Figure 1.
(A–C) Blood was recovered from naive, bled, P.c. adami-infected and PHZ-treated female BALB/c mice prior to and every day following treatment for assessment of hemoglobin (Hb) and percentages of reticulocytes (CD71+). (D) Kinetics of parasitemia in P. c. adami BALB/c mice. (E) The lowest Hb concentrations (g/dL) and (F) highest reticulocytosis measured in mice are represented. Data are mean ± SEM from 4–7 mice per group, compiled and compared using a one–way ANOVA and Tukey's multiple test. *P<0.05, **P<0.01, ***P<0.001 are comparisons to naive; εεP<0.01, εεεP<0.001 are comparisons to bled; ψP<0.05, ψψψP<0.001 are comparisons between PHZ-treated and Plasmodium-infected mice.
Figure 2.
Naive, bled, P.c. adami-infected and PHZ-treated female BALB/c mice (4–6 weeks old) were euthanized and the trabecular portions of tibia were scanned. (A) Representative scan from the trabecular portion of tibia. (B–D) Ratio of bone volume (BV) on total volume (TV), trabecular spacing (Tb.Sp) were compared between each groups and Tb.Sp were plotted against BV/TV. Results are mean ± SEM from two independent experiments (n = 5–6 mice per group) compiled and compared to control mice using a one–way ANOVA and Tukey's mutiple comparison test. *P<0.05, ***P<0.001 compared to naive and εP<0.05, εεP<0.01 compared to bled.
Figure 3.
Naive, bled, P.c. adami-infected and PHZ-treated (n = 5–6) female BALB/c mice (4–6 weeks old) were euthanized and the trabecular portion of tibias were scanned. A) The trabecular number (Tb.N) was compared between each groups and plotted against the BV/TV. Results are mean ± SEM from two independent experiments (n = 5–6 mice per group) compiled and compared to control mice using a one –way ANOVA and Tukey's mutiple comparison test. ***P<0.001 compared to naive and εP<0.05, εεεP<0.001 compared to bled.
Figure 4.
Bone marrow erythroblast subsets.
Fresh bone marrow cell suspensions from the tibia of naive, bled, P.c. adami-infected and PHZ-treated female BALB/c mice (4–6 weeks old) were stained with anti-CD71-FITC and anti-Ter119-PE antibody for analysis of stage specific erythroblasts. Ter119+ cells were gated in R1 whereas a R2 region selected proerythroblasts (A). Cells within the R1 gate (Ter119 high) were further analyzed with respect to their CD71 and forward scatter (FSC) profiles (B) and the percentages of proerythroblasts (C), basophilic erythroblasts (R3) (D), basophilic/polychromatic erythroblasts (R4) (E) and late polychromatophilic erythroblasts (R5) (F) were estimated. Data are mean ± SEM from n = 4–5 mice per group and are compared to naive mice (*P<0.05, **P<0.01, ***P<0.001), bled mice (εP<0.01, εεP<0.01, εεεP<0.001) or between Plasmodium-infected and PHZ-treated mice (ΨΨΨP<0.001) using a one– way ANOVA and Tukey's multiple test.
Figure 5.
Reactive oxygen species in the bone marrow and serum heme levels.
Naive, bled, P.c. adami-infected and PHZ-treated female BALB/c mice (4–6 weeks old) were euthanized. (A) Serum levels of heme were assessed as described in Material and Methods. (B) Bone marrow cells were recovered from the tibia by flushing and cell aliquots were stained with anti-F4-80 biotin/strepatavidin PERCP and then labelled with 2′,7′ dichlorodihydrofluorescein diacetate (H2-DCFDA). The mean cellular fluorescence was measured and analyzed in F4-80+ cells (B) and F4-80− cells (C) in a FACScan cytofluorometer. Results are mean ± SEM from 4 mice per group and are compared using a one–way ANOVA and Tukey's multiple test. *P<0.05, **P<0.01 and ***P<0.001 are comparisons to naive; εP<0.05, εεP<0.01 and εεεP<0.001 are comparisons to bled mice.
Figure 6.
Erythroid blast forming units (BFU-E) in tibial bone marrow and spleen of mice with anemia.
Naive, bled, P.c. adami-infected and PHZ-treated female BALB/c mice (4–6 weeks old) were euthanized and BFU-Es were determined in 12-days bone marrow (A) and spleen (B) cultures using MethoCult® 03434 media. Data are mean ± SEM from 4 mice per group and compared to naive (**P<0.001; ***P<0.01) or to bled mice (εεεP<0.001) using a one–way ANOVA and Tukey's multiple test.
Figure 7.
Bone remodeling markers and osteoclastogenesis in anemic mice.
Naive, bled, P.c. adami-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). Results are mean ±SEM from 4–10 mice per group, compared by a one –way ANOVA and a Tukey's mutiple comparison test. *P<0.05, **P<0.01, ***P<0.001 are comparisons to naïve mice; εP<0.05, εεP<0.01, εεεP<0.001 are comparisons to bled mice; ΨΨΨP<0.001 represents comparison between PHZ-treated and Plasmodium-infected mice.
Figure 8.
Bone histochemical analysis of tibia.
Naive, bled, P.c. adami-infected and PHZ-treated female BALB/c mice (4–6 weeks old) were euthanized and tibias were used for Von Kossa and tartrate-resistant acid phosphatase (TRAP) staining on representative bone sections.