Neuronal Apoptosis in an in Vitro Model of Photochemically Induced Oxidative Stress

Abstract

In neurons, oxidative stress can be triggered by neurotransmitter-linked mechanisms and may lead to apoptotic cell death. A simple and reproducible model of inducing oxidative stress is needed to elucidate mechanisms which link oxidative stress and neuronal apoptosis. We report here a method of inducing apoptosis in cell cultures by loading them with a photosensitive dye, rose bengal, and exposing the cultures to light, a procedure which generates reactive singlet oxygen. We used this model in primary culture of rat cerebellar granule neurons, and in a nonneuronal human embryonic kidney 293 cell line. We have measured the following: (a) metabolic activity of the mitochondria by quantitative staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), (b) DNA fragmentation by quantitative in situ terminal deoxynucleotidyl transferase assay, and (c) cell viability by a trypan blue exclusion test. The oxidative stress caused an early impairment of mitochondrial function (MTT assay). This was followed by DNA fragmentation and ultimately by cell death. Protection was obtained with an inhibitor of macromolecular synthesis, anisomycin, and with antioxidant, vitamin E. This model can be used to study the mechanism of oxidative stress-triggered neuronal apoptosis, and it may help in discovering new targets for neuroprotective drugs.