Keratin particle-induced osteolysis: a mouse model of inflammatory bone remodeling related to cholesteatoma.

We implanted keratin and poly(methyl methacrylate) (PMMA) particles to the surface of mouse calvariae to produce a quantitative, localized, inflammatory bone remodeling similar to that seen in cholesteatoma. Both types of particles resulted in increased osteoclast density compared with controls. Osteoclasts infiltrated from marrow and vascular spaces and were active at the periphery of these spaces leading to significant bone remodeling, as demonstrated by the incorporation of bone-labelling fluorophores. Osteoclasts were rarely found on the surface of the calvariae, and mineral apposition rate at the ventral surface was not altered in keratin-implanted animals compared with nonoperated controls. While not useful for the study of the root cause of cholesteatoma, this model will allow the study ofpathologic bone remodeling related to cholesteatoma in a genetically defined animal.

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.

Expression patterns of cytokeratins in retraction pocket cholesteatomas.

OBJECTIVES: To investigate the patterns of cytokeratin (CK) expression in retraction pocket cholesteatoma. STUDY DESIGN: An animal model study. METHODS: Retraction pocket cholesteatomas were induced by electrocautery of the eustachian tube orifice in 24 mongolian gerbils. They were divided into normal and cholesteatoma groups of clinical stages I to IV. The antibodies to pan-cytokeratin CK 1/10, CK 5/6, CK 4, and CK 13/16 were used for immunohistochemical staining. The intensity of staining in each group as measured with densitometry was compared regarding anatomical sites and clinical stages. RESULTS: In retraction pocket cholesteatoma, CK expression was altered only at focal sites such as the pars tensa of the tympanic membrane. The change of CK expression was observed only at certain stages of cholesteatoma formation. In keratinocytes from cholesteatomas, CK 13/16 was overexpressed compared with control specimens, indicating hyperproliferation. The site with the most prominent change in retraction pocket cholesteatoma was somewhat different from that in canal ligation cholesteatoma in a previous study. CONCLUSIONS: The results suggested that aural cholesteatoma is a disease with a spectrum of pathological conditions and that the transmigration and hyperproliferation process of squamous epithelium occurs in areas adjacent to the cholesteatoma.

Chronic bacterial rhinosinusitis: description of a mouse model.

OBJECTIVES: To survey normal murine sinonasal anatomy and to create a mouse model for chronic bacterial rhinosinusitis. DESIGN: Anatomic, histologic, and pathophysiologic study displaying normal murine sinonasal anatomy and surgically created unilateral sinonasal inflammation. SUBJECTS: Twenty-one 6-week-old, male C57BL/6 mice. INTERVENTIONS: Animals that underwent unilateral maxillary sinus ostial obstruction using Merocel nasal packing, animals with unilateral Bacteroides fragilis inoculation alone, and animals with both ostial obstruction and bacterial inoculation were examined at 4 weeks for histologic evidence of chronic sinonasal inflammation. Experimental interventions were compared with contralateral control sinuses within each animal and with normal and sham-operated controls. RESULTS: Normal mouse paranasal sinuses include maxillary sinuses, ethmoid air cells, and respiratory-type epithelium. In experimental animals, the lateral maxillary sinus wall, nasal septum, and superior turbinelle of the maxillary sinus were examined histologically. Epithelial thickening and disarray, goblet cell hyperplasia, inflammatory infiltrates, and sinonasal fibrosis were present in the experimental sinuses of animals packed with Merocel alone or Merocel with bacterial inoculation. Changes seen with Merocel and bacteria were more dramatic than those with Merocel alone. Sham-operated controls and sinuses inoculated with bacteria alone did not differ significantly from the sinuses of normal animals. CONCLUSION: Unilateral maxillary sinus ostial obstruction using Merocel nasal packing along with B fragilis inoculation results in a persistent, localized bacterial rhinosinusitis in mice.

Invasiveness of fibroblasts from experimental cholesteatomas.

