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. 2021 Mar 1;10:e66532. doi: 10.7554/eLife.66532

Figure 2. Sensory nerve innervation in subchondral bone decreased with PTH treatment.

(A) Immunofluorescence analysis of calcitonin gene-related peptide+ (CGRP+) (green) sensory nerve fibers in subchondral bone of sham-operated, PTH-treated DMM, and vehicle-treated DMM mice at week 8 after surgery. DAPI stains nuclei blue. Upper: low-magnification images (orange dotted line outlines the contour of the tibial subchondral bone), scale bar: 300 μm; bottom: high-magnification images. Scale bar: 50 μm. (B) Quantitative analysis of the density of CGRP+ sensory nerve fibers in tibial subchondral bone at week 8 after sham or DMM surgery (n = 8/group). (C, D) Immunofluorescence and quantitative analysis of substance P+ (SP+) (red) nerve fibers in subchondral bone at week 8 after sham or DMM surgery (n = 8/group). DAPI stains nuclei blue. Scale bar: 50 μm. (E, F) Representative images of immunofluorescence co-staining and quantitative analysis of the CGRP+ sensory nerves (green) and endomucin+ (EMUN) vessel (red) in the subchondral bone at week 8 after sham or DMM surgery (n = 8/group). Scale bar: 50 μm. (G, H) Immunofluorescence and quantitative analysis of CGRP+ (green) nerve fibers in synovium at week 8 after sham or DMM surgery. DAPI stains nuclei blue. Scale bar: 50 μm. n = 8/group. (I) μCT images of the tibial subchondral bone medial compartment (coronal) at week 8 after sham or DMM surgery. Arrowhead indicates the osteophyte. Scale bar: 500 μm. (J) Total volume measurement of osteophytes from the tibial plateau of sham-operated, PTH-treated DMM, and vehicle-treated DMM mice. (n = 8/group). (K) Immunohistochemical analysis of cyclooxygenase 2+ (COX2+) cells in the tibial subchondral bone at week 4 after DMM surgery. Arrowhead indicates the positive cells. Scale bar: 50 μm. (L) Quantitative analysis of COX2+ cells in mouse tibial subchondral bone (both bone marrow and subchondral bone matrix) (n = 8/group). (M) Quantitative analysis of PGE2 in subchondral bone determined by enzyme-linked immunosorbent assay (ELISA) (n = 8/group). *p<0.05, **p<0.01. NS, no significant difference.

Figure 2—source data 1. Raw data of CGRP staining, SP staining, EMUN staining, CGRP staining in synovium, quantification osteophyte volume, COX2 staining, and quantification of level of PGE2.

Figure 2.

Figure 2—figure supplement 1. PTH treatment significantly reduced CGRP-positive nerve fibers in subchondral bone.

Figure 2—figure supplement 1.

(A) Immunofluorescence analysis of co-staining of P2 × 3+ (red) and CGRP+ (green) nerve fibers in tibial subchondral bone at week 8 after sham or DMM surgery. DAPI stains nuclei blue. Scale bar: 50 μm. (B) Immunofluorescence analysis of co-staining of PIEZO2+ (red) and CGRP+ (green) nerve fibers in tibial subchondral bone at week 8 after sham or DMM surgery. DAPI stains nuclei blue. Scale bar: 50 μm. (C–D) Quantitative analysis of the density of P2 × 3+ and PIEZO2+ nerve fibers in tibial subchondral bone at week 8 after sham or DMM surgery (n = 8/group).
Figure 2—figure supplement 1—source data 1. Raw data of P2 × 3 staining and PIEZO2 staining.