Abstracts

Rationale: Epilepsy is a common and disabling neurological manifestation of the genetic disorder, Tuberous Sclerosis Complex (TSC). Brain inflammation has been implicated in contributing to epileptogenesis in acquired epilepsy due to brain injury, but the potential role of inflammatory mechanisms in genetic epilepsies is relatively unexplored. In this study, we investigated activation of inflammatory mediators, particularly cytokines and chemokines, and tested the effects of anti-inflammatory treatment on epilepsy in a mouse model of TSC. Methods: Two to four-week-old GFAP-Cre-Tsc1-Floxed conditional knockout mice (Tsc1CKO mice) and littermate controls were used for all experiments, with brains harvested at various ages for different studies. Quantitative RT-PCR evaluated mRNA expression of a panel of cytokine and chemokines in whole brain extracts and astrocyte cultures. Based on the RT-PCR results, a subset of inflammatory proteins was selected for analysis by western blot, particularly IL-1beta and CXCL10. In addition, immunohistochemical studies of IL-1beta were performed in four-week old mice, with double-labeling for neurons, astrocytes, and microglia with NeuN, GFAP, and Iba1, respectively. Finally, in separate experiments, three-week old mice were treated with the green tea extract, epicatechin-3-gallate (ECG), an inhibitor of IL-1beta and CXCL10, for one week for western blot studies, and for up to twenty weeks for video-EEG studies to monitor seizures. Results: In screening a panel of inflammatory markers by RT-PCR, the mRNA levels of CCL2, IL-1beta, IFN-gamma, CXCL10 and IL-6 were increased, while CXCL12 was decreased, in the brains of Tsc1CKO mice compared to control mice, at four weeks of age, which is around the time seizures start in Tsc1CKO mice. However, only the mRNA level of IL-1beta, CCL2 and CXCL10 were increased in the brains of two-week-old Tsc1CKO mice and in astrocyte cultures from Tsc1CKO mice. Rapamycin treatment significantly down-regulated the mRNA levels of CCL2, IL-1beta and CXCL10 in the brains of Tsc1CKO mice. Western blot analysis confirmed that the protein levels of IL-1beta and CXCL10 were significantly elevated in Tsc1CKO brains, which was also reversed by rapamycin. Immunohistochemical studies with double-labeing showed that IL-1beta was increased primarily in GFAP-positive cells in the Tsc1CKO mice. ECG treatment partially reversed the increased protein expression of IL-1beta and CXCL10 by Western blot and corresponding improved survival and slightly decreased seizure frequency of Tsc1CKO mice. Conclusions: The expression of proinflammatory mediators, including cytokines and chemokines, are increased in Tsc1CKO mice, predominantly within astrocytes. Treatment with an inhibitor of IL-1beta and CXCL10 decreased these inflammatory markers and correspondingly improved survival and decreased seizure frequency in Tsc1CKO mice. These findings suggest that inflammatory mechanisms may be involved in epileptogenesis in the genetic epilepsy, TSC.