Evaluation of autophagy and cell death in osteocytes of alveolar bone in ovariectomized female rats treated with estrogen

Abstract

Estrogen plays important functions on osteocytes viability and its deficiency leads to decrease in the number of these cells and subsequent increase in bone resorption. There are evidences that autophagy also plays important roles on osteocyte viability and that estrogen plays a role in the regulation of autophagy in some cell types; in osteocytes, however, this mechanism is still unknown. The effect of estrogen on osteocyte viability remains in constant debate and its role in the regulation of autophagy in osteocytes, as well as its role on bone remodeling has not been investigated. Thus, the purpose of this study is to evaluate the influence of estrogen in the regulation of autophagy and cell death of osteocytes; in addition, we intend to investigate a possible correlation between the process of cell death of osteocytes and the remodeling of alveolar bone in ovariectomized rats. Sixty-six adult female Wistar rats (aging 4 months) will constitute the SHAM-operated (SHAM) or ovariectomized (OVX) groups; 24 days after the surgical procedures, the animals will be distributed into 3 groups with 18 animals each, and the body weight will be monitored weekly. Group I (SHAM) and Group II (OVX-Control): the animals will receive subcutaneously vehicle solution (corn oil) during 15, 30 and 45 days. Group III (OVX-EST): the animals will receive subcutaneous injections of 30µg/Kg/day of estrogen (diethylstilbestrol, Sigma-Aldrich) during 15, 30 and 45 days. Afterwards, the animals will be euthanized, fragments of maxilla (right and left) containing alveolar bone of the first molars will be removed and fixed in formaldehyde (pH 7,4). After decalcification in EDTA, the samples will be processed for light microscopy and submitted to histomorphometric and immunohistochemical analysis. Some samples will be fixed in glutaraldehyde/formaldehyde and processed for analysis under transmission electron microscopy. In order to verify the process of autophagy in osteocytes, markers will be used (BECN1, MAP LC3A e SQSTM1); the process and regulation of apoptosis will be investigated by the TUNEL method and immunohistochemical detection of cleaved caspase-3 and bcl2, as well as by ultrastructural images. Also, a possible relationship between autophagy and apoptosis processes will be investigated as well as whether there is a relationship between osteocytes death and bone resorption. (AU)