Abstracts

Rationale: Delayed neuronal death is a well-established phenomenon in the hippocampus and other brain regions. The process is of interest as a means to explain clinical deterioration after acute brain injury. However, mechanisms underlying delayed neuronal death and its relationship to apoptosis remain poorly understood. Recently, new fluorophores and microscopy techniques have made possible more detailed explorations of this process. We describe our initial findings in an in vitro preparation in which this process can be studied. Methods: We evaluated the death of neurons in a chronically epilepticin vitropreparation 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 vitrountil 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. Results: We report the following sequence of events in delayed neuronal death following the substantial trauma involved in brain slice preparation. The first detectable event was loss of participation in network activity and mild, sustained elevation of cytoplasmic Ca²⁺. The second stage was marked by activation of caspases evidenced by FLICA staining. During the second stage, fluorescence of transgenic fluorophores was lost. In the third stage, neurons admitted AM dyes including SBFI-AM and Fura-AM. The fourth stage was marked by steady increases in cytoplasmic Na⁺concentrations from 10 mM to concentrations approaching that of the extracellular solution. Cytoplasmic Ca²⁺remained elevated during this interval. During this fourth stage, cytoplasmic membrane damage could be demonstrated by Annexin V staining, retraction of dendrites and axons was completed, and condensation of nuclear chromatin visualized by NucBlue became progressively evident. Throughout the fourth stage, TMRM staining, pharmacological antagonists, and ion-sensitive fluorophores confirmed ongoing glycolysis and mitochondrial respiration and ATP production, sodium transport via Na⁺/K⁺ATPases, and secondary transport including cation-Cl^-cotransport and Na⁺/Ca²⁺exchange. Key events in the fifth and final stage included microglial engulfment (as indicated by isolectin staining), sharp rises in Na⁺and Ca²⁺concentrations, and terminal cell shrinkage. Conclusions: We describe an in vitro model of delayed neuronal cell death in the developing hippocampus.  Dying cells were rapidly removed from neuronal circuits, and thus are unlikely to contribute directly to seizures after brain injury. However, they continued to consume oxygen and glucose, which might be in short supply in the penumbra of injury. Delayed neuronal death was characterized by a prolonged phase of progressively increasing membrane permeability that could be blocked, without altering the course of neuronal death. Funding: NIH Grant 5R37NS077908-07