Fig. 1. Skeletal unloading decreases bone volume (A) and reduces osteoblastogenesis as measured by the extent of bone covered with osteoblasts (osteoblast surface) in the tibia metaphysis (B). Data are the means ± S.E. (n = 5 per group). * Indicates a significant difference with corresponding loaded rats (P < 0.05).

Fig. 2. Microphotographs showing that skeletal unloading induces an immediate increase in osteoblast and osteocyte apoptosis in unloaded rat tibia metaphysis at 2 days of suspension (c) compared to loaded bone (b). Apoptotic osteoblasts and osteocytes were identified by Tunel staining (e, f). DNase treatment of histological sections was used as positive control (d) whereas negative control showed no staining (a) (a–d: × 125; e, f: × 1000).

Fig. 3. Time-related effect of skeletal unloading on osteoblast and osteocyte apoptosis in rat bone. The number of Tunel-positive osteoblasts (A) and osteocytes (B) was determined in loaded and unloaded tibia metaphysis. Data are the means ± S.E. (n = 5 per group). * Indicates a significant difference with corresponding loaded rats (P < 0.05).

Fig. 4. Skeletal unloading decreases α5β1 integrin expression in the rat tibia metaphysis in vivo. Tibia metaphysis from unloaded (Unl) and loaded (Ld) rats were subjected to Western blot analysis using specific antibodies against α5 and β1 integrins. Autoradiographs were scanned and quantifications were corrected for loading using β-actin. The results are expressed as % of the corresponding data in loaded bone and given as means ± S.E.

Fig. 5. Skeletal unloading does not affect FAK and ERK signaling in metaphyseal bone. Tibia metaphysis from unloaded (Unl) and loaded (Ld) were subjected to immunoprecipitation (IP) analysis using anti-total or phospho-FAK antibodies (A) or western blot analysis using anti-total or phospho-ERK1/2 antibodies (B). The data are expressed as the ratio of phosphorylated/total kinase. Autoradiographs were scanned and quantifications were corrected for loading using β-actin. The results are expressed as % of the corresponding data in loaded bone and given as means ± S.E.

Fig. 6. Skeletal unloading alters PI3K-Bcl-2 signaling in metaphyseal bone. Tibia metaphysis from unloaded (Unl) and loaded (Ld) were subjected to immunoprecipitation analyses using anti-total or phospho-PI3K antibodies (A) or Western blot analysis using anti-Bax or anti-Bcl-2 antibodies (B). The data are expressed as the ratio of phosphorylated/total kinase (A) or Bax/Bcl-2 ratio (B). Autoradiographs were scanned, quantifications were corrected for loading using β-actin and the results are given as % data in loaded bone (means ± S.E.).

Lack of change in surrenal gland weight and plasma corticosterone levels in unloaded rats compared to loaded rats

Mean ± S.E. of 5 values; NA: not available.