Abstracts

Rationale: Acute brain injury is frequently complicated by both electrographic seizures and cytotoxic edema. The mechanisms underlying neuronal death and volume shifts in these circumstances are poorly understood. Recently, new fluorophores and microscopy techniques have made possible more detailed explorations of these processes. Methods: We evaluated the death of neurons in a chronically epileptic in vitro preparation in which multiphoton microscopy could be performed over a period of several days. Organotypic hippocampal slice cultures were made from wild-type C57BL/6J mice, and imaged with transgenic fluorophores as well as the Na+ dye SBFI-AM. 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 moribund neurons from the analysis. Results: Immediately post-trauma, a transient rise in neuronal [Na+]i was observed. A subpopulation of neurons then underwent a stereotyped sequence of events culminating in cell death. First, the emission of transgenically expressed fluorescent proteins was permanently reduced. Next, SBFI-AM permeated the neuronal membrane and stained the cytosol. Dendritic retraction and a sustained elevation of [Na+]i occurred next. Finally, the cytosolic and nuclear membranes admitted PI, and the cell volume was markedly reduced. Neurons with highest values of [Na+]i were most likely to stain for propidium iodide. In neurons at the stage of admitting SBFI-AM, [Na+]i could be measured. Acute perfusion of 10 M of the Na+/K+ ATPase inhibitor ouabain or 100 M of the KCC2/NKCC1 antagonist furosemide increased [Na+]i. 10 M of the NKCC1 antagonist bumetanide or 100 M of the Na+/Ca2+ exchange antagonist benzamil also increased [Na+]i, indicating that NKCC1 and the Na+/Ca2+ exchanger operate in the reverse of their canonical directions by exporting Na+ and, presumably, the cotransported ions. Inhibition of COX-2 via perfusion of 10 M of the selective non-steroidal anti-inflammatory drug celecoxib significantly lowered [Na+]i, as did inhibition of the pore-forming Bax protein via perfusion of 200 M of the Bax-inhibiting peptide V5. This suggests that an apoptotic pathway leads to the insertion of permeability pores in the cytoplasmic membrane, which may be responsible for the rise in [Na+]i and related changes. Conclusions: Overall, a stereotypical sequence of events preceded neuronal death by at least several days, beginning with quenched emission of fluorescent proteins, admission of AM dyes, dendritic retraction, elevation in [Na+]i , and terminal cell shrinkage. ATPase activity and secondary ion transport remained robust throughout this process. We are currently testing whether the mitigation of elevated [Na+]i reflects a translationally useful neuroprotective effect or a specific effect on [Na+]i. Funding: NINDS 2 R37 NS077908 05A1