- Email: [email protected]
Effect of aspirin on phase gradient of 2F1–F2 distortion product otoacoustic emissions
Hearing Research 205 (2005) 44–52 www.elsevier.com/locate/heares
Effect of aspirin on phase gradient of 2F1–F2 distortion product otoacoustic emissions M. Parazzini a
a,*
, A.J. Hall b, M.E. Lutman b, S. Kapadia
b
Istituto di Ingegneria Biomedica ISIB, CNR, Milan, Piazza Leonardo da Vinci 32, 20133 Milano, Italy b Institute of Sound and Vibration Research, University of Southampton, UK Received 3 September 2004; accepted 28 February 2005 Available online 28 March 2005
Abstract It is well known that aspirin consumption temporarily reduces overall otoacoustic emission (OAE) amplitude in humans. However, little is known about changes in the separate components of distortion product otoacoustic emissions (DPOAE), which may be distinguished by examining phase gradients. The effects of aspirin on the phase gradient of the DPOAE 2F1–F2 obtained with fixed frequency ratio sweeps were studied longitudinally in a group of twelve subjects in whom a temporary hearing loss was induced by aspirin consumption. DPOAE were recorded daily for two days pre-aspirin consumption, during the three days of aspirin consumption and two days afterwards. DP-grams were recorded over a restricted frequency range centered on 2, 3, 4 and 6 kHz with the following stimulus levels: L1/L2 of 60/50–80/70 in 10-dB steps. The effects of aspirin on the phase gradients varied between the subjects and across frequency: the general trend was that the phase gradient became steeper across successive sessions for the higher frequencies, while no significant effect was found at the lower frequencies. These results suggest that aspirin may have more persistent effects on cochlear function than are disclosed by measurements of hearing threshold level or DPOAE amplitude. Particularly, DPOAE phase gradient appears to be increased by aspirin consumption and has not recovered two days after cessation of aspirin intake, despite almost complete recovery of DPOAE amplitude and hearing threshold levels. These findings may suggest differential effects on the distortion and reflection mechanisms considered to underlie DPOAE generation. Ó 2005 Elsevier B.V. All rights reserved. Keywords: Distortion product otoacoustic emissions; Aspirin; Phase gradient
1. Introduction The drug aspirin (acetylsalicylic acid) is one of the salicylates and is known to affect the mechanical response of the cochlea (Brown et al., 1993). This effect Abbreviations: DP, distortion product; DPOAE, distortion product otoacoustic emissions; FFT, fast Fourier transform; HTL, hearing threshold level; OAE, otoacoustic emissions; OHC, outer hair cell; SD, standard deviation; SNR, signal to noise ratio; TEOAE, transientevoked otoacoustic emissions * Corresponding author. Tel.: +39 0223993359; fax: +39 0223993367/ 60. E-mail address: [email protected] (M. Parazzini). 0378-5955/$ - see front matter Ó 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2005.02.010
may be the consequence of the changes in turgidity and motility that have been observed in isolated mammalian outer hair cells (OHC) perfused with aspirin (Shehata et al., 1991; Tunstall et al., 1994; Russell and Schauz, 1995). Different studies (for a review on auditory sensorineural alterations induced by salicylate, see Cazals, 2000) show that clear alterations are produced on otoacoustic emissions (OAE) by salicylate. The most conspicuous effect is the disappearance of spontaneous OAE (McFadden and Plattsmier, 1984; Long and Tubis, 1988a; Wier et al., 1988; Long et al., 1991). However, the data do not indicate, either for amplitude or time course, clear correlations between the reduction
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
and/or disappearance of spontaneous OAE and the loss of absolute hearing sensitivity. Studies of evoked OAE (Long and Tubis, 1988b; Janssen et al., 2000) indicate reductions in response amplitude and associated threshold elevation, corresponding with loss of absolute hearing sensitivity. The reductions in the distortion product otoacoustic emission (DPOAE) amplitude become more pronounced as the levels of the stimuli are lowered (Kujawa et al., 1992; Fitzgerald et al., 1993). Detailed measures of various DPOAE components and/or characteristics further revealed more subtle changes in different aspects of nonlinear cochlear functioning (Stypulkowski, 1990; Brown et al., 1993; Frank and Kossl, 1996). Both studies of isolated OHC and studies of human and animal data show that alterations induced by salicylate appear to be dose-dependent and reversible (Cazals, 2000). The reversible nature of the aspirin-induced hearing loss makes it an attractive tool as it allows the experimenter to compare performance in the same subject both in the absence and in the presence of hearing loss (Beveridge and Carlyon, 1996). While it is known that significant reductions in DPOAE amplitude are a sign of functional or structural damage to OHC (Kemp, 1978), the relationship between DPOAE phase and cochlear status is still not well understood. Previous studies suggest that the phase gradient against frequency, obtained using fixed frequency ratio sweeps, is consistent with a combination of two different DPOAE emission components (Knight and Kemp, 1999, 2000, 2001). Knight and Kemp refer to these as placed- and wave-fixed mechanisms, based on the assumed site of generation. The general assumption with a wave-fixed mechanism is that the emission is generated by distortion at a site that is an integral part of and moves smoothly with the stimulus travelling wave envelope as stimulus frequency is swept (Shera and Guinan, 1999). For the 2F1–F2 distortion product (DP), the wave-fixed component is considered to be generated close to the F2 place on the basilar membrane and reaches the ear canal via a travelling wave propagating in the reverse direction along the basilar membrane. The phase at any point moving with the travelling wave envelope changes little; therefore, any OAE contribution from that point would have a shallow phase gradient using fixed frequency ratio sweeps. Distortion generated at the F2 place also propagates in the forward direction to the DP place, where it may be reflected. Zweig and Shera (1995) have proposed a series of reflecting or scattering sites existing along the basilar membrane and a mechanism of coherent reflection involving the sharply tuned basilar membrane excitation pattern. As a stimulus is swept in frequency and its excitation pattern moves along the basilar membrane, the stimulus phase at the reflection site will change, thus changing the OAE phase
45
and creating a steep gradient.1 Phase gradients may also be considered in terms of group delay, where a shallow slope corresponds to a short delay relative to the primary stimuli, while a steep slope corresponds to a longer delay, with phase gradient proportional to delay. Because these two components are generated by different processes, at different sites along the basilar membrane, it is of fundamental interest to understand how they each change with cochlear damage. Steep and shallow phase gradients have been observed in the 2F1–F2 DP (Knight and Kemp, 1999) depending on whether a large or small frequency ratio is used. In particular, for a small frequency ratio, the phase gradient is steep, consistent with a predominantly place-fixed emission mechanism, while with a larger frequency ratio, the phase gradient becomes shallow and is more consistent with a wave-fixed mechanism. The reasons for this are not yet understood fully, although Knight and Kemp (2001) propose a model that suggests that the propagation of DP travelling waves is biased by the shapes of the primary travelling waves. For the more widely spaced primary frequencies commonly used to measure the 2F1–F2 DP, the reverse travelling wave in the F2 frequency region is promoted so that the wave-fixed component tends to dominate the response, thus explaining the shallow phase gradients observed with larger frequency ratios. When aspirin interferes with cochlear function, it is unclear whether it affects the place-fixed or wave-fixed mechanism, or both. Because the two mechanisms are fundamentally different, as described above, it is plausible that aspirin may affect either or both. It is therefore of interest to know whether aspirin produces a steeper or shallower phase gradient of the 2F1–F2 DP in parallel with the well known effect of reduction of the amplitude of the same DPOAE component. Previous studies using aspirin in humans have measured amplitude but have not reported on phase. Hence, the aim of the analysis reported here was to investigate effects of aspirin on DPOAE phase. These findings may throw some light on whether aspirin affects primarily wave-fixed or placefixed mechanisms of OAE generation, or in other words whether it affects primarily distortion or reflection mechanisms. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institute of Sound and Vibration Research Human Experimentation Safety and Ethics Committee.
1
Whether the two components are categorised by the assumed site of generation, wave- or place-fixed, or by the assumed mechanism, ‘‘distortion’’ or ‘‘reflection’’ is a subtlety beyond the scope of this work. For simplicity, they are referred to as wave- and place-fixed, following the usage of Knight and Kemp (1999).
