Blood-labyrinth barrier and fluid dynamics of the inner ear.

Under normal conditions, the inner ear possesses remarkably stable homeostatic mechanisms for the maintenance of functional integrity of the inner ear fluid. The inner ear fluid maintains its homeostasis by a variety of regulatory mechanisms such as an ion transport system, a blood-labyrinth barrier, and a constant blood supply. Highly regulated transport of ions into and out of the inner ear provides for the maintenance of inner ear fluid composition necessary for auditory transduction. Any disturbance in one of these mechanisms can disrupt homeostasis expressed by ionic, osmotic, or metabolic imbalance between the compartments. Free radicals, stress hormones, noise exposure, and aminoglycoside antibiotics may induce short- and long-term effects on cellular function of the auditory or vestibular system (or both) and serve as a triggering mechanism for abrupt functional disturbances of inner ear fluid ion homeostasis. In this article, we present a comprehensive review of the mechanisms underlying inner ear fluid homeostasis necessary for normal auditory function and factors that can disrupt homeostasis and lead to functional disturbances, namely sensorineural hearing loss, tinnitus, and vertigo.

Expression of inducible nitric oxide synthase (iNOS) in middle ear epithelial cells by IL-1beta and TNF-alpha.

OBJECTIVE: The importance of nitric oxide (NO) in the development of mucoid middle ear effusion (MMEE) has been reported, but the mechanism regulating NO release is unclear. We hypothesized that middle ear epithelial cells (MEEC) are an important source of NO and that cytokines may be responsible for inducible nitric oxide synthase (iNOS) mRNA expression in middle ear epithelial cells. This study aims to identify and localize iNOS in middle ear epithelium, and to characterize the effects of cytokines IL-1beta and TNF-alpha on the expression and regulation of iNOS in rat middle ear epithelial cells. METHODS: In vitro study: 40 Healthy adult Sprague-Dawley rats weighing 200-250 g were used as donors of MEEC. Cultured MEEC were exposed to IL-1beta (10 ng/ml), TNF-alpha (5 ng/ml) or PBS (negative control) stimulation for 16 h. In vivo study: A total of 45 healthy adult Sprague-Dawley rats weighing 200-250 grams were used for this study. The tympanic bullae were exposed bilaterally by a submandibular approach. Animals were equally divided into three groups and inoculated with either 250 ng of IL-1beta, 250 ng of TNF-alpha or PBS. A PBS group served as control. Expression of iNOS mRNA in MEEC from both in vivo and in vitro studies was determined by RT-PCR using specific primers. Expression of iNOS protein in MEEC was determined by immunocytochemistry and Western blot using specific anti-iNOS antibody. RESULTS: Primary culture of rat MEEC was positively stained by cytokeratin antibody, but not by vimentin, indicating the epithelial origin of the cultured cells. RT-PCR revealed that cultured MEEC without treatment of IL-1 beta or TNF-alpha did not express iNOS mRNA whereas cultured MEEC treated with IL-1beta or TNF-alpha for 16 h expressed iNOS mRNA. Both immunocytochemistry and Western blot demonstrated the expression of iNOS protein in the majority of cultured MEEC treated with IL-1beta or TNF-alpha for 16 h, whereas the expression of iNOS protein was not detectable in MEEC without treatment. Expression of iNOS protein in vivo was observed in middle ear mucosa treated with IL-1beta and TNF-alpha by immunohistochemistry. CONCLUSION: Expression of iNOS mRNA and iNOS protein is induced in MEEC following the treatment of cytokines IL-1beta or TNF-alpha both in vivo and in vitro. The results of the present study demonstrate that rat MEEC possess the capacity to express iNOS after IL-1beta and TNF-alpha stimulation.

Induction of mucin gene expression in middle ear of rats by tumor necrosis factor-alpha: potential cause for mucoid otitis media.

