3H). ED1 immunohistochemistry revealed an increased number of immunopositive cells in the marrow of specimens from the eldecalcitol group (compare Fig. 4A to B). To assess to what degree such cells were committed to the osteoclastic lineage, double immunostaining for cathepsin K/ED1 was carried out and made evident the distinction between macrophages and osteoclasts (Figs. 4C–D). Higher-magnified light microscopy revealed that the bone marrow of eldecalcitol-treated specimens has a great number of macrophages with inclusion bodies (Fig. 4E), while TEM further envisioned many lysosomes in these macrophages (Fig. 4F). Quantification of cathepsin K-negative/ED1-positive cells identified a

statistically significant increase after eldecalcitol administration when compared

to OVX group (Fig. 4G). In order to investigate whether the increased presence of macrophages in the marrow Ganetespib solubility dmso was due to enhanced apoptosis after eldecalcitol administration, we conducted TUNEL staining. Quantification of TUNEL-stained cells showed that the number of apoptotic cells is the lowest in eldecalcitol group (Fig. 5A). After administering eldecalcitol or vehicle to OVX rats, our main findings were: 1) with eldecalcitol administration, osteoblasts accumulate and synthesize new bone on top of smooth cement lines in process known as bone minimodeling; 2) eldecalcitol appears to affect osteoblastic differentiation and activation instead of stimulating preosteoblastic Fluorouracil molecular weight proliferation; 3) treatment with eldecalcitol lowers osteoclast number and diminishes osteoclastic activity/functionality, without promoting osteoclast apoptosis; and 4) eldecalcitol administration may favor the macrophage differentiation cascade on the expense of cells that would otherwise become osteoclasts. Therefore, eldecalcitol indirectly promotes a bone formation process known as minimodeling, but appears to exert Amino acid its bone-protective

effects mainly by affecting osteoclastic number and function. It may do so by favoring the macrophage lineage while hampering final osteoclastic differentiation, since there is an increased macrophage population in the bone marrow of eldecalcitol-treated specimens that cannot be explained by enhanced apoptosis. In agreement with previous reports on the action of vitamin D analogs [23], [26], [32] and [33], this experiment showed that eldecalcitol can successfully prevent bone loss after ovariectomy. Our histological, histomorphometrical and femoral BMD analyses did demonstrate the recovery of bone structural parameters in OVX rats administered with eldecalcitol (Table 1). Interestingly, neither osteoblast and osteoid surface nor any of the kinetic bone parameters’ values were positively affected by eldecalcitol; in fact, the values obtained for eldecalcitol and Sham groups were very similar.