Abstracts

Rationale: Recent studies suggest that free radical scavenging during acute phase of status epilepticus (SE) can reduce neuronal loss and prevent aberrant neurogenesis in short intervals after insult (Mishra et al, Scientific Reports 2016). There are however no data on long term effects and functional consequences of this treatment. Methods: The free radical scavenger N-tert-butyl-?-phenylnitrone (PNB) was administered twice in a total dose of 200 mg/kg during LiCl/pilocarpine SE in juvenile, 25-day-old rats. Three months later, cognitive abilities and emotional status were assessed using Morris Water maze (MWM), elevated plus maze (EPM) and capture resistance (CR) test. To assess severity of epilepsy animals were video/EEG monitored for 5 days (7/24). Neuropathological changes were quantified using morphometry and unbiased stereology. Aberrant neurogenesis was evaluated with prospero-like homeobox protein 1 (Prox1) immunohistochemistry. The functional maturity of these granule cells was determined by double labeling with Prox1 and neuronal specific nuclear protein (NeuN). Newly born neurons were detected with doublecortin (DCX) immunohistochemistry. Data from behavioral tests and histological analysis were correlated to find out possible association between changes in neurogenesis and functional alterations. Results: PBN administration resulted in partial protection of the hippocampus. The hippocampal volume was enhanced by 31.4% and number of hilar neurons was by 41.6% higher compared to untreated SE controls. Protection of the hippocampus was associated with improved behavior in the MWM test. Negatively, PBN exacerbates emotional impairment and epilepsy. PBN animals spent more time in the enclosed arms of EPM (78.4 vs. 34.1%; p=0.003) and exhibited higher resistance in the CR test (score 3.30.7 vs. 2.20.4; p>0.001). Interestingly, the number of Prox1-ir cells co-expressing NeuN was enhanced by 36.3% in PBN animals (p=0.046) and number of Prox1/NeuN-ir cells was significantly associated with level of anxiety (r=0.723, p < 0.001). PBN also increased severity of epilepsy. In the PBN group, seizure frequency (mean number of seizures per 24h) was 28.910.1, while in the untreated animals it was 4.71.3 (p=0.046). There was a correlation between seizure frequency and the number of Prox1-ir cells (r=0.551, p=0.021) or Prox1/NeuN-ir cells (r=0.684, p=0.006). The total number of DCX-ir neurons in the DG (i.e. in the subgranular zone, granular cell layer and hilus) was 17% lower in PBN animals than in controls (2431222 vs. 2918311), but this difference was not significant. The total number of DCX-ir neurons was negatively associated with seizure frequency (r=-0.538, p=0.045). Conclusions: These findings suggest that PBN causes increased numbers of postnatally born granule cells to aberrantly migrate to the dentate hilus where they appear to mature faster. This raises the intriguing possibility that these aberrant neurons may abnormally integrate into hippocampal circuitry and produce both excessive excitability leading to more severe epilepsy and behavioral comorbidities. Funding: This study was supported by grants LH 15025 of the Ministry of Education and P304/16-04726S of the Czech Science Foundation.