46
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
2. Materials and methods Results presented here were obtained from an additional analysis of existing data recorded in another study (Hall, unpublished PhD thesis), where the aim was to explore the relationship between changes in hearing threshold level (HTL) and changes in cochlear amplifier gain inferred from OAE measurements (both TEOAE and DPOAE). Changes were induced by causing a temporary hearing loss using aspirin. As the experiment was not designed to explore phase gradients the methods have some limitations. Nevertheless, given the difficulties in conducting experiments where normal participants are administered a drug with potential side effects, the data were utilised for this preliminary exploratory study. 2.1. Subjects and drug schedule Twelve male subjects, aged 19–38 years, took part in the experiment. Subjects were given a list of contraindications to aspirin, which if occurring in any individual would preclude participation in the study. Subjects had HTL better than or equal to 20 dB at all standard audiometric frequencies. All had normal middle ear function with bone conduction thresholds within 5 dB of air conduction thresholds averaged across 0.5, 1, 2 and 4 kHz, normal tympanometry and normal otoscopy. Subjects took 11.7 g of aspirin over a period of 72 h spanning four calendar days (975 mg twice on the first day, four times each on the second and third days and twice on the fourth). They consumed food with each dose in accordance with recommended practice. They were asked to refrain from alcohol consumption during the entire aspirin consumption period and to fill in a diary to confirm their intake of aspirin. To facilitate correct dosage, subjects were given a dose box containing the number of aspirin tablets to be taken at each session on each day for the course of the experiment. Salicylate concentration in the blood was monitored on days 3–5 and generally increased over the dosage period. Maximum concentration ranged from 0.5 to 1.5 mmol/l across subjects (mean value 0.89 mmol/l). 2.2. DPOAE measurements The distortion product 2F1–F2 was recorded using laboratory apparatus designed specifically for the purpose. Primary frequencies are denoted by F1 and F2, while primary levels are denoted by L1 and L2. An Etymotic ER-10B+ microphone probe and pre-amplifier (+40 dB) were used for recording ear canal sound pressure. Two Etymotic ER-2 insert earphones were used to deliver the primary tone stimuli to the subject via the probe. The amplified microphone signal was digitised (16-bit resolution, 32.768-kHz sample rate) by an exter-
nal A/D and D/A converter unit (Institute of Hearing Research DSP remote converter module) that also generated the primaries. Signal processing by custom software running on a TMS-320 DSP card converted consecutive 62.5-ms epochs of the microphone signal to the frequency domain by performing FFT with a bin width of 16 Hz. The complex FFT was averaged after rejection of epochs in which the estimated noise level was greater than 10 dB SPL in the frequency range close to the DP. The amplitude and phase of the DP were estimated from the real and imaginary FFT components corresponding to the single bin centred on the DP frequency. Noise at the DP frequency was estimated by averaging the power in 10 spectral lines either side of the DP. Recording of a DPOAE for a particular frequency was stopped after a minimum number of epochs had been acquired and a minimum SNR was reached; these minima were set at 20 epochs and a SNR of 10 dB. If neither criterion was met, averaging was curtailed after 100 non-rejected epochs. DP-grams were recorded over restricted frequency ranges with the following stimulus parameters: L1/L2 of 60/50–80/70 in 10-dB steps. The F2/F1 ratio was 1.22 and F2 frequency ranges were 1904–2096, 2896– 3088, 3904–4096, 5904–6096 Hz with sweeps having steps of 48 Hz. We refer to these as mini DP-grams. The presence of any spontaneous OAE was checked for by asynchronous power averaging of the microphone signal FFT in the absence of stimulation using the same apparatus. HTL and DPOAE were measured daily: two days before aspirin consumption (sessions pre-1 and pre-2), during the three days of aspirin consumption and two days post-treatment. All measures were obtained from both ears. The raw phase data obtained from the DPOAE recording were referenced to the primary stimuli by subtracting 2/1 /2, where /1 and /2 are phases measured at the probe microphone for the primaries at F1 and F2, respectively. As phase can only be defined within the range ±180°, the phase data sequences were ‘‘unwrapped’’ to avoid phase jumps of more than 180° between two consecutive measurements. The phase was unwrapped by adding or subtracting multiples of 360° so as to minimize adjacent phase steps. Subsequently, the unwrapped DPOAE phase data of each mini DPgram were plotted as a function of F2 and the gradient of the best-fit line was computed. 2.3. Statistical analysis The Kolmogorov–Smirnoff test indicated that the phase gradient data were approximately normally distributed so parametric statistical analysis was performed. The first step was to quantify the test–retest repeatability (i.e., stability) of the DPOAE phase
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
gradients when normal hearing subjects are tested. One indicator of repeatability between measures is the within-subject SD on replication. This was estimated by dividing the standard deviation of the difference between the phasepgradients obtained in the two pre-aspirin sesffiffiffi pffiffiffi sions by 2. The reason for dividing by 2 is because the standard deviation of the difference incorporates the pooled uncertainty of the two measurements and if each replication has the same uncertainty (within-subject variance) the difference has double the variance. Throughout this study, repeatability is expressed in term of replication SD estimated in this way. To test if there were any significant changes in the phase gradient due to the aspirin consumption at each frequency, repeated measures analyses of variance were performed. These analyses focus on within-subject variations rather than the differences between subjects. Day of testing was the single within-subject factor and all subjects were included in each analysis. If day of testing was significant, a pairwise multiple comparison test, with a Bonferroni adjustment to the criterion of significance, was performed to evaluate differences among
47
specific means. To estimate if there was a linear, quadratic or cubic trend with respect to day of testing, a polynomial contrast was used. In addition, we compared the pre-aspirin measures (mean of session pre-1 and pre-2), with those from the three sessions of drug consumption (asp-1, asp-2, and asp-3) and the two sessions after drug consumption (post-1, post-2). All statistical analysis was performed using SPSS for Windows version 11.5.
3. Results All twelve subjects completed the full dosage of aspirin and the seven DPOAE test sessions. However, due to some missing recordings in some subjects, the data set comprised 21 mini DP-grams at 2 kHz for each recording session and for each stimulus level, and 22 mini DPgrams at 3, 4 and 6 kHz. Fig. 1 shows the mean (±1 SD) DPOAE amplitude (averaged across frequency) for the mini DP-grams centred on 2, 3, 4 and 6 kHz for the three L1 levels. Sessions
Fig. 1. Mean changes (±1 SD) in DPOAE amplitude for the mini DP-grams centred on 2 (12 subjects, 21 ears), 3, 4 and 6 kHz (12 subjects, 22 ears) at each L1 level across sessions 1–7. Session 1–2 are the two pre-aspirin sessions, sessions 3–5 are the period of aspirin consumption and sessions 6–7 are the two post-aspirin sessions.
48
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
1 and 2 represents the two pre-aspirin sessions. Sessions 3, 4 and 5 are the three sessions during aspirin consumption. Sessions 6 and 7 are the two post-aspirin sessions. In all the figures, data include both the left and right ears. The general trend of the DP amplitude for all the frequencies and levels is a statistically significant reduction during the aspirin period and almost complete recovery within the two days after the cessation of the intake, as anticipated from previous studies. There was no statistically significant difference between the amplitudes measured on the pre-1 or pre-2 and post-2 sessions.
ing to the method previously described. Table 1 summarizes the replication SD of the phase gradients obtained for each F2 frequency range and for each L1. On the basis of the values in Table 1 (less than 0.5°/Hz), it appears that the DPOAE phase gradients were repeatable across time for all frequencies and levels, with the poorest repeatability at the lowest L1 level (except 6 kHz) and the better repeatability at higher L1 levels. Moreover, a paired sample T-test performed on the data for sessions pre-1 and pre-2 was not statistically significant (p > 0.05) for any frequency or for any level.
