Late-phase allergy and eustachian tube dysfunction.

OBJECTIVE: This study investigated the role of late-phase allergy in the development of otitis media with effusion. METHODS: Brown Norway rats were sensitized to ovalbumin and later challenged transtympanically. Eustachian tube ventilatory function was assessed 2, 4, 8, 24, 28, and 32 hours postchallenge by measuring passive opening and closing pressures, active clearance of positive and negative middle ear pressure, and mucociliary clearance. RESULTS: The results demonstrate that exposure to transtympanic allergen induces eustachian tube dysfunction and subsequent formation of effusion. Allergic animals showed significant increases in passive and active opening pressures, as well as a decreased ability to actively clear middle ear pressure. Finally, the mucociliary was significantly impaired in all sensitized rats exposed to transtympanic allergen. CONCLUSION: These findings demonstrate that late-phase allergy leads to significant eustachian tube dysfunction and subsequent formation of effusion by impairing the ventilatory and clearance functions of the eustachian tube.

Function of the eustachian tube after weekly exposure to pepsin/hydrochloric acid.

OBJECTIVE: To determine the effects of repeated pepsin/hydrochloric acid (HCl) exposure on the eustachian tube (ET). STUDY DESIGN AND SETTING: ET function was studied in 22 rats. Group I (control) rats received transtympanic phosphate buffered saline solution; groups II (0.5 mg/ml) and III (2.0 mg/ml) received transtympanic pepsin/HCl. Test solutions were applied on day 0 with ET function evaluated on days 1, 2, 3, and 7 after exposure. Each 7-day period represents 1 cycle; all groups underwent 4 cycles. ET function was evaluated using passive opening and closing pressure, and active clearance of positive and negative pressure tests. RESULTS: Rats exposed to pepsin/HCl had elevated passive opening pressures and a decreased ability to clear positive and negative pressure. A temporal relationship exists. CONCLUSION: The results suggest middle ear exposure to pepsin/HCl leads to ET dysfunction in rats, and that this dysfunction is enhanced with repeated exposures. SIGNIFICANCE: Gastroesophageal reflux may induce ET dysfunction.

Action of histamine on eustachian tube function.

INTRODUCTION: The role of allergy in eustachian tube dysfunction is controversial. In this study, allergy was simulated by exposure to histamine, and eustachian tube function testing was performed in an experimental rat model. METHODS: Ventilatory function was assessed by measuring passive opening and closing pressures of the eustachian tube after challenge with either transtympanic or intranasal histamine. The mucociliary clearance time of the tubotympanum was assessed by observing dye transport from the middle ear to the nasopharynx after challenge with either transtympanic histamine or control solution. RESULTS: There was a statistically significant increase in passive opening and closing pressures with transtympanic histamine versus intranasal histamine. In addition, mucociliary clearance times of the tubotympanum after transtympanic histamine showed a statistically significant increase when compared with those after transtympanic control solution. CONCLUSIONS: Transtympanic histamine exposure causes eustachian tube dysfunction in the rat by increasing passive opening and closing pressures of the eustachian tube and impairing mucociliary clearance time.

Nitric oxide is a mediator of the late-phase response in an animal model of nasal allergy.

The presence of nitric oxide (NO) in the nose is well documented; however, the role of this molecule in nasal physiology is still poorly understood. Our laboratory has previously demonstrated that NO is a mediator of the immediate secretory response to an intranasal histamine challenge in a rat model of nasal allergy. Histamine challenge, however, does not elicit a late-phase response (LPR). To study the role of NO in the LPR, we developed a model of nasal allergy in which brown Norway rats are actively sensitized to the allergen ovalbumin and later challenged intranasally with either phosphate-buffered saline solution (vehicle), ovalbumin in vehicle, or ovalbumin and the NO synthase inhibitor N -nitro-l -arginine methyl ester. In each experiment, nasal lavage samples were collected 30, 120, 240, and 360 minutes after challenge. Lavage samples were analyzed for albumin content by ELISA, inflammatory cell concentration with a hemocytometer, and evidence of inflammation by light microscopy. Blocking NO synthesis with N -nitro-l -arginine methyl ester significantly inhibited both albumin exudation and inflammatory cell influx into the nasal cavity during the LPR. These data suggest that NO plays a role in the LPR of nasal allergy.

