Abstracts

Rationale: Severe head trauma has been shown to cause a disruption in neuronal transmembrane ion gradients and can lead to epileptic seizure activity. We have recently provided evidence supporting the idea that post-traumatic accumulation of intracellular Cl- results in a shift in the action of GABA (which mediates Cl- flux through its receptors) from hyperpolarizing to depolarizing. This leads to reduced inhibition, enhanced propagation of neuronal firing, and may contribute to early post-traumatic seizures. Charge balance dictates that traumatic increases in intracellular Cl- must be accompanied by either a loss of another intracellular anion (unlikely because the only other mobile anion, HCO3-, is strongly buffered) or a gain of an accompanying cation. Gain of a cation could underlie cytotoxic edema, the abnormal neuronal swelling that is known to accompany epilepsy. Our objective was therefore to test for changes in intracellular cation concentration, beginning with Na+. Methods: The experiments were performed on acute hippocampal slices (~300 µm in thickness), which were prepared from juvenile wild-type C57BL/6J mice whose ages ranged from P13 to P26. We recently demonstrated that acute hippocampal slices are an excellent model of traumatic axonal injury. The slices were incubated with the Na+-sensitive dye SBFI for 4 to 5 hours prior to imaging. Following calibration of the dye, two-photon imaging was used to excite SBFI at both Na+-sensitive and -insensitive wavelengths, allowing for the ratiometric determination of the intracellular Na+ concentration. Results: SBFI fluorescence of neurons in the CA1 and CA3 regions of the hippocampus indicated a significantly higher Na+ concentration than has been reported in undamaged neurons. Specifically, neuronal intracellular Na+ was highest near the cut surface of the slice, and lowest in the deepest neurons. Conclusions: These data indicate that trauma results in significant increases in intracellular Na+. Our initial correlations of intracellular Na+ and Cl- support the hypothesis that Na+ is the principal cation accompanying the traumatic increases in intraneuronal Cl-. If so, then increases in intracellular NaCl after brain injury could engender cerebral edema, depolarizing shifts in the reversal potential for GABA, and early epileptic seizures.