Abstracts

Rationale: Recent reports have shown an increase in expression of the system xc- glutamate-cystine antiporter (Sx_c-) in reactive astrocytes and microglia in several models of CNS injury. As inflammation is also observed in temporal lobe epilepsy (TLE), this antiporter is a potential target for novel antiseizure drugs (ASDs). Corneal kindled mice display secondarily generalized partial seizures that have high predictive ability, supporting the model s use in early evaluations of investigational ASDs. In addition, Theiler s murine encephalomyelitis virus (TMEV) infection induces seizures accompanied by neuronal death, astrogliosis, microglial activation, and the development of spontaneous recurrent seizures. However, little is known about what changes in Sx_c- expression may occur in these two animal models of TLE. Thus, the purpose of this study was to examine changes in Sx_c- expression so as to test the hypothesis that increases in expression of this transporter in astrocytes and microglia contribute to the pathology observed in epileptogenesis.Methods: Male C57BL/6 mice were injected intracerebrally with PBS or TMEV (2 X 10⁶ pfu) and sacrificed 3 days post-injection. CF1 mice were stimulated to a minimum of 2 daily 5 consecutive stage 5 seizures for 2 days, and sacrificed 24 hours later. Age-matched control CF1 mice were not stimulated. All mice were transcardially perfused with PBS followed by 4% paraformaldehyde, and 20 m hippocampal slices were collected on a microtome. For immunohistochemistry, sections from each treatment group were batch processed with antibodies against the Sx_c- subunit (xCT), and either astrocyte (GFAP), microglia (Iba1), or neuronal proteins (NeuN). Images were acquired in CA1 and the immediately superficial cortex with a confocal microscope, and evaluated for colocalization and expression levels using ImageJ.Results: Sx_c- was widely expressed in neurons throughout the hippocampus and cortex of CF1 control and kindled mice, as well as C57BL/6 mice injected with PBS. In TMEV-infected mice, neuronal Sx_c- levels were markedly decreased in CA1 due to viral tropism and significant cell death. Whereas GFAP and IBA1 expression were increased in the TMEV group, there was no detectable colocalization of Sx_c- with GFAP or IBA1 in either CA1 or cortex. Corneal kindled and control CF1 mice were not observed to have notably different microglial morphology, increased IBA1 expression, or expression of Sx_c- in microglia and astrocytes. However, increased GFAP staining in the hippocampus was noted in kindled animals, suggesting the presence of inflammation.Conclusions: This data indicates that Sx_c- is highly expressed in neurons throughout the hippocampus and posterior parietal association areas under basal conditions. The noted absence of Sx_c- expression in either astrocytes or microglia in any treatment group was surprising. However, these findings are significant to the development of directed pharmacotherapies targeting mechanisms of glutamate regulation and seizure generation in epilepsy. This work was supported by Contract #271201100029C, NINDS.