Allergy increases susceptibility to otitis media with effusion in a rat model. Second place--Resident Clinical Science Award 1998.

To investigate the possible relationship between allergy and otitis media with effusion (OME), we investigated the hypothesis that allergen presentation to the middle ear causes functional disruption of the eustachian tube predisposing to the development of OME. Thirteen of 19 Brown-Norway rats were sensitized to ovalbumin, and the remaining 6 served as nonallergic controls. To mimic subclinical exposure to allergen, we transtympanically injected ovalbumin at a dose (0.01 mg) that produced no changes detectable by otologic examination. Next, both allergic and nonallergic rats were exposed to transtympanic injection of either low-dose (10 microg/mL) or high-dose (100 microg/mL) lipopolysaccharide to simulate bacterial exposure. The allergic rats were found to have larger middle ear effusions when exposed to high-dose lipopolysaccharide as compared with the nonallergic controls. This response could be inhibited by diphenhydramine. We conclude that allergen presentation to the middle ear of allergic rats causes eustachian tube dysfunction predisposing to OME.

Systemic reactivation of otitis media with effusion in a rat model.

OBJECTIVE: This study addresses the interaction of bacterial antigens, specifically peptidoglycan-polysaccharide (PG-PS) and lipopolysaccharide (LPS), in the induction and reactivation of mucoid middle ear effusions. METHODS: Twenty-seven rats underwent eustachian tube obstruction before inoculation of the middle ear bulla with PG-PS. Three weeks later, after resolution of all middle ear effusions, 6 rats were randomly selected and euthanized as the first control group (control I). The remaining 21 animals were randomly assigned to 3 groups that received intravenous injections of Krebs Ringer (control II), PG-PS, and LPS, respectively. These rats were euthanized 2 days after intravenous injection. Middle ear mucin production and histologic changes were measured in all animals. RESULTS: The mean concentrations of mucin were 0.94 +/- 0.52 mg/mL, 0.41 +/- 0.87 mg/mL, 16.33 +/- 3.67 mg/mL, and 1.15 +/- 0.41 mg/mL in the control I, control II, PG-PS, and LPS groups, respectively. Thus the mean concentration of mucin in the middle ear lavage samples was significantly greater in rats that were injected intravenously with PG-PS than in rats in other groups (P < 0.05). Histologic analyses demonstrated a greater degree of goblet cell hyperplasia in the PG-PS group than in other groups. CONCLUSIONS: This is the first animal model of recurring otitis media with effusion in which a systemic injection of PG-PS was used to reactivate a middle ear effusion in rats previously primed with a transtympanic injection of PG-PS. This study suggests that after otitis media with effusion has resolved, it may be reactivated by the presence of bacterial antigens and/or cytokines in the systemic circulation.

Mucin production in the middle ear in response to lipopolysaccharides.

OBJECTIVE: This study examined the response of middle ear tissue to establish the lowest dose of lipopolysaccharide to induce mucin production in a rat otitis media model. METHODS: Twenty-six male Sprague-Dawley rats' eustachian tubes were obstructed before transtympanic inoculation of the bulla tympanica with 35 microL of Krebs Ringer or 1, 10, 100, or 1000 microgram/mL lipopolysaccharide. After 7 days the effusion and a lavage were collected for mucin ELISA measurement, and tissue was collected for histologic evaluation. RESULTS: Mucin secretion was significantly increased in the 100 microgram/mL 51.20 +/- 13.6 microgram/mL (SE) and 1000 microgram/mL 69.42 +/- 8.57 microgram/mL groups when compared with the Krebs Ringer control group 1.84 +/- 0.28 microgram/mL (P < 0.05). Histologic evaluation shows goblet cell metaplasia and hyperplasia in the middle ear epithelium in the 1000 and 100 microgram/mL groups. CONCLUSIONS: The histology and ELISA results suggest that a middle ear effusion is generated with a dose of lipopolysaccharide as low as 100 microgram/mL.

Sodium nitroprusside/nitric oxide causes apoptosis in spiral ganglion cells.

