Caracterização do sistema purinérgico na formação hipocampal de ratos submetidos ao modelo de epilepsia do lobo temporal induzida por pilocarpina
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Arquivos
Data
2006
Autores
Doná, Flávia [UNIFESP]
Orientadores
Fernandes, Maria Jose da Silva [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
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Resumo
A pilocarpina é um agonista colinérgico que, quando injetada em altas doses
(i.p.) a ratos, reproduz as principais características da epilepsia do lobo temporal
(ELT) humana. O animal exibe diferentes padrões de comportamentos, incluindo
status epilepticus (fase aguda), normalização comportamental (fase latente) e
finalmente crises espontâneas e recorrentes (fase crônica).
Os objetivos desse estudo foram analisar por microdiálise e HPLC a
concentração de ATP e seus metabólitos na formação hipocampal de ratos que se
encontram nas fases aguda e crônica do modelo experimental de ELT induzida por
pilocarpina, e avaliar através de imunoblot e imuno-histoquímica, os receptores
P2X2,4,7 na formação hipocampal desses animais, incluindo o grupo latente.
O procedimento de microdiálise foi aplicado para determinação da
concentração de purinas liberadas durante o status epilepticus (SE) em amostras
coletadas nos seguintes tempos: Basal (3 amostras antes de qualquer tratamento),
após a administração de metilescopolamina (usada para minimizar os efeitos
periféricos da pilocarpina) e 30 min. 1H, 2H, 3H e 4H após a aplicação da pilocarpina.
Os animais crônicos foram estudados nos períodos interictal e ictal. As amostras foram
analisadas por HPLC com detecção por fluorescência.
Animais de três grupos experimentais (G12H; GLat; GCro) e respectivos
controles, foram empregados para o estudo dos receptores P2X2,4,7, através de
imunoblot e imuno-histoquímica. O imunoblot foi revelado por quimiluminescência. A
imuno-histoquímica foi feita com cortes fixados e flutuantes em tampão, usando
anticorpo biotinilado e kit ABC e revelação com diaminobenzidina.
O estudo bioquímico revelou um aumento de ADP, AMP e adenosina durante
o SE. Na fase crônica, evidenciou-se uma redução de todas as purinas no período
interictal, e um aumento das mesmas no período ictal, sendo significante para o ATP
e adenosina. Em relação aos receptores estudados por imuno-histoquímica e
imunoblot, observou-se uma intensa redução dos P2X4 versus CT no período crônico
(35%, p<0.001, ANOVA), um aumento do P2X7 nas fases aguda e crônica (G12H: 20%,
p<0,05; GCRO: 18% p<0,05, ANOVA).Com base nos resultados, conclui-se que o metabolismo das purinas está
alterado na fisiopatologia da ELT induzida por pilocarpina, por exemplo, na fase
aguda o aumento na concentração de ADP, AMP e de adenosina, pode estar refletindo
um aumento na taxa metabólica do ATP pelas ectonucleotidases, como um mecanismo
compensatório inibitório. No entanto, o ATP antes de ser metabolizado pode estar
ativando receptores P2X7, e exercendo um papel importante na citotoxicidade. Já na
fase crônica, a redução das purinas, infere principalmente um comprometimento na
atividade inibitória mediada pela adenosina, que por sua vez pode facilitar a
susceptibilidade às crises epilépticas.
