPase activity maintenance in the injected striatum with GA. Since GM1 and muscimol prevented neurotoxic effects induced by GA, we investigated the GM1 action after intrastriatal administration of pentylenetetrazole (PTZ), a GABAA receptor antagonist. GM1 treatment prevented seizures, Na+,K+-ATPase inhibition, and increase of TBARS and protein carbonyl induced by PTZ (1.8 mol/striatum) in the rats striatum. Furthermore, these data suggest that Na+,K+-ATPase and GABAA receptor-mediated mechanisms may play important roles in GA-induced seizures and in their prevention by GM1. / O monossialogangliosídeo (GM1) é um componente natural de membrana plasmática que está envolvido no crescimento, reconhecimento e diferenciação celular, além de proteger o SNC da ação dos radicais livres. No presente estudo investigou-se o efeito do GM1 sobre a atividade das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD) e glutationa peroxidase (GPx), assim como na quimiluminescência e capacidade antioxidante total (TRAP) em córtex cerebral de ratos machos adultos ex vivo e in vitro. A administração sistêmica de GM1 (50 mg/kg, i.p.; duas doses: 24 horas e 30 minutos antes do sacrifício) reduziu a quimiluminescência e aumentou significativamente a atividade da CAT ex vivo. A adição de GM1 (103-104 nM) ao meio de incubação diminuiu a atividade da CAT in vitro. Estes resultados sugerem que o efeito neuroprotetor do GM1 não é devido à ação antioxidante intrínseca deste glicoesfingolipídeo, mas devido ao aumento secundário das defesas antioxidantes e/ou uma redução da geração de radicais livres. Além disso, avaliamos se o GM1 tinha efeito neuroprotetor em um modelo experimental da acidemia glutárica, um erro inato do metabolismo caracterizado pelo acúmulo tecidual de ácido glutárico (GA) e alterações neurológicas, como degeneração estriatal e convulsões. A administração de GM1 preveniu as convulsões, o aumento da produção dos marcadores do dano oxidativo (carbonilação protéica total e substâncias reativas do ácido tiobarbitúrico - TBARS) e a inibição da atividade da Na+,K+-ATPase induzidas pelo GA (4 mol/2 µl) em estriado de ratos. Além disso, os episódios convulsivos induzidos por GA apresentaram uma correlação significativa com a inibição da atividade da Na+,K+-ATPase no estriado injetado, mas não com os níveis dos marcadores do estresse oxidativo. A adição de GM1 (50 200  ao meio de incubação preveniu a inibição da Na+,K+-ATPase, mas não reduziu o dano oxidativo induzido por GA (6 mM) in vitro. A administração intraestriatal de muscimol (46 pmol/0,5 l), um agonista de receptor GABAA, mas não dos antagonistas de receptores glutamatérgicos, MK-801 (3 nmol/0,5 l) e DNQX (8 nmol/0,5 l), preveniu as convulsões e a inibição da atividade da Na+,K+-ATPase induzidas por GA. A proteção do GM1 e muscimol contra as convulsões induzidas por GA apresentou uma correlação significativa com a manutenção da atividade da Na+,K+-ATPase no estriado injetado com GA. Desde que o GM1 e o muscimol preveniram os efeitos neurotóxicos induzidos pelo GA, investigou-se a ação do GM1 após a administração intraestriatal de pentilenotetrazol (PTZ), um antagonista de receptores GABAA. O tratamento com GM1 preveniu as convulsões, o dano oxidativo e a inibição da atividade da Na+,K+-ATPase induzidas por PTZ (1,8 µmol/2 µl). Esses dados sugerem que a atividade da Na+,K+-ATPase e mecanismos mediados pela ativação de receptores GABAérgicos podem ser de grande importância para a atividade convulsiva induzida por GA, bem como nos mecanismos de neuroproteção induzidos pelo GM1.