OBJECTIVE: In the cochlea, excitatory amino acid receptor overstimulation induces toxicity in spiral ganglion neurons by an unknown mechanism. In the central nervous system, excitatory amino acid-induced toxicity is mediated by nitric oxide, which induces apoptosis in neurons. This study tested the hypothesis that cochlear nitric oxide-mediated toxicity is the result of induction of apoptosis in spiral ganglion neurons. METHODS: The cochleas of 15 gerbils randomly assigned to different groups were perfused for 30 minutes with a test solution of 1 mmol/L sodium nitroprusside, a nitric oxide donor, or a control solution of artificial perilymph. Animals were killed at varying times, including 2, 3, 4, 8, and 18 hours after perfusion. DNA fragmentation or in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling analysis was done on cochleas for detection of apoptosis. RESULTS: Analysis by both techniques demonstrated marked apoptotic cell changes in spiral ganglion neurons of sodium nitroprusside-treated cochleas evident 4 to 8 hours after perfusion, as compared with minimal to no evidence of apoptosis in spiral ganglion neurons of control specimens. CONCLUSIONS: Exposure to high levels of nitric oxide induces apoptosis in spiral ganglion neurons. Because apoptosis is a delayed, potentially reversible cell death pathway, this may present an opportunity for intervention to prevent or attenuate hearing damage induced by excitotoxic stimuli.

Inhibition of nitric oxide synthase causes elevation of hearing thresholds.

OBJECTIVE: Nitric oxide mediates the effects of excitatory amino acids in the central nervous system. The excitatory amino acids are thought to be the neurotransmitters at the cochlear hair cell-afferent nerve synapse. Nitric oxide synthase is present in spiral ganglion cells. This study investigated the role of nitric oxide in cochlear neurotransmission. METHODS: In gerbils, cochlear compound action potential thresholds were recorded before and after cochlear perfusions with control solutions of artificial perilymph solution and test solutions of S-methyl-L-thiocitrulline (MTC), a competitive inhibitor of nitric oxide synthase. Cochleas were also preperfused with L-arginine before perfusion with a mixture of MTC/L-arginine (to overcome competitive inhibition by MTC with L-arginine, the natural substrate of nitric oxide synthase). RESULTS: Cochlear perfusion with MTC caused significant elevations of compound action potential threshold of 51 dB as opposed to insignificant elevations of only 10 dB in control animals. An insignificant threshold shift of 9 dB was observed when L-arginine was coperfused with MTC. CONCLUSIONS: Nitric oxide is involved in neurotransmission/neuromodulation in the cochlea. Because nitric oxide is both a mediator of neurotoxicity and an initiator of apoptosis in the central nervous system, nitric oxide may play a role in these processes in the cochlea.

Role of tumor necrosis factor and interleukin-1 in endotoxin-induced middle ear effusions.

In a rat model, we investigated the role of tumor necrosis factor (TNF) and interleukin-1 (IL-1) in endotoxin-induced middle ear effusions (MEEs). After the eustachian tube was obstructed, the middle ear was transtympanically injected with 35 microL of either 1) 1 mg/ mL lipopolysaccharide (LPS); 2) LPS and 100 micrograms TNF binding protein (TNFbp); 3) LPS and 1 microgram IL-1 receptor antagonist (IL-1ra); or 4) LPS, TNFbp, and IL-1ra. Every 2 hours, the fluid within the middle ear was collected, and the quantity of albumin in the fluid, an index of vascular leakage, was determined by enzyme-linked immunosorbent assay. After 6 hours, the middle ear was fixed for histologic analysis. The TNFbp significantly attenuated vascular extravasation into the middle ear. The IL-1ra did not significantly alter effusion development. These results indicate that TNF, but not IL-1, is a mediator of LPS-induced MEE. Therefore, TNFbp may represent a novel approach to the treatment of otitis media with effusion.

Nitric oxide is a mediator of neurogenic vascular exudation in the nose.

