IMMUNOCYTOCHEMICAL DISCRIMINATION BETWEEN EARLY AND LATE S-PHASE - A NEW APPROACH TO THE STUDY OF GINGIVAL EPITHELIUM PROLIFERATION IN RATS

THE RENEWAL OF THE FREE GINGIVAL MARGIN epithelium in rats was studied evaluating 5-bromodeoxyuridine (BrdU) incorporation in proliferating cells by means of an immunocytochemical method. We found a close corre spondence between Light and electron microscopy patterns of BrdU incor poration at a nuclear level. BrdU was localized in the inner interchro matin regions in cells starting DNA synthesis, while it was localized in the peripheral heterochromatin domains in cells terminating the S p hase. This possibility of discriminating cells in early S phase from c ells in late S is able to provide far more information as to the time at which a labeled cell starts proliferation than that obtainable with H-3-thymidine autoradiography. This, in turn, permits detection of ce lls that start proliferation in a wide period of time by means of a si ngle BrdU administration. Rats treated at 7 a.m. demonstrated higher p roliferation than rats treated at 7 p.m., supporting the existence of circadian variations in the epithelial renewal. Proliferative events t ake place by consecutive activation of at least three replication wave s, producing clusters of labeled cells which could be observed in rats sacrificed at 10 a.m. In rats treated once with BrdU at 7 a.m., the c lusters were localized in both the basal and suprabasal layer of the e pithelium; in rats further injected with BrdU at the same time, the cl usters increased in size, progressively extending throughout the epith elium. In this way, the renewal of the free gingival margin epithelium does not proceed randomly, but by consecutive activation of discrete units or clusters of basal cells, which then extend to the upper layer s. This can be followed at a morphological level as a progression of l abeled cells, which move from the basal layer to the epithelium surfac e in approximately 82-85 hours.