Figure 4.

Methionine enkephalin (MENK) suppressed osteocyte apoptosis induced by compression force loading. (A) Histological analysis of the parietal bones at 6 hours after application of compressive force was performed by H&E staining. MENK was injected s.c. into the parietal bone area prior to application of compressive force. A magnified picture of the ROI is shown in the lower right corner of each picture. Scale bar = 100 μm, and 10 μm in insets. (B) TUNEL staining and subsequent counterstaining with hematoxylin of the parietal bones were performed. All four ROIs in a section in each group are shown. Arrowheads indicate TUNEL‐positive osteocytes. Scale bar = 10 μm. (C) TUNEL‐positive ratio of osteocytes was calculated (four animals per group). (D) Caspase‐3 immunohistochemistry and subsequent counterstaining with hematoxylin of the parietal bones were performed. All four ROIs in a section in each group are shown. Arrowheads indicate caspase‐3‐positive osteocytes. Scale bar = 10 μm. (E) Caspase‐3‐positive ratio of osteocytes was calculated (four animals per group). Comp = compressive force loading. *P < 0.05, **P < 0.01.