Rhinorrhea is a troublesome symptom of rhinitis seen commonly by otolaryngologists. The sources of nasal fluid production are glandular secretions and exudation from submucosal blood vessels. This study was designed to investigate the role of nitric oxide in neurogenically mediated vascular exudation in the nose. A rat model of the nasonasal reflex was developed in which one nasal cavity was challenged with histamine while albumin exudation was measured on the contralateral side. Histamine challenge was associated with a significant rise in albumin leakage, indicating an increase in vascular permeability. Perfusion with a nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester (L-NAME)) in the nasal cavity contralateral to nasal challenge was found to block albumin exudation on that side. This inhibition was overcome by the addition of L-arginine, the natural substrate of nitric oxide synthase, to the perfusate. Treatment of the ipsilateral nasal cavity with L-NAME did not significantly decrease the contralateral response. These findings indicate that NO is an important mediator of the effector arm of the nasonasal reflex that increases vascular permeability but is not involved in the sensory nerve afferent pathway. Further elucidation of the role of NO in nasal physiology may lead to novel pharmacotherapeutic approaches to the treatment of allergic and nonallergic rhinorrhea.

Nitric oxide mediates mucin secretion in endotoxin-induced otitis media with effusion.

The mechanisms that regulate mucin release in chronic otitis media with effusion, a leading cause of hearing loss in children, remain largely unknown. We developed an animal model using Sprague-Dawley rats to determine the factors responsible for mucin production in chronic otitis media with effusion. N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of nitric oxide synthase, was used to investigate the role of nitric oxide in mucin secretion by the middle ear epithelium. All rats underwent eustachian tube obstruction. In the first set of rats, the middle ear was then injected transtympanically with 35 microl of either 300 mOsm Krebs-Ringer bicarbonate buffer (control group) or 1 mg/ml lipopolysaccharide in Krebs-Ringer (experimental group 1). In a second set of rats, the middle ear space was injected with lipopolysaccharide and then infused at a continuous rate for 7 days with either Krebs-Ringer (experimental group 2) or 1 mmol/L L-NAME in Krebs-Ringer (experimental group 3) through an osmotic infusion pump. After 7 days the volume of effusion and the quantity of mucin collected were significantly greater in lipopolysaccharide-exposed ears than in controls. In addition, antimucin immunostaining demonstrated mucous cell hyperplasia in response to lipopolysaccharide. The lipopolysaccharide-induced production of mucin and mucous cell hyperplasia was inhibited in ears treated with lipopolysaccharide and L-NAME. These results suggest that nitric oxide is a mediator in the pathway of mucin secretion in chronic otitis media with effusion.

The role of glucocorticoids in endotoxin-mediated otitis media with effusion.

OBJECTIVE: To understand the actions of glucocorticoids on the development and progression of endotoxin-mediated otitis media with effusion. METHODS AND DESIGN: The middle ears of 20 Sprague-Dawley rats were exposed to 35 microL of either 300-mOsm Krebs-Ringer solution (control; n = 5) or lipopolysaccharide, 1 mg/mL, dissolved in Krebs-Ringer solution (n = 15). Among the group that received lipopolysaccharide, 10 rats were randomly selected to receive dexamethasone (1 mg/kg intramuscularly), either 2 hours (n = 5) or 24 hours (n = 5) before the introduction of lipopolysaccharide. Middle ear fluid was sampled after 2, 4, and 6 hours of exposure. OUTCOME MEASURES: Middle ear fluid volume and albumin content were determined as measures of vascular extravasation. Histological sections of the middle ear mucosa were used to quantify the degree of leukocyte exudation. Data were analyzed by 1- or 2-way analysis of variance with the significance level set at P < .05. RESULTS: Lipopolysaccharide exposure caused a significant increase in the mean +/- SEM middle ear fluid volume from 29.9 +/- 0.99 to 45.8 +/- 1.4 microL between the 2- and 6-hour samplings. Lipopolysaccharide exposure caused a significant increase in the albumin content of middle ear fluid from 70.1 +/- 19.2 to 271.0 +/- 93.1 micrograms between the 2- and 6-hour samplings. Both increases were significant compared with controls. Lipopolysaccharide also caused a significant increase in leukocytes localized to the middle ear mucosa. Pretreatment of animals with dexamethasone for either 2 or 24 hours inhibited the lipopolysaccharide-induced changes. There were no differences between 2- and 24-hour pretreatment with dexamethasone. CONCLUSIONS: Dexamethasone inhibits the development of endotoxin-induced otitis media with effusion.

Consumption of a high-galactose diet induces diabetic-like changes in the inner ear.

