Abstracts

Rationale: Pilocarpine-induced status epilepticus (SE) activates the JAK/STAT pathway in the hippocampus. Increased transcription of Cyclin D1, a G1 cell-cycle regulator, after SE is mediated by STAT3. Early administration of WP1066, a STAT3 inhibitor, blocks increases in pSTAT3 protein levels; reduces increases in Cyclin D1 mRNA; and delays progression of epileptogenesis. Cell-cycle activation has been associated with activation and proliferation of microglia and astroglia and secondary neurodegeneration following brain injury. These distinctive properties suggest that increases in Cyclin D1 may be important to the development of epilepsy and associated comorbidities. Methods: Potential increases in Cyclin D1 mRNA and protein were evaluated in microdissected dentate gyrus (DG), CA1, and CA3 via RT-PCR and western blot 24h, 48h, 72h, and 1 week after pilocarpine-induced SE. To assess whether early inhibition of STAT3 phosphorylation could suppress increased expression of Cyclin D1 in hippocampal subregions during early epileptogenesis, RT-PCR and Western blot were used to quantify mRNA and protein levels of Cyclin D1 24 h after SE and WP1066 (50 mg/kg at onset of SE and 50 mg/kg 45 minutes later) or vehicle treatment. Results: Cyclin D1 mRNA increases at 24h, peaks at 48 h, and is still increased at 1 week in all regions. Following WP1066 administration, Cyclin D1 mRNA expression was ~59% lower in the DG, ~35% lower in CA1, and ~56% lower in CA3 24 h after SE onset relative to tissue from vehicle-treated rats (p<0.05). Cyclin D1 protein increases significantly in a time-dependent manner in all three regions of the hippocampus. These increases peak 72 h after SE, at which point Cyclin D1 protein increased about ~29 times in DG, ~9 times in CA1, and ~18 times in CA3 (p<0.05). WP1066 (50 mg/kg) injected i.p. at the onset of SE and again 45 minutes later reduced SE-induced increases of Cyclin D1 by ~48% 24 h after onset of SE in DG (p<0.05), but not in CA1 and CA3. Current studies are evaluating what cell types express Cyclin D1 and whether it participates in mechanisms of secondary injury following prolonged seizures. Conclusions: These results suggest that 100 mg/kg total WP1066 treatment at SE reduces seizure-induced increases in Cyclin D1 mRNA and protein in the dentate gyrus. This mechanism may contribute to the ability of WP1066 to successfully slow the acquisition of increasing seizure frequencies over time inherent to post-SE models. Immunohistochemical analysis of Cyclin D1 protein expression following SE (and in response to STAT3 inhibition) in neurons, astrocytes, microglia, and oligodendrocytes is required to further evaluate this hypothesis, and better understand the histopathological role of Cyclin D1 in epileptogenesis.