3.1. Repeatability of the phase gradient
3.2. Relationship between the phase gradient and aspirin consumption
The repeatability of the phase gradients of 2F1–F2 DP over sessions pre-1 and pre-2 was calculated accordTable 1 Replication SD (session pre-1 vs. session pre-2) of the phase gradients (degree/Hz) L1 dB SPL
2 kHz
3 kHz
4 kHz
6 kHz
60 70 80
0.50 0.37 0.37
0.54 0.27 0.36
0.48 0.29 0.31
0.38 0.16 0.38
The relationship between the phase gradient and aspirin consumption varied among subjects and across frequencies: the general trend was an increase in (negative) phase gradient across successive sessions, particularly for the high frequencies. Fig. 2 shows examples of the phase gradient recorded around 3 and 4 kHz for the three L1 levels as a function of the recording session. These graphs clearly show that the phase gradient appears to be increased by aspirin consumption and has
Fig. 2. Examples of the trend of the phase gradient at 3 and 4 kHz at each L1 level across sessions 1–7 for some subjects. For details of the sessions see Fig. 1.
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
not recovered after cessation of aspirin intake. The apparent increase in negative phase in two examples in Fig. 2 on the last day of testing is beyond the range of change to be expected and may reflect errors arising from phase unwrapping. In all four examples, the initial phase gradient is close to zero, indicating a predominantly wave-fixed DP. The phase gradient increases sharply to indicate a substantially place-fixed DP. (A phase gradient of 2°/Hz corresponds to a group delay of approximately 5.5 ms, which suggests a predominantly place-fixed DP at these frequencies, 3 and 4 kHz.) These examples show the most marked changes occurring, but there were still significant changes present on the average of the subjects. Fig. 3 shows the mean (over 21 or 22 mini DP-grams) and the standard deviation of the phase gradients recorded at the F2 frequency ranges centred on 2, 3, 4 and 6 kHz for the three L1 levels. In general, the mean phase gradients are intermediate between suggesting predominantly wave-fixed or predominantly place-fixed behaviour. This arises from pooling data across subjects having different patterns. Fig. 3 (top left panel) shows that aspirin consumption has no mean effect on the phase gradient recorded
49
around 2 kHz at any L1 stimulus level. This observation was confirmed by the statistical analysis. By contrast, the other panels of Fig. 3 suggest a general trend for the phase gradient to become steeper across successive sessions. This was confirmed by statistical analysis, which showed that the linear within-subject contrast was statistically significant (p < 0.05) in most cases. These results are shown in detail in the last column of Table 2. In the same table all the results of the statistical analysis performed on the data at 3, 4, and 6 kHz are summarised. In Table 2 (third column) the paired group comparisons that are statistically significant (p < 0.05) are reported. It can be seen that only for 4 and 6 kHz was it possible to identify significant mean differences between specific sessions. Moreover, these differences were between pre-aspirin and post-aspirin sessions (pre-v-post-1 and pre-v-post-2 for 4 kHz at L1 = 80 dB SPL and pre-v-post-1 for 6 kHz at L1 = 70 dB SPL). There was only one instance in which there was a significant change shown during the aspirin consumption period (pre-v-asp-3 for 4 kHz at L1 = 80 dB SPL). It is known that DPOAE are characterized by high inter-subject variability in amplitude and frequency dispersion
Fig. 3. Mean change (±1 SD) of the phase gradient at 2 (12 subjects, 21 ears), 3, 4, 6 kHz (12 subjects, 22 ears) at each L1 stimulus level across sessions 1–7. For details of the sessions see Fig. 1.
50
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
Table 2 Results of the statistical analysis F2 kHz
L1 dB SPL
Pairwise comparisons
Within-subjects polynomial contrast
3
60 70 80
ns ns ns
Cubic (p = 0.018) Linear (p = 0.009) ns
4
60 70 80
6
60 70
ns pre asp-3 (p = 0.04) pre post-1 (p = 0.018) pre post-2 (p = 0.005) pre post-1 (p = 0.003)
80
Linear (p = 0.04) Linear (p = 0.024) Linear (p < 0.001)
Linear (p = 0.014) Linear (p = 0.037) Quadratic (p = 0.007) Linear (p = 0.044)
‘‘ns’’ means that no statistical significant effects were found; a blank cell means that the test was not performed.
(McCoy et al., 1990; Gaskill and Brown, 1990), while they are stable across time within individual ears. Therefore, the large error bars in Fig. 3 primarily relate to inter-subject variability rather than the reliability of measurements within individuals.
4. Discussion A temporary sensory hearing loss, in the range 7 to 21.5 dB (Hall, unpublished PhD thesis) was elicited in these twelve normally hearing subjects. This range of aspirin induced-shifts in hearing threshold is consistent with results reported in other papers (Long and Tubis, 1988a,b; Brown et al., 1993; Carlyon and Butt, 1993; Beveridge and Carlyon, 1996). In all cases the hearing threshold levels returned to their pre-aspirin values. Changes in cochlear function were monitored using mini DP-grams. Changes in DP amplitude were generally consistent with the hearing threshold data, showing a reduction during aspirin consumption. DP amplitude returned to pre-aspirin levels in all cases in the sense that there were no statistically significant differences between pre- and post-aspirin levels. However, it can be seen in Fig. 1 that the mean DP amplitudes may not have quite returned the pre-aspirin levels. In this report, we focus on the effect of aspirin on the phase gradient of the 2F1–F2 DP. While OAE amplitude reduction is an obvious and well-known effect of aspirin, there appear to be no previous studies that explore the secondary effect on phase gradient. The present study therefore focused on phase effects, despite the fact that they are less marked than the primary effects on amplitude. The reason is that phase gives some insight into which component of the DP may be changing. The effects of aspirin on the phase gradients varied between the subjects and across frequencies. The largest mean change was at 3 kHz and amounted to a change of approximately 0.5°/Hz, from approximately 1.0 to
1.5°/Hz (see Fig. 3). More extreme individual examples are shown in Fig. 2, where Subject 3 shows a change from approximately 0 to 2°/Hz. This represents a change from an entirely wave-fixed DP (0°/Hz) to a predominantly place-fixed DP. Knight and Kemp (1999) show phase gradients centred on 2 kHz for various primary frequency ratios for a single subject. The steepest gradient, corresponding presumably to a place-fixed DP, has a slope of approximately 2°/Hz. This observation is corroborated by studies involving a larger number of subjects in our own laboratory. Arguably, the phase gradient associated with a place-fixed component at 3 or 4 kHz would be less steep due to the shorter group delay at higher frequencies. This underlines the fact that the changes in phase gradient with aspirin consumption, although numerically small, represent a substantial change towards predominance of the placefixed component. The general trend was that the phase gradients for the higher F2 frequencies became steeper across successive sessions, while no significant effect was found at the lowest frequency. This frequency dependence is consistent with the greater sensitivity of the human cochlea to damage at high frequencies, and also is consistent with the greater reductions in DPOAE amplitude in the present study at the same frequencies. The differences between sessions for which the effect of the drug was significant were between the pre- and the post-aspirin consumption (Table 2), revealing a persistent effect of the drug without indication of recovery by the end of the 7-day observation period. It is important to bear in mind that the corresponding DPOAE amplitude (Fig. 1) decreased during aspirin consumption and returned almost to normal within the two days after the cessation of consumption. These reductions and recoveries of the DP amplitude across aspirin treatment are consistent with previous studies (Kujawa et al., 1992; Fitzgerald et al., 1993) and are more evident at lower stimulus level where OAEs are more sensitive to
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
change. Also the induced hearing loss completely recovered within the last two sessions. Moreover, the shift in amplitude and in phase gradient from the average of the two pre-aspirin days, was not significantly correlated for any F2 frequency range or for any L1. It should be noted that the stimulus levels and the frequencies for which the differences between the pre- and post-aspirin data are statistically significant are among the ones characterized by a high repeatability. Technical improvements to the data collection method, such as increasing averaging times, may improve repeatability and allow a better comparison of changes across stimulus frequencies and levels. The increase in phase gradient across sessions without subsequent recovery, contrasting with HTL and DP amplitude measures in the same subjects where there is substantial or complete recovery, may possibly be explained by changes in the DPOAE generation mechanism under the influence of aspirin. The 2F1–F2 DPOAE in humans for F2/F1 = 1.22 appears to be dominated by the wave-fixed component (Knight and Kemp, 1999, 2000). However, the detailed part of Knight and KempÕs work was restricted to only two subjects and more extensive work in our laboratory suggests that there is substantial variation amongst subject. It is probably more correct to state that there tends to be a predominance of the wave-fixed component. Whereas the DP amplitude depends on the vector summation of amplitudes of the wave-fixed and place-fixed components, the DP phase depends on their relative amplitudes. The DP phase will tend towards the phase of the component with the greater amplitude. Hence, an increase in the place-fixed component with a decrease in the wave-fixed component could theoretically result in an increase in (negative) phase gradient without a change in the overall amplitude. The model presented by Knight and Kemp (2001) might provide a speculative explanation for this phenomenon. The proposed biasing of the DP travelling waves according to the travelling waves shapes of the primary stimuli is considered to promote the wave-fixed component for larger frequency ratios. If aspirin were to alter these shapes, it is feasible that the wave-fixed component might be reduced and the place-fixed component increased, without a net change in the DP amplitude. Other explanations might be sought from alternative mechanisms that have been suggested for DPOAE generation, involving an interaction between the second harmonic of F1 and F2 (Fahey et al., 2000). These suggestions require more detailed modelling work and further experimentation, which is outside the scope of this study. The study reported here was not designed specifically to measure phase gradient and has some shortcomings. Furthermore, the finding of a persistent change in phase gradient was found incidentally, rather than being hypothesis driven. Further study designed specifically
51
to test the hypothesis that aspirin affects phase gradient is required to confirm or refute the preliminary findings presented here. Such study should include separation of place-fixed and wave-fixed components, for example using the method described by Kalluri and Shera (2001).
