Papel da enzima fosfatidilinositol-3-quinase (PI3K) sobre crises convulsivas e epileptogênese em modelo animal de epilepsia induzido por pilocarpina

Abstract

Introduction: Temporal Lobe Epilepsy (TLE) is the most common form of epilepsy in adults. The experimental model of epilepsy induced by pilocarpine (PILO) induces behavioral and pathophysiological changes in rodents very similar to those seen in humans with TLE. However, this model is associated with a high mortality of the animals. Although few studies have shown that intrahippocampal injection of PILO rat is a reliable model of TLE, no study has been conducted so far in mice. The phosphatidylinositol-3 kinase (PI3K) / protein kinase B (Akt) / mammalian target of rapamycin (mTOR) pathway has been associated with several pathologies in the Central Nervous System (CNS). However, the specific role of PI3K in the pathophysiology of epilepsy is poorly understood. Therefore, in the present study, we investigated the involvement of PI3K enzyme on seizures and on the production of neurotrophic factors and inflammatory mediators as well as the expression of microglia and hippocampal neurogenesis in mice subjected to microinjection of intrahippocampal PILO. Methods: Experiments were performed in adult male mice C57Bl/6 (WT) and PI3k-/- (10-12 weeks of age). Guide cannulas were implanted in the left and right hippocampus, using stereotactic surgery and then was awaited 5 days for recovery animals. The status epilepticus (SE) was induced by bilateral intrahippocampal microinjection of PILO (20g per site/200nL) or saline as control animals and WT PI3k-/-. Seizures were analyzed for 90 minutes and classified according to the scale set of Racine (1972). There was also observed changes in memory of WT and PI3k-/- animals, 72h post-SE. 24 hours after induction of SE, hippocampus were removed and prefrontal cortex to determine the neurotrophic factors, BDNF and NGF, as well as of inflammatory mediators, IL-2, IL-4, IL-6, IL-10, TNF- , IFN- and IL-17A. To evaluate the expression of microglial cells (Iba-1) and neurogenesis (doublecortin - DCX), the brains were removed after intracardiac perfusion, 72 h after SE induction. Analyses Statistics were performed using GraphPad Prism 5 and expressed as mean ± SEM (P <0.05). Results: We observed behavioral changes post-SE. Levels of BDNF were significantly increased in the hippocampus of WT and PI3K-/- mice after microinjection of PILO. In the prefrontal cortex, only animals PI3K-/- showed significant increase in BDNF levels. Moreover, levels of NGF in the hippocampus of WT and PI3K-/- animals not were altered by administration of PILO. In the levels of inflammatory mediators, there was an increase in IL-6 levels in the hippocampus of PI3K-/- animals and, in the prefrontal cortex, in both groups; IL- 10 WT in the hippocampus of animals, TNF- in the hippocampus of WT animals, and IFN- in the hippocampus of both groups. Moreover, there were increase in the expression of microglial cells in WT animals, as well as a decrease in the hippocampal neurogenesis in PI3K-/- animals, after the induction of SE. Conclusions: The intrahippocampal microinjection of PILO induced seizures, as well as biochemical changes observed in the model intraperitonaeal injection of PILO, with the advantage of low rates of mortality. Moreover, we observed several changes related to the PI3K pathway.