Vitamin D deficiency leads to sensory and sympathetic denervation of the rat synovium

Abstract

Vitamin D deficiency is associated with increased susceptibility to inflammatory arthritis. Sensory and sympathetic synovial nerves are critical to the development of inflammatory arthritis and spontaneously degenerate in the early phases of disease. These nerves contain vitamin D receptors and vitamin D influences nerve growth and neurotrophin expression. We therefore examined the density of synovial nerves and neurotrophin-containing cells in vitamin D-deficient rats. Seven-week-old Sprague-Dawley rats were fed either control or vitamin D-deficient diets for 4weeks. Knee synovium sections extending from the patella to the meniscus were immunostained for total nerves, myelinated and unmyelinated nerves, sympathetic nerves, peptidergic and non-peptidergic sensory nerves, and neurotrophins and immune cell markers. In control rats, intimal innervation by unmyelinated sensory fibers was denser than subintimal innervation. In contrast, sympathetic innervation was confined to the subintima. Many sensory axons contained markers for both peptidergic and non-peptidergic nerves. Nerve growth factor (NGF) was primarily expressed by intimal CD163-negative type B synoviocytes, while neurturin, a ligand selective for non-peptidergic sensory neurons, was expressed by synovial mast cells. In vitamin D-deficient rats, there were significant reductions in sensory nerves in the intima and sympathetic nerves in the subintima. While there was no significant change in NGF-immunoreactivity, the number of neurturin-expressing mast cells was significantly reduced in the intima, suggesting that intimal reductions in sensory nerves may be related to reductions in neurturin. Vitamin D deficiency therefore may increase susceptibility to inflammatory arthritis by depleting sensory and sympathetic synovial nerves as a result of reduced synovial neurotrophin content.

Keywords: arthritis; nerves; sensory; sympathetic; synovium; vitamin D.

Fig. 1

Nerves and vasculature in the rat synovium. In this immunofluorescently-labeled section of knee synovium from a control rat, the pan-neuronal marker, protein gene product 9.5 (PGP9.5, green), shows the location of nerves in relationship to the vascular marker smooth muscle actin (SMA, red). A white line superimposed on the image separates the intima (left) from the subintima (right). Larger SMA-ir vessels are found in the subintima, while the intima contains smaller microvasculature. The SMA-ir vasculature in both the subintima and intima are frequently associated with PGP9.5-ir nerves (arrows). There are also free nerve endings in the intima that appear to terminate near the intima/cavity junction (arrow heads). Scale=100μm

Fig. 2

Nerves in knee synovium of control and vitamin D deficient (VD-) rats. Representative images are shown of immunofluorescently-labeled nerves in knee synovium (A,B,D,E,G,H) from control (A,D,G) and VD- (B,E,H) rats, and graphical representation of nerve density quantitation (C,F,I). Synovial sections were immunostained for PGP9.5 (A-C), Neurofilament H (NFH, D-F) and peripherin (G-I). A white line superimposed on images separates the intima (left) from the subintima (right) (A,B,D,E,G,H). Arrows point toward selected nerves in the subintima and arrowheads point toward selected intimal nerves. Data are presented as mean±s.e.m (C,F,I). # p<0.05 between intima and subintima of rats on the same diet by two-way ANOVA. * p<0.05 between control and VD- rats within the intima or subintima by two-way ANOVA. Scale=50μm

Fig. 3

Sympathetic and sensory nerves in knee synovium of control and vitamin D deficient (VD-) rats. Representative images are shown of immunofluorescently-labeled nerves in knee synovium (A,B,D,E,G,H,J,K) from control (A,D,G,J) and VD- (B,E,H,K) rats, and graphical representation of nerve density quantitation (C,F,I,L). Synovial sections were immunofluorescently-labeled for tyrosine hydroxylase (TH, A-C), Calcitonin gene-related peptide (CGRP, D-F), substance P (SP,G-I), and GDNF family receptor alpha 2 (GFRα2, J-K). A white line has been superimposed on images to separate the intima (left) from the subintima (right) (A,B,D,E,G,H,J,K). Arrows point toward selected nerves in the subintima and arrowheads point toward selected intimal nerves. Data are presented as mean±s.e.m (C,F,I,L). # p<0.05 between intima and subintima of rats on the same diet by two-way ANOVA. * p<0.05 between control and VD- rats within the intima or subintima by ANOVA. Scale=50μm

