Abstracts

Rationale: The aim of our study was to investigate the role of interleukin1-beta (il1b) in the pilocarpine rat model using RNA interference as a functional tool. Methods: Small interfering RNA molecules (siRNA) targeting the interleukin-1b gene (il1b) and and its endogenous antagonist (il1ra) were conjugated with a glycoprotein derived from the rabies virus (RVG-9R) to ensure optimal delivery into the central nervous system. Eight-week-old male rats were divided into four groups: i) a control group injected with buffer; ii) a second control group injected with an irrelevant siRNA; and iii) and iv) two experimental groups injected with siRNA directed against the two target genes (il1b and il1ra). SiRNA complexes were delivered by intravenous injection in the caudal vein. Intravenous injections were performed 48 h prior to induction of the pilocarpine model. Results: Transvascular delivery of siIl1b and siIl1ra promoted significant gene silencing in the brain 48h post-injection (p.i.), with the highest silencing effect observed at 72h p.i. SiRNA injections provided a dose-response curve, and successful gene knockdown was achieved in four out of five brain regions analyzed. In addition, no evidence of off-target gene effects was observed and gadolinium-MRI showed no disruption of the blood-brain-barrier after the siRNA injections. Phenotypic analysis of the animals which had il1-b silenced showed a significant decrease in the latency for the first seizure (p<0.05) as well as for the status epilepticus (SE) (p<0.05). In contrast, the animals which had il1ra silenced (il1-b endogenous antagonist) had a significant increase in latency for the first seizure (p<0.05) as well as for SE (p<0.05). Furthermore, we found that by silencing il1b a down-regulation of Slc1a3 was also obtained. Slc1a3 is one of the most important transporter proteins involved in glutamate re-uptake in the synaptic cleft. In addition, histological analysis of tissue obtained from animals in the chronic phase showed a significant decrease in neuronal cell death in the hippocampus of animals which had been previously silenced for il1ra (il1\b endogenous antagonist). Conclusions: Our results indicate that il1b may have a protective effect in the early stages of the pilocarpine rat model. In addition, this effect seems to be mediated by glutamate regulation in the synaptic cleft.