Abstracts

Rationale: Post-traumatic increases in intracellular Cl- can shift the action of GABA from hyperpolarizing to depolarizing, which could lead to disinhibition and early post-traumatic epileptic seizures. Although the mechanism for Cl- accumulation is not known, increases in intracellular Cl- anions are likely to be balanced by an increase in intracellular cations, and the consequent salt accumulation could underlie cytotoxic edema.Methods: We investigated post-traumatic changes in intracellular Na+ concentration ([Na+]i) using organotypic hippocampal slice cultures from wild-type C57BL/6J mice, imaged with the Na+ dye SBFI. Organotypic slice cultures were prepared on P6 and incubated in vitro until use, with SBFI added 24 hours prior to imaging. Two-photon imaging was used to excite SBFI at both Na+-sensitive and -insensitive wavelengths, allowing for the ratiometric determination of the [Na+]i. Propidium iodide (PI), an indicator of cell death, was used as a costain in most experiments to exclude damaged neurons from the analysis.Results: Hippocampal neurons exhibited a broad distribution of [Na+]i values, and in many cases [Na+]i was significantly higher than has been reported in undamaged neurons. All values of [Na+]i were stable for hours. Neurons with highest values of [Na+]i were more likely to stain for propidium iodide, suggesting that [Na+]i elevation is an early and possibly progressive biomarker of eventual neuronal death. Population studies indicated that high [Na+]i values were more common immediately after slicing trauma, and returned to low levels within ~2 days. At longer incubation times, during which slices become epileptic, [Na+]i again became elevated, returning to physiological levels at 20 DIV, at which age seizure intensity diminished. Acute perfusion of 10 µM of the Na+/K+ ATPase inhibitor ouabain or 100 µM of the KCC2/NKCC1 antagonist furosemide increased [Na+]i. 10 µM of the NKCC1 antagonist bumetanide or 100 µM of the Na+/Ca2+ exchange antagonist benzamil also increased [Na+]i, indicating that in the days after trauma NKCC1 and the Na+/Ca2+ exchanger operate in the reverse of their canonical directions by exporting Na+ and, presumably, the cotransported ions. Application of 8 μM fluorescein conjugated to large dextran molecules indicated severe membrane disruption in only a small number of neurons, but a moderate amount of damage in a larger number of neurons, particularly as epileptogenesis progresses. Perfusion of 10 μM of the selective non-steroidal anti-inflammatory drug celecoxib significantly reduced [Na+]i in nearly all neurons, and also caused significant reductions in the power, frequency, and duration of seizures.Conclusions: Overall, elevated [Na+]i is a promising new biomarker for compromise of neuronal membrane permeability, which precedes many traditional indicators of epileptic activity and ictal cell death. The ability of celecoxib to mitigate elevated [Na+]i thus represents a prime therapeutic target for the early treatment of epilepsy.