Fig. 4

Colocalization of CGRP and GFRα2 immunoreactivity in synovial nerves. Representative images are shown of immunofluorescently-labeled nerves in knee synovium (A-F) from control (A-C) and vitamin D deficient (VD-, D-F) rats. Synovial sections were immunofluorescently-labeled for both GDNF family receptor alpha 2 (GFRα2, A&D) and calcitonin gene-related peptide (CGRP, B&E) and shown in merged images (C&F). A white line has been superimposed on images to separate the intima (left) from the subintima (right) (C&F). Arrowheads point toward selected nerves with GFRα2-only labeling, while arrows point toward selected nerves with CGRP and GFRα2 co-labeling. Scale=50μm

Fig. 5

Confocal volume view images showing CGRP and GFRα2 within the same axons. Representative images of a group of axons within the synovial intima of a control rat (A-H). Synovial sections were stained with DAPI (A&E) and immunofluorescently-labeled for both GDNF family receptor alpha 2 (GFRα2, B&F) and calcitonin gene-related peptide (CGRP, C&G). D&H show merged images. Volume view images are shown from top-down (A-D) and side (E-H) views. In A-D, a white line has been superimposed on images to separate the subintima (above) from the intima (below) (C&F). GFRα2 and CGRP appear to be located within many of the same nerve fibers.

Fig. 6

Nerve growth factor (NGF) in knee synovium of control and vitamin D deficient (VD-) rats. Representative images are shown of NGF-labeled cells in knee synovium (A,B,C) from control (A,B) and VD- (C) rats, and graphical representation of NGF-ir density (D). Synovial sections were immunofluorescently labeled for NGF (green, A-C), CD163-ir (red) macrophages/type A synoviocytes (A), and stained with DAPI (blue, A-C). A white line has been superimposed on images to separate the intima (left) from the subintima (right) (B&C). Data are presented as mean±s.e.m (D). # p<0.05 between intima and subintima of rats on the same diet by two-way ANOVA. * p<0.05 between control and VD- deficient rats within the intima or subintima by two-way ANOVA. Scale=50μm

Fig. 7

Neurturin in knee synovium of control and vitamin D deficient (VD-) rats. An image from a synovial section from control rat was immunofluorescently-labeled for neurturin and superimposed over a phase contrast image of the same area (A) and subsequently stained with Giemsa, indicative of mast cells (B). A synovial section from a control rat was immunoflourescently-labeled for neurturin (green), mast cell tryptase (red), and DAPI (blue, C). Representative images are shown of neurturin-ir (green) cells in the in knee synovium from control (D) and vitamin D deficient (VD-, E) rats, and graphical representation neurturin-ir cell density (F). A DAPI nuclear stain (blue) shows the tissue morphology and a white line has been superimposed on images to separate the intima (left) from the subintima (right). Data are presented as mean±s.e.m (F). # p<0.05 between intima and subintima of rats on the same diet by two-way ANOVA. * p<0.05 between control and VD- rats within the intima or subintima by two-way ANOVA. Scale=50μm

Fig. 8

Immune cells in the synovium. Representative images are shown of immunofluorescently-labeled immune cells in knee synovium (A,B,D,E) from control (A,D) and vitamin D deficient (VD-, B,E) rats, and graphical representation of immune cell density quantitation (C,F). Synovial sections were immunofluorescently-labeled for CD163 (macrophages, (A,B) or mast cell tryptase (MCT, D,E) and stained with DAPI (A,B,D,E). A white line has been superimposed on images to separate the intima (left) from the subintima (right) (A,B,D,E). Data are presented as mean±s.e.m (C,F). # p<0.05 between intima and subintima of rats on the same diet by two-way ANOVA. * p<0.05 between control and VD- rats within the intima or subintima by two-way ANOVA. Scale=50μm