L’objectif premier de notre projet était d’établir un modèle animal de douleur chronique orofaciale, lequel pourrait imiter la sensibilité retrouvée chez les patients souffrant de douleur orofaciale myalgique. Nous avons procédé à des injections intramusculaires de saline acide (2 injections à 2 jours d’intervalle pH 4.0) pour induire une sensibilisation mécanique des mucles massétérins. La réponse nocifensive a été mesurée à l’aide de filaments de von Frey avant et après ces injections dans des rats Sprague-Dawley. Par la suite, le potentiel analgésique de différents antagonistes des récepteurs glutamatergiques fût évalué par l’injection intramusculaire de ces antagonistes à différents moments. Nos résultats suggèrent que deux injections de saline acide, produisent une hypersensibilité mécanique signalée par l’augmentation du nombre de réponses à l’application de filaments de von Frey. Cet effet dure plusieurs semaines et est bilatéral, même lorsque les injections sont unilatérales, indiquant qu’une composante centrale est forcément impliquée. Toutefois, une composante périphérique impliquant les récepteurs glutamatergiques semble présider le tout puisque les antagonistes glutamatergiques, appliqués de façon préventive empêchent le développement de l’hypersensibilité. Cependant, le maintien de cette hypersensibilité doit dépendre de mécanismes centraux puisque l’application d’antagonistes une fois la sensibilisation induite, ne diminue en rien le nombre de réponses obtenues. Ce modèle semble approprié pour reproduire une hypersensibilité musculaire durable de bas niveau. Nos données indiquent que les récepteurs glutamatergiques périphériques participent à l’induction de cette hypersensibilité de longue durée. Nous croyons que ce modèle pourra éventuellement contribuer à une meilleure compréhension des mécanismes à l’origine des myalgies faciales persistantes. / The first objective of this project was to establish an animal model of chronic orofacial pain, which could mimic symptoms of patients suffering from orofacial myalgia. We used acidic saline injections (2 injections, 2 days apart at pH 4.0) in masseteric muscles to induce mechanical hypersensitivity. Nocifensive behavior was measured before and after the injections using von Frey filaments in male Sprague Dawley rats. Later, the potential analgesic effect of glutamate receptors antagonists was measured by intramuscular administration of these antagonists at different times. Our results suggest that two injections of acidic saline produce a mechanical hypersensitivity as reflected by the increased number of responses to applications of von Frey filaments. This effect lasts several weeks and is bilateral, even when the injections are unilateral, indicating that a central component must be involved. However, the initial stage of induction of this hypersensitivity involves peripheral glutamate receptors since injection of their antagonists before the second acidic saline injection prevents development of the nocifensive response, whereas their injection at later times is ineffective in blocking development of the response. This model based on a double injection of acidic saline seems appropriate to reproduce low intensity, long-lasting muscle pain. Our data suggests that peripheral glutamate receptors are involved in the induction of this long-term hypersensitivity. We believe that this model may contribute to a better understanding of the mechanisms behind persistent orofacial muscle pain.

L’objectif premier de notre projet était d’établir un modèle animal de douleur chronique orofaciale, lequel pourrait imiter la sensibilité retrouvée chez les patients souffrant de douleur orofaciale myalgique. Nous avons procédé à des injections intramusculaires de saline acide (2 injections à 2 jours d’intervalle pH 4.0) pour induire une sensibilisation mécanique des mucles massétérins. La réponse nocifensive a été mesurée à l’aide de filaments de von Frey avant et après ces injections dans des rats Sprague-Dawley. Par la suite, le potentiel analgésique de différents antagonistes des récepteurs glutamatergiques fût évalué par l’injection intramusculaire de ces antagonistes à différents moments. Nos résultats suggèrent que deux injections de saline acide, produisent une hypersensibilité mécanique signalée par l’augmentation du nombre de réponses à l’application de filaments de von Frey. Cet effet dure plusieurs semaines et est bilatéral, même lorsque les injections sont unilatérales, indiquant qu’une composante centrale est forcément impliquée. Toutefois, une composante périphérique impliquant les récepteurs glutamatergiques semble présider le tout puisque les antagonistes glutamatergiques, appliqués de façon préventive empêchent le développement de l’hypersensibilité. Cependant, le maintien de cette hypersensibilité doit dépendre de mécanismes centraux puisque l’application d’antagonistes une fois la sensibilisation induite, ne diminue en rien le nombre de réponses obtenues. Ce modèle semble approprié pour reproduire une hypersensibilité musculaire durable de bas niveau. Nos données indiquent que les récepteurs glutamatergiques périphériques participent à l’induction de cette hypersensibilité de longue durée. Nous croyons que ce modèle pourra éventuellement contribuer à une meilleure compréhension des mécanismes à l’origine des myalgies faciales persistantes. / The first objective of this project was to establish an animal model of chronic orofacial pain, which could mimic symptoms of patients suffering from orofacial myalgia. We used acidic saline injections (2 injections, 2 days apart at pH 4.0) in masseteric muscles to induce mechanical hypersensitivity. Nocifensive behavior was measured before and after the injections using von Frey filaments in male Sprague Dawley rats. Later, the potential analgesic effect of glutamate receptors antagonists was measured by intramuscular administration of these antagonists at different times. Our results suggest that two injections of acidic saline produce a mechanical hypersensitivity as reflected by the increased number of responses to applications of von Frey filaments. This effect lasts several weeks and is bilateral, even when the injections are unilateral, indicating that a central component must be involved. However, the initial stage of induction of this hypersensitivity involves peripheral glutamate receptors since injection of their antagonists before the second acidic saline injection prevents development of the nocifensive response, whereas their injection at later times is ineffective in blocking development of the response. This model based on a double injection of acidic saline seems appropriate to reproduce low intensity, long-lasting muscle pain. Our data suggests that peripheral glutamate receptors are involved in the induction of this long-term hypersensitivity. We believe that this model may contribute to a better understanding of the mechanisms behind persistent orofacial muscle pain.