Vitamin D deficiency promotes skeletal muscle hypersensitivity and sensory hyperinnervation

Abstract

Musculoskeletal pain affects nearly half of all adults, most of whom are vitamin D deficient. Previous findings demonstrated that putative nociceptors ("pain-sensing" nerves) express vitamin D receptors (VDRs), suggesting responsiveness to 1,25-dihydroxyvitamin D. In the present study, rats receiving vitamin D-deficient diets for 2-4 weeks showed mechanical deep muscle hypersensitivity, but not cutaneous hypersensitivity. Muscle hypersensitivity was accompanied by balance deficits and occurred before onset of overt muscle or bone pathology. Hypersensitivity was not due to hypocalcemia and was actually accelerated by increased dietary calcium. Morphometry of skeletal muscle innervation showed increased numbers of presumptive nociceptor axons (peripherin-positive axons containing calcitonin gene-related peptide), without changes in sympathetic or skeletal muscle motor innervation. Similarly, there was no change in epidermal innervation. In culture, sensory neurons displayed enriched VDR expression in growth cones, and sprouting was regulated by VDR-mediated rapid response signaling pathways, while sympathetic outgrowth was not affected by different concentrations of 1,25-dihydroxyvitamin D. These findings indicate that vitamin D deficiency can lead to selective alterations in target innervation, resulting in presumptive nociceptor hyperinnervation of skeletal muscle, which in turn is likely to contribute to muscular hypersensitivity and pain.

Figures

Figure 1.

Behavioral comparisons of control (○), VD−/+Ca (■), and VD− (▿) rats. A, Changes in deep tissue pressure sensitivity, measured as the maximum force (g) required to elicit an attempted limb withdrawal or vocalization to mechanical compression of the calf muscle. B, There were no changes in cutaneous mechanical sensitivity as measured by the percentage of withdrawal responses to the application of a 4 g monofilament to the plantar surface of the hindpaw. C, Changes in balance were measured by the number of hindpaw slips while traversing an elevated 2.5-cm-diameter, 110-cm-long beam. VD− and VD−/+Ca rats failed to improve, while control rats improved compared to initial performance. D, There were no changes in forelimb grip strength, as measured by the force (g) required to break the rats grip. E, F, Assessment of locomotor activity during a 5 min recording using a force-plate actimeter revealed no changes in distance traveled (E) or number of rearing events (F). *p < 0.05 compared to control in same week, #p < 0.05 compared to VD− in same week, ##p < 0.5 compared to week 0 within the same group.

Figure 2.

Images of hematoxylin and eosin-stained lateral gastrocnemius muscle and tibial growth plate sections. A–C, Hematoxylin and eosin-stained transverse sections of the lateral gastrocnemius of rats receiving control (A), VD−/+Ca (B), or VD− (C) diets. There were no discernible differences between groups. Scale bar, 100 μm for A–C. D–F, Sagittal sections (20 μm) of tibial growth plates from rats receiving control (D), VD−/+Ca (E), or VD− (F) diets. There were no discernible differences between groups. Scale bar, 20 μm for D–F.

Figure 3.

Comparisons of innervation densities of the lateral gastrocnemius muscles of rats after 4 weeks on control, VD−/+Ca, or VD− diets. A, There were no changes in the volumes of the calf muscles as measured by fluid displacement. B–E, There were significant increases in the percentage of nerve area per field area of peripherin-immunoreactive (ir) fibers (B) and CGRP-ir sensory peptidergic nerves (C) in muscles from vitamin D-deficient rats, but not VMAT2-ir sympathetic (D) or NFH-ir myelinated (E) nerve fibers. F, There were also no changes in neuromuscular junction (NMJ) area as measured by α-bungarotoxin binding reactivity (−r).

Figure 4.

Immunoreactive nerves in gastrocnemius muscle. Transverse sections of the lateral gastrocnemius of rats receiving control (A, D, G, J), VD−/+Ca (B, E, H, K), or VD− (C, F, I, L) diets were immunostained for peripherin (A–C, green), CGRP (D–F, red), VMAT2 (G–I, red), or NFH (J–L, red) and bungarotoxin (J–L, green). Scale bar (in J), 50 μm for all panels.

Figure 5.

Comparisons of innervation densities of footpad epidermis in rats after 4 weeks on control, VD−/+Ca, or VD− diets. Sagittal sections of footpads from rats receiving control (A, D), VD−/+Ca (B, E), or VD− (C, F) diets were immunostained for PGP 9.5 (A–C, green) or CGRP (D–F, red). There were no significant differences in the number of intra-epidermal nerve fibers per millimeter (G, H, IENF; number of fiber nerves crossing the dermal–epidermal junction) or the total nerve fiber density (I, J) within the epidermis in sections immunofluorescently labeled with PGP (G, I) or CGRP-ir (H, J). Scale bar (in F), 100 μm in all panels.

Figure 6.

1,25(OH)2D3 acts directly on peripherin-ir sensory nerves to regulate axonal growth. A, B, Primary DRG cultures immunostained for peripherin (A) and vitamin D receptors (B, VDR), showing that VDR is expressed by cultured sensory neurons and localized to both the nucleus and the cytoplasm in cultures without added 1,25(OH)2D3 (scale bar, 50 μm). C–E, At a higher magnification, peripherin-ir neurites (C) contain the growth cone marker GAP43 (D) localized to the axon tip, and this colocalizes with VDR (E), which also extends into the filopodia. Scale bar (in E), 5 μm for C–E. F–H, Representative images of neurons treated with 0 (F), 20 (G), or 100 pm (H) 1,25(OH)2D3. Scale bar (in H), 50 μm for F–H. I, Fold change in peripherin-ir neurite area per neuron at various 1,25(OH)2D3 concentrations. Results reveal a biphasic curve with the peak at 20 pm. The shaded area corresponds to 1,25(OH)2D3 levels reported in patients with severe pain (Gloth et al., 1991). J, DRG cultures treated with JN, a selective agonist for membrane/cytoplasm-localized rapid response VDRs, and stained for peripherin (•) or NFH (□). While there was no significant change in NFH-ir neurite area /neuron, there was a biphasic response to JN, with a peak at 20 pm in peripherin-ir axons. *p < 0.05.

Figure 7.

Sympathetic neurons express VDRs, but 1,25(OH)2D3 has no effect on neurite outgrowth. A, Sympathetic neurons of SCGs were immunofluorescently labeled for VDR. VDR was localized to the nucleus and cytoplasm of selected neurons. B–E, Primary SCG cultures immunofluorescently stained for peripherin (B, D) and VDR (C, E). Scale bars: (in B) B, C, 50 μm; (in D) D, E, 5 μm. VDR was localized throughout the cell body (C), neurite (C, E), and growth cone (E). F, Primary SCG cultures were grown for 48 h with 0, 20, or 100 pm 1,25(OH)2D3 and immunofluorescently labeled with peripherin. There were no significant changes in neurite area/neuron.