A method to assess the accuracy of sonotubometry for detecting Eustachian tube openings.

Sonotubometry is a simple test for Eustachian tube (ET) opening during a maneuver. Different sonotubometry configurations were suggested to maximize test accuracy, but no method has been described for comparing sonotubometry test results with those for a definitive measure of ET opening. Here, we present such a method and exemplify is use by an accuracy assessment of a simple sonotubometry configuration. A total of 502 data-sequences from 168 test sessions in 103 adult subjects were analyzed. For each session, subjects were seated in a pressure chamber and relative middle ear over- and under-pressures created by changing chamber pressure. At each pressure, the test sequence of bilateral tympanometry, bilateral sonotubometry while the subject swallowed twice, and bilateral tympanometry was done. Tympanometric data were expressed as the fractional gradient equilibrated (FGE) by swallowing and sonotubometric signals were analyzed to record the shape of detected sound signals. Tympanometric and sonotubometric tubal opening assignments were analyzed by cross-correlation. For the data sequences with FGE = 0 (n = 32) evidencing no tubal opening and one (n = 249) evidencing definitive tubal opening, detection of a sonotubometry sound signal during a swallow had a sensitivity and specificity of 74.2 and 65.6 % for identifying ET openings and an accuracy of 73.3 % for assigning ET opening/non-opening by swallowing. Measures of sound signal shape were significantly different between those groups. This protocol allows a sonotubometry accuracy assessment for detecting ET openings. For the test configuration used, accuracy was moderate, but this should improve as more sophisticated sonotubometry test configurations are evaluated.

CO(2) gas exchange across the human tympanic membrane is not appreciably affected by pathology.

Past in vivo studies in humans showed that the tympanic membrane (TM) is permeable to physiological gases. Animal studies show that transTM CO(2) conductance is increased by TM pathology. The objective of the study was to determine if transTM CO(2) exchange in humans is affected by atrophic and sclerotic pathologies. The study used an ear canal (EC) probe (ECP) constructed from a custom-fitted acrylic body, a glass capillary tube enclosing an oil meniscus to maintain ambient ECP + EC pressure and a silica glass microtube linked to a mass spectrometer (MS) for measuring gas composition that was hermetically sealed within the ear canal of the test ear. ECP + EC volume was measured and gas samples taken at 10 min intervals for 1 h. The fractional CO(2) pressure measured in the ECP + EC for each sample was regressed on time and the slope of the function multiplied by the ECP + EC volume and divided by the estimated transTM CO(2) gradient at the start of the experiment to yield transTM CO(2) conductance (microL/min/Pa). Data were complete for 15 normal, 13 sclerotic and 9 atrophic TMs. The average (+std) transTM CO(2) conductances were 1.76 × 10(-4) + 7.27 × 10(-5), 2.26 × 10(-4) + 1.5 × 10(-4) and 2.36 × 10(-4) + 1.14 × 10(-4) microL/min/Pa/TM for the normal, sclerotic and atrophic TMs, respectively. A pairwise comparison of data for the normal and atrophic TMs under the directional hypothesis of a greater CO(2) exchange rate for thinner TMs approached statistical significance (P = 0.07). A similar pairwise comparison for the sclerotic and normal TMs did not approach statistical significance (P = 0.28). The effect of TM pathologies on CO(2) conductance was limited.

Interaction of pneumococcal phase variation, host and pressure/gas composition: virulence expression of NanA, HylA, PspA and CbpA in simulated otitis media.

