In situ detection of apoptosis in dental and periodontal tissues of the adult mouse using annexin-V-biotin

An early event in apoptosis is exposure of phosphatidylserine, an aminophos pholipid normally present in the inner leaflet of the plasma membranes, at the outer leaflet of the plasma membrane facing the extracellular space, An nexin V (Anx-V) is a 35-kDa protein with high affinity for phosphatidylseri ne, which can be applied to detect apoptosis. We injected biotin-labelled A nx-V intravenously in adult mice and examined the tissue distribution of An x-V-labelled cells in dental and periodontal tissues using ABC-peroxidase h istochemistry. In the continuously erupting incisors, strong and frequent i mmunostaining was observed in transitional stage and late maturation stage ameloblasts with less frequent staining in preameloblasts, Frequency of sta ining in odontoblasts and pulp cells was low but increased slightly at olde r stages of dentinogenesis. Labelling was also seen in phagocytic or phagoc ytic-like cells in the enamel organ and pulp. A positive staining was furth ermore found in fibroblasts of the periodontal ligament in continuously eru pting incisors and in fully erupted molar teeth. Staining intensity and the number of positive cells were enhanced by antigen retrieval using high-pre ssure cooking. We conclude that Anx-V-biotin labels dental cells in early s tages of cell death and indirectly cells that have ingested labelled apopto tic cells during the course of the experiment. The data confirm that during amelogenesis most cell death occurs in transitional stage and late maturat ion stage ameloblasts. Thus, label ling with Anx-V is a useful marker for s tudying cell death and the dynamics of clearance of apoptotic cells during tooth development.