Gap Detection Deficits in Chinchillas with Selective Carboplatin-Induced Inner Hair Cell Loss

Abstract

Temporal resolution is essential for processing complex auditory information such as speech. In hearing impaired persons, temporal resolution, often assessed by detection of brief gaps in continuous sound stimuli, is typically poorer than in individuals with normal hearing. At low stimulus presentation levels, hearing impaired individuals perform poorly but the deficits are greatly reduced when the sensation level of the stimuli are adjusted to match their normal hearing peers. In the present study, we evaluated the effect of selective inner hair cell loss on gap detection in chinchillas treated with carboplatin, an anticancer drug that selectively damages inner hair cells and afferents in this species. Treatment with carboplatin-induced inner hair cell loss of ~ 70 % but had little effect on audiometric thresholds in quiet and produced no evidence of outer hair cell loss. In contrast, selective inner hair cell loss had a significant effect on gap detection ability across a wide range of presentation levels. These results suggest that gap detection tasks are more sensitive to inner hair cell pathology than audiometric thresholds.

INTRODUCTION

In mammals, hearing sensitivity depends on the normal function of two distinct sensory cell types in the cochlea. Inner hair cells (IHC) serve as the primary conduit of acoustic energy to the central auditory nervous system (CANS) via extensive connections with type-I afferents (> 90 %). In contrast, outer hair cells (OHC) are innervated by the remaining 10 % and there is no evidence that OHC transmit acoustic information to the CANS (Spoendlin 1975). OHC, however, are essential for low thresholds as these provide active cochlear amplification via electromotility (Allen 1980; Johnstone et al. 1986; Preyer and Gummer 1996) and play an essential role in frequency tuning. When OHC are damaged, thresholds increase and tuning decreases. (McGill and Schuknecht 1976; Dallos and Harris 1978; Stebbins et al. 1979; Patuzzi et al. 1989; Ohlms et al. 1991),(Cody and Russell 1985; Borg 1987; Davis et al. 1989; Hamernik et al. 1989; McFadden et al. 2002; Davis et al. 2005). In contrast, chinchillas with carboplatin-induced, selective IHC loss show little if any elevation in thresholds until IHC loss is profound (~ 80 %) (Lobarinas et al. 2013) but show poorer thresholds in competing background broadband and narrowband noise (Lobarinas et al. 2016). One plausible explanation for these deficits is that degraded temporal resolution reduces the opportunities to detect signals in a fluctuating competing background noise. To assess whether temporal resolution is negatively affected by selective IHC loss, we evaluated gap detection in broadband noise (BBN) before and after carboplatin treatment in chinchillas with no evidence of hearing loss as assessed by thresholds in quiet.

MATERIALS AND METHODS

Subjects

Ten, healthy, adult, 1–2-year-old male chinchillas were used (400–600 g). Thresholds in quiet and gap detection were assessed before and after treatment a single dose of 75 mg/kg carboplatin (i.p.), a treatment previously shown to produce moderate to severe IHC loss (Hofstetter et al. 1997a, b). Subjects were housed in custom individual cages in a temperature-controlled room with a 12-h light/dark cycle. Animals had free access to food and water. All procedures were approved by the University at Buffalo’s (5 animals) and University of Florida’s (5 animals) IACUC committees. Group 1 represents data from animals at the University at Buffalo and Group 2 data represents data collected at the University of Florida.