Streptococcus pneumoniae, a leading cause of otitis media (OM), adapts to the host environment and undergoes spontaneous intra-strain phase variations in colony morphology. Transparent (T) phase variants are more efficient in colonizing the nasopharynx while the opaque (O) phase variants exhibit greater virulence during systemic infections. We recently demonstrated that T phase variants exhibited a higher growth rate and greater epithelial adherence and destruction than did O phase variants during interactions with human middle ear (ME) epithelial cells. This study was to delineate the underlying molecular mechanisms. Human ME epithelial cells were preconditioned for 24 h under one of the three simulated ME gas/pressure conditions designed to reflect those for 1) normal ME, 2) ME with Eustachian tube obstruction (ETO) and 3) ME with tympanostomy tube (TT) placement; subsequently exposed to a dose (10(7) CFU/ml) of either T or O phase variants of S. pneumoniae (6A), and then incubated for 1h and 3 h. Gene expressions coding for pneumococcal NanA, HylA, PspA, and CbpA virulence factors in inoculum, epithelium-attached and free-floating bacteria were assayed using real-time PCR. Result showed significantly higher basal expression levels for NanA and HylA in T inoculums than in O inoculum. Furthermore, striking differences between the two phase variants were observed in the forms of the inocula, significantly higher expression levels for PspA in T inoculum, but for CbpA in O inoculum. The TT condition enhanced the molecular activities of NanA, HylA, and PspA virulence factors in epithelium-attached T phase variants and fluid-floating O variants, followed by the ETO condition. Our study suggests that the pneumococcal virulence of the phase variations are modulated by the pathological ME environment and host-pathogen interaction during the pathogenesis of OM.

Directional asymmetry in the measured nitrous oxide time constant for middle ear transmucosal gas exchange.

OBJECTIVES: Simple, 2-compartment mathematical models of middle ear (ME) transmucosal gas exchange reproduce observed ME pressure behavior. These models require input of an experimentally determined, lumped-parameter exchange constant for each represented gas species. Previous model applications assumed directional asymmetry for those parameters, which has not been experimentally validated. METHODS: As a surrogate for the inert gas nitrogen (N2), for which exchange is too slow to be measurable, the nitrous oxide (N2O) transmucosal exchange constant for 16 ears of 8 monkeys was measured for positive and negative ME blood N20 gradients. RESULTS: The paired exchange constants for each ear were highly correlated, but the ME-blood/blood-ME exchange constant ratio was approximately 13. Modeling shows this asymmetry to depend on the value of the arterial-venous/arterialME ratio, a variable in the exchange constant for perfusion-limited gases. CONCLUSIONS: These results support an asymmetric rate of transmucosal N20 and, by extension, N2 exchange for the ME. Because the primary controlling parameter for ME pressure behavior in the absence of eustachian tube opening is the rate of transmucosal N2 exchange, this effect needs to be incorporated into the simple 2-compartment exchange models for predictive accuracy. The gradient ratio dependence suggests that parameter-free modeling may require treating the ME mucosa as having a distributed gradient for certain gas species.

Nasal prostaglandin challenge increases N2O exchange from blood to middle ear.

Nasal inflammation (NI) resulting from allergy or virus infection is causally associated with otitis media. Impaired Eustachian tube (ET) function consequent to NI may mediate this relationship. Moreover, the functional demand placed upon the ET for gas supply may be increased by NI, a hypothesis tested here. A total of five experiments were done at a minimum 2-week interval on four monkeys. For each experiment, the monkey was anesthetized and acclimated for 60 min. Then, the monkey was breathed with a 40% N2O gas mixture for 60 min followed by air breathing for 100 min. Fifteen minutes into air breathing, the nose was challenged with one of the five substances: saline, PGD2, capsaicin, histamine or bradykinin. Throughout, middle ear (ME) pressures were recorded at 5-min intervals. All pressure-time curves had a similar form consisting of a curvilinear decrease during acclimation, a linear increase during N2O breathing and a linear decrease during air breathing. The rates of pressure increase were ear-specific and not different across experiments. Compared to saline, the rate of pressure decrease was greater for challenge with all the substances and significantly greater after PGD2 challenge. Like N2, N2O is an inert gas whose transmucosal exchange is perfusion-limited for the ME. The measured pressure change is linearly related to the volume gas exchange between ME and blood. Therefore, nasal challenge with PGD2 and perhaps the other substances increases transmucosal inert gas exchange. ME to blood N2 transfer rate determines the physiological demand placed upon the ET for gas supply. That demand is increased by nasal exposure to certain inflammatory mediators.

Middle ear pressure change during controlled breathing with gas mixtures containing nitrous oxide.