5. Conclusions The original aim of this analysis was to investigate the effect of aspirin on DPOAE phase, in parallel with the well-known effect of reduction of the amplitude of some DPOAE components. As anticipated from other studies, data presented here show that the primary effect of aspirin consumption on DPOAE was an amplitude reduction during the aspirin period and a recovery after the end of the drug intake. However, the findings presented here also suggest that aspirin may have secondary effects on cochlear function that are more persistent than the primary effects disclosed by measurements of hearing threshold level or DPOAE amplitude. Specifically, DPOAE phase gradient appears to be increased by aspirin consumption and has not recovered two days after cessation of aspirin consumption, despite almost complete recovery of DPOAE amplitude and hearing threshold levels. The increase in phase gradient infers a change from a predominantly wave-fixed (distortion) mechanism to a predominantly place-fixed (reflection) mechanism of DPOAE generation. It may not be safe to infer that effects of aspirin have disappeared purely on the basis of recovery of DPOAE amplitude and hearing threshold level.
Acknowledgements This work was supported by a Marie Curie Training Site Fellowship to the first author (Contract Number HPMT-CT-2000-00034) and by a research grant from Defeating Deafness to the second and third authors.
References Beveridge, H.A., Carlyon, R.P., 1996. Effects of aspirin on human psychophysical tuning curves in forward and simultaneous masking. Hear. Res. 99, 110–118. Brown, A.M., Williams, D.M., Gaskill, S.A., 1993. The effect of aspirin on cochlear mechanical tuning. J. Acoust. Soc. Am. 93, 3298–3307. Carlyon, R.P., Butt, M., 1993. Effects of aspirin on human auditory filters. Hear. Res. 66, 233–244. Cazals, Y., 2000. Auditory sensori-neural alterations induced by salicylate. Progr. Neurobiol. 62, 583–631. Fahey, P.F., Stagner, B.B., Lonsbury-Martin, B.L., Martin, G.K., 2000. Nonlinear interactions that could explain distortion product interference response areas. J. Acoust. Soc. Am. 108, 1786– 1802.
52
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
Fitzgerald, J.J., Robertson, D., Johnstone, B.M., 1993. Effects of intracochlear perfusion of salicylate on cochlear microphonic and other auditory response in the guinea pig. Hear. Res. 67, 147–156. Frank, G., Kossl, M., 1996. The acoustic two-tone distortion 2f1–f2 and 2f2–f1 and their possible relation to changes in the operating point of the cochlear amplifier. Hear. Res. 98, 104–115. Gaskill, S.A., Brown, A.M., 1990. The behaviour of otoacoustic distortion product 2f1–f2 from the human ear and its relation to auditory sensitivity. J. Acoust. Soc. Am. 88, 821–839. Janssen, T., Boege, P., Oestreicher, E., Arnold, W., 2000. Tinnitus and 2f1–f2 distortion product otoacoustic emissions following salicylate overdose. J. Acoust. Soc. Am. 107, 1790–1792. Kalluri, R., Shera, C.A., 2001. Distortion-product source unmixing: a test of the two-mechanism model for DPOAE generation. J. Acoust. Soc. Am. 109, 622–637. Kemp, D.T., 1978. Stimulated otoacoustic emission from within the human auditory system. J. Acoust. Soc. Am. 64, 1386–1391. Knight, R.D., Kemp, D.T., 1999. Relationship between DPOAE and TEOAE amplitude and phase characteristics. J. Acoust. Soc. Am. 106, 1420–1435. Knight, R.D., Kemp, D.T., 2000. Indications of different distortion product otoacoustic emission mechanism from a detailed f1, f2 area study. J. Acoust. Soc. Am. 107, 457–473. Knight, R.D., Kemp, D.T., 2001. Wave and place fixed maps of the human ear. J. Acoust. Soc. Am. 109, 1513–1525. Kujawa, S.G., Fallon, M., Bobbin, R.P., 1992. Intracochlear salicylate reduces low-intensity acoustic and cochlear microphonic distortion products. Hear. Res. 64, 73–80. Long, G.R., Tubis, A., 1988a. Investigations into the nature of the associations between threshold microstructure and otoacoustic emissions. Hear. Res. 36, 125–138. Long, G.R., Tubis, A., 1988b. Modifications of spontaneous and evoked otoacoustic emissions and associated psychoacoustic
microstructure by aspirin consumption. J. Acoust. Soc. Am. 84, 1343–1353. Long, G.R., Tubis, A., Jones, K.L., 1991. Modelling synchronization and suppression of spontaneous otoacoustic emissions using Van der Pol oscillators: effects of aspirin administration. J. Acoust. Soc. Am. 89, 1201–1212. McCoy, M.J., Franklin, D.J., Lonsbury-Martin, B.L., Martin, G.R., 1990. Evoked otoacoustic emissions: test–retest reliability. ASHA Monogr., 37–156. McFadden, D., Plattsmier, H.S., 1984. Aspirin abolishes spontaneous otoacoustic emissions. J. Acoust. Soc. Am. 76, 443–448. Russell, I.J., Schauz, C., 1995. Salicylate ototoxicity: effects on the stiffness and electromotility of outer hair cells isolated from the guinea pig cochlea. Audit. Neurosci. 1, 309–319. Shera, C.A., Guinan, J.J., 1999. Evoked otoacoustic emissions arise by two fundamentally different mechanisms: a taxonomy for mammalian OAEs. J. Acoust. Soc. Am. 105, 782–798. Shehata, W.E., Brownell, W.E., Dieler, R., 1991. Effect of salicylate on shape, electromotility and membrane characteristics of isolated outer hair cells from guinea pig cochlea. Acta Oto-Laryngol. (Stockholm) 111, 707–718. Stypulkowski, P.H., 1990. Mechanisms of salicylate ototoxicity. Hear. Res. 46, 113–145. Tunstall, M.J., Ashmore, J.F., Gale, J.E., 1994. The effects of salicylate on the properties of isolated outer hair cells from the guinea-pigs cochlea. Br. J. Audiol. 27, 332–333. Wier, C.C., Pasanen, E.G., McFadden, D., 1988. Partial dissociation of spontaneous otoacoustic emissions and distortion products during aspirin consumption in humans. J. Acoust. Soc. Am. 84, 230–237. Zweig, G., Shera, C.A., 1995. The origin on the periodicity in the spectrum of evoked otoacoustic emissions. J. Acoust. Soc. Am. 98, 2018–2047.
