Papel dos receptores TRPA1 e TRPV1 em modelos de crises epilépticas

Abstract

Epilepsy is a neurological disorder that affects millions of people around the world, while temporal lobe epilepsy (ELT) accounts for 40% of all epilepsy cases, being the most common form in adults. The family of transient potential receptors (TRP) are a group of cation channels involved in different physiological processes, such as ion homeostasis. Calcium accumulation in the neurons of the hippocampus has been associated with the etiology of epilepsy, which motivates the study of the TRP family, whose channels are highly permeable to this ion. The aim of this study was to evaluate the effects of TRPA1 and TRPV1 blockers on seizures, histological, and biochemical changes, besides glutamate release and cytosolic calcium content in model of pilocarpine or pentylenetetrazole -induced seizures or in cortex synaptosomal preparations. For this, C57Bl/6 animals (8-12 weeks of age) received intrahippocampal pilocarpine injection (40 μg), 30 minutes after intraperitoneal injection of TRPA1, TRPV1 blocker or vehicle. Blockers of the TRPA1, AP18 receptor (1, 3 and 10 mg/Kg) and TRPV1 receptor, SB366791 (0.1; 0.3 and 1 mg/Kg) were used in the in vivo experiments. Concentrations of 0.1; 1; 10 and 30 μM were used in the preparation of synaptosomes for both blockers. Twenty-four hours after pilocarpine-induced status epilepticus (SE), animals were euthanized to collect brain tissues. Half of their brains were removed and processed, for further histological techniques to evaluate neuronal death and viability, microglia and astrocyte staining. The other half of the animal’s brains were removed for further analysis of BDNF and cytokines levels, besides the protein kinase B (Akt) and extracellular-signal-regulated kinase (Erk) activation. The seizures induced by pilocarpine were evaluated according to Racine’s scale of seizures. Strong association between AP18 and the development of SE was revealed, as well as the blocker and tonic-clonic seizures. AP18 (3 mg/Kg) increased the seizures classified as 3, 4 and 5. On the other hand, AP18 (10 mg/Kg) increased the median latency for the animals to develop the SE as compared with pilocarpine group. Pilocarpine increased the labeling of neurons in the degeneration process evidenced by Fluoro-Jade C (FJC) staining, which was exacerbated by the administration of AP18 (1 and 3 mg/kg). Surprisingly, the drug induced a dichotomous response in microgliosis induced by pilocarpine, as it exacerbated in dentate gyrus (DG) with AP18 (3 mg/kg) and decreased in CA1 with doses of 1 and 3 mg/kg of AP18. We observed a reduction in astrocyte reactivity with AP18 (1 and 10 mg/kg) induced by pilocarpine, while the dose of 3 mg/kg of AP18 increased this marking in DG. In addition, AP18 reduced the levels of BDNF, TNF-α and CX3CL1, and enhanced IL-10 levels compared to the pilocarpine group. In relation to TRPV1, strong association was observed between SB366791 and the development of the SE. The frequency of SE and tonic-clonic seizures in the SB366791 group (0.1 and 0.3 mg/Kg) was higher than the pilocarpine group. The behavioral seizure was accompanied by an enhancement of neuronal death. AP18 and SB366791 did not change any of the analysis with cortical synaptosomes preparations. Finally, in the pentylenetetrazole-induced seizures AP18 and SB366791 did not change the latency for SE development. The present study showed that the systemic administration of the TRPA1 and TRPV1 blockers, AP18 or SB366791, respectively, increased the severity of epileptic seizures and neuronal damage resulting from the SE induced by the intra-hippocampal injection of pilocarpine. Our results suggested that AP18 and SB366791 show pro-convulsive effects in the pilocarpine model, but not in pentylenetetrazole- induced seizures. Further studies will allow the characterization of the underlying mechanisms of these channels in TLE.