Abstracts

Rationale: Drug-resistant epilepsy often starts with an isolated prolonged convulsion in early life, a remission period, followed by recurring intractable seizures. Early life seizures can have both acute and long-lasting effects on the rapidly developing brain. Several studies have investigated changes in microRNAs (miRNAs) in pre-clinical models of status epilepticus, specifically focusing on hippocampus. miRNAs are small RNA molecules that post-transcriptionally regulate gene expression. In this study we used a "two-hit" seizure model to study the epileptogenic effect of early life seizures and to understand the effect of early life seizures on miRNA expression in both cortex and hippocampus. Methods: Mice were subjected either to febrile seizure (FS; lipopolysaccharide + hyperthermia) or to kainate-induced status epilepticus (KA) on postnatal day (P) 15, with saline (PBS) treated mice serving as controls (n=5 in each treatment). These mice were sacrificed 24h after treatment. A second set of mice were subjected to two hits: FS, KA or PBS on P15 followed by KA on P45 and then sacrificed 24h after treatment (n=5 in each treatment). Cortex and hippocampus were dissected from all mice and flash frozen. Total RNA was isolated using the mirVana miRNA isolation kit. High-resolution quantification of miRNA expression was performed using small RNA-Seq, from 1 g of total RNA, on an Illumina High Seq 2000 (24 samples per lane, ~ 10 million reads per sample). Statistical analyses including Principal Component Analysis, ANOVA, and clustering were performed using the Partek Genomics Suite software package. Network and pathway analyses were performed using Ingenuity Pathway Analysis (IPA) software. Results: Principal Component Analysis of miRNA expression in "two-hit" adult mice revealed distinct profiles for each brain region. Interestingly, miRNA expression changes in the cortex was significantly larger than the hippocampus in each of the treatment groups. In the cortex, > 200 miRNAs showed differential expression (p < 0.05, fold change >2 or <-2) while only very few miRNAs were differentially expressed in the hippocampus across groups. In addition, the experience of an early life seizure had a lasting effect on the brain; there were significant differences in miRNA expression between FS-KA and control animals (PBS-KA) with 15 miRNAs showing >3-fold differences in the cortex. Contrary to mRNAs, miRNAs have a smaller dynamic range in expression levels, making a 2-3 fold change significant. We observed that the miRNA dysregulation after KA treatment at P45 was also dependent on the type of seizure trigger at P15. This is in contrast to P15 mice, where the variation in miRNA expression seemed largely independent of the nature of the trigger. Pathway analyses revealed cortical miRNAs upregulated in adult mice with prior exposure to an early life seizure to be involved in cellular proliferation and cellular stemness. Conclusions: Our data indicate that an early life seizure has a sustained effect by altering miRNA expression in the cortex and that only limited changes are seen in the hippocampus. Further studies are needed to evaluate the effect of this miRNA dysregulation on brain development. Funding: University of Minnesota College of Pharmacy Grants Award Program