Silencing of P2X7R by RNA interference in the hippocampus can attenuate morphological and behavioral impact of pilocarpine-induced epilepsy

Abstract

Cell signaling mediated by P2X7 receptors (P2X7R) has been suggested to be involved in epileptogenesis, via modulation of intracellular calcium levels, excitotoxicity, activation of inflammatory cascades, and cell death, among other mechanisms. These processes have been described to be involved in pilocarpine-induced status epilepticus (SE) and contribute to hyperexcitability, resulting in spontaneous and recurrent seizures. Here, we aimed to investigate the role of P2X7R in epileptogenesis in vivo using RNA interference (RNAi) to inhibit the expression of this receptor. Small interfering RNA (siRNA) targeting P2X7R mRNA was injected into the lateral ventricles (icv) 6 h after SE. Four groups were studied: Saline-Vehicle, Saline-siRNA, Pilo-Vehicle, and Pilo-siRNA. P2X7R was quantified by western blotting and neuronal death assessed by Fluoro-Jade B histochemistry. The hippocampal volume (edema) was determined 48 h following RNAi. Behavioral parameters as latency to the appearance of spontaneous seizures and the number of seizures were determined until 60 days after the SE onset. The Saline-siRNA and Pilo-siRNA groups showed a 43 and 37% reduction, respectively, in P2X7R protein levels compared to respective vehicle groups. Neuroprotection was observed in CA1 and CA3 of the Pilo-siRNA group compared to Pilo-Vehicle. P2X7R silencing in pilocarpine group reversed the increase in the edema detected in the hilus, suprapyramidal dentate gyrus, CA1, and CA3; reduced mortality rate following SE; increased the time to onset of spontaneous seizure; and reduced the number of seizures, when compared to the Pilo-Vehicle group. Therefore, our data highlights the potential of P2X7R as a therapeutic target for the adjunct treatment of epilepsy.

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Acknowledgments

This work was supported by the Brazilian funds from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, nos. 142743/2010-0 and 248728/2012-1), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES). We thank Prof. Henning Ulrich who kindly provided the material for tests.

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Correspondence to Maria José da Silva Fernandes.

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All experimental procedures were performed under the supervision and with the approval of our internal Ethics Committee (Federal University of São Paulo, CEP N. 0961/10). Animal protocols were conducted in accordance with national and international legislation (Guidelines of the Brazilian College of Animal Experimentation, COBEA; NIH Guide for Care and Use of Laboratory Animals) and the experiments followed the principles outlined in the Basel Declaration [45].

Conflict of interest

Rebeca Padrão Amorim declares that she has no conflict of interest.

Michelle Gasparetti Leão Araújo declares that she has no conflict of interest.

Jorge Valero declares that he has no conflict of interest.

Iscia Lopes-Cendes declares that she has no conflict of interest.

Vinicius Davila Bitencourt Pascoal declares that he has no conflict of interest.

João Oliveira Malva declares that he has no conflict of interest.

Maria José da Silva Fernandes declares that she has no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Amorim, R.P., Araújo, M.G.L., Valero, J. et al. Silencing of P2X7R by RNA interference in the hippocampus can attenuate morphological and behavioral impact of pilocarpine-induced epilepsy. Purinergic Signalling 13, 467–478 (2017). https://doi.org/10.1007/s11302-017-9573-4

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Keywords

  • Temporal lobe epilepsy
  • P2X7 purinergic receptors
  • RNA interference
  • Pilocarpine
  • Hippocampus