Image analysis of blastema cell proliferation in denervated limb regenerates of the newt, Pleurodeles waltlii M.

When denervated at the medium bud stage, limb blastemas of the newt, Pleurodeles waltlii Michah, stop growing. In order to better understand the role of nerves in the cell cycle in blastemas, we studied the distribution of mesenchymal cells in the G0-1, G1, S, G2 and M phases 48 and 96 h after denervation. The cell-cycle phases were determined by examining Feulgen-stained nuclei using a SAMBA 200 (System for Analytical Microscopy in Biological Applications) cell image processor. The cell nuclei were automatically analyzed by calculating 18 parameters related to the densitometry and texture of chromatin, and the shape of each nucleus. Cell-cycle phases were classified according to the unsupervised recognition method using a SAMBA 200 system as proposed by Moustafa and Brugal for cell-kinetics analysis. The classification obtained was tested against the results of stepwise linear discriminant analysis performed according to the method of Giroud. Our results show that, in blastemas 96 h after denervation, the percentage of cells in the S, G2, and M phases decreases significantly, while the percentage of G1 and G0-1 cells increases (+ 51% for G1 cells; + 30% for G0-1 cells). Thus, it appears that denervation of medium-bud-stage limb blastemas promotes the lengthening of G1 and premature exiting of cells from the cycle into the G0-1 phase. These results show that nerves (i.e., neurotrophic factor) regulate cell kinetics during newt limb regeneration by maintaining blastema mesenchymal cells in the cell-cycle.