The change in middle ear pressure while breathing gas mixtures containing N(2)O was studied in four monkeys. At each of three experimental sessions, monkeys were anesthetized, acclimated for 60 min, breathed with room air for 60 min, and then breathed with 5, 10, or 20% N(2)O for 60 min. Middle ear pressure, rectal temperature, and vital signs were recorded throughout. The time constant for blood-middle ear N(2)O exchange was calculated from these data. Middle ear pressure decreased during acclimation, was stable during air breathing, and increased during N(2)O breathing. The rate of pressure change was similar for both ears of each animal and was directly related to N(2)O percent. The calculated time constant ranged from 0.003 to 0.008 min(-1) across animals but was not different for a given ear across sessions. These results show that breathing gas mixtures containing N(2)O causes predictable and quantifiable increases in middle ear pressure.

Rate of nitrous oxide exchange across the middle ear mucosa in monkeys before and after blockage of the mastoid antrum.

OBJECTIVES: We tested the hypothesis that mastoid volume buffers the rate of change in middle ear pressure caused by transmucosal, inert gas exchange. STUDY DESIGN: Twelve monkeys were randomly assigned to group 1 or group 2. Right ears of group 1 had sham surgery and of group 2 had obstruction of the mastoid antrum. Before and after surgery, the time constant for transmucosal N(2)O exchange was estimated from N(2)O breathing experiments. The hypothesis predicts that the postoperative time constant measured for right ears of group 2 but not group 1 is greater than that measured before surgery. RESULTS: Mastoid antrum block significantly decreased right middle ear volume but did not affect the time constant for transmucosal N(2)O exchange. CONCLUSION: A mastoid gas-reserve function is not supported by the experimental data. SIGNIFICANCE: These results for monkeys and the theory developed to explain the effect of mastoid volume on transmucosal inert gas exchange suggest that the results for previous experiments in humans interpreted as evidencing a mastoid gas-reserve function are consistent with alternative explanations.

Tympanometry accurately measures middle ear underpressures in monkeys.

Tympanometry is useful for evaluating middle ear (ME) status, but its accuracy in estimating true ME pressure has been questioned. We evaluated the accuracy of tympanometry in 6 monkeys. Direct application and measurement of ME pressure were achieved with a probe introduced into the mastoid antrum, and tympanometry was done over a large range of applied ME pressures. For all ears, tympanometric pressure was a linear function of applied pressure. At large overpressures, the tympanometric pressure was approximately 40 mm H2O greater than the applied pressure, but there was little error in the measurement for applied underpressures. The measurement error was proportional to the ME pressure multiplied by the ratio of the extant volume displacement of the tympanic membrane to ME volume. These results show that in monkeys, tympanometry provides an accurate, relatively unbiased estimate of ME underpressure and suggest that the measurement error for tympanometry can be predicted for MEs of other species.

Middle ear gas exchange in the air phase.

OBJECTIVE: The rate of gas exchange between the tympanum and mastoid is important for middle ear pressure regulation. The best-studied model of middle ear pressure regulation is the cynomolgus monkey. The aim of this study was to determine the time required for the equilibration of tympanum and mastoid partial pressures for two inert gases, argon and helium, in cadaveric cynomolgus monkey middle ears. MATERIAL AND METHODS: In separate experiments on 5 air-dried temporal bones from monkeys, helium or argon was introduced into the tympanum to bring the total pressure to 300 mmH2O above ambient and the partial pressures of all relevant gases in the tympanum and mastoid were measured using mass spectrometry at 0, 1, 3, 5, 10 or 15 min. RESULTS: The average ratios of tympanum:mastoid partial pressure at 0 min were 4.7 +/- 1.9 and 3.9 +/- 2.0 for helium and argon, respectively. By 5 min those ratios had decreased to 1.2 +/- 0.2 and 1.1 +/- 0.1, respectively, suggesting approximate equilibrium. CONCLUSIONS: Air-phase gas exchange between the tympanum and mastoid is rapid. Partial-pressure gradients across the middle ear will be dissipated quickly and are not an important contributor to middle ear pressure regulation in the studied model.

Gas diffusion across the tympanic membrane in chinchillas: effect of repeated perforations.

