IN-SITU NICK-TRANSLATION DETECTS FOCAL APOPTOSIS IN THYMUSES OF GLUCOCORTICOID-TREATED AND LIPOPOLYSACCHARIDE-TREATED MICE

In this study we used in situ nick-translation to analyze apoptotic ev ents in the thymus and in cultured thymocytes at the level of individu al cell nuclei. In vitro nuclear DNA strand breaks were observed 3 hr after exposure of thymocytes to dexamethasone (Dex) in 30% of cells an d increased to 78% after 15 hr. In sections of 10-day-old mouse thymus , single cells with DNA strand breaks were dispersed throughout the co rtex and to a lesser degree in the medulla. In contrast, a large numbe r of clusters of apoptotic cells were seen in the thymic cortex 3-18 h r after injection of Dex or lipopolysaccharide (LPS). After 48 hr apop totic cells were no longer detactable. Positive signals correlated wit h the detection of DNA ladders of multimers of about 180 BP size on ag arose gels. Electron microscopy confirmed the presence of apoptotic ce ll clusters and showed that apoptotic foci were located around capilla ries in LPS-injected animals. We conclude that in situ nick translatio n is a suitable method to detect apoptotic nuclei in cultured cells an d 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 affec ts cell clusters, possibly due to high local concentrations of apoptos is inducers.