Effect of aspirin on phase gradient of 2F1–F2 distortion product otoacoustic emissions M. Parazzini a
a,*
, A.J. Hall b, M.E. Lutman b, S. Kapadia
b
Istituto di Ingegneria Biomedica ISIB, CNR, Milan, Piazza Leonardo da Vinci 32, 20133 Milano, Italy b Institute of Sound and Vibration Research, University of Southampton, UK Received 3 September 2004; accepted 28 February 2005 Available online 28 March 2005
Abstract It is well known that aspirin consumption temporarily reduces overall otoacoustic emission (OAE) amplitude in humans. However, little is known about changes in the separate components of distortion product otoacoustic emissions (DPOAE), which may be distinguished by examining phase gradients. The effects of aspirin on the phase gradient of the DPOAE 2F1–F2 obtained with fixed frequency ratio sweeps were studied longitudinally in a group of twelve subjects in whom a temporary hearing loss was induced by aspirin consumption. DPOAE were recorded daily for two days pre-aspirin consumption, during the three days of aspirin consumption and two days afterwards. DP-grams were recorded over a restricted frequency range centered on 2, 3, 4 and 6 kHz with the following stimulus levels: L1/L2 of 60/50–80/70 in 10-dB steps. The effects of aspirin on the phase gradients varied between the subjects and across frequency: the general trend was that the phase gradient became steeper across successive sessions for the higher frequencies, while no significant effect was found at the lower frequencies. These results suggest that aspirin may have more persistent effects on cochlear function than are disclosed by measurements of hearing threshold level or DPOAE amplitude. Particularly, DPOAE phase gradient appears to be increased by aspirin consumption and has not recovered two days after cessation of aspirin intake, despite almost complete recovery of DPOAE amplitude and hearing threshold levels. These findings may suggest differential effects on the distortion and reflection mechanisms considered to underlie DPOAE generation. Ó 2005 Elsevier B.V. All rights reserved. Keywords: Distortion product otoacoustic emissions; Aspirin; Phase gradient
1. Introduction The drug aspirin (acetylsalicylic acid) is one of the salicylates and is known to affect the mechanical response of the cochlea (Brown et al., 1993). This effect Abbreviations: DP, distortion product; DPOAE, distortion product otoacoustic emissions; FFT, fast Fourier transform; HTL, hearing threshold level; OAE, otoacoustic emissions; OHC, outer hair cell; SD, standard deviation; SNR, signal to noise ratio; TEOAE, transientevoked otoacoustic emissions * Corresponding author. Tel.: +39 0223993359; fax: +39 0223993367/ 60. E-mail address: [email protected] (M. Parazzini). 0378-5955/$ - see front matter Ó 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2005.02.010
may be the consequence of the changes in turgidity and motility that have been observed in isolated mammalian outer hair cells (OHC) perfused with aspirin (Shehata et al., 1991; Tunstall et al., 1994; Russell and Schauz, 1995). Different studies (for a review on auditory sensorineural alterations induced by salicylate, see Cazals, 2000) show that clear alterations are produced on otoacoustic emissions (OAE) by salicylate. The most conspicuous effect is the disappearance of spontaneous OAE (McFadden and Plattsmier, 1984; Long and Tubis, 1988a; Wier et al., 1988; Long et al., 1991). However, the data do not indicate, either for amplitude or time course, clear correlations between the reduction
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
and/or disappearance of spontaneous OAE and the loss of absolute hearing sensitivity. Studies of evoked OAE (Long and Tubis, 1988b; Janssen et al., 2000) indicate reductions in response amplitude and associated threshold elevation, corresponding with loss of absolute hearing sensitivity. The reductions in the distortion product otoacoustic emission (DPOAE) amplitude become more pronounced as the levels of the stimuli are lowered (Kujawa et al., 1992; Fitzgerald et al., 1993). Detailed measures of various DPOAE components and/or characteristics further revealed more subtle changes in different aspects of nonlinear cochlear functioning (Stypulkowski, 1990; Brown et al., 1993; Frank and Kossl, 1996). Both studies of isolated OHC and studies of human and animal data show that alterations induced by salicylate appear to be dose-dependent and reversible (Cazals, 2000). The reversible nature of the aspirin-induced hearing loss makes it an attractive tool as it allows the experimenter to compare performance in the same subject both in the absence and in the presence of hearing loss (Beveridge and Carlyon, 1996). While it is known that significant reductions in DPOAE amplitude are a sign of functional or structural damage to OHC (Kemp, 1978), the relationship between DPOAE phase and cochlear status is still not well understood. Previous studies suggest that the phase gradient against frequency, obtained using fixed frequency ratio sweeps, is consistent with a combination of two different DPOAE emission components (Knight and Kemp, 1999, 2000, 2001). Knight and Kemp refer to these as placed- and wave-fixed mechanisms, based on the assumed site of generation. The general assumption with a wave-fixed mechanism is that the emission is generated by distortion at a site that is an integral part of and moves smoothly with the stimulus travelling wave envelope as stimulus frequency is swept (Shera and Guinan, 1999). For the 2F1–F2 distortion product (DP), the wave-fixed component is considered to be generated close to the F2 place on the basilar membrane and reaches the ear canal via a travelling wave propagating in the reverse direction along the basilar membrane. The phase at any point moving with the travelling wave envelope changes little; therefore, any OAE contribution from that point would have a shallow phase gradient using fixed frequency ratio sweeps. Distortion generated at the F2 place also propagates in the forward direction to the DP place, where it may be reflected. Zweig and Shera (1995) have proposed a series of reflecting or scattering sites existing along the basilar membrane and a mechanism of coherent reflection involving the sharply tuned basilar membrane excitation pattern. As a stimulus is swept in frequency and its excitation pattern moves along the basilar membrane, the stimulus phase at the reflection site will change, thus changing the OAE phase
45
and creating a steep gradient.1 Phase gradients may also be considered in terms of group delay, where a shallow slope corresponds to a short delay relative to the primary stimuli, while a steep slope corresponds to a longer delay, with phase gradient proportional to delay. Because these two components are generated by different processes, at different sites along the basilar membrane, it is of fundamental interest to understand how they each change with cochlear damage. Steep and shallow phase gradients have been observed in the 2F1–F2 DP (Knight and Kemp, 1999) depending on whether a large or small frequency ratio is used. In particular, for a small frequency ratio, the phase gradient is steep, consistent with a predominantly place-fixed emission mechanism, while with a larger frequency ratio, the phase gradient becomes shallow and is more consistent with a wave-fixed mechanism. The reasons for this are not yet understood fully, although Knight and Kemp (2001) propose a model that suggests that the propagation of DP travelling waves is biased by the shapes of the primary travelling waves. For the more widely spaced primary frequencies commonly used to measure the 2F1–F2 DP, the reverse travelling wave in the F2 frequency region is promoted so that the wave-fixed component tends to dominate the response, thus explaining the shallow phase gradients observed with larger frequency ratios. When aspirin interferes with cochlear function, it is unclear whether it affects the place-fixed or wave-fixed mechanism, or both. Because the two mechanisms are fundamentally different, as described above, it is plausible that aspirin may affect either or both. It is therefore of interest to know whether aspirin produces a steeper or shallower phase gradient of the 2F1–F2 DP in parallel with the well known effect of reduction of the amplitude of the same DPOAE component. Previous studies using aspirin in humans have measured amplitude but have not reported on phase. Hence, the aim of the analysis reported here was to investigate effects of aspirin on DPOAE phase. These findings may throw some light on whether aspirin affects primarily wave-fixed or placefixed mechanisms of OAE generation, or in other words whether it affects primarily distortion or reflection mechanisms. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institute of Sound and Vibration Research Human Experimentation Safety and Ethics Committee.
1
Whether the two components are categorised by the assumed site of generation, wave- or place-fixed, or by the assumed mechanism, ‘‘distortion’’ or ‘‘reflection’’ is a subtlety beyond the scope of this work. For simplicity, they are referred to as wave- and place-fixed, following the usage of Knight and Kemp (1999).