Mucoid otitis media (MOM) is characterized by viscous fluid, high in mucin concentration, which accumulates in the middle ear cavity. Recent studies suggest that initial infection in the middle ear cleft may be key to the development of MOM. However, factors of the initial infection attributed to the stimulation of mucin production are not clearly understood. This study demonstrated that tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine in mucoid effusion, markedly increased Muc2 mucin mRNA expression in middle ear epithelium, in a time- and dose-dependent manner. Parallel to this was a marked increase in mucin glycoprotein in middle ear fluid. Also, TNF-alpha demonstrated an autocrine and/or paracrine effect on the expression of endogenous TNF-alpha gene in the middle ear, which may contribute to the production of mucin in this study. These findings suggest that TNF-alpha plays an important role in the development of MOM by stimulating mucin metabolism.

Immunohistochemical localization of collagen types I, III, and IV, laminin, fibronectin, and keratin in the endolymphatic sac.

We have employed immunohistochemistry to obtain baseline information on the molecular constituents of the extracellular matrix (ECM) of the endolymphatic duct (ED) and endolymphatic sac (ES) of the chinchilla. The results demonstrated that collagen types I and III were distributed in the subepithelial layer in the ED and ES, type IV collagen and laminin in the basement membranes, and fibronectin in the subepithelial layer and partly in the conglomerated cells in the ES. Collagen type III was diffusely distributed in the whole subepithelial layer of the ES, whereas collagen type I was concentrated densely in the deep layer of the interstitium, although gradually, the cuboidal epithelium in the ES was transformed into a flatter type in the ED. The epithelial cells of the ED and ES were clearly positive for keratin. This study deals, in particular, with the normal distribution of ECM components of the ED and ES of the chinchilla.

Effect of stress-related hormones on inner ear fluid homeostasis and function.

HYPOTHESIS: Induction of suprathreshold levels of stress-related hormone by systemic administration of epinephrine can change inner ear fluid homeostasis and function. BACKGROUND: Meniere's disease is frequently associated with high levels of anxiety and other forms of psychological disturbance. Most clinicians agree that emotional stress or severe anxiety can precipitate relapse or aggravate the symptoms. In general, it is known that stress-related hormones such as epinephrine, norepinephrine, vasopressin, aldosterone, and cortisol are released into systemic circulation in response to stress. Significantly higher levels of plasma norepinephrine and vasopressin in patients with Meniere's disease have been reported. METHODS: Concentrations of sodium and potassium in perilymph were measured by a flame photometer after systemic infusion of epinephrine (6.3 microg/min for 3 hours). Control animals were treated with equal volumes of 0.9% physiologic saline. Compound action potentials (CAP) elicited by brief tone bursts were measured before and 3 hours after the infusion of epinephrine. For chronic studies, epinephrine (10 microg/d/kg) was given by osmotic pump implanted subcutaneously for 1, 2, 3, and 4 weeks, respectively. Click- and tone-evoked auditory brain responses (ABRs) were measured at 1, 2, 3, and 4 weeks after epinephrine administration. RESULTS: Concentrations of sodium and potassium increased significantly in perilymph (p < 0.001 and p < 0.01) after epinephrine infusion over controls. The osmolality increased significantly in serum and perilymph after epinephrine infusion. The CAP threshold was significantly elevated at all frequencies. The shift of the CAP threshold caused by epinephrine tended to be larger at higher frequencies. In chronic studies, epinephrine administration caused a transient 20 to 45 dB threshold shift that increased with time and was relatively constant across frequency. CONCLUSIONS: There is good evidence to suggest that stress-related hormones such as epinephrine can alter inner ear fluid homeostasis and auditory function. This study confirmed this hypothesis and illuminated the processes of alteration by demonstrating specific changes in perilymph composition and auditory function.

Immunohistochemical distribution of extracellular matrix components and keratin in experimentally induced otitis media.

