Abstracts

Rationale: Neuronal death terminates with microglial engulfment of apoptotic neurons or necrotic neuronal debris. At present estimates of neuronal death are based on the number of neurons that are visibly undergoing the death process. This number is a function of three variables: the rate at which neurons are entering the death process, the visibility of neurons during the process (which depends largely on the membrane permeability delimiting the rate of biomarker uptake), and the rate at which they are exiting the process via microglial engulfment. 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 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 intracellular sodium concentrations ([Na+]i). Results: We report the following sequence of events in delayed neuronal death following the substantial trauma involved in brain slice preparation. Neurons entering the death process quenched fluorescent proteins (such as TurboRFP) concurrent with elevated caspase activity (visualized with FLICA). Due to their increased membrane permeability (visualized with Annexin V and PO-PRO-1) these neurons rapidly took up AM ester dyes such as SBFI, lost their neuronal processes, and exhibited evidence of DNA degradation (as seen with chromatin staining). The mitochondria and membrane transporters of dying neurons remained active for as long as two weeks after entering this pathway. Throughout this period, the permeability of the neuronal membranes gradually increased, as evidenced by a progressive increase in [Na+]i, and eventually reached the point where polar dyes such as propidium iodide (PI) entered the nucleus. The death process ended with microglial engulfment. The three variables were then studied during exposure to conditions in the developing nervous system considered to be neurotoxic (ethanol) or neuroprotective (kynurenate and tetrodotoxin). Ethanol did not affect fluorescent protein quenching but did alter [Na+]i, whereas kynurenate and tetrodotoxin did not affect [Na+]i but did alter PI positivity and microglial engulfment. Conclusions: We describe an in vitro model of delayed neuronal cell death in the developing hippocampus after brain injury, and conclude that current assays of neuronal death are misleading in this setting. Sequential assays of cellular fluorescent protein expression provide the most robust measure of changes in the number of viable neurons. Funding: NIH Grant 5R37NS077908-07