46
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
2. Materials and methods Results presented here were obtained from an additional analysis of existing data recorded in another study (Hall, unpublished PhD thesis), where the aim was to explore the relationship between changes in hearing threshold level (HTL) and changes in cochlear amplifier gain inferred from OAE measurements (both TEOAE and DPOAE). Changes were induced by causing a temporary hearing loss using aspirin. As the experiment was not designed to explore phase gradients the methods have some limitations. Nevertheless, given the difficulties in conducting experiments where normal participants are administered a drug with potential side effects, the data were utilised for this preliminary exploratory study. 2.1. Subjects and drug schedule Twelve male subjects, aged 19–38 years, took part in the experiment. Subjects were given a list of contraindications to aspirin, which if occurring in any individual would preclude participation in the study. Subjects had HTL better than or equal to 20 dB at all standard audiometric frequencies. All had normal middle ear function with bone conduction thresholds within 5 dB of air conduction thresholds averaged across 0.5, 1, 2 and 4 kHz, normal tympanometry and normal otoscopy. Subjects took 11.7 g of aspirin over a period of 72 h spanning four calendar days (975 mg twice on the first day, four times each on the second and third days and twice on the fourth). They consumed food with each dose in accordance with recommended practice. They were asked to refrain from alcohol consumption during the entire aspirin consumption period and to fill in a diary to confirm their intake of aspirin. To facilitate correct dosage, subjects were given a dose box containing the number of aspirin tablets to be taken at each session on each day for the course of the experiment. Salicylate concentration in the blood was monitored on days 3–5 and generally increased over the dosage period. Maximum concentration ranged from 0.5 to 1.5 mmol/l across subjects (mean value 0.89 mmol/l). 2.2. DPOAE measurements The distortion product 2F1–F2 was recorded using laboratory apparatus designed specifically for the purpose. Primary frequencies are denoted by F1 and F2, while primary levels are denoted by L1 and L2. An Etymotic ER-10B+ microphone probe and pre-amplifier (+40 dB) were used for recording ear canal sound pressure. Two Etymotic ER-2 insert earphones were used to deliver the primary tone stimuli to the subject via the probe. The amplified microphone signal was digitised (16-bit resolution, 32.768-kHz sample rate) by an exter-
nal A/D and D/A converter unit (Institute of Hearing Research DSP remote converter module) that also generated the primaries. Signal processing by custom software running on a TMS-320 DSP card converted consecutive 62.5-ms epochs of the microphone signal to the frequency domain by performing FFT with a bin width of 16 Hz. The complex FFT was averaged after rejection of epochs in which the estimated noise level was greater than 10 dB SPL in the frequency range close to the DP. The amplitude and phase of the DP were estimated from the real and imaginary FFT components corresponding to the single bin centred on the DP frequency. Noise at the DP frequency was estimated by averaging the power in 10 spectral lines either side of the DP. Recording of a DPOAE for a particular frequency was stopped after a minimum number of epochs had been acquired and a minimum SNR was reached; these minima were set at 20 epochs and a SNR of 10 dB. If neither criterion was met, averaging was curtailed after 100 non-rejected epochs. DP-grams were recorded over restricted frequency ranges with the following stimulus parameters: L1/L2 of 60/50–80/70 in 10-dB steps. The F2/F1 ratio was 1.22 and F2 frequency ranges were 1904–2096, 2896– 3088, 3904–4096, 5904–6096 Hz with sweeps having steps of 48 Hz. We refer to these as mini DP-grams. The presence of any spontaneous OAE was checked for by asynchronous power averaging of the microphone signal FFT in the absence of stimulation using the same apparatus. HTL and DPOAE were measured daily: two days before aspirin consumption (sessions pre-1 and pre-2), during the three days of aspirin consumption and two days post-treatment. All measures were obtained from both ears. The raw phase data obtained from the DPOAE recording were referenced to the primary stimuli by subtracting 2/1 /2, where /1 and /2 are phases measured at the probe microphone for the primaries at F1 and F2, respectively. As phase can only be defined within the range ±180°, the phase data sequences were ‘‘unwrapped’’ to avoid phase jumps of more than 180° between two consecutive measurements. The phase was unwrapped by adding or subtracting multiples of 360° so as to minimize adjacent phase steps. Subsequently, the unwrapped DPOAE phase data of each mini DPgram were plotted as a function of F2 and the gradient of the best-fit line was computed. 2.3. Statistical analysis The Kolmogorov–Smirnoff test indicated that the phase gradient data were approximately normally distributed so parametric statistical analysis was performed. The first step was to quantify the test–retest repeatability (i.e., stability) of the DPOAE phase
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
gradients when normal hearing subjects are tested. One indicator of repeatability between measures is the within-subject SD on replication. This was estimated by dividing the standard deviation of the difference between the phasepgradients obtained in the two pre-aspirin sesffiffiffi pffiffiffi sions by 2. The reason for dividing by 2 is because the standard deviation of the difference incorporates the pooled uncertainty of the two measurements and if each replication has the same uncertainty (within-subject variance) the difference has double the variance. Throughout this study, repeatability is expressed in term of replication SD estimated in this way. To test if there were any significant changes in the phase gradient due to the aspirin consumption at each frequency, repeated measures analyses of variance were performed. These analyses focus on within-subject variations rather than the differences between subjects. Day of testing was the single within-subject factor and all subjects were included in each analysis. If day of testing was significant, a pairwise multiple comparison test, with a Bonferroni adjustment to the criterion of significance, was performed to evaluate differences among
47
specific means. To estimate if there was a linear, quadratic or cubic trend with respect to day of testing, a polynomial contrast was used. In addition, we compared the pre-aspirin measures (mean of session pre-1 and pre-2), with those from the three sessions of drug consumption (asp-1, asp-2, and asp-3) and the two sessions after drug consumption (post-1, post-2). All statistical analysis was performed using SPSS for Windows version 11.5.
3. Results All twelve subjects completed the full dosage of aspirin and the seven DPOAE test sessions. However, due to some missing recordings in some subjects, the data set comprised 21 mini DP-grams at 2 kHz for each recording session and for each stimulus level, and 22 mini DPgrams at 3, 4 and 6 kHz. Fig. 1 shows the mean (±1 SD) DPOAE amplitude (averaged across frequency) for the mini DP-grams centred on 2, 3, 4 and 6 kHz for the three L1 levels. Sessions
Fig. 1. Mean changes (±1 SD) in DPOAE amplitude for the mini DP-grams centred on 2 (12 subjects, 21 ears), 3, 4 and 6 kHz (12 subjects, 22 ears) at each L1 level across sessions 1–7. Session 1–2 are the two pre-aspirin sessions, sessions 3–5 are the period of aspirin consumption and sessions 6–7 are the two post-aspirin sessions.
48
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
1 and 2 represents the two pre-aspirin sessions. Sessions 3, 4 and 5 are the three sessions during aspirin consumption. Sessions 6 and 7 are the two post-aspirin sessions. In all the figures, data include both the left and right ears. The general trend of the DP amplitude for all the frequencies and levels is a statistically significant reduction during the aspirin period and almost complete recovery within the two days after the cessation of the intake, as anticipated from previous studies. There was no statistically significant difference between the amplitudes measured on the pre-1 or pre-2 and post-2 sessions.
ing to the method previously described. Table 1 summarizes the replication SD of the phase gradients obtained for each F2 frequency range and for each L1. On the basis of the values in Table 1 (less than 0.5°/Hz), it appears that the DPOAE phase gradients were repeatable across time for all frequencies and levels, with the poorest repeatability at the lowest L1 level (except 6 kHz) and the better repeatability at higher L1 levels. Moreover, a paired sample T-test performed on the data for sessions pre-1 and pre-2 was not statistically significant (p > 0.05) for any frequency or for any level.
