Immunohistochemical detection of Caspase 3, RANKL and OPG in apoptotic osteoclasts.

Apoptosis is a complex process characterized by cytoplasmatic expression of sequentially-activated enzymes named caspases. Intrinsic and extrinsic signaling pathways of apoptosis culminate in the activation of caspase 3 (C3) and its expression in apoptotic osteoclasts (Ocs). has been described. The presence of mRNA encoding RANKL in Ocs has been described in only one work and OPG in Ocs has never been described because of, according to the bibliography, osteoclastic-lineage cells do not express OPG. Therefore, the objective of this study was to evaluate the expression of C3, RANKL and OPG in Ocs and Ocs with apoptotic morphology in order to determine whether apoptosis produces changes in the expression of these proteins. Materials and Methods: Female Wistar rats weighing 160 g were used. To induce apoptosis in the Ocs, the animals were injected with monosodic olpadronate (OPD, Gador SA) via i.p. for 5 weeks and vehicle was injected to controls. On the 6th week of the experiment, the animals were sacrificed and the mandibles were ressected, decalcified and processed to obtain sections oriented mesio-distally of the lower first molar. Immunohistochemical detection of C3, RANKL and OPG were performed. Under bright field microscopy with immersion objective, Ocs were classified into 4 categories, according to the positive or negative Immunohistochemical detection (C3 + / - RANKL + / - OPG + / -) and the presence or absence of apoptotic morphology (AM + /-AM). The statistical analysis was performed using the Mc Nemar´s chi square test and Bayes conditional probability. Results: C3: 315 Ocs were classified and was found a direct association between the expression of C3 and AM. RANKL: 264 Ocs were classified and also found association between AM and the expression of RANKL. OPG: 327 Ocs were classified and was not found association between the expression of OPG and the AM, while some apoptotic Ocs expressed OPG.
Conclusion: The apoptotic Oc express not only the specific marker of apoptosis C3, but also RANKL. RANKL is a protein related to recruitment and differentiation of Ocs. This finding has not been described and the expression of RANKL in apoptotic Ocs could act as a positive signal for to recruit new Ocs.
UBACyT O013, Gador SA