Effects of catecholamines on calvarial bone resorption in vitro.

Many important diseases in otolaryngology manifest through abnormal bone remodeling or destruction. The mechanisms for such pathological remodeling remain poorly understood. Bone is known to be innervated by norepinephrine-containing sympathetic nerves, and sympathectomy is known to induce bone resorption. The role, however, of norepinephrine as a potential bone-modulatory substance is unknown. Using the calvarial calcium release assay, we conducted the following experiment to evaluate the bone-modulatory activity of norepinephrine, the alpha-agonist octopamine, and the beta-agonist isoproterenol. Each agent was tested at 2 concentrations with and without parathyroid hormone. Norepinephrine was found to have no effect on calcium release. In contrast, octopamine at 10(-8) mol/L exerted a significant stimulatory effect on calcium release, and isoproterenol at 10(-6) mol/L exerted a significant inhibitory effect on parathyroid hormone-induced calcium release. The investigation suggests that a bimodal, concentration-dependent, receptor-specific model for catecholamine-mediated modulation of bone resorption may operate in calvarial bone.

A unique case of the rapid onset of a large cystic hygroma in the adult.

Cystic hygroma is an uncommon lymphatic tumor seen rarely in adults, with less than 100 cases reported in the literature. The etiology and pathophysiology of this lesion is still in question. The majority of cystic hygromas occur in the head and neck, particularly in the posterior triangle. Although cystic hygromas tend to enlarge progressively over a span of weeks or months, relatively rapid enlargement over a span of days has been described. We present the unique case of an adult woman who experienced sudden onset of a large cystic hygroma in the neck without history of antecedent swelling, infection, or trauma. Successful surgical removal of the hygroma was performed. A brief review of the literature is presented.

Effect of chemical sympathectomy with 6-hydroxydopamine on osteoclast activity in the gerbilline middle ear bulla.

HYPOTHESIS: Chemical sympathectomy with 6-hydroxydopamine (HDA) increases middle ear bulla bone resorption in the Mongolian gerbil. BACKGROUND: Many diseases of the middle ear have pathologic processes linked to abnormal bone remodeling. Numerous factors controlling bone remodeling have been identified. An understanding of these factors and their role in pathologic remodeling is therefore essential. Sympathectomy, induced both surgically and pharmaceutically, is known to increase middle ear bone resorption, suggesting a role for the central nervous system in bone metabolism. This effect, however, may be confounded by hemodynamic changes induced by hemicranial surgical sympathectomy or by uncertainty in the action of pharmaceutical agents on the sympathetic nervous system. In this experiment, a third modality with unique properties, chemical sympathectomy with HDA, was used to quantify further the effect of sympathectomy on middle ear bone remodeling. METHODS: Eight gerbils designated experimental received intraperitoneal injections of HDA (75 mg/kg) for 1 week, whereas eight animals designated control received similar injections of saline. One week after injections, the animals were euthanized and bulla bone samples were analyzed histomorphometrically to determine osteoclastic activity. In addition, to assess for any direct effects on bone metabolism, the activity of HDA was determined in vitro using the calvarial calcium release assay. RESULTS: The in vitro study found HDA to have no direct stimulatory activity on calcium release. The in vivo study showed HDA to increase osteoclastic activity significantly in middle ear bone. CONCLUSION: HDA-induced sympathectomy increases bone resorption in gerbilline middle ear bone.

First place--resident basic science award 1999. Effects of leukotriene and cyclo-oxygenase inhibition on adaptive bone remodeling in the middle ear.