HYPOTHESIS: Cultured fibroblasts derived from experimental gerbil cholesteatoma tissue exhibit an invasive phenotype in comparison with normal fibroblasts. BACKGROUND: Aural cholesteatomas are enlarging accumulations of keratin debris caused by keratinizing squamous epithelium in the middle ear. They characteristically result in the destruction of adjacent tissues, specifically bone erosion. The mechanisms by which cholesteatomas relentlessly invade the structures of the temporal bone are varied, but it has been suggested that one factor contributing to the aggressive nature of cholesteatomas is the transformation of resident fibroblasts into an invasive phenotype. METHODS: The ability of cultured normal and cholesteatoma fibroblasts to invade a basement membrane matrix in a Boyden chamber assay was examined. RESULTS: Less than 1% of gerbil fibroblasts invaded the matrix, compared with almost 10% of the invasive HT-1080 fibrosarcoma cells. Normal and cholesteatoma fibroblasts did not differ from each other in their invasive potential. CONCLUSION: Normal fibroblasts and fibroblasts from induced cholesteatomas do not exhibit the invasive phenotype characteristic of true neoplastic cells.

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.

Pathophysiology of otosclerosis.

OBJECTIVE: To review current knowledge of the pathophysiology of otosclerosis and to review hypotheses for the amelioration of this disease. DATA SOURCES: Review of the literature and experimental observations by the authors. CONCLUSIONS: Otosclerosis is a localized disease of bone remodeling within the otic capsule of the human temporal bone. Unlike other similar bone diseases, it does not occur outside of the temporal bone. These lesions seem to begin by resorption of stable otic capsule bone in adults, followed by a reparative phase with bone deposition. There are clearly genetic factors that lead to this disease, but measles virus infection and autoimmunity also may play contributing roles. Surgical correction of the conductive hearing loss is highly effective, but nonsurgical intervention has not yet been shown to prevent or slow the disease. Of the factors that may inhibit this process, fluorides, cytokine inhibitors, and bisphosphonates, third-generation bisphosphonates appear to hold the most promise.

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.

Middle ear reconstructive techniques.

Middle ear reconstructive techniques have increased to such an extent that the otolaryngologist is faced with a bewildering number of surgical options. The literature, however, has yet to demonstrate any technique to be optimal in every surgical scenario. This is because of the lack of direct comparisons between techniques and dissimilar criteria for measuring success. This article uses hearing results and the ability to resist extrusion as the principle measures of success, and provides middle ear reconstructive techniques proven to be successful for commonly faced surgical dilemmas.

Cytochrome P-450 inhibition blocks bone resorption in vitro and in vivo.

Although the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism have been shown to lead to osteoclastic resorption, the cytochrome P450 pathway has not been implicated. We investigated the effects of the cytochrome P450 pathway in IL-1beta-induced calcium release from cultured mouse calvaria in vitro in the presence of clotrimazole, a cytochrome P450 inhibitor, or L-N(G)-arginine methyl ester, a nitric oxide synthase inhibitor. Clotrimazole inhibited calcium release in a dose-dependent manner; however, L-N(G)-arginine methyl ester did not inhibit resorption. These results suggest that cytochrome P450 may be another possible mediator of IL-1beta-induced bone resorption in vitro. In the in vivo portion of the study, clotrimazole was administered in the gerbil model of adaptive bone modeling. Clotrimazole inhibited osteoclast surface; however, it did not reduce the osteoclast number, mean erosion surface per osteoclast, mineralization surface, or mineral-apposition rate. These results suggest that clotrimazole may inhibit the activation of osteoclasts and that cytochrome P450-dependent enzymes may be related to osteoclast activation in vivo.

Inhibition of nitric oxide synthase blocks osteoclastic bone resorption in adaptive bone modeling.