Equipment

For group 1, subjects were placed in a restraining yoke that held the subject in a fixed, standing position in a calibrated sound field within a single-walled sound booth (inside dimensions: L × W × H 91.4 × 101.6 × 193 cm, Industrial Acoustics Company Inc. Bronx, NY) lined with 3-in. acoustic foam. A micro-switch mounted on the restraining yoke was used to record the behavioral response of the animal. The behavioral response consisted of a 1 cm upward movement on the restraining yoke that closed a micro-switch which generated +5 V pulse that was delivered to the input of an A/D converter (TDT Smartport PI2) and recorded by custom psychophysical software. This response was used throughout all phases of the behavioral experiment. Two silver disk electrodes covered with conductive paste (Synapse Conductive Electrode Cream, SYN 1505) were taped on to the shaved tail of the chinchilla; electrodes were placed on either side of the tail near the base. Brief current pulse (1–5 mA, 500 ms on, 500 ms off) could be delivered to the electrodes using a constant current generator (Coulbourn Instruments, Precision Regulated Animal Shocker E13–14) connected to an input/output module (TDT Smartport PI2) controlled by a personal computer running custom software. A calibrated speaker (Realistic Minimus-7 40-2030A, 40w 50–20,000 Hz) was located at the level of the subjects’ head approximately 50 cm from the left ear (270 degrees azimuth). A safety light (40 watt light bulb) was mounted on the wall of the sound booth in front of the animals (0 degrees azimuth) at a distance of 55 cm. The light could be turned on and off using an input/output module (TDT Smartport PI2) controlled through the computer. A small buzzer was mounted on the restraining yoke and could be turned on and off using the input/output module (TDT Smartport PI2) controlled through the computer.

For group 2, subjects were placed in an acoustically transparent (stainless steel bar), operant chamber with dimensions, 31.75 (w) × 34.29 (H) × 25.4 (D) cm, (Med Associates, 007-VPX), housed within a sound-attenuating cubicle (Med Associates, ENV-018V) lined with acoustic foam. The sound-attenuating cubicles resided in a single-walled sound-attenuating booth (WhisperRoom, MDL 4870S). Within each chamber, a photobeam (Med-Associates, ENV-253SD) was placed and centered 19.54 cm above a stainless steel bar grid floor wired for scrambled foot shock delivery (Med-Associates, ENV-005A-QD and ENV-414S, 2–5 mA scrambled foot shock). The output of the photobeam was fed to an adjustable single channel IR controller (Med-Associates, ENV-253B) and then to a real-time processor (Tucker Davis Technologies, RP2.1) connected to a personal computer running custom software. A calibrated speaker (Foxtex, FE127E) placed on the top of the operant chamber (26 cm) was used to deliver all acoustic stimuli. A piezo buzzer (Radio Shack, 273–059) and house lights (Med-Associates, ENV-215M) were used to provide feedback during testing.

Study Design

The overall aim of the study was to evaluate the effect of selective IHC loss on gap detection in BBN. A within-subjects design was implemented with pre-carboplatin baseline assessments followed by a post-carboplatin assessment of thresholds in quiet and gap detection conditions.

Psychophysical Methods

Hearing thresholds were obtained using a shock avoidance conditioning procedure we have described previously (Lobarinas et al. 2013), and similar to that described in earlier reports (Blakeslee et al. 1978; Salvi et al. 1978; Giraudi et al. 1980; Giraudi-Perry et al. 1982; Salvi and Arehole 1985; Lobarinas et al. 2013).

Thresholds in Quiet

Thresholds were assessed by presenting tone bursts (500 ms duration, 5 ms rise/fall time) presented at 0.5, 1, 2, 4, 8, and 11.3 kHz. Tonal stimuli were created with custom software (group 1) or a Matlab script (group 2) in conjunction with RPVDS software running a real-time processor, RP2.1 (Tucker Davis Technologies). Loudspeaker output was calibrated using a 0.5-in. microphone (Larson Davis 2559) and a sound level meter (Larson Davis 800B).

Each experimental session contained approximately 100–120 trials. During each trial, six tone bursts (500 ms on/500 ms off, 5 ms rise/fall time) were presented. If the subject reared and closed the microswitch (group 1) or broke the plane of the photobeam (group 2) within the first four of the six tone bursts, the tone burst signal was terminated, the house light stayed on, and the response was recorded as correct (Hit). However, if the subject failed to respond within the first four tone bursts, either a tail shock (group 1) or foot shock (group 2) and a buzzer were turned on, house lights were turned off and the trial was recorded as an incorrect response (Miss).

During misses, the combination of the shock stimulus and buzzer immediately elicited a response that turned off the shock (Escape). Maximum shock time was limited to 2 s. When tone-burst intensity levels fell below 20 dB SPL, only the buzzer and light-off condition (shock was turned off) signaled a Miss.

