Pain in fibrous dysplasia: identifying nociceptive mechanisms in a preclinical model

Abstract

Pain is a common symptom of fibrous dysplasia (FD), a rare mosaic disorder characterized by fibro-osseous lesions in the bone. Despite the prevalence of pain in FD patients, there is little knowledge about the nociceptive mechanisms and few efficacious treatments. As such, understanding FD pain is essential for patient care. The overall aim of this study was to identify nocifensive behaviors and potential underlying mechanisms in a transgenic mouse model of FD, previously shown to display high face and translational validity. Significant nocifensive behaviors were observed in FD mice (male and female), compared to control mice in the burrowing, grid hanging, home cage activity, and wheel running assays. These changes corresponded to lesion development, as visualized by X-ray imaging. Behavioral deficits improved when analgesics were administered, indicating a nociceptive origin. Tibias and femurs from FD mice demonstrated characteristic FD lesions and the presence of mono- and multi-nucleated CD68+ cells, calcitonin gene-related peptide sensory nerve fibers, and vascularization. Lumbar dorsal root ganglia from male FD mice displayed increased staining for activating transcription factor-3 and tyrosine hydroxylase neurons. No difference was observed in the spinal cords between the FD and control groups for glial cell presence and neuropeptide expression. Bone marrow stromal cells were obtained from FD and control mice and cultured in vitro. FD cells developed an increased concentration of inflammatory cytokines (IL-6, tumor necrosis factor-alpha), chemokines (monocyte chemoattractant protein, keratinocyte chemoattractant/human growth-regulated oncogene), and nerve growth factor as compared to controls. Taken together, this study demonstrated for the first time that nociceptive mechanisms such as axonal growth in FD lesions, nerve injury, and inflammation may contribute to FD pain, and it provides a foundation for conducting further studies of pain- and disease-modifying therapeutics for FD patients.

Keywords: bone pain; fibrous dysplasia; in vivo; mechanism; pain.

Figure 1

Behavior changes in FD and control mice. (A) Burrowing behavior in female mice over time with ibuprofen (30 mg/kg) and vehicle treatment. (B) Burrowing behavior in male mice. (C) AUC of home cage activity in female mice over time. (D) AUC of wheel running distance in females over time. (E) AUC of home cage activity in male mice over time. (F) AUC of wheel running distance in males over time. (G) Grid hanging behavior in female mice before treatment. (H) Grid hanging behavior in female mice after morphine (10 mg/kg) and vehicle treatment at D11. (I) Grid hanging behavior in male mice. Except wheel running and home cage activity where n represents a cage, n represents 1 mouse; female mice were housed 2 per cage and males housed 1 per cage. Statistics: A-H, two-way ANOVA with Tukey’s multiple comparison correction; I, one-way ANOVA. Graphs are individual values with bars representing median ± IQR. ^*p < .05. ^**p < .01. ^***p < .001. ^****p < .0001. Abbreviations: AUC, area under the curve; BL, baseline; D, day; FD, fibrous dysplasia.

Figure 2

Histological hematoxylin and eosin staining and micro-CT in FD mice compared to control mice. (A, B) H&E histological staining of bone tissue. (C-J) Representative micro-CT images of the distal femur (C-F) and proximal tibia and fibula (G-J) of FD (D, F, H, J) and control (C, E, G, I) mice, post-euthanasia. (K-N) micro-CT analysis of BMD (K, L) and BV/TV (M, N) comparing FD mice and control mice. Unpaired Student t-test used to compare groups and graphs are individual values with bars representing median ± IQR. ^*p < .05. ^**p < .01. ^***p < .001. ^****p < .0001. Abbreviations: BV/TV, bone volume fraction; FD, fibrous dysplasia.

Figure 3

Representative images of IHC staining of bone innervation and vascularity in FD mice (A, B, C, E, G, I, K, M, O) and control mice (D, F, H, J, L, N). (A-E) CD68⁺ and DAPI staining demonstrating cells from the monocyte lineage. Asterisk demonstrates mononuclear cells (eg, monocytes, macrophages, pre-osteoclasts) and arrowhead demonstrates multinuclear cells (eg, osteoclasts); (F, G) PGP 9.5 and DAPI staining; (H, I) CGRP⁺ and DAPI staining; (J, K) NF200 and DAPI staining; (L, M) TH and DAPI staining; (N, O) Endomucin and DAPI staining. Mouse details: (A, B, C, E, I) female FD mouse, 14-16 wk old; (D, H, J, L, N) female control mouse, 18-20 wk old; (F) female control mouse, 14-16 wk old; (G, K) female FD mouse, 13-15 wk old; (M, O) male FD mouse, 14-16 wk old. Abbreviations: ♀, female; ♂, male; CD68⁺, cluster of differentiation 68; CGRP⁺, calcitonin gene-related peptide; DAPI, 4′,6-diamidino-2-phenylindole; FD, fibrous dysplasia; IHC, immunohistochemistry; NF200, neurofilament 200 kDa; PGP 9.5, protein gene product 9.5; TH, tyrosine hydroxylase.

Figure 4

Protein expression change in DRGs from FD and control mice. Representative IHC images of DRGs from control (A, C, F, H) and FD (B, D, G, I) mice, both female (A, B, F, G) and male (C, D, H, I). (A-D) ATF3 staining of DRGs; (F-I) TH staining of DRGs; (E) graph demonstrating proportion of DRG cell bodies with ATF3 staining in female and male mice; (J) graph demonstrating proportion of TH-positive axons in DRGs from female and male mice. Unpaired Student t-test used to compare groups and graphs are individual values with bars representing median ± IQR. ^*p < .05. ^***p < .001. Abbreviations: ATF3, activating transcription factor 3; DRG, dorsal root ganglion; FD, fibrous dysplasia; TH, tyrosine hydroxylase.

Figure 5

Neuropeptide expression and glial cell presence in spinal cords from FD and control mice (female and male). Neuropeptide and GFAP were measured using CTCF and microglia (Iba1⁺ and Pp38) were measured using cells/area. Unpaired Student t-test used to compare groups and graphs are individual values with bars representing median ± IQR. No significant differences observed. Abbreviations: CTCF, corrected total cell fluorescence; FD, fibrous dysplasia; GFAP, glial fibrillary acidic protein; Iba1⁺, ionized calcium-binding adaptor molecule 1; Pp38, phospho-p38 mitogen-activated protein kinase.

Figure 6

In vitro cAMP assay (A), NGF ELISA (B), and multiplex assay (C-K). FD cells and control cells were activated with doxycycline (10 μg/mL) or vehicle (phosphate buffered saline) for 24 hr with IBMX. Intracellular cAMP was assessed using cAMP Gs dynamic kit and protein factors were assessed using MSD U-PLEX assay. Two-way ANOVA with Tukey’s multiple comparison test. Graphs are individual values with bars representing median ± IQR. ^****p < .0001. Abbreviations: cAMP, cyclic adenosine monophosphate; FD, fibrous dysplasia; IBMX, 3-isobutyl-1-methylxanthine; NGF, nerve growth factor.