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.

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.

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.

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.

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.

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.

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.

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.