The distribution of collagen types I, III, and IV and of laminin, fibronectin, and keratin was studied in otitis media experimentally induced by Streptococcus pneumoniae in the chinchilla. The expression of interstitial collagen types I and III and of fibronectin was increased in the subepithelial space that was thickened by inflammation in the acute period of infection. The expression of collagen type IV in the subepithelial space could be seen in the early period. The epithelial cells in the middle ear changed from flat cuboidal to pseudostratified columnar in pneumococcus-inoculated ears, and the number of keratin-positive epithelial cells in the middle ear increased remarkably after infection. These results indicate that changes in epithelial cell differentiation effected by the extracellular matrix correlate with changes in expression of keratin. It is proposed that the extracellular matrix may contribute to tissue repair in the middle ear after bacterial infection by interfering with cell proliferation of epithelial cells and fibroblasts.

Early-response cytokine expression in adult middle ear effusions.

Various cytokines are presently known to be associated with the regulation of inflammatory responses. In pediatric otitis media, cytokines that correlate with various degrees of inflammation are present in middle ear effusions as inflammatory mediators. The present study was undertaken to examine the potential role of the early-response cytokines, interleukin-1beta and tumor necrosis factor-alpha, in adult otitis media. Fifty-nine adults with otitis media underwent tympanocentesis, and the effusion specimens were analyzed for the presence of both cytokines by enzyme-linked immunosorbent assay methods. Eighty-eight percent of the effusions were serous in nature. Sixty-seven percent of the patients had a known history of head and neck malignancy and radiation to the temporal bone. Twelve percent of the effusions were positive for interleukin-1beta expression, compared with 85% of effusions in children with otitis media. Eight percent of the effusions contained tumor necrosis factor-alpha, compared with 85% of those collected in pediatric otitis media. All of the specimens that contained tumor necrosis factor-alpha also contained interleukin-1beta. In the present study, there was no correlation with head and neck malignancy/radiation or the clinical degree of inflammation with the presence of either cytokine. We conclude that adult otitis media is associated with lower expression of an acute inflammatory response, as judged by the levels of interleukin-1beta and tumor necrosis factor-alpha in the effusions. Additionally, adult otitis probably represents a less severe and more chronic inflammatory state in comparison with pediatric otitis media. Further analysis of inflammatory mediators in adult otitis media is necessary to evaluate the contribution of cytokines in relation to various etiologic factors.

Increase of mucous glycoprotein secretion by tumor necrosis factor alpha via a protein kinase C-dependent mechanism in cultured chinchilla middle ear epithelial cells.

Tumor necrosis factor alpha (TNF-alpha), originally defined by its antitumoral activity, is now recognized as a polypeptide mediator of inflammatory and cellular immune response. Recent studies have demonstrated that TNF-(alpha exists in the fluid of otitis media with effusion and, therefore, suggested its possible role in the pathogenesis of mucus hypersecretion. In this study, the effects of TNF-alpha on mucous glycoprotein (MGP) secretion from cultured chinchilla middle ear epithelial cells were examined, and TNF-alpha was found to stimulate MGP secretion in a time- and concentration-dependent manner. The action of TNF-alpha on MGP secretion was significantly and dose-dependently inhibited by TNF-alpha monoclonal antibody; this finding is suggestive of its specificity on MGP secretion. The addition of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperidine (H-7) to the culture significantly blocked TNF-alpha-induced MGP secretion, while the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) did not. This suggests that TNF-alpha stimulates MGP secretion via a protein kinase C-dependent mechanism.

Two types of K+ currents in marginal cells cultured from rat stria vascularis.

Membrane currents in marginal cells cultured from rat stria vascularis were studied using the whole-cell patch-clamp technique. Two types of voltage-dependent whole-cell currents were observed in the voltage range from -150 mV to +50 mV: an outwardly rectifying current and an inwardly rectifying current. The outwardly rectifying current, which was activated by depolarizing pulses more positive than -30 mV, was sensitive to TEA (20 mM) and relatively not to Ba2+ (0.5 mM). Tail current analysis revealed that the outward currents were primarily K+-selective. The conductance of the current was half-maximal at 0.5 mV and a substantial portion of current was not inactivated by the depolarizing prepulses from -30 mV to +20 mV. The inwardly rectifying current with rapid exponential activation was observed with hyperpolarizing voltage pulses. The zero-current potential of this current was dependent on extracellular K+ concentration. In contrast to the outwardly rectifying current, this current was blocked by extracellular application of Ba2+, not by TEA. The conductance of this current increased with the increase in external K+ concentration. Our data suggest that marginal cells cultured from rat stria vascularis express at least two types of voltage-dependent K+ currents which may serve as K+ secretory pathways into endolymph.