OBJECTIVE: Earlier studies documented a slow diffusion of gas across the tympanic membrane (TM) in humans and animals. However, structural changes caused by repeated TM perforations could affect gas diffusion rates. This possibility was evaluated using a chinchilla model. METHODS: In six chinchillas, the right TM was perforated four times at 2-week intervals; the left TM served as a control. Approximately 12 weeks after the fourth perforation and when the right TMs had healed, a probe was introduced into the external canal and sealed to the environment. For 120 min, pressure in the probe was continuously monitored and probe gas was sampled at 10 min intervals and analyzed for composition by online mass spectrometry. Percent compositions for CO(2) and O(2) were calculated and these data were recast as partial-pressure versus time functions. The information contained in those functions was used to estimate time-constants for transTM O(2) and CO(2) diffusion. RESULTS: In all experiments, CO(2) percent composition in the probe increased as a curvilinear function of time while that for O(2) decreased linearly. For all animals and both gases, the rate of change in probe partial-pressure was greater for the right TM. Average values of the right and left time-constants were (6.7 +/- 2.4) x 10(-3) and (4.2 +/- 2.2) x 10(-3)min(-1)TM(-1) for CO(2), and (2.3 +/- 0.8) x 10(-3) and (1.5 +/- 0.5) x 10(-3)min(-1)TM(-1) for O(2). Between side differences in these time-constants were statistically significant (P < 0.05, 2-tailed, paired, Student's t-test). The average right/left, transTM time-constant ratio was 1.66 +/- 0.43 for CO(2) and 1.61 +/- 43 for O(2); both were significantly different from a value of 1.0 (P < 0.05, 2-tailed Student's t-test). CONCLUSION: These results document an increased rate of diffusive gas exchange across TMs that had been repeatedly perforated. This effect may be caused by structural thinning secondary to scar formation and could have implications for middle ear pressure regulation in ears with a history of repeated myringotomies and/or tympanostomy tube insertions.

Pressure chamber tests of eustachian tube function document lower efficiency in adults with colds when compared to without colds.

CONCLUSIONS: Fractional gradient equilibrated (FGE) for ears with applied positive but not negative middle ear (ME)-ambient pressure gradients is highly sensitive to a cold-like illness (CLI). OBJECTIVE: The sequential development of eustachian tube (ET) dysfunction, ME under-pressure, and otitis media (OM) characterizes many children during a CLI. If linked, OM burden would be lessened by interventions that promote/preserve good ET function during a CLI. Evaluating this requires a quantitative ET function test for MEs with an intact tympanic membrane responsive to a CLI. METHODS: Pressure chamber testing of ET function was performed at +200 and -200 daPa in 3 groups of adults: group I, 21 subjects with an extant CLI and groups II and III, 14 and 57 adults, respectively, without a CLI. ME-chamber pressure gradient was recorded by tympanometry before and after the subject swallowed twice. ET functional efficiency was quantified as the FGE, which was then compared among groups using a Mann-Whitney U test. RESULTS: At chamber pressures of 200 daPa, the ME-chamber pressure gradient was negative, and FGE was low and not different among groups. At chamber pressures of -200 daPa that gradient was positive, and FGE was significantly higher in groups II and III when compared with group I.

Eustachian tube opening measured by sonotubometry is poorer in adults with a history of past middle ear disease.

OBJECTIVE: Test the hypothesis that active Eustachian tube opening efficiency as measured by sonotubometry is higher in adults with no extant middle-ear disease and no history of previous otitis media (Group-1) when compared to adults with no middle-ear disease but a positive history for otitis media (Group-2). METHODS: Eustachian tube function for 1 ear of 33 otherwise healthy adult subjects, 16 assigned to Group-1 and 17 to Group-2, was tested by sonotubometry using a standard protocol. For each test, the sound envelopes for 3 swallows were abstracted independently by 2 observers from the data stream and 7 descriptive parameters related to sound envelope "shape" were calculated. Inter-relatedness among the values for the parameters was explored using correlation analysis. The contributions of swallow, observer and group to the variance in each parameter were evaluated for significance using a General Linear Model. RESULTS: The shape parameters reflecting envelope height, area and rise and fall rates were highly inter-correlated, but those reflecting envelope widths were not. There was no effect of "swallow" on any of the parameters; but there was a significant "observer" effect on all measures of envelope width, greater for observer-2, and a significant "group" effect for 5 of the 7 shape parameters, all greater in Group-1. CONCLUSIONS: Quantifiable measures of the sound signal "shape" recorded by sonotubometry during swallowing were significantly different between the 2 groups of subjects. This is interpretable as evidencing a more efficient Eustachian tube opening-function in adults with healthy middle ears who do not have a previous history of otitis media when compared to similar adults with a history of prior otitis media. Inefficient Eustachian tube function as children may not be completely resolved by adulthood increasing adult otitis media risk when Eustachian tube function is down-graded by extant upper respiratory diseases that provoke nasopharyngeal inflammation.