Abnormal bone remodeling is associated with important otolaryngologic diseases. In such diseases, the mechanisms of osteoclastic control underlie the pathologic processes. It is known that strain applied to auditory bullae induces bone resorption-an effect mediated by prostaglandins and blocked by cyclo-oxygenase inhibitors. It is also known that cyclo-oxygenase inhibition shunts arachidonic acid into alternate metabolic pathways, mainly the lipoxygenase pathway with leukotriene production. The role of these metabolites in adaptive bone remodeling is unknown. Using the gerbilline bulla as a model, we infused BW755c (dual lipoxygenase/cyclo-oxygenase inhibitor) and L-663,536 (5-lipoxygenase inhibitor) into animals undergoing middle ear pressurization. After 7 days, the bulla bones were harvested, and osteoclasts were quantified histomorphometrically. The results showed that neither treatment altered pressure-induced resorption. However, BW755c significantly increased resorption in unpressurized bone when compared with control values. Because BW775c blocks both lipoxygenase and cyclo-oxygenase pathways, the results suggest an alternate pathway in middle ear bone resorption.

Sympathectomy, which induces membranous bone remodeling, has no effect on endochondral long bone remodeling in vivo.

Sympathectomy has been shown to induce resorption within the membranous middle ear bone of gerbils. It is unknown whether sympathectomy exerts a similar effect on endochondral long bone. In the present study, guanethidine sulfate (GS) and 6-hydroxydopamine (HDA) were administered to gerbils to induce sympathectomy. One week later, samples of middle ear bulla bone and radial long bone were harvested and assessed for osteoclastic activity. Histomorphometric analysis showed both pharmacologic sympathectomy with GS and chemical sympathectomy with HDA significantly increased the osteoclast counts and osteoclast surfaces of bulla bone samples but not radial long bone samples, respectively. In contrast, HDA but not GS increased the osteoclast profile area of both long bone and membranous bone samples when compared with vehicle-treated controls. Sympathectomy, induced both chemically and pharmacologically, thus has been shown to increase resorption in membranous bone but not endochondral long bone in the gerbilline model.

Effect of pharmacological sympathectomy on osteoclastic activity in the gerbilline auditory bulla in vivo.

Bone destruction causes hearing loss in various middle ear disorders. The mechanisms of such pathological remodeling are unknown. Unilateral surgical sympathectomy is known to induce resorption within mandibular and auditory bulla bone. Explanation of the cause of this effect, however, may be confounded by hemodynamic changes induced by hemicranial sympathectomy and by uncertainty as to the neuroanatomical origins of sympathetic fibers. In this study, gerbils were infused with guanethidine sulfate (GS) to evaluate the in vivo effects of systemic sympatholysis on auditory bone remodeling. In addition, to discount any direct osteolytic effect, GS was assessed of its bone resorbing activity in vitro by means of the calvarial calcium release assay. The in vitro study revealed GS to have no effect on calcium release. The in vivo study revealed GS to increase both the osteoclast surface and number. Guanethidine-induced sympathectomy has thus been shown to increase remodeling in gerbilline auditory bone, while no direct osteolytic effect could be measured in vitro.

In vivo effects of surgical sympathectomy on intramembranous bone resorption.

Bone modeling and remodeling are highly regulated processes in the mammalian skeleton. The exact mechanism by which bone can be modeled at a local site with little or no effect at adjacent anatomic sites is unknown. Disruption of the control of modeling within the temporal bone may lead to various bone disease such as otosclerosis, osteogenesis imperfecta, Paget's disease of bone, fibrous dysplasia, or the erosion of bone associated with chronic otitis media. One possible mechanism for such delicate control may be related to the ubiquitous and rich sympathetic innervation of all periosteal surfaces. Previous studies have indicated that regional sympathectomy leads to qualitative alterations in localized bone modeling and remodeling. In this study, unilateral cervical sympathectomy resulted in significant increases in osteoclast surface and osteoclast number within the ipsilateral bulla of experimental animals. The mechanisms by which sympathectomy leads to increased local bone loss is unknown. Potential mechanisms include disinhibition of resorption, secondary to the elimination of periosteal sympathetics, as well as indirect vascular effects.

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 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: Forty-eight Mongolian gerbils were placed into four groups: group 1 received intraperitoneal injections of 6-hydroxydopamine at 75 micrograms/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 micrograms/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 histomorphometry 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 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