Abstracts

Rationale: Animal models which use convulsant drugs, such as pilocarpine, are one of the most used tools to study the mechanisms involved in mesial temporal lobe epilepsy (MTLE). However these models are based in the induction of an initial episode of status epilepticus (SE), usually leading to extensive extra-hippocampal lesions, which are events not commonly observed in patients with MTLE. It has been observed that some animals which receive pilocarpine but do not develop SE can display long term spontaneous recurrent seizures and more discrete SNC lesions. Nevertheless, the mechanisms responsible for the occurrence of spontaneous seizures and how different hippocampus subfields are affected in this SE-free model are unknown. Transcriptome analyzes using high-throughput next generation sequencing (HTNS) offers the possibility of profiling gene expression with significant reduced cost, greater sensibility for detecting rare transcripts and elimination of hybridization artifacts that may affect traditional microarray experiments. In the present study we analyzed by HTNS the gene expression profile of the dentate gyrus (DG) of SE-free rats presenting long term spontaneous seizures after the administration of pilocarpine.Methods: Eight-weeks old male Wistar rats received methylscopolamine (1mg/kg, i.p.), followed 30 minutes later by pilocarpine (320 mg/kg, i.p.). Control animals received the same dose of methylscopolamine followed by saline administration. Only animals that did not developed SE, defined as continuous generalized level 3-5 Racine scale seizures, were used in this study. Rats were video-monitored 24 hours for 6 months after pilocarpine administration and at the end of this period, they were euthanized in a CO2 chamber. The brain was quickly removed and frozen in liquid nitrogen. Frozen sections were produced in a cryostat (Leica) and mounted in PEN covered glass slides (Zeiss). Slides were Nissl stained, and the DG was laser microdissected using Zeiss PALM LCM. RNA was extracted from microdissected samples obtained from 3 pilocarpine treated rats and 3 control animals, using RNAeasy microkit (Qiagen). The purified RNA was processed with TruSeq RNA Sample Preparation kit (Illumina) for the production of cDNA libraries. HTNS experiments were performed in a HISeq 2000 (Ilumina). Sequence alignment was performed using TopHat and gene expression quantification with HTseq.Results: We found a total of 122 genes differentially expressed in pilocarpine treated animals as compared to controls: 35 were up-regulated and 87 were down-regulated.Conclusions: Although the DG apparently does not present morphological changes in this SE-free experimental model, the observed transcriptional changes indicate that more subtle molecular and physiological mechanisms are indeed present and these more subtle changes are likely to be responsible for the occurrence of spontaneous seizures. This work was supported by FAPESP (grant 2011/50680-2). All animal procedures were approved by the UNICAMP Ethics Committee on Animal Experimentation (prot 2903-1).