Radioautographic studies on dna synthesis of the lungs of aging salamanders

Authors:Tetsuji Nagata
Int J Biol Med Res. 2011; 2(3): 702-708  |  PDF File

Abstract

The DNA synthesis and morphological changes of the lungs of the salamanders, Hynobiusnebulosus, were studied by light microscopic radioautography. The lung tissues of 21 salamanders in 7 groups at various aging stages, from juvenile animals at 4, 6 and 8 weeks after metamorphosis, young adults at 3, 8 and 12 months after metamorphosis, and senescent adults at 5 years after metamorphosis were used for this study. They were injected intraperitoneally with 3H-thymidine (370 KBq/g body weight), and after 1 hr the lung tissues were fixed in buffered 2.5% glutaraldehyde solution for 2 hr and postfixed in 1% osmium tetroxide solution for 1 hr. The tissues were embedded in epoxy resin Quetol-812. Thick sections were cut at 2 mm on a Porter-Blum MT-6000 ultramicrotome and were coated with Konica NR-M2 emulsion by a dipping procedure for light microscopic radioautography. After exposure for 2 months, the radioautographs were developed in SDX-1 developer, stained with 0.1% toluidine blue and were observed with an Olympus Vanox light microscope, and analyzed quantitatively. As the results, the labeling indices in the nuclei of the pneumocytes, the mucous cells, the basal cells and the ciliated cells in the superficial layer as well as the fibroblasts and the endothelial cells in the deep layer changed due to aging. The labeling indices of the 3 types of cells in the superficial layer, the mucous cells, the pneumocytes and the basal cells were high from 4 weeks to 8 weeks but dropped down at 8, 12 and 60 months. These results showed that the 3 types of cells proliferated in the early stages of development from 4 to 8 weeks and completed the development in the adult stages. The labeling index of the ciliated cells was very low, which showed that they had no activity to proliferate but replaced by the immature basal cells which differentiated to the ciliated cells. To the contrary, the labeling indices of the 2 cell types in the deep layer, the fibroblasts and the endothelial cells, were lower than the cells in the superficial layer and kept very low levels at 8, 12 and 60 months after metamorphosis. Thus, it was concluded that the 4 types of cells in the superficial layer belonged to the renewing cell population and the 2 types of cells in the deep layer to the stable cell population.