Dexamethasone inhibits mucous glycoprotein secretion via a phospholipase A2-dependent mechanism in cultured chinchilla middle ear epithelial cells.

Inhibition or attenuation of mucous hypersecretion in middle ear epithelium is a key step toward resolution of mucoid otitis media. Mucous hypersecretion induced by platelet-activating factor (PAF) in cultured Chinchilla middle ear epithelial cells is dependent on arachidonic acid metabolites via PAF receptors, suggestive of the role of phospholipase A2 (PLA2) in mucous glycoprotein (MGP) secretion. In this study, dexamethasone added to cultured Chinchilla middle ear epithelial cells inhibited baseline and PAF-induced MGP secretion in a concentration-dependent manner. A definite time lag (16 h) was observed between administration of dexamethasone and MGP inhibition. This inhibition was reversed by the addition of exogenous PLA2 (the rate-limiting enzyme of arachidonic acid metabolism) and actinomycin D (an inhibitor of mRNA synthesis). This suggests that dexamethasone inhibits baseline and PAF-induced MGP secretion via a PLA2-dependent mechanism.

Tumor infiltrating lymphocytes in squamous cell carcinoma of the head and neck: mechanisms of enhancement using prostaglandin synthetase inhibitors.

Indomethacin has been shown clinically to inhibit growth of SCCHN (Panje, 1981). This inhibition appears to be due to augmentation of cellular immunity. The inhibitory effect of indomethacin may act by limiting tumor associated prostaglandin E2 production, thereby allowing return of costimulatory cytokines by antigen presenting cells. This would have the net result of relief from host unresponsiveness and promotion of B-cell and CTL differentiation, allowing the individual to mount an effective response. The enhancement of tumor infiltrating lymphocytes in SCCHN seen with indomethacin administration could presumably be further augmented when given in combination with cytokine therapy. Future investigation may allow the biochemical staging of an individuals' tumor to determine the optimal combination of cytokine therapy and prostaglandin inhibition through selective use of NSAID's. The effect of NSAID manipulation of prostaglandin and leukotriene metabolism on prevention of metastatic disease in SCCHN has yet to be studied. Given that a preselected, potentially responsive subset of immunocytes exists within the tumor tissue and lymph nodes, the development of the LAK phenomenon in TIL's and tumor draining lymph nodes from surgical specimens is a viable and exceedingly interesting area for future investigations in autologous LAK immunotherapy. The potential exists to harvest a preselected population of tumor infiltrating (Boscia, 1988) or tumor draining immunocytes (McKinnon, 1990). These can then potentially be returned to a state of antigen responsiveness with a combination of cytokine exposure (e.g. rIL-2) and systemic cytokine therapy. With subsequent inhibition of tumor associated prostaglandin synthesis by the systemic administration of prostaglandin synthesis inhibitors, it may be possible to successfully alter the host response to tumor.

Effect of lipoxygenase inhibition on mucous glycoprotein secretion from chinchilla middle ear epithelial cells in vitro.

Lipoxygenase is an enzyme that metabolizes arachidonic acid down to leukotrienes. Recent studies have shown that the enzyme is implicated in mucous glycoprotein (MGP) secretion stimulated by inflammatory mediators in the airways, suggesting its possible role in secretion of MGP from middle ear epithelial cells. To investigate a correlation between MGP secretion and the arachidonic acid metabolites, we examined the effects of nordihydroguaretic acid (NDGA, both a cyclooxygenase and lipoxygenase inhibitor), low-dose indomethacin (an inhibitor of cyclooxygenase), and A63162 (an inhibitor of lipoxygenase) on MGP secretion in cultured chinchilla middle ear epithelial cells. It was found that lipoxygenase inhibition led to reduction of MGP secretion from cultured chinchilla middle ear epithelial cells, while cyclooxygenase inhibition did not. Both cyclooxygenase and lipoxygenase inhibition resulted in profound blockage of MGP secretion in baseline and platelet activating factor-stimulated MGP secretion. It was concluded, therefore, that MGP secretion was linked to arachidonic acid metabolites, especially lipoxygenase products.

