A fluorescence confocal assay to assess neuronal viability in brain slices

Abstract

Hippocampal slice models are used to study the mechanisms of ischemia-induced neurotoxicity and to assess the neuroprotective potential of novel therapeutic agents. A number of morphological and functional endpoints are available to assess neuronal viability. The slice model also allows the study of selectively vulnerable neuronal populations within the same preparation. The fluorescence procedure described here provides a method of assessing the viability of neurons in rat hippocampal slices exposed to hypoxic-hypoglycemic conditions. Control and/or treated slices that had been subjected to a 10 min oxygen-glucose deprivation insult are double stained with calcein-AM (4 μM), which stains live cells green, and ethidium homodimer (6 μM), which stains the nucleus of dead cells red. The stained slices are then imaged using confocal microscopy. Vulnerable neurons in the CA1 region of slices deprived of oxygen and glucose became increasingly permeant to ethidium homodimer over the 4 h reperfusion period. Exposure to low Ca 2+ concentration (0.3 mM) or the N-, P- and Q-type Ca 2+ channel antagonist MVIIC (100 nM), which have been shown to be neuroprotective in this model of ischemia using field evoked post-synaptic potential (EPSP) measures as an endpoint, were also shown to be protective using the fluorescence assay.