Eustachian tube function in young children without a history of otitis media evaluated using a pressure chamber protocol.

CONCLUSIONS: Protocol limitations were identified and accounted for in the analysis. Percent gradient equilibrated (PGE) was affected by driving gradient direction in a similar manner to other efficiency measures. A finer resolution of possible age-related changes in eustachian tube opening efficiency is expected with the application of more sophisticated statistical models to the complete dataset at study end. OBJECTIVE: To report the results of an interim analysis for an ongoing study designed to characterize the age-related changes in eustachian tube opening efficiency measured using a pressure chamber protocol in children without a history of middle ear disease. METHODS: To date, 41 children aged 3 years without a history of otitis media have been enrolled in a longitudinal study of the age-related changes in eustachian tube function and evaluated at yearly intervals between 3 and 7 years of age. Eustachian tube opening efficiency, the percent of the applied pressure gradient equilibrated by swallowing, was measured by repeat tympanometry during a pressure chamber protocol. Data (120 tests) were analyzed using an ANOVA with variance partitioned by age (3 through 6 years), gradient direction (positive/negative), and ear (left/right). RESULTS: PGE was higher for left ears and positive driving gradients, but was not different among age groups.

Transmucosal O2 and CO2 exchange rates for the human middle ear.

OBJECTIVE: Estimate the transmucosal CO(2) and O(2) rate-constants for adult middle ears (MEs). METHODS: Ten adults with healthy MEs had a unilateral myringotomy. A custom-fitted acrylic mold with a valved line to a mass spectrometer (MS) and central tube coupled to a 3-way valve and connected to a pressure transducer (the probe) was sealed with adhesive glue within the ipsilateral ear-canal. A second 3-way valve was attached to the probe valve, a flow-regulated tank gas source and paired syringes. Volumes of the ME and probe were measured. On sequential days, the probe+ME was washed for 15-min with 6% O(2), Balance N(2) and 25% O(2), 6% CO(2), Balance N(2) to create transmucosal CO(2) and O(2) gradients, respectively. After washing, the probe+ME was isolated from the gas source, and baseline and 10-min gas samples were obtained for MS analysis of gas partial-pressures. The rates of change in ME CO(2) and O(2) pressures were divided by their established transmucosal gradients to yield CO(2) and O(2) rate-constants. RESULTS: The average (±STD) transmucosal CO(2) and O(2) rate-constants were 0.062 ± 0.034 (N=10, range: 0.032-0.119) and 0.011 ± 0.009 (N=8, range: 0.002-0.032)mmHg/min/mmHg, respectively. The average half-life for the CO(2) and O(2) gradient was 11.1 and 61.6 min. The average CO(2):O(2) rate-constant ratio was 8.1 ± 4.0 (N=8, range: 3.6-14.6). CONCLUSIONS: For adult human MEs, transmucosal CO(2) exchange is rapid and much faster than transmucosal O(2) exchange. The estimated CO(2)/O(2) rate-constant ratio for the human ME is not consistent with that predicted for diffusion-limited gas exchange across a water-based barrier.

Interaction of phase variation, host and pressure/gas composition: pneumococcal gene expression of PsaA, SpxB, Ply and LytA in simulated middle ear environments.