Diabetes mellitus is a disease that affects multiple organ systems. In our laboratory it has been shown that there is a significant loss of outer hair cells in genetically diabetic rats. Galactosemia can also produce diabetic-like changes. This study was performed to demonstrate whether these changes also occur in the cochlea. Three groups of Sprague-Dawley rats were used and fed either a control diet, a 50% galactose diet, or a 50% galactose diet with the addition of an aldose reductase inhibitor. After 6 months the animals were killed, and the cochleas were removed, fixed, and stained. Diabetes-induced damage was assessed by counting the hair cells and calculating the neuroganglion cell density. The histopathologic changes induced by galactose were manifested as outer hair cell loss and a decrease in neuroganglion cell density. Control animals had the least amount of hair cell loss and the greatest neuroganglion cell density of all three groups. Galactose-only animals demonstrated the most pronounced changes in both hair cell loss and neuroganglion cell degeneration; however, only changes of neuroganglion cell density in the basal turn were significant. The addition of an aldose reductase inhibitor provided inconclusive results in both hair cell determination and neuroganglion cell density; however, generally the inhibitor partially prevented the damage produced by galactose. These results suggest that a high-galactose diet can induce diabetic-like changes in the cochlea.

Immunohistochemical characterization of nitric oxide synthase activity in squamous cell carcinoma of the head and neck.

This study was designed to investigate the presence of nitric oxide in human squamous cell carcinoma of the head and neck. We localized the activity of nitric oxide synthase in these tumors through immunohistochemical analysis using antibodies to L-citrulline (a byproduct of nitric oxide synthase), to inducible nitric oxide synthase, and to constitutive nitric oxide synthase. We found presence of inducible enzyme in squamous cells throughout these tumors, with the highest intensity staining occurring directly around keratin pearls. Our findings suggest that inducible nitric oxide synthase activity is present in squamous cell carcinomas of the head and neck, leading us to conclude that inducible nitric oxide synthase may play a significant role in tumor growth.

An assessment of cochlear hair-cell loss in insulin-dependent diabetes mellitus diabetic and noise-exposed rats.

OBJECTIVE: The purpose of this study was to investigate if insulin-dependent diabetes mellitus causes degenerative changes in the inner ear and whether these changes are exacerbated by noise exposure. METHODS: Insulin-dependent diabetes mellitus was induced in male rats using streptozotocin (65 mg/kg of body weight, intravenously). Half the animals were exposed to 95 dB of random noise for 12 hours per day over a period of 6 months. The cochleae were removed, fixed, decalcified, dissected, and the hair cells counted. RESULTS: A significant loss of outer hair cells was exhibited in both noise-exposed groups; however, although there was no significant difference between these two groups, the noise-exposed diabetic animals had significant loss in more turns than did the noise-exposed control animals. The diabetic animals were not statistically different from the control animals. CONCLUSION: These results suggest that insulin-dependent diabetes mellitus may increase the hair-cell loss caused by noise overstimulation.

Nitric oxide in the rat vestibular system.

Nitric oxide is known to function as a neurotransmitter in the central nervous system. It is also known to be involved in the central nervous system excitatory amino acid neurotransmission cascade. Activation of excitatory amino acid receptors causes an influx of calcium, which activates nitric oxide synthase. The resulting increase in intracellular nitric oxide activates soluble guanylate cyclase, leading to a rise in cyclic guanosine monophosphate. The excitatory amino acids glutamate and aspartate are found in the vestibular system and have been postulated to function as vestibular system neurotransmitters. Although nitric oxide has been investigated as a neurotransmitter in other tissues, no published studies have examined the role of nitric oxide in the vestibular system. Neuronal NADPH-diaphorase has been characterized as a nitric oxide synthase. This enzyme catalyzes the conversion of L-arginine to L-citrulline, producing nitric oxide during the reaction. We used a histochemical stain characterized by Hope et al. (Proc Natl Acad Sci 1991;88:2811) as specific for neuronal nitric oxide synthase to localize the enzyme in the rat vestibular system. An immunocytochemical stain was used to examine rat inner ear tissue for the presence of the enzyme's end product, L-citrulline, thereby demonstrating nitric oxide synthase activity. Staining of vestibular ganglion sections showed nitric oxide synthase presence and activity in ganglion cells and nerve fibers. These results indicate the presence of active nitric oxide synthase in these tissues and suggest modulation of vestibular neurotransmission by nitric oxide.