In this study, the auditory bulla of the gerbil was pressurized, leading to active modeling of the bone of the bulla wall with a significant increase in osteoclast surface and mineral apposition rate. Systemic infusion of L-N(G)-nitro-arginine-methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), inhibited this modeling process. The percentage osteoclast surface (Oc.S/BS) on the inner surface bulla wall was significantly reduced in the L-NAME-treated animals when compared with pressurized saline-treated bullae. Fluorescent bone surface (BSf) mineral apposition rates (MAR) and bone formation rate (BFR) were not significantly different in the pressurized bullae when the L-NAME group was compared with the control (vehicle only) group. However, L-NAME significantly suppressed BSf in the unpressurized bullae. Therefore, it is likely that nitric oxide is a mediator of osteoclastic resorption due to adaptive bone modeling through one or more of the isoforms of NOS.

Adaptive bone modeling in the middle ear is substance-P dependent.

HYPOTHESIS: Localized bone modeling in the middle ear is substance-P dependent. BACKGROUND: Processes of local bone modeling and remodeling in the middle and inner ear lead to destructive processes such as otosclerosis and chronic otitis media. The cellular events associated with these processes are known, but the mechanisms of the control and activation of the involved cells are not. The authors hypothesized that one of the control mechanisms of local bone modeling is related to the action of a neuropeptide, substance-P and that capsaicin, which depletes substance-P, would block modeling in the gerbil model of adaptive bone modeling. METHODS: One middle ear of each of 24 Mongolian gerbils was pressurized to 10 mmHg to induce adaptive bone modeling. Half of the animals were pretreated with capsaicin and half received vehicle alone. At the end of the 5-day experimental period, the bulla was studied histomorphometrically for osteoclastic and osteoblastic activity. RESULTS: Capsaicin pretreatment inhibited the percent of bone occupied by osteoclasts on the inner surface of the bulla and the rate of mineralization of bone on the outer surface of the bulla. CONCLUSIONS: It is likely that substance-P is a mediator of localized adaptive bone modeling in vivo. Processes of bone modeling and remodeling in the middle and inner ear may be under neural control.

Experimental models of aural cholesteatomas in Mongolian gerbils.

Mongolian gerbils have a remarkable propensity for the development of aural cholesteatoma; canal cholesteatomas develop spontaneously in aged animals. In the present study, cholesteatomas were produced in a single species, Meriones unguiculatus, by three different methods of induction: 1) external canal ligation (ECL), 2) eustachian tube obstruction (ETO) by electrocautery, and 3) application of propylene glycol (PG) into the middle ear. Each method of induction resulted in cholesteatomas with characteristic features and success rates. With ECL, cholesteatomas could be induced in all ligated ears; stage III and IV cholesteatomas were seen in 4 of 12 ears after 8 weeks. With ETO, cholesteatomas were seen in 7 of 9 animals 8 weeks after induction. With PG, cholesteatomas developed in 2 of 7 animals 4 weeks after induction, although keratin accumulation in the external auditory canal could be found in all animals injected. Although epithelial hyperplasia of the external auditory canal was most prominent in the ECL group, it was also seen in the ETO and PG groups. Keratin accumulation in the ECL or PG group was much greater than that in the ETO group. Thickening of the tympanic membrane, which was most prominent in the PG group, was quite variable in the other groups. Adhesions of the tympanic membrane began at the prominence of the cochlea in the ECL group. In contrast, adhesions in the PG group usually began at the superior bulla, the area in which PG was applied. Each of the three methods of inducing cholesteatoma may be helpful in investigating different clinical aspects of this disease.

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.

Unusual lesions of the internal auditory canal.

Tumor types other than acoustic neuromas are uncommonly encountered in the cerebellopontine angle, and unusual lesions limited to the internal auditory canal (IAC) are given more rare. We present five patients with unusual tumors of the IAC including two meningiomas, two arachnoid cysts, and a hemangioma. These unusual tumors, and lipomas, are discussed. Particular attention is paid to the pathophysiology of the lesions and treatment recommendations. The differential diagnosis of intracanalicular lesions using magnetic resonance imaging is also presented.