For group 1, each session trial was presented using a random intertrial interval (ITI) that ranged from 20 to 60 s. A Modified Method of Limits with a 10 dB down, 5 dB up step size was implemented in order to determine thresholds in quiet. Testing began using a high stimulus level (> 60 dB SPL). Within a trial, if a subject produced a correct response, the tone-burst intensity was decreased by 10 dB on the subsequent trial. However, failure to respond resulted in a 5 dB intensity increase on the next trial. The false alarm rate (rate of responding when no stimulus was presented) was measured using blank trials (5–10 % of the trials) to allow for monitoring of false positives and correct rejection.

For group 1, threshold at each frequency was operationally defined as the intensity at which two out of three reversals (66 %) occurred. The time needed to measured threshold across all stimulus frequencies in a daily session was 50–60 min. The daily thresholds were averaged across 5 days to determine stable baseline pure-tone thresholds at each frequency. Daily threshold measures were considered valid if the false alarm rate was less than 10 % during the session. For group 2, the same 66 % criterion was used, however, the software used a constant stimulus paradigm, whereby the presentation level was randomized but a fixed number of trials at each duration level were presented (6 trials/frequency/intensity).

Gap Detection

Gap detection was assessed by inserting silent gaps in an ongoing BBN carrier. Each trial consisted of six gaps in a BBN carrier presented at 500 ms intervals. As described in the previous section, if the subject reared and broke the plane of the photobeam within the first four of the six gap presentations, the house light stayed on and the response was recorded as correct.

Failure to respond within the first four gaps resulted in shock delivery and/or buzzer activation. Gap duration began at 256 ms and was systematically decreased to 128, 64, 32, 16, 12, 8, 6, 4, 2, and 0 ms following each correct response. For group 1, when a miss was detected, the gap duration was increased by one gap step until correct responses were recorded. For group 2, a method of constant stimuli was used with 6 presentations at each level and intensity. Threshold was determined as the gap duration at which two out of three (66 %) gap step reversals occurred for Group 1 or when gaps were detected four out of six times (66 %) for group 2. The procedure was tested with gaps in BBN presented at 75, 65, 50, and 40 dB SPL for group 1 and 60, 50, and 40 dB SPL for group 2.

Cochleograms

At the end of the post-carboplatin hearing assessments, subjects were sacrificed with an overdose of carbon dioxide, decapitated, and the cochleae removed for histological analysis to determine the extent of hair cell loss (Trautwein et al. 1996; Hofstetter et al. 1997a, b; Ding et al. 1999a, b). Both cochleae were removed carefully, the round window and oval window opened, and a solution of succinate dehydrogenase (SDH) (2.5 ml, 0.2 M sodium succinate, 2.5 ml, 0.2 M phosphate buffer, pH 7.6, and 5 ml 0.1 % tetranitro blue tetrazolium) was perfused through the round window. The cochleae were then immersed in SDH and incubated at 37 °C for 45 min. The cochlea was post-fixed with 10 % formalin and stored in this fixative for 24 h. The basilar membrane containing the organ of Corti was dissected from the apex to the base as a flat surface preparation, mounted in glycerin on glass slides, and cover slipped. The organ of Corti was analyzed by light microscopy (Zeiss Standard) at × 400 magnification. Successive segments (0.24 mm) were analyzed for missing IHC and OHC. Hair cells were counted as present if the SDH stained cell bodies were present and visible. Cochleograms were constructed for each ear of each animal and the percent missing hair cells was plotted as a function of distance from the apex. Percent hair cell loss was determined from laboratory norms established from cochlea obtained from normal chinchillas (n = 9, 1–2 months of age). Percent distance from the apex was also converted to frequency using a cochlear frequency-place map (Greenwood 1990). Cochleograms were constructed for both ears; however, no significant differences in the size or pattern of hair cell loss were observed for the right versus left ears. Therefore, the data presented in the “RESULTS” section show the hair cell lesions from the left ear.

