First Place — Student Research Award 1995: A Mechanism for Sympathectomy-Induced Bone Resorption in the Middle Ear

BACKGROUND: Recent investigations have demonstrated a link between sympathectomy and osteoclast-mediated bone resorption. The exact nature of this link, however, is unknown, We hypothesize that substance P, a potent vasoconstrictive neuropeptide found in peripheral sensory fibers, including those innervating bone, is the mediator of this phenomenon. To test this theory, the effects of substance P on in vitro calcium release from cultured neonatal mouse calvaria were assessed. In addition, an in vivo study was conducted whereby gerbils were injected with capsaicin to eliminate substance P-containing fibers before sympathectomy with 6-hydroxydopamine. If the effects of 6-hydroxydopamine were eliminated by prior administration of capsaicin, the role of sensory nerves in sympathectomy-induced resorption would be strongly implicated.

IN VITRO STUDY: Substance P at 10⁻⁸ mol/L was incubated with eight newborn Swiss-Webster mouse hemicalvarial explants and compared with explants incubated in control media alone. The neonatal mice were euthanized at day 3, and their hemicalvaria were preincubated in 2 ml of stock media without treatment for 24 hours at 36.5° C as a stabilization period. After the stabilization period, the stock media were replaced with 2 ml of fresh control media or media containing substance P at 10⁻⁸ mol/L. A similar experiment was performed with the addition of indomethacin at 5 × 10⁻⁷. The explants were then incubated for 72 hours with gassing every 12 hours with a mixture of O₂, N₂, and CO₂. At the end of the 72-hour period, the media were analyzed for calcium content by atomic absorption spectrophotometry and compared by one-way analysis of variance with Bonferroni-corrected post hoc tests.

IN VSVO STUDY: Forty-eight Mongolian gerbils were placed into four groups: group 1 received intraperitoneal injections of 6-hydroxydopamine at 75 μg/gm body weight on days 1, 2, 6, 7, and 8; group 2 received identical injections of hydroxydopamine, but 12 hours after receiving subdermal injections of capsaicin at 50 μg/gm body weight; group 3 received only subdermal injections of capsaicin; and group 4 received only saline injections to serve as controls. Seven days after treatment, the animals were euthanized, and the ventral wall of each animal's right bulla was resected and quantified for osteoclast number and surface with a computer-based histomor-phometry system. Analysis was then made by one-way analysis of variance with Bonferroni-corrected post hoc tests.

RESULTS: The results of the in vitro study revealed that substance P at 10⁻⁸ mol/L (11.05 ± 3.37 μg/ml) induced significant calcium release from cultured neonatal mouse calvaria when compared with control bone incubated in base media alone (0.92 ± 2.85 μg/ml, p < 0.01). The process was completely inhibited by 5.0 × 10⁻⁷ indomethacin. The results of the in vivo study showed 6-hydroxydopamine treatment significantly increased both the osteoclast number (NOc/TL = 3.14 ± 1.33/mm) and the osteoclast surface (OcS/BS = 16.04% ± 6.95%) of bone when compared with bone from saline-treated controls (NOc/TL = 1,77 ± 0.79/mm, p < 0.01; OcS/BS = 8.88% ± 4.15%, p < 0.01), These 6-hydroxydopamine-induced increases were eliminated, however, in animals pretreated with capsaicin before sympathectomy (NOc/TL = 1.86 ± 0.68/mm, p > 0.05; OcS/BS = 9.92 ± 3.73, p > 0.05), whereas treatment with capsaicin alone had no effect when compared with bone from saline-treated controls (NOc/TL = 2.02 ± 0.50/mm, p > 0.05; OcS/BS = 10.28% ± 2.62%, p > 0.05), Substance P has thus been shown to induce calcium release from membranous bone in vitro, whereas capsaicin, a substance P-specific sensory neurolytic chemical, eliminates the in vivo osteoclast-inductive effects of 6-hydroxydopamine when given 12 hours before treatment. The results indicate that substance P is capable of inducing resorption and that substance P-containing sensory nerves are required for the induction of resorption after sympathectomy in the gerbil.