Abstracts

Rationale: Many studies have implicated glial-mediated inflammatory changes that occur after status epilepticus (SE) in the subsequent development of epilepsy.  While this makes anti-inflammatory strategies an attractive target for therapeutic intervention, the appropriate timing for such an intervention is unclear.  The aim of this work is to define the timing of the inflammatory response through the TLR4/IL-1ß pathway regulated via pro-inflammatory miR-155 and anti-inflammatory miR-146a in both male and female mouse models of kainic acid-induced SE. Methods: SE was induced in 7 to 8 week-old male and female C57BL/6J mice (12 males and 12 females at each time point) using intraperitoneal injections of 20 mg/kg kainic acid, which produced a clinical intermittent SE phenotype.  The onset of SE was defined as the first Class 3 seizure as defined using a modified Racine Scale.  The intensity of the SE episode was estimated by the total number of discrete class 5 seizures observed.  After 2 hours, the SE was aborted with administration of diazepam, and hippocampal tissue was harvested at 3 hr, 18 hr, 24 hr, 48 hr and 72 hr after diazepam administration.  RNA was isolated using TRIzole (Life Technologies) followed by qRT-PCR analysis to define the steady-state expression levels of miR-155 and miR-146a at these time points.  In addition, the mRNA levels of SOCS1 and TRAF6, known inflammation-related mRNA targets of miR-155 and miR-146a, respectively, were compared to the changes observed in miR-155 and miR-146a. Results: We observed a 3-3.5 fold increase in the steady state expression levels of miR-155 in both male and female mice after SE, but the time to peak level is 24 hours in the male mice and 48 hours in the female mice (Figure 1a and 1c).  There was a direct association between the intensity of the episode of SE, as defined by the number of class V seizures, and the expression levels of miR-155.  There was a decrease in the steady state expression of the anti-inflammatory miR-146a at 18 hours in both male and female mice that returned to baseline control levels by 24 hours, although the effect appeared to be more pronounced in the females.  The level of SOCS1 mRNA peaked in response early in inflammation but decreased to control levels after the peak expression of miR-155.  TRAF6 mRNA showed consistent steady-state expression levels, as the levels of miR-146a were only observed to show a minimal decrease (Figure 1b and 1d). Conclusions: MiRNA-155 shows a time-dependent increase in steady-state expression within 24-48 hours of SE, but the timing is different between males and females.  The overall expression levels appear to correlate with the clinical intensity of the episode of SE and with changes in one of its inflammation-regulating targets, SOCS1.  Both males and females exhibit a small decrease in the relative expression of miR-146a at 18 hours after SE, but the steady-state expression levels of the miRNA-146a target, TRAF6, remain unchanged.  Future work will focus on determining the source of the differences in the inflammatory response induced by SE between the male and female mice in addition to other inflammation-regulating targets. Funding: Cincinnati Children’s Hospital Medical Center, Division of Neurology