Data Analysis

All subjects were evaluated for thresholds in quiet and for gaps in continuous BBN. Mean data for the subjects were analyzed using a 2-way repeated measures analysis of variance (ANOVA) to ascertain the effect carboplatin on thresholds as a function of frequency and gap detection as a function of presentation level. All statistical comparisons used an alpha level of 0.05 and post hoc analysis was performed using Tukey tests to avoid type-I errors associated with multiple comparisons. Sigma Stat 12.5 and Develve 4.7 were used for all statistical analyses. All results are presented as mean ± standard deviation (SD).

RESULTS

Moderate and severe carboplatin-induced IHC Loss

The 75 mg/kg carboplatin treatment produced a mean IHC loss of ~ 70 % for groups 1 and 2. There was no evidence of significant OHC loss; results that were consistent with previous publications (Trautwein et al. 1996; Hofstetter et al. 1997a, b; Wang et al. 1997; Ding et al. 1999b; Lobarinas et al. 2013). Figure 1 shows the post-mortem mean IHC and OHC loss for both groups of animals. The percent hair cell loss is plotted as a function of the frequency regions tested behaviorally (~ 10 % intervals centered on each of the test frequencies). Across animals, the IHC loss was evident along most of the basilar membrane. A two-way repeated measures ANOVA (2 W-RM-ANOVA) showed no statistically significant difference for IHC loss between groups 1 and 2 (F(1,69) = 0.0507, p = 0.828) and no group differences for OHC loss (F(1,69) = 0.299, p = 0.6). When both groups were combined, there was a statistically significant difference between IHC and OHC loss, (F(1,139) = 70.279, p < 0.001)

Fig. 1.

Mean inner and outer hair cell loss is shown as a function of test frequency for groups 1 and 2. Carboplatin (75 mg/kg) produced inner hair cell loss of 40–80 %. In contrast, there was no evidence of outer hair cell loss. Data are plotted as mean ± standard deviation

Thresholds in Quiet

The mean baseline and post-carboplatin thresholds (Fig. 2) for both groups were in general agreement with previous results in untreated chinchillas (Miller 1970; Blakeslee et al. 1978; Salvi et al. 1978; Lobarinas et al. 2013) and in chinchillas with carboplatin-induced IHC loss (Lobarinas et al. 2013).

Fig. 2.

Mean thresholds in quiet are shown as a function of test frequency for groups 1 and 2 before and after carboplatin. There was significant increase in thresholds post-carboplatin treatment from 2000 to 8000 Hz. However, the change in thresholds was 4–8 dB, a level much lower than the presentation levels of the carrier noise in the gap conditions (40–75 dB SPL). Data are plotted as mean ± standard deviation

Post-carboplatin (21d), thresholds increased by 4–8 dB in the 2000–8000 Hz range. A 2W-RM-ANOVA showed no significant differences in thresholds between groups before carboplatin treatment (F(1,69) = 0.0463, p = 0.835) or after treatment (F(1,69) = 0.252, p = 0.629). When both groups were combined there was a small but statistically significant increase in thresholds after carboplatin treatment at 2000, 4000, and 8000 Hz (F(1,139) = 5.245, p = 0.048).

Carboplatin Treatment

Following baseline pure-tone and gap detection threshold assessment, each subject was treated with a single 75 mg/kg intraperitoneal (i.p.) injection of carboplatin (Sigma C2538, cis-Diammine 1,1-cyclobutanedicarboxylate platinum) dissolved in 5 ml of saline. The animals then received daily 10 ml subcutaneous injections of saline for 2 days following carboplatin treatment. Following a 21-day recovery period, thresholds in quiet and gap detection were reevaluated. The duration of the experiments, including the recovery period, were 15–18 weeks.