OBJECTIVE: Streptococcus pneumoniae, a leading cause of otitis media (OM), undergoes spontaneous intra-strain variations in colony morphology. Transparent (T) variants are more efficient in colonizing the nasopharynx while opaque (O) variants exhibit greater virulence during systemic infections. This study was intended to delineate the underlying molecular mechanisms by which the predominant S. pneumoniae variant efficiently infects the middle ear (ME) mucosa. METHODS: Human ME epithelial cells were preconditioned for 24h under one of the three gas/pressure conditions designed to simulate those for (1) normal ME (NME), (2) ME with Eustachian tube obstruction (ETO) and (3) ME with tympanostomy tube placement (TT), and then were incubated with ∼ 10(7)CFU/ml of either T or O variants of S. pneumoniae (6A) for 3h. Relative expression levels of genes encoding virulence factors, PsaA (surface adhesion), SpxB (pyruvate oxidase), Ply (pneumolysin), and LytA (autolysin) were assessed separately in epithelium-attached and supernatant bacteria 3h post infection using real-time PCR. RESULTS: Basal levels of the virulence molecules in inocula were comparable between two variants. However, relative expression levels of the gene transcripts were significantly induced in epithelium-attached T variants 3h after infection. Comparing with NME and TT conditions, ETO environment produced the largest effect on the differential expression of the virulence genes in the infected ME epithelial cells between T (induced) and O (suppressed) phenotypic pneumococci. CONCLUSIONS: T variant is a predominant phenotype responsible for the pathogenesis of pneumococcal OM.

Repair of the tympanic membrane with urinary bladder matrix.

OBJECTIVES/HYPOTHESIS: To test urinary bladder matrix (UBM) as a potential treatment for tympanic membrane (TM) healing and regeneration. STUDY DESIGN: This prospective pilot study was designed to provide both qualitative and semiquantitative assessment of temporal and spatial healing events in the chinchilla model of chronic TM perforations with and without UBM patching. METHODS: Bilateral myringotomies were performed and repeated as necessary to create subtotal perforations over an 8-week period. Myringoplasty was then performed, with left TMs serving as controls and right TMs receiving UBM patches. TMs were excised at 4 weeks, 8 weeks, and 12 weeks. Fixed tissue samples were characterized for gross morphology, then processed for microscopic evaluation. RESULTS: Chronic perforations were maintained with one or more repeated myringotomies. Although both control and patched TMs were thicker than native tissue, patched TMs were transparent and uniform in thickness without any inclusions. UBM patches were readily degraded and replaced by newly deposited and organized host tissue that recapitulated the native TM layers. CONCLUSIONS: UBM scaffolds were an effective biological scaffold for TM closure and tissue remodeling, leading to thicker than normal anatomy but otherwise normal morphology. Future studies are required to determine functional and temporal outcomes as well as alternative patch orientations. The results show particular promise as a superior alternative means of reconstructing not only chronic TM perforations but also dimeric TMs associated with retraction pockets and atelectasis. Laryngoscope, 2009.

Interaction of pneumococcal phase variation and middle ear pressure/gas composition: an in vitro model of simulated otitis media.

Streptococcus pneumoniae, a leading cause of otitis media (OM), undergoes spontaneous intra-strain variations in colony morphology. Transparent (T) variant is more efficient in colonizing the nasopharynx while the opaque (O) variant exhibits greater virulence during systemic infections. We hypothesized that changes in middle ear (ME) gas pressure/composition during Eustachian tube (ET) dysfunction and the treatment of that dysfunction, e.g., tympanostomy tube (TT) insertion, play a role in selecting the S. pneumoniae variant that can efficiently colonize/infect the ME mucosa. Human ME epithelial cells were preconditioned for 24h under one of three conditions that simulated (1) normal ME, (2) ME with ET obstruction (ETO) and (3) ME with TT; subsequently exposed to a dose (approximately 10(7)CFU/ml) of either T or O variant of S. pneumoniae, and then incubated for 1h and 3h. Under the simulated ETO and TT conditions, T variant exhibited a higher growth rate and greater epithelial adherence and killing than did O variants. Attachment of T variant to epithelial cells was documented by scanning electron microscopy. These results suggest that the T variant is more highly adapted to various ME environments than the O variants.