Bone remodeling markers and osteoclastogenesis in anemic mice.

<p>Naive, bled, <i>P.c. adami</i>-infected and PHZ-treated female BALB/c mice (4–6 weeks old) were euthanized eight days after treatment. Sera were assessed for concentrations of (A) osteocalcin (OCN), (B) procollagen type I N-terminal propeptide (PINP), (C) carboxy-terminal collagen crosslinks (CTX) and (D) Tartrate-resistant acid phosphatase (TRAP) 5b. Bone marrow cells were recovered from the tibia by flushing, an aliquot of each bone marrow cell suspension was stained with anti F4-80-PE antibody for estimation of osteoclast F4-80⁺ progenitors by flow cytometry (E). The remaining cells were allowed to adhere overnight after which non adherent cells were stimulated with recombinant M-CSF and RANK-L for 6 days for induction of osteoclastogenesis, and the numbers of multinucleated ostoclasts (>4 nuclei, TRAP+ cells) was determined by TRAP/Giemsa staining and confocal microscopy (D). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046101#s3" target="_blank">Results</a> are mean ±SEM from 4–10 mice per group, compared by a one –way ANOVA and a Tukey's mutiple comparison test. *<i>P</i><0.05, <i>**P</i><0.01, <i>***P</i><0.001 are comparisons to naïve mice; ^ε<i>P</i><0.05, ^εε<i>P</i><0.01, ^εεε<i>P</i><0.001 are comparisons to bled mice; ^ΨΨΨ<i>P</i><0.001 represents comparison between PHZ-treated and <i>Plasmodium</i>-infected mice.