Gap Detection

Gap detection thresholds were measured in BBN over a range of intensities from 40 to 75 dB SPL for group 1 and 40–60 dB SPL for group 2 before and after carboplatin treatment. Mean gap detection thresholds for group 1 are shown in Fig. 3 as a function of BBN intensity. Gap detection thresholds increased as BBN presentation level decreased. Before carboplatin treatment, gap detection thresholds ranged from 3.9 ms at 75 dB SPL BBN to 7.5 ms at 40 dB SPL. Following carboplatin treatment, thresholds also increased as a function of intensity. At 40 dB SPL, the mean gap detection thresholds increased from approximately 7 ms to about 17 ms. By contrast, the mean gap detection threshold at 75 dB SPL increased from 3.9 to 7 ms. A 2W-RM-ANOVA showed a statistically significant change in gap detection thresholds following carboplatin treatment (F(1,39) = 7.889, p = 0.048) and a significant interaction effect of treatment with BBN presentation level (F(3,39) = 15.832, p < 0.001).

Fig. 3.

Mean percent correct detection of gaps are shown for group 1 as a function of gap duration for 75, 65, 50, and 40 dB SPL carrier noise levels. Gap detection thresholds were operationally defined at the 66 % criteria (2 out of 3 reversals). Post-carboplatin thresholds show increases at all presentation levels with the largest changes at 40 dB SPL. Data are plotted as mean ± standard deviation

The data presented for group 2 were derived from a modification to both the original paradigm as described in the “MATERIALS AND METHODS” section (Yoke restraint vs. photobeam) as well as updated software. The updated software allowed us to present multiple trials for each gap duration and to create input output functions for each duration. Figure 4 a, b, and c show percent correct as a function of gap duration for 60, 50, and 40 dB SPL noise carrier levels.

Fig. 4.

Mean percent correct detection of gaps are shown as a function of gap duration for a 60 dB SPL, b 50 dB SPL, and c 40 dB SPL carrier noise levels. Gap detection thresholds were operationally defined at the 66 % criteria. Post-carboplatin thresholds showed significant increases at all presentation levels. At 40 dB SPL animals could not reach the 66 % criterion. Data are plotted as mean ± standard deviation.

Because the data for group 2 failed the normality test, a Cox-box linear transformation was applied to normalize the data for analysis with a 2W-RM_ANOVA (Develve 4.7). A 2W-RM-ANOVA for the 60 dB SPL presentation level showed a significant effect of gap duration (F(5,59) = 119.745, p < 0.001) and carboplatin treatment (F(1,39) = 29.393, p = 0.006) and an interaction effect of gap duration and carboplatin treatment (F(5,59) = 2.658, p = 0.053). For the 50 dB SPL presentation level, duration (F(5,59) = 57.285, p < 0.001), carboplatin treatment (F(1,59) = 13.023, p = 0.023), and interaction of treatment and duration (F(5,39) = 3.251, p = 0.026) were also significant. Finally, for the 40 dB SPL presentation, post-treatment results showed that even at longer gap durations, animals could not reach 66 % correct. A 2W-RM-ANOVA showed significant effects for duration (F(5,59) = 58.902, p < 0.001], carboplatin treatment (F(1,39) = 8.301, p = 0.045) and an interaction effect of gap duration and carboplatin treatment (F(5,59) = 3.465, p = 0.02).

Summary of Results

The overall results show that large IHC lesions had minimal effect on thresholds in quiet but significant effects on gap detection at all presentation levels. These results suggest that gap detection is more sensitive to IHC pathology than thresholds in quiet. Thresholds in quiet (audiometric thresholds) are the current and most widely used clinical correlates of inner ear functional status.

DISCUSSION

The aim of this study was to evaluate the perceptual consequence of selective IHC loss on an index of temporal resolution in animals with near normal thresholds. To accomplish this goal, psychophysical methods were used to evaluate tone thresholds in quiet and the detection of gaps in otherwise continuous noise. The measures were obtained before and after a selective IHC lesion using carboplatin. The magnitude of the IHC lesion was assessed at the end of the psychophysical studies by anatomical confirmation of IHC and OHC loss as a function of location along the basilar membrane. The within subjects experimental paradigm allowed all the animals in the study to serve as their own controls across all experimental measures.

