Figure 3

Surgical nerve ablation at the nerve entry site induces bone loss via reduced bone formation. (A) Schematic diagram of surgical nerve ablation. Nerve fibers entering the tibia were surgically ablated at the entry site. (B) Representative Osteo-DISCO images of Sox10-positive nerves inside the tibia in the sham operation group and nerve ablation group (upper) and % nerve/bone ratio: percentage of nerve fibers in tibia bone area on the Z-stack image (lower). The number of Sox10-positive nerves was remarkably reduced in the nerve ablation group. Scale bars: 500 µm. These data are presented as the means ± SEMs (n = 6). P values were obtained using a two-tailed paired t test. (C) Representative images of immunofluorescence staining for CGRP, TH, in both groups. Scale bars: 50 µm. CGRP: calcitonin gene-related peptide, TH: tyrosine hydroxylase, EMCN: endomucin. (D) Micro-CT images of proximal tibiae in both groups showing the decrease in bone mass in the nerve ablation group. (E) Micro-CT analysis of trabecular bone in proximal tibiae showing a significant decrease in bone volume and trabecular number in the nerve ablation group. BV/TV: bone volume/tissue volume, Tb.Th: trabecular thickness, Tb.N: trabecular number, Tb.Sp: trabecular separation. (F) Bone histomorphometric analysis of trabecular bone in proximal tibiae showing reduced bone formation in the nerve ablation group. BFR/BS: bone formation rate/bone surface, N.Ob/BS: osteoblast number/bone surface, Ob.S/BS: osteoblast surface/bone surface, N.Oc/BS: osteoclast number/bone surface, Oc.S/BS: osteoclast surface/bone surface. All data are presented as the means ± SEMs (n = 10). P values were obtained using a two-tailed paired t test (E and F).