Fig 1.
General view of the mandible periosteum from control animals.
A. Enzymohistochemistry staining for alkaline phosphatase revealing the cells of the osteogenic lineage. This layer was used to delineate the osteogenic compartment (zone 1) from the non-osteogenic compartment (zone 2). B. Toluidine blue staining. The arrowheads point out the mast cells present in the non-osteogenic compartment (2). At the interface with the mineralized bone matrix, the arrows delineate the osteoid seam. The double arrows separate the two compartments. Same magnification in A and B, bar: 100 μm.
Fig 2.
Effect of sympathectomy on proNGF, TrkA and tPA in the osteogenic compartment.
A. 17-day control animal. proNGF is expressed extracellularly in the osteogenic compartment (arrows), within the osteoid seam (asterisk) and on the fibers at the periphery of the compartment (arrowheads). B. 17-day sympathectomy. The compartment is almost completely depleted in proNGF. Noticeably, the proNGF content of the osteoid seam was maintained in the sympathectomized animals (asterisk). Magnification bar (A and B): 50 μm. (C) Immunocontrol; no primary antibody used. Bar = 50 μm. Time-course changes in proNGF distribution in the osteogenic layer (D), in proNGF-positive fibers (E), and in proNGF/osteoid seam thicknesses ratio (F). * P<0.05, **P≤0.02, ***P<0.01, ****P<0.005 vs the corresponding controls. °° P<0.02 vs the 7-days sympathectomized animals. Values are mean ± SEM. Effects after 17 days of sympathectomy on TrkA (G and H) and tPA (I and J) immunostainings. Magnification bars: 50 μm. Ct: control animal; Symp: sympathectomized animal. The double arrows delineate the osteogenic compartment.
Fig 3.
Semaphorin3a expression in the osteogenic compartment.
A. 17-day control. Most cells were immunopositive in the osteogenic layer. Osteocytes subjacent to the cortical surface also expressed Sema3a (arrows). B. 17-day sympathectomy. Only a few scarce cells expressed the marker, most osteocytes were no longer immunopositive. Magnification bar (A and B): 50 μm. C and D: Changes with time in Sema3a-positive osteogenic cells (C) and osteocytes (D). *P<0.05, ***P<0.01 vs the corresponding controls. °°° P<0.002 versus the 7-day sympathectomy group. Immunostaining for neuropilin-1 in a control animal (E) and in a 17-days sympathectomized animal (F). Sympathectomy strongly reduced receptor expression. Arrowheads point out the osteogenic cells and arrows, the osteocytes. Magnification bar (E and F): 50 μm. Ct: control animal; Symp: sympathectomized animal. The double arrows delineate the osteogenic compartment.
Fig 4.
Only mast cells in the non-osteogenic compartment were immunostained. A. Control animal. B. 17-day sympathectomy. VIP treatment of sympathectomized (C) or intact (D) animals. VIP10-28 treatment (E). Sympathectomy caused mast cell activation and ßNGF release, as did VIP10-28 treatment. VIP treatments stabilized the cells. F to I. Different stages of mast cell activation and ßNGF depletion. When mast cells are activated, their granule content is released outside the cell, thus the immunostaining for the marker progressively decreases within the cell. Magnification bars, A to E: 50 μm; F to I: 10 μm. Variations in ßNGF-positive activated/total mast cells ratio, J: time-related changes and K: effect of the different treatments on the activated/total mast cells ratio. *P<0.05, ** P<0.01, ***P<0.005 vs the corresponding controls. ° P<0.05 vs the 7-day sympathectomy. § P<0.01 vs sympathectomy. # P<0.005 vs VIP10-28 group. Values are mean ± SEM.
Fig 5.
Effect of VIP treatments or VIP antagonism.
Expression of proNGF in the osteogenic layer (A), in peripheral fibers (B), on proNGF/osteoid thicknesses ratio (C). Expressions of TrkA (D) and of semaphorin 3a in the osteogenic cells (E) and the osteocytes (F). *P<0.05, **P≤0.02, ***P <0.01, ****P<0.002 vs the corresponding controls. °P<0.01, °°P<0.001 vs sympathectomy. Values are mean ± SEM.
Fig 6.
Variations in CGRP-immunostaining and expression.
Immunoreactive sensory fibers and osteogenic cells expressed CGRP. A. 17-day control animal (Ct). CGRP fibers (arrowheads) were located at the boundary between the two compartments. B. Day-17 sympathectomized animal (Symp). C. VIP-treated sympathectomized animal (Symp+VIP). D. VIP-treated intact animal (VIP). E. VIP10-28-treated animal (VIP10-28). Same magnification from A to E, bar: 50 μm. (F) Immunocontrol; no primary antibody used. Bar = 50 μm. In the osteogenic compartment, CGRP expression increased as soon as day 7 in the sympathectomy group, and continued so at day 17 (G). VIP treatment of sympathectomized animals restored the expression profile of controls. Treating intact animals with VIP10-28 or VIP increased CGRP expression (H). In the non-osteogenic compartment, sympathectomy increased sensory fibers (I). Effects of treatments on the sympathectomized and intact animals on CGRP-IR expression (J). *P<0.05, **P<0.01 vs the corresponding controls. °P<0.05 vs the sympathectomized animals. # P<0.05 vs the VIP-treated cultures. Values are mean ± SEM. Immunostaining for substance P, another marker of sensory fibers. K. Control animal. L. Sympathectomized animal. Same magnification in K and L, bar: 50 μm. The double-headed arrows delineate the osteogenic compartment.
Fig 7.
Schematic representation of the crosstalk between nerve fibers and osteogenic cells in the periosteum.
VIP (blue solid arrows) synthesized by the sympathetic cholinergic fibers (SCF) induces the release by the osteogenic cells (purple outline) of proNGF (green dotted arrows), which is stored in the extracellular matrix. proNGF has a trophic (+) effect on sympathetic fibers and osteogenic cells. VIP also induces the release by osteogenic cells and osteocytes of semaphorin 3a (red broken arrows) that repulses (-) sympathetic and sensory fibers (wavy lines, SF) out the osteogenic compartment. VIP stabilizes (=) mast cells (MC, grey filling) to prevent excess release of ßNGF, which is trophic for sensory fibers. VIP induces CGRP (brown) expression by osteogenic cells. VIP unavailability after sympathectomy or VIP10-28 treatment disrupted this metabolic network, favored sensory fiber penetration in the osteogenic compartment, decreased proNGF and semaphorin 3a synthesis, enhanced ßNGF release by mast cells and sprouting of sensory fibers in the non-osteogenic compartment. The double arrows delineate the osteogenic compartment (OC) and the non-osteogenic compartment (NOC).