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. 2020 May 15;21(10):3521.
doi: 10.3390/ijms21103521.

Functional Block of Interleukin-6 Reduces a Bone Pain Marker but Not Bone Loss in Hindlimb-Unloaded Mice

Hiroki Wakabayashi, Gaku Miyamura, Nobuto Nagao  1 , Sho Kato  1 , Yohei Naito  1 , Akihiro Sudo  1
Affiliations

Functional Block of Interleukin-6 Reduces a Bone Pain Marker but Not Bone Loss in Hindlimb-Unloaded Mice

Hiroki Wakabayashi et al. Int J Mol Sci. .

Abstract

Interleukin-6 (IL-6) is widely accepted to stimulate osteoclasts. Our aim in this study was to examine whether the inhibitory effect of IL-6 on bone loss and skeletal pain associated with osteoporosis in hindlimb-unloaded (HU) mice in comparison with bisphosphonate. Eight-week-old male ddY mice were tail suspended for 2 weeks. Starting immediately after reload, vehicle (HU group), alendronate (HU-ALN group), or anti-IL-6 receptor antibody (HU-IL-6i group) was injected subcutaneously. After a 2-week treatment, pain-related behavior was examined using von Frey filaments. The bilateral distal femoral and proximal tibial metaphyses were analyzed three-dimensionally with micro-computed tomography. Calcitonin gene-related peptide (CGRP) expressions in dorsal root ganglion (DRG) neurons innervating the hindlimbs were examined using immunohistochemistry. HU mice with tail suspension developed bone loss. The HU mice showed mechanical hyperalgesia in the hindlimbs and increased CGRP immunoreactive neurons in the L3-5 DRG. Treatment with IL-6i and ALN prevented HU-induced mechanical hyperalgesia and upregulation of CGRP expressions in DRG neurons. Furthermore, ALN but not IL-6i prevented HU-induced bone loss. In summary, treatment with IL-6i prevented mechanical hyperalgesia in hindlimbs and suppressed CGRP expressions in DRG neurons of osteoporotic models. The novelty of this research suggests that IL-6 is one of the causes of immobility-induced osteoporotic pain regardless improvement of bone loss.

Keywords: CGRP; hindlimb-unloading; interleukin-6; osteoporosis; osteoporotic pain; tail suspension.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Effect of pain-related behavior by anti-IL-6 antibody and alendronate (ALN) administration in hindlimb-unloaded (HU) mice: (A) Paw withdrawal threshold (g). (B) 50% paw withdrawal threshold by the up-down method (g). (C) Withdrawal frequency stimulation (%). Top, bottom, and middle lines of the graph correspond to the 75th percentile, 25th percentile, and median, respectively. Cross represents mean. Each circle represents an outlier (n = 8 in each group). * p < 0.05, *** p < 0.005, and **** p < 0.001.
Figure 1
Figure 1
Effect of pain-related behavior by anti-IL-6 antibody and alendronate (ALN) administration in hindlimb-unloaded (HU) mice: (A) Paw withdrawal threshold (g). (B) 50% paw withdrawal threshold by the up-down method (g). (C) Withdrawal frequency stimulation (%). Top, bottom, and middle lines of the graph correspond to the 75th percentile, 25th percentile, and median, respectively. Cross represents mean. Each circle represents an outlier (n = 8 in each group). * p < 0.05, *** p < 0.005, and **** p < 0.001.
Figure 2
Figure 2
Immunohistochemical analysis of Calcitonin gene-related peptide (CGRP) expression in dorsal root ganglion (DRG) neurons: (A) CGRP expression in the DRG neurons (Scale bar is 50 μm). The ratios of CGRP-immunoreactive L3 (B), L4 (C), and L5 (D) DRG neurons (%). Top, bottom, and middle lines of the graph correspond to the 75th percentile, 25th percentile, and median, respectively. Cross represents mean. Each circle represents an outlier (n = 8 in each group). * p < 0.05 and ** p < 0.01.
Figure 3
Figure 3
Micro-CT analyses of the distal femoral metaphysis and the proximal tibial metaphysis: Three-dimensional images of the distal femoral metaphysis (A) and the proximal tibial metaphysis (B–D) BV/TV (bone volume/tissue volume) (%), (E,F) Tb.N (trabecular number) (/mm), (G,H) Tb.Th (trabecular thickness) (μm), and (I,J) Tb.Sp (trabecular separation) (μm). Top, bottom, and middle lines of the graph correspond to the 75th percentile, 25th percentile, and median, respectively. Cross represents mean. Each circle represents an outlier (n = 8 in each group). * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001.
Figure 4
Figure 4
Histological analysis of hindlimb bone: Tartrate-resistant acid phosphatase (TRAP) staining of the proximal tibial metaphysis, Scale bar is 100 μm (A). Histological analysis of the number of osteoclasts/bone perimeter (N.Oc./B.Pm) in the distal femoral metaphysis (B) and the proximal tibial metaphysis (C) (/mm). Top, bottom, and middle lines of the graph correspond to the 75th percentile, 25th percentile, and median, respectively. Cross represents mean. Each circle represents an outlier (n = 8 in each group). * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001.
Figure 4
Figure 4
Histological analysis of hindlimb bone: Tartrate-resistant acid phosphatase (TRAP) staining of the proximal tibial metaphysis, Scale bar is 100 μm (A). Histological analysis of the number of osteoclasts/bone perimeter (N.Oc./B.Pm) in the distal femoral metaphysis (B) and the proximal tibial metaphysis (C) (/mm). Top, bottom, and middle lines of the graph correspond to the 75th percentile, 25th percentile, and median, respectively. Cross represents mean. Each circle represents an outlier (n = 8 in each group). * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001.
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