Pilocarpine, a muscarinic cholinergic agonist when inject at high doses (i.p.) in rats, reproduces the main characteristics of Human Temporal Lobe Epilepsy (TLE). The animal exhibits different patterns of behavior, including status epilepticus (acute phase), electrophysiological and behavioral normalization (latent phase) and spontaneous recurrent seizures (chronic phase). The objectives of this study were to analyze by means of microdyalysis and HPLC, the concentration of ATP and its metabolites ADP, AMP and adenosine in the hippocampal formation of rats during the acute and chronic phases of pilocarpine model, and evaluating by western blotting and immunohistochemistry, the receptors P2X2,4,7 in the hippocampal formation of these animals. The microdyalysis procedure used to determine the purines concentration in the acute period, was taken according to the following periods of time: Basal (three samples before treatment onset), after methylscopolamine injection (used in order to minimize the peripheral effects of pilocarpine) and 30 min. 1h, 2h, 3h and 4h after pilocarpine injection. Chronic animals were studied during the interictal and ictal period. The samples were analyzed by a fluorescence detector coupled to HPLC. Animals from three different experimental groups (Group 12h = G 12h, Latent Group = G Lat, Chronic Group = G Cro) and their respective controls were utilized to analyze the purinergic receptors P2X2,4,7, by means of western blotting and immunohistochemistry. The immunohistochemistry was achieved with fixated slices floating on buffer solution, using biotinylated antibody, ABC kit and development with diaminobenzidine. The biochemical study revealed an increase of ADP, AMP and adenosine concentrations during the SE. In contrast, a reduction of all purines was detected in the hippocampal formation during the interictal period of chronic rats. Rats studied during the ictal phase, exhibited high concentrations of ATP and adenosine. The immunohistochemistry and western blotting analysis showed a great reduction of P2X4, versus CT in the chronic period (35%, p<0.001, ANOVA), and an increase of P2X7, in the acute and chronic phases (G12H: 20%, p<0.05; GCRO: 18% p<0.05, ANOVA). According to these findings, we can conclude that the metabolism of purines is altered in the hippocampal formation of rats in pilocarpine-induced epilepsy. In the acute phase, the increase of ATP metabolites may reflect an increase in the metabolism rates of ATP by the ectonucleotidases, possibly as a compensatory inhibitory mechanism. On the other hand, before ATP is hydrolyzed, it may activate P2X7 purinoceptor, and consequently, intensify hyperexcitability and cytotoxicity cascades. A decrease in all purines analyzed in the chronic phase could suggest an energy metabolism dysfunction in the epileptic hippocampus. The decrease in the adenosine concentrations might facilitate the onset of spontaneous and recurrent seizures.
Pilocarpine, a muscarinic cholinergic agonist when inject at high doses (i.p.) in rats, reproduces the main characteristics of Human Temporal Lobe Epilepsy (TLE). The animal exhibits different patterns of behavior, including status epilepticus (acute phase), electrophysiological and behavioral normalization (latent phase) and spontaneous recurrent seizures (chronic phase). The objectives of this study were to analyze by means of microdyalysis and HPLC, the concentration of ATP and its metabolites ADP, AMP and adenosine in the hippocampal formation of rats during the acute and chronic phases of pilocarpine model, and evaluating by western blotting and immunohistochemistry, the receptors P2X2,4,7 in the hippocampal formation of these animals. The microdyalysis procedure used to determine the purines concentration in the acute period, was taken according to the following periods of time: Basal (three samples before treatment onset), after methylscopolamine injection (used in order to minimize the peripheral effects of pilocarpine) and 30 min. 1h, 2h, 3h and 4h after pilocarpine injection. Chronic animals were studied during the interictal and ictal period. The samples were analyzed by a fluorescence detector coupled to HPLC. Animals from three different experimental groups (Group 12h = G 12h, Latent Group = G Lat, Chronic Group = G Cro) and their respective controls were utilized to analyze the purinergic receptors P2X2,4,7, by means of western blotting and immunohistochemistry. The immunohistochemistry was achieved with fixated slices floating on buffer solution, using biotinylated antibody, ABC kit and development with diaminobenzidine. The biochemical study revealed an increase of ADP, AMP and adenosine concentrations during the SE. In contrast, a reduction of all purines was detected in the hippocampal formation during the interictal period of chronic rats. Rats studied during the ictal phase, exhibited high concentrations of ATP and adenosine. The immunohistochemistry and western blotting analysis showed a great reduction of P2X4, versus CT in the chronic period (35%, p<0.001, ANOVA), and an increase of P2X7, in the acute and chronic phases (G12H: 20%, p<0.05; GCRO: 18% p<0.05, ANOVA). According to these findings, we can conclude that the metabolism of purines is altered in the hippocampal formation of rats in pilocarpine-induced epilepsy. In the acute phase, the increase of ATP metabolites may reflect an increase in the metabolism rates of ATP by the ectonucleotidases, possibly as a compensatory inhibitory mechanism. On the other hand, before ATP is hydrolyzed, it may activate P2X7 purinoceptor, and consequently, intensify hyperexcitability and cytotoxicity cascades. A decrease in all purines analyzed in the chronic phase could suggest an energy metabolism dysfunction in the epileptic hippocampus. The decrease in the adenosine concentrations might facilitate the onset of spontaneous and recurrent seizures.
Descrição
Citação
DONÁ, Flávia. Caracterização do sistema purinérgico na formação hipocampal de ratos submetidos ao modelo de epilepsia do lobo temporal induzida por pilocarpina. 2006. 111 f. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2006.