3.1. Repeatability of the phase gradient
3.2. Relationship between the phase gradient and aspirin consumption
The repeatability of the phase gradients of 2F1–F2 DP over sessions pre-1 and pre-2 was calculated accordTable 1 Replication SD (session pre-1 vs. session pre-2) of the phase gradients (degree/Hz) L1 dB SPL
2 kHz
3 kHz
4 kHz
6 kHz
60 70 80
0.50 0.37 0.37
0.54 0.27 0.36
0.48 0.29 0.31
0.38 0.16 0.38
The relationship between the phase gradient and aspirin consumption varied among subjects and across frequencies: the general trend was an increase in (negative) phase gradient across successive sessions, particularly for the high frequencies. Fig. 2 shows examples of the phase gradient recorded around 3 and 4 kHz for the three L1 levels as a function of the recording session. These graphs clearly show that the phase gradient appears to be increased by aspirin consumption and has
Fig. 2. Examples of the trend of the phase gradient at 3 and 4 kHz at each L1 level across sessions 1–7 for some subjects. For details of the sessions see Fig. 1.
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
not recovered after cessation of aspirin intake. The apparent increase in negative phase in two examples in Fig. 2 on the last day of testing is beyond the range of change to be expected and may reflect errors arising from phase unwrapping. In all four examples, the initial phase gradient is close to zero, indicating a predominantly wave-fixed DP. The phase gradient increases sharply to indicate a substantially place-fixed DP. (A phase gradient of 2°/Hz corresponds to a group delay of approximately 5.5 ms, which suggests a predominantly place-fixed DP at these frequencies, 3 and 4 kHz.) These examples show the most marked changes occurring, but there were still significant changes present on the average of the subjects. Fig. 3 shows the mean (over 21 or 22 mini DP-grams) and the standard deviation of the phase gradients recorded at the F2 frequency ranges centred on 2, 3, 4 and 6 kHz for the three L1 levels. In general, the mean phase gradients are intermediate between suggesting predominantly wave-fixed or predominantly place-fixed behaviour. This arises from pooling data across subjects having different patterns. Fig. 3 (top left panel) shows that aspirin consumption has no mean effect on the phase gradient recorded
49
around 2 kHz at any L1 stimulus level. This observation was confirmed by the statistical analysis. By contrast, the other panels of Fig. 3 suggest a general trend for the phase gradient to become steeper across successive sessions. This was confirmed by statistical analysis, which showed that the linear within-subject contrast was statistically significant (p < 0.05) in most cases. These results are shown in detail in the last column of Table 2. In the same table all the results of the statistical analysis performed on the data at 3, 4, and 6 kHz are summarised. In Table 2 (third column) the paired group comparisons that are statistically significant (p < 0.05) are reported. It can be seen that only for 4 and 6 kHz was it possible to identify significant mean differences between specific sessions. Moreover, these differences were between pre-aspirin and post-aspirin sessions (pre-v-post-1 and pre-v-post-2 for 4 kHz at L1 = 80 dB SPL and pre-v-post-1 for 6 kHz at L1 = 70 dB SPL). There was only one instance in which there was a significant change shown during the aspirin consumption period (pre-v-asp-3 for 4 kHz at L1 = 80 dB SPL). It is known that DPOAE are characterized by high inter-subject variability in amplitude and frequency dispersion
Fig. 3. Mean change (±1 SD) of the phase gradient at 2 (12 subjects, 21 ears), 3, 4, 6 kHz (12 subjects, 22 ears) at each L1 stimulus level across sessions 1–7. For details of the sessions see Fig. 1.
50
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
Table 2 Results of the statistical analysis F2 kHz
L1 dB SPL
Pairwise comparisons
Within-subjects polynomial contrast
3
60 70 80
ns ns ns
Cubic (p = 0.018) Linear (p = 0.009) ns
4
60 70 80
6
60 70
ns pre asp-3 (p = 0.04) pre post-1 (p = 0.018) pre post-2 (p = 0.005) pre post-1 (p = 0.003)
80
Linear (p = 0.04) Linear (p = 0.024) Linear (p < 0.001)
Linear (p = 0.014) Linear (p = 0.037) Quadratic (p = 0.007) Linear (p = 0.044)
‘‘ns’’ means that no statistical significant effects were found; a blank cell means that the test was not performed.
(McCoy et al., 1990; Gaskill and Brown, 1990), while they are stable across time within individual ears. Therefore, the large error bars in Fig. 3 primarily relate to inter-subject variability rather than the reliability of measurements within individuals.
4. Discussion A temporary sensory hearing loss, in the range 7 to 21.5 dB (Hall, unpublished PhD thesis) was elicited in these twelve normally hearing subjects. This range of aspirin induced-shifts in hearing threshold is consistent with results reported in other papers (Long and Tubis, 1988a,b; Brown et al., 1993; Carlyon and Butt, 1993; Beveridge and Carlyon, 1996). In all cases the hearing threshold levels returned to their pre-aspirin values. Changes in cochlear function were monitored using mini DP-grams. Changes in DP amplitude were generally consistent with the hearing threshold data, showing a reduction during aspirin consumption. DP amplitude returned to pre-aspirin levels in all cases in the sense that there were no statistically significant differences between pre- and post-aspirin levels. However, it can be seen in Fig. 1 that the mean DP amplitudes may not have quite returned the pre-aspirin levels. In this report, we focus on the effect of aspirin on the phase gradient of the 2F1–F2 DP. While OAE amplitude reduction is an obvious and well-known effect of aspirin, there appear to be no previous studies that explore the secondary effect on phase gradient. The present study therefore focused on phase effects, despite the fact that they are less marked than the primary effects on amplitude. The reason is that phase gives some insight into which component of the DP may be changing. The effects of aspirin on the phase gradients varied between the subjects and across frequencies. The largest mean change was at 3 kHz and amounted to a change of approximately 0.5°/Hz, from approximately 1.0 to
1.5°/Hz (see Fig. 3). More extreme individual examples are shown in Fig. 2, where Subject 3 shows a change from approximately 0 to 2°/Hz. This represents a change from an entirely wave-fixed DP (0°/Hz) to a predominantly place-fixed DP. Knight and Kemp (1999) show phase gradients centred on 2 kHz for various primary frequency ratios for a single subject. The steepest gradient, corresponding presumably to a place-fixed DP, has a slope of approximately 2°/Hz. This observation is corroborated by studies involving a larger number of subjects in our own laboratory. Arguably, the phase gradient associated with a place-fixed component at 3 or 4 kHz would be less steep due to the shorter group delay at higher frequencies. This underlines the fact that the changes in phase gradient with aspirin consumption, although numerically small, represent a substantial change towards predominance of the placefixed component. The general trend was that the phase gradients for the higher F2 frequencies became steeper across successive sessions, while no significant effect was found at the lowest frequency. This frequency dependence is consistent with the greater sensitivity of the human cochlea to damage at high frequencies, and also is consistent with the greater reductions in DPOAE amplitude in the present study at the same frequencies. The differences between sessions for which the effect of the drug was significant were between the pre- and the post-aspirin consumption (Table 2), revealing a persistent effect of the drug without indication of recovery by the end of the 7-day observation period. It is important to bear in mind that the corresponding DPOAE amplitude (Fig. 1) decreased during aspirin consumption and returned almost to normal within the two days after the cessation of consumption. These reductions and recoveries of the DP amplitude across aspirin treatment are consistent with previous studies (Kujawa et al., 1992; Fitzgerald et al., 1993) and are more evident at lower stimulus level where OAEs are more sensitive to
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
change. Also the induced hearing loss completely recovered within the last two sessions. Moreover, the shift in amplitude and in phase gradient from the average of the two pre-aspirin days, was not significantly correlated for any F2 frequency range or for any L1. It should be noted that the stimulus levels and the frequencies for which the differences between the pre- and post-aspirin data are statistically significant are among the ones characterized by a high repeatability. Technical improvements to the data collection method, such as increasing averaging times, may improve repeatability and allow a better comparison of changes across stimulus frequencies and levels. The increase in phase gradient across sessions without subsequent recovery, contrasting with HTL and DP amplitude measures in the same subjects where there is substantial or complete recovery, may possibly be explained by changes in the DPOAE generation mechanism under the influence of aspirin. The 2F1–F2 DPOAE in humans for F2/F1 = 1.22 appears to be dominated by the wave-fixed component (Knight and Kemp, 1999, 2000). However, the detailed part of Knight and KempÕs work was restricted to only two subjects and more extensive work in our laboratory suggests that there is substantial variation amongst subject. It is probably more correct to state that there tends to be a predominance of the wave-fixed component. Whereas the DP amplitude depends on the vector summation of amplitudes of the wave-fixed and place-fixed components, the DP phase depends on their relative amplitudes. The DP phase will tend towards the phase of the component with the greater amplitude. Hence, an increase in the place-fixed component with a decrease in the wave-fixed component could theoretically result in an increase in (negative) phase gradient without a change in the overall amplitude. The model presented by Knight and Kemp (2001) might provide a speculative explanation for this phenomenon. The proposed biasing of the DP travelling waves according to the travelling waves shapes of the primary stimuli is considered to promote the wave-fixed component for larger frequency ratios. If aspirin were to alter these shapes, it is feasible that the wave-fixed component might be reduced and the place-fixed component increased, without a net change in the DP amplitude. Other explanations might be sought from alternative mechanisms that have been suggested for DPOAE generation, involving an interaction between the second harmonic of F1 and F2 (Fahey et al., 2000). These suggestions require more detailed modelling work and further experimentation, which is outside the scope of this study. The study reported here was not designed specifically to measure phase gradient and has some shortcomings. Furthermore, the finding of a persistent change in phase gradient was found incidentally, rather than being hypothesis driven. Further study designed specifically
51
to test the hypothesis that aspirin affects phase gradient is required to confirm or refute the preliminary findings presented here. Such study should include separation of place-fixed and wave-fixed components, for example using the method described by Kalluri and Shera (2001).