Treatment of chronic tympanic-membrane perforations with a platelet-derived releasate.

PURPOSE: Tympanic membrane perforations are very common and often require surgical treatment. Recent studies have suggested that growth factors may be an effective nonsurgical alternative for treating chronic perforations. The purpose of this study was to assess the efficacy of a platelet releasate in the treatment of chronic nonhealing perforations in the chinchilla model. METHODS: Bilateral perforations were created in 47 chinchillas by excising 80% of the tympanic membrane with a thermal myringotomy knife. Bilateral perforations > 50% of its surface area persisted for 10 weeks in 34 animals, and unilateral perforations > 50% of its surface area, in nine animals. Only animals with bilateral chronic perforations were included in this study. After deepithelializing the perforation and packing the middle ear and external ear canals with Gelfoam, we treated the perforations with either platelet releasate or buffered saline. Each animal served as its own control. RESULTS: The tympanic membranes were evaluated over a 12-week period by microscopy, photography, tympanometry, and histology. No statistical difference between treated and control ears in the incidence of perforation closure was evident. Histologically, the treated tympanic membranes consistently had a thicker fibrous layer than the controls. CONCLUSIONS: These data suggest that platelet releasate is not effective in enhancing closure of chronic tympanic membrane perforations in the chinchilla model.

Inhibition of head and neck tumor cell growth with arachidonic acid metabolism inhibition.

Head and neck tumors are known to synthesize arachidonic acid metabolites. The authors have postulated that these substances may confer growth advantage to cancer cells, and by interfering with arachidonic acid metabolism squamous cancer growth may be altered. The tongue derived squamous carcinoma cell line, SCC-25, was treated with three leukotriene synthesis inhibitors and indomethacin. A dose-dependent decrease in DNA synthesis occurred with leukotriene inhibition, but not prostaglandin inhibition. All leukotriene synthesis inhibitors produced a dramatic and immediate effect ( > 70% inhibition by 4 hours) without cytotoxicity ( > 90% trypan blue exclusion). Cell populations at 96 hours were decreased when compared to control populations. In conclusion, leukotrienes or other lipoxygenase products may play a role as growth factors for squamous cell carcinoma, and arachidonic acid inhibition may be a novel target for chemotherapeutic intervention.

Vascular effects of sustained-release fibroblast growth factors.

Since the half-life of most angiogenic growth factors is several hours or less, sustained-release delivery would be optimal for their future clinical use. Two fibroblast growth factors, basic fibroblast growth factor (bFGF) and endothelial cell growth factor (ECGF), were delivered in two sustained-released modalities (poloxamer 407 and a gelatin sponge [Gelfoam]) to attempt to increase soft tissue vascularity. In vitro bioactivity of ECGF-poloxamer formulations was also tested on endothelial cell cultures. Among vascular-compromised skin flaps in rabbits, ECGF-poloxamer (N = 26), bFGF-poloxamer (N = 5), ECGF-poloxamer (N = 9, irradiated), and bFGF-Gelfoam flaps (N = 22) did not demonstrate significant differences in viability and vascularity compared to controls (p > .05). Irradiation had a detrimental effect on both flap vascularity and viability (p = .02). Future efforts for sustained delivery of angiogenic proteins are critical in order to make them clinically useful in wound healing.

Effects of platelet-activating factor (PAF) receptor blockage on mucous glycoprotein secretion in cultured chinchilla middle ear epithelium.