Control of the mucosal microcirculation in the upper respiratory tract.

This study was designed to investigate the regulatory mechanisms of the mucosal microvascular network in the upper respiratory tract. Tracheal mucosal circulation was observed using a specially constructed chamber that allowed direct microscopic visualization of mucosal arterioles. Solutions of increasing hypertonicity (500 and 900 mOsm) applied to the tracheal epithelium resulted in increasing dilation of the underlying mucosal arterioles (p < 0.001). N(omega)-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L), a specific inhibitor of nitric oxide synthesis, added to a hypertonic solution inhibited dilation of mucosal arterioles (p < 0.001). Addition of the substrate for nitric oxide synthesis, L-arginine (0.6 mmol/L) to the hypertonic solution containing L-NAME resulted in dilation of mucosal arterioles once again. These data demonstrate that nitric oxide is a crucial mediator in the response of mucosal arterioles to the hypertonic stimulus presented to the epithelial surface of the trachea. Further elucidation of the control of the mucosal microcirculation in the upper respiratory tract could be implemented in new treatment for pathologic processes of the upper respiratory tract such as mucosal congestion and edema.

Ciprofloxacin. Use as a topical otic preparation.

Most common topical otic preparations have been shown to cause sensorineural hearing loss and hair-cell damage in experimental animals. Ciprofloxacin is a relatively new fluoroquinolone with excellent activity against Pseudomonas and methicillin-resistant Staphylococcus aureus. Recent studies have shown oral ciprofloxacin to be effective in the treatment of chronic serous otitis media and malignant external otitis. However, this drug has never been used as a topical otic preparation. Thirty-five albino female guinea pigs were used to investigate the ototoxicity of topical ciprofloxacin hydrochloride. Bilateral transbullae drug delivery tubes were placed and auditory brain-stem response thresholds were recorded at 20, 16, 8, and 4 kHz before treatment and 21 days after the completion of treatment. Two groups of guinea pigs were used. In group 1 (positive controls), five guinea pigs had 0.1 mL of neomycin sulfate administered in one ear while the opposite (control) ear received 0.1 mL of 0.9% sodium chloride solution; in group 2, 30 guinea pigs received 0.75% ciprofloxacin ophthalmic solution and 0.9% sodium chloride solution in the control ear. All drugs were given twice a day for 7 consecutive days. All results were evaluated with paired, two-tailed t test and Hotelling's T2 test, and calculation of power was performed on all nonsignificant results. No significant ototoxic reaction was observed; small increases in hearing thresholds occurred at 4 (5.65 +/- 8.25 dB [mean +/- SD]) and 8 kHz (3.70 +/- 6.63 dB [mean +/- SD]) in the ciprofloxacin-treated ears; however, no significant hair-cell loss was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Inner ear damage secondary to diabetes mellitus. II. Changes in aging SHR/N-cp rats.

The congenic spontaneous hypertensive/National Institutes of Health (Bethesda, Md)-corpulent rat (SHR/N-cp) is a model for non-insulin-dependent diabetes mellitus. A previous study in our laboratory found significant loss of outer hair cells (OHC) in diabetic rats at 5.0 months of age. Our present study was designed to further evaluate the effects of the diabetic state on the inner ear in 10.5-month-old rats. The following comparisons were made: diabetic vs euglycemic control animals; obese vs lean phenotypes; and sucrose vs starch as the source of dietary carbohydrate. Cochleas were removed, fixed, stained, mounted on slides, and analyzed for OHC loss. We found a significant OHC loss in the cochleas of all diabetic animals. No statistical difference was found when comparing obese and lean phenotypes. Increased OHC loss was observed in all sucrose-fed vs starch-fed diabetic animals, although this increase was not statistically significant. Compared with an earlier study, an increase in OHC loss was also noted in the 10.5-month-old lean SHR/N-cp animals. Our results indicate that there is a relationship between non-insulin-dependent diabetes mellitus and inner ear damage and suggest that OHC loss is related to hyperglycemia and a genetic predisposition for glucose intolerance.