Thresholds in Quiet

Baseline audiograms were obtained prior to carboplatin treatment. Thresholds in quiet ranged from 2 to 8 dB SPL from 250 to 11,300 Hz. The thresholds in the current study were in good agreement with our previous findings (Lobarinas et al. 2013) and earlier reports from other labs using behavioral methods (Miller 1970; Heffner and Heffner 1991). Following carboplatin treatment, there were minimal effects on thresholds in quiet. These results confirm our previous findings that puretone thresholds in quiet are relatively insensitive to moderate to severe IHC loss.

Gap Detection

The main findings of this study were that, although the degree of IHC loss had little impact on thresholds in quiet, loss of IHC significantly degraded gap detection performance. These results suggest that gap detection is more sensitive to IHC pathology and may partially explain findings in humans with normal thresholds but impaired gap detection. It also suggests that in some cases the site of lesion for these deficits is likely originating in the cochlea and not as a result of central temporal processing deficits. Because the gap detection deficits were observed at suprathreshold levels, this type of testing might provide additional information regarding the status of IHC even in individuals with threshold elevation. Changes in thresholds correlate well with OHC loss but not IHC loss. Gap detection deficits could help explain differences in hearing ability among individuals with similar audiograms.

With respect to our findings, there are two possible explanations for the observed effects of carboplatin on gap detection. The first is that the loss of IHC could negatively impact neural synchrony and thus partially impair gap temporal cues. This possibility, however, is unlikely given published reports on the effects of carboplatin on the auditory brainstem response (ABR). Carboplatin-induced IHC loss has been shown to reduce ABR wave-I amplitudes, suggesting, a reduction in cochlear output but has not been shown to impair the morphology or latency of the waveforms (El-Badry and McFadden 2009). Moreover, correlates of later ABR waveforms show compensation for amplitude and in some cases have greater amplitude even when wave-I amplitudes are reduced (Qiu et al. 2000). In preliminary ongoing behavioral experiments, we have not found an effect of carboplatin on measures of temporal summation.

A second possibility is that the loss of IHC may disrupt intensity coding.

Our data show poorer gap detection at all suprathreshold levels but, the differences were more robust at lower presentation levels, suggesting a presentation level effect. We are currently working on experiments evaluating suprathreshold intensity discrimination to determine if these could provide insight into the observed deficits on gaps.

We are also interested in the relationship between degree of IHC loss and the gap detection deficits. However, when reviewing the current data we did not observe any relationship between the degree of IHC loss and gap detection deficits. This may be in large part due to the use of a single dose that produced robust (> 55 %) IHC loss, the variability of IHC loss, and the small sample size of our experiments. We have additional ongoing experiments with larger cohorts evaluating the effect of lower doses of carboplatin that produce less IHC loss to determine if we can establish more reliable relationship between IHC loss and functional deficits. These experiments will help guide additional measures that may show even greater sensitivity to IHC loss.

IHC Loss to Perceptual Changes

Currently, there are no widely accepted functional tests of IHC loss readily available to clinicians but increasing test sensitivity to IHC dysfunction could provide significant diagnostic value in relation to functional impairment and differential diagnosis. Such tests could help differentiate central hearing loss from peripheral hearing loss in cases of disproportionally poor speech perception and poor hearing in noise with seemingly normal hearing sensitivity.

The results presented here highlight the limitations of testing thresholds in quiet as an assay of IHC integrity and underscore the importance of assessing suprathreshold auditory function as a routine clinical measure to better evaluate overall hearing ability and identify hidden hearing deficits. The use of simple non-speech tasks such as gap-detection makes the test suitable for special populations, for a broad range of ages, and for individuals with poor receptive language proficiency.

Abbreviations

ABR

Auditory brainstem response

CANS

Central auditory nervous system

BBN

Broadband noise

dB

Decibel

IHC

Inner hair cell

OHC

Outer hair cell

SD

Standard deviation

SEM

Standard error of the mean

SPL

Sound pressure level

SDH

Succinate dehydrogenase

Author Contributions

EL and DD performed data collection, analysis, and all authors contributed to writing the manuscript.