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 degrees 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 x 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 VIVO STUDY: RESULTS: The results of the in vitro study revealed that substance P at 10(-8) mol/L (11.05 +/- 3.37 micrograms/ml) induced significant calcium release from cultured neonatal mouse calvaria when compared with control bone incubated in base media alone (0.92 +/- 2.85 micrograms/ml, p < 0.01). The process was completely inhibited by 5.0 x 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 ne
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