Apoptosis is developmentally regulated in rat growth plate

Abstract

Apoptosis occurs in the growth plate during normal and abnormal longitudinal growth. To investigate the role of apoptosis during growth plate maturation, apoptosis and apoptosis-related proteins were studied in rat tibial growth plates at 2, 4, 8, and 11 wk of age. Apoptosis was studied by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end-labeling (TUNEL) method, and immunohistochemistry was used to detect p53, caspase-3 and -6, the antiapoptotic proteins Bcl-2 and Bcl-x, and the proapoptotic proteins Bax and Bad. In all age groups studied, most apoptotic chondrocytes were terminal hypertrophic chondrocytes (THPCs) with a significant increase during development. At 2 wk, 0.108 +/- 0.026 THPCs were found to be apoptotic per millimeter of growth plate width; at 4 wk, 0.355 +/- 0.048; at 8 wk, 0.394 +/- 0.043; and at 11 wk, 1.084 +/- 0.069 (p < 0.001; 11 wk vs 2, 4, and 8 wk). THPCs were negative for p53 immunoreactivity at 2 and 4 wk, whereas some THPCs were positive at 8 and 11 wk. Caspase-3 and -6 were found in proliferative and early hypertrophic cells at 2 wk, whereas mature hypertrophic cells and THPCs were negative. At later stages of development, mature hypertrophic cells and THPCs were stained for both caspase-3 and -6. Bcl-2 and Bcl-x were present in proliferative and early hypertrophic cells at 2 wk, whereas at older ages a decrease in staining was observed. At 2 wk of age, Bax and Bad immunoreactivities were localized in proliferative and early hypertrophic cells, whereas at 8 and 11 wk many mature hypertrophic cells and THPCs were immunoreactive for Bax and Bad. Our results show that apoptosis is developmentally regulated in the rat growth plate. In older animals, with decreased growth rate and growth plate height, apoptosis is significantly increased, especially in THPCs.