Abstracts

Rationale: The mechanisms underlying cytotoxic cerebral edema remain largely unknown. For example, it is not clear to whether primary injury is necessary for neuronal swelling, or whether the necrotic death of neighbors contributes to subsequent neuronal injury and swelling. We utilized the photosensitization conferred by trangenically expressed fluorophores to study how the acute necrosis of a single neurons affected surrounding neurons. Methods: We used two photon microscopy and the transgenically-expressed chloride fluorophore Clomeleon to explore consequences of death of neighboring neurons on intraneuronal chloride ([Cl ]i) changes in hippocampal organotypic slice cultures. Two photon laser irradiation to the soma of single CA1 pyramidal neuron exclusively lysed the targeted cell with a predictable dose-response function. Results: Lysis of the target neuron was followed by an immediate [Cl-] increase in surrounding neurons. The anatomical distribution of neurons with elevated [Cl-]i increased over over time. The time course of [Cl-]i elevation differed from neuron to neuron, with some neurons exhibiting large increases that persisted over 60 minutes. Membrane depolarization-induced passive Cl- influx contributed to the [Cl-]i elevation, because the [Cl-]i elevation was reduced by GABAA receptor antagonists. However, a substantial [Cl-]i elevation could still be observed in the presence of GABAA receptor antagonists, indicating that other elements of [Cl-]i homeostasis were altered in these neurons. Clomeleon imaging combined with membrane permeable fluorochrome-labeled inhibitors of caspase activity (FLICA) staining and imaging was used to analyze the correlation between [Cl-]i elevation and undergoing apoptosis. The FLICA probe was detected in cells near the target cell at 60 minutes after the breakdown of the target cell. In contrast, the cells with persistent [Cl-]i increase farther away from the target were not labeled by FLICA probe, suggesting that mechanisms for changing [Cl-]i homeostasis can occur without activation of the apoptotic cascade. Conclusions: These results imply that other mechanisms besides GABA-gated Cl flux or acute damage contribute to the disruption of [Cl-]i homeostasis in the neighbors of necrosed neurons. We are currently testing the role of hydrolases released from the necrotic neuron, and increasing the rigor of the analyses of Cl- dynamics by combining Cl studies with Ca2+ and pH imaging.