5. Conclusions The original aim of this analysis was to investigate the effect of aspirin on DPOAE phase, in parallel with the well-known effect of reduction of the amplitude of some DPOAE components. As anticipated from other studies, data presented here show that the primary effect of aspirin consumption on DPOAE was an amplitude reduction during the aspirin period and a recovery after the end of the drug intake. However, the findings presented here also suggest that aspirin may have secondary effects on cochlear function that are more persistent than the primary effects disclosed by measurements of hearing threshold level or DPOAE amplitude. Specifically, DPOAE phase gradient appears to be increased by aspirin consumption and has not recovered two days after cessation of aspirin consumption, despite almost complete recovery of DPOAE amplitude and hearing threshold levels. The increase in phase gradient infers a change from a predominantly wave-fixed (distortion) mechanism to a predominantly place-fixed (reflection) mechanism of DPOAE generation. It may not be safe to infer that effects of aspirin have disappeared purely on the basis of recovery of DPOAE amplitude and hearing threshold level.
Acknowledgements This work was supported by a Marie Curie Training Site Fellowship to the first author (Contract Number HPMT-CT-2000-00034) and by a research grant from Defeating Deafness to the second and third authors.
References Beveridge, H.A., Carlyon, R.P., 1996. Effects of aspirin on human psychophysical tuning curves in forward and simultaneous masking. Hear. Res. 99, 110–118. Brown, A.M., Williams, D.M., Gaskill, S.A., 1993. The effect of aspirin on cochlear mechanical tuning. J. Acoust. Soc. Am. 93, 3298–3307. Carlyon, R.P., Butt, M., 1993. Effects of aspirin on human auditory filters. Hear. Res. 66, 233–244. Cazals, Y., 2000. Auditory sensori-neural alterations induced by salicylate. Progr. Neurobiol. 62, 583–631. Fahey, P.F., Stagner, B.B., Lonsbury-Martin, B.L., Martin, G.K., 2000. Nonlinear interactions that could explain distortion product interference response areas. J. Acoust. Soc. Am. 108, 1786– 1802.
52
M. Parazzini et al. / Hearing Research 205 (2005) 44–52
Fitzgerald, J.J., Robertson, D., Johnstone, B.M., 1993. Effects of intracochlear perfusion of salicylate on cochlear microphonic and other auditory response in the guinea pig. Hear. Res. 67, 147–156. Frank, G., Kossl, M., 1996. The acoustic two-tone distortion 2f1–f2 and 2f2–f1 and their possible relation to changes in the operating point of the cochlear amplifier. Hear. Res. 98, 104–115. Gaskill, S.A., Brown, A.M., 1990. The behaviour of otoacoustic distortion product 2f1–f2 from the human ear and its relation to auditory sensitivity. J. Acoust. Soc. Am. 88, 821–839. Janssen, T., Boege, P., Oestreicher, E., Arnold, W., 2000. Tinnitus and 2f1–f2 distortion product otoacoustic emissions following salicylate overdose. J. Acoust. Soc. Am. 107, 1790–1792. Kalluri, R., Shera, C.A., 2001. Distortion-product source unmixing: a test of the two-mechanism model for DPOAE generation. J. Acoust. Soc. Am. 109, 622–637. Kemp, D.T., 1978. Stimulated otoacoustic emission from within the human auditory system. J. Acoust. Soc. Am. 64, 1386–1391. Knight, R.D., Kemp, D.T., 1999. Relationship between DPOAE and TEOAE amplitude and phase characteristics. J. Acoust. Soc. Am. 106, 1420–1435. Knight, R.D., Kemp, D.T., 2000. Indications of different distortion product otoacoustic emission mechanism from a detailed f1, f2 area study. J. Acoust. Soc. Am. 107, 457–473. Knight, R.D., Kemp, D.T., 2001. Wave and place fixed maps of the human ear. J. Acoust. Soc. Am. 109, 1513–1525. Kujawa, S.G., Fallon, M., Bobbin, R.P., 1992. Intracochlear salicylate reduces low-intensity acoustic and cochlear microphonic distortion products. Hear. Res. 64, 73–80. Long, G.R., Tubis, A., 1988a. Investigations into the nature of the associations between threshold microstructure and otoacoustic emissions. Hear. Res. 36, 125–138. Long, G.R., Tubis, A., 1988b. Modifications of spontaneous and evoked otoacoustic emissions and associated psychoacoustic
microstructure by aspirin consumption. J. Acoust. Soc. Am. 84, 1343–1353. Long, G.R., Tubis, A., Jones, K.L., 1991. Modelling synchronization and suppression of spontaneous otoacoustic emissions using Van der Pol oscillators: effects of aspirin administration. J. Acoust. Soc. Am. 89, 1201–1212. McCoy, M.J., Franklin, D.J., Lonsbury-Martin, B.L., Martin, G.R., 1990. Evoked otoacoustic emissions: test–retest reliability. ASHA Monogr., 37–156. McFadden, D., Plattsmier, H.S., 1984. Aspirin abolishes spontaneous otoacoustic emissions. J. Acoust. Soc. Am. 76, 443–448. Russell, I.J., Schauz, C., 1995. Salicylate ototoxicity: effects on the stiffness and electromotility of outer hair cells isolated from the guinea pig cochlea. Audit. Neurosci. 1, 309–319. Shera, C.A., Guinan, J.J., 1999. Evoked otoacoustic emissions arise by two fundamentally different mechanisms: a taxonomy for mammalian OAEs. J. Acoust. Soc. Am. 105, 782–798. Shehata, W.E., Brownell, W.E., Dieler, R., 1991. Effect of salicylate on shape, electromotility and membrane characteristics of isolated outer hair cells from guinea pig cochlea. Acta Oto-Laryngol. (Stockholm) 111, 707–718. Stypulkowski, P.H., 1990. Mechanisms of salicylate ototoxicity. Hear. Res. 46, 113–145. Tunstall, M.J., Ashmore, J.F., Gale, J.E., 1994. The effects of salicylate on the properties of isolated outer hair cells from the guinea-pigs cochlea. Br. J. Audiol. 27, 332–333. Wier, C.C., Pasanen, E.G., McFadden, D., 1988. Partial dissociation of spontaneous otoacoustic emissions and distortion products during aspirin consumption in humans. J. Acoust. Soc. Am. 84, 230–237. Zweig, G., Shera, C.A., 1995. The origin on the periodicity in the spectrum of evoked otoacoustic emissions. J. Acoust. Soc. Am. 98, 2018–2047.