In situ nick-translation detects focal apoptosis in thymuses of glucocorticoid- and lipopolysaccharide-treated mice.

Abstract

In this study we used in situ nick-translation to analyze apoptotic events in the thymus and in cultured thymocytes at the level of individual cell nuclei. In vitro nuclear DNA strand breaks were observed 3 hr after exposure of thymocytes to dexamethasone (Dex) in 30% of cells and increased to 78% after 15 hr. In sections of 10-day-old mouse thymus, single cells with DNA strand breaks were dispersed throughout the cortex and to a lesser degree in the medulla. In contrast, a large number of clusters of apoptotic cells were seen in the thymic cortex 3-18 hr after injection of Dex or lipopolysaccharide (LPS). After 48 hr apoptotic cells were no longer detectable. Positive signals correlated with the detection of DNA ladders of multimers of about 180 BP size on agarose gels. Electron microscopy confirmed the presence of apoptotic cell clusters and showed that apoptotic foci were located around capillaries in LPS-injected animals. We conclude that in situ nick translation is a suitable method to detect apoptotic nuclei in cultured cells and on cryostat sections. With this method we could demonstrate that in vivo spontaneous apoptosis occurs in single dispersed thymocytes, also including the medulla, whereas experimentally induced apoptosis affects cell clusters, possibly due to high local concentrations of apoptosis inducers.