Platelet-activating factor (PAF) is a naturally occurring phospholipid that acts as a pleiotropic mediator of inflammation via specific membrane receptors. It has been demonstrated in recent years that PAF is a strong secretagogue of mucous glycoprotein (MGP) in airways and middle ear epithelium. MGP secretion accompanies otitis media with effusion (OME) and prolongs the course of this disease by increasing the viscoelasticity of the fluid. It is important, therefore, to inhibit the pathological secretion of MGP in the treatment of otitis media. In the current study, we investigated the effects of PAF receptor inhibitor WEB 2170 BS on in vitro MGP secretion. At concentrations of 100 mu M, PAF significantly stimulated MGP secretion. WEB 2170 BS significantly inhibited this PAF-simulated MGP secretion at concentrations of 2000 mu M. The action of WEB 2170 BS was concentration-dependent. However, it did not affect MGP secretion stimulated by IL-1beta, suggesting that WEB 2170 BS inhibits PAF-stimulated MGP secretion, specifically. It was noted that WEB 2170 BS did not completely eliminate MGP secretion induced by PAF even though a high concentration was used. The fact that WEB 2170 BS alone does not exhibit an inhibitory or stimulatory effect on the secretion of MGP suggests that WEB 2170 BS competitively binds to PAF receptors and possesses less affinity to PAF receptors than PAF.

Mucociliary activity and histopathology of sinus mucosa in experimental maxillary sinusitis: a comparison of systemic administration of antibiotic and antibiotic delivery by polylactic acid polymer.

To evaluate the efficacy of antibiotic delivered by polyactic acid (PLA) polymer in sinusitis, we induced maxillary sinusitis in 32 New Zealand white rabbits by occluding the sinus ostium and inoculating the sinus cavity with Streptococcus pneumoniae. The rabbits were divided into three groups consisting of group 1 (control group, 8 rabbits), which was treated only by reopening the ostium; group 2, which was treated by both reopening the ostium and injecting ampicillin intramuscularly (40 mg/kg/day in three divided doses, 12 rabbits); and group 3 (12 rabbits) in which a piece of PLA-polymer ampicillin (0.326 mg) sheet (1.5 x 1.5 cm) was placed within the sinus after re-establishing ostial patency. The light microscopic findings such as epithelial ulceration, loss of cilia, infiltration of inflammatory cells, and edema were less pronounced in group 2 and minimal in group 3. The electron microscopic findings such as swelling of mitochondria and endoplasmic reticulum and protruded cytoplasm were severest in the control group, followed by groups 2 and 3. The mucociliary transport speed measured at the medial wall of the maxillary sinus was highest in group 3. The results of this study suggest that treatment with PLA-polymer ampicillin may have a better efficacy in maxillary sinusitis than that with systemic administration of ampicillin.

Effect of platelet-activating factor on secretion of mucous glycoprotein from chinchilla middle ear epithelial cells in vitro.

Platelet-activating factor (PAF) is a naturally occurring phospholipid that acts as a pleiotropic mediator and mediates cell-cell reactions under physiological and pathological conditions. Recently, it has been shown that PAF is a strong secretagogue of mucous glycoprotein in the airways, suggesting its role in mucous glycoprotein secretion and the pathogenesis of otitis media with effusion. In the current study, we examined the effect of PAF on mucous glycoprotein secretion in cultured chinchilla middle ear epithelial cells. PAF at 1 microM significantly stimulated mucous glycoprotein secretion from cultured chinchilla middle ear epithelial cells. This action was concentration-dependent, with secretions reaching near maximum when the cells were incubated with PAF at 100 microM. In a time-dependent study, PAF demonstrated an initial rapid stimulation of mucous glycoprotein secretion, followed by a gradual increase thereafter. A six-fold increase was seen in the first 2 h compared with controls. Cycloheximide, a protein synthesis inhibitor, demonstrated an inhibitory effect on PAF-stimulated mucous glycoprotein secretion in this study. These findings suggest that PAF plays an important role in the pathogenesis of otitis media with effusion by stimulating mucous glycoprotein secretion in vitro.