Abstracts

Rationale: We investigated the relationship between acute neuronal necrosis and secondary neuronal injury and swelling in neighboring neurons. We utilized the photosensitization conferred by trangenically expressed fluorophores to study how the acute necrosis of a single neuron affected the chloride concentration in the cytoplasm of surrounding neurons.Methods: We used two photon microscopy and the transgenically-expressed chloride fluorophore Clomeleon to explore consequences of death of a neighboring neuron on the intraneuronal chloride ([Cl–]i) in hippocampal pyramidal cells in organotypic slice cultures. Two photon laser irradiation to the soma of single CA1 pyramidal neuron exclusively lysed the targeted cell. Cell damage was assessed by morphological changes as well as caspase activation using FLICA staining.Results: Lysis of the target neuron was followed by an immediate [Cl-] increase in surrounding neurons; sub-lytic radiation had no effect on neighbors. The anatomical distribution of neurons with elevated [Cl-]i increased 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 concomitant with caspase activation. The [Cl-]i increase and subsequent cell damage was exclusively triggered by ionic influx because they were never observed under the perfusion of NaCl free solution. When extracellular NaCl was replaced by Nagluconate (High Na+ , low Cl- solution), lysis of the target neuron did not cause [Cl-]i increase or swelling of neighbors, although caspase was activated in cells near the target. In contrast, low Na+, high Cl- solutions (NaCl replaced by Choline chloride) or NaCl + kynurenic acid had only modest effects on post-lytic [Cl-]i increase in neighbors. These results highlight the critical role of Cl- influx for cytotoxic cell swelling and damage after nearby neuronal lysis. The influx pathway of Cl- is still being elucidated. Pharmacological blockade of GABAA receptor, cation-chloride co- transporters, and anion channels/exchangers including chloride-sulfate transporters did not reduce the [Cl-]i increase. The anion channel blockers NPPB and DIDS actually increased neighboring neurons’ [Cl-]i increase.Conclusions: This novel, readily-studied model demonstrates that cytotoxic edema and injury can occur as purely secondary phenomena in initially uncompromised neurons after acute brain injury. Pharmacological results indicate that swelling occurs via Cl- influx pathways that are distinct from known Cl- channels or transporters. Ion substitution experiments indicate that [Cl-]i increase is independent of Na+ influx, suggesting that the counter ion may be potassium. We will continue to utilize this model to explore mechanisms of cytotoxic edema in hopes of developing new therapeutic approaches.