Acta Oto-Laryngologica. 2015; 135: 35–41
ORIGINAL ARTICLE
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Utility of auditory steady-state and brainstem responses in age-related hearing loss in rats
RICARDO SANZ-FERNÁNDEZ1, CAROLINA SÁNCHEZ-RODRIGUEZ2, JOSÉ JUAN GRANIZO3, ENRIQUE DURIO-CALERO1 & EDUARDO MARTÍN-SANZ1 1
Department of Otolaryngology, University Hospital of Getafe, Madrid,Spain, 2Fundación para la Investigación Biomédica, University Hospital of Getafe, Madrid, and 3Clinical Epidemiology Unit, Hospital Infanta Cristina, Parla, Madrid, Spain
Abstract Conclusions: The results support the idea that auditory steady-state response (ASSR) is a more accurate test for studying age-related hearing loss (ARHL) in Sprague-Dawley rats. Differences in the rat middle ear may explain the variations of the click properties, with a displacement of the energy toward the 8 and 10 kHz frequencies compared with humans. Objectives: The purpose of this study was to evaluate ARHL in older and younger Sprague-Dawley rats using auditory clicks and tone burst with auditory brainstem response (ABR), in addition to ASSR. Methods: This was a prospective cohort study with 50 animals divided into 5 groups based on their age in months. A total of 100 registers were elicited from each one of the 3 auditory measurements systems in an electrically shielded, double-walled, sound-treated cabin. Nine frequencies, from 0.5 to 16 kHz were analyzed with the auditory steady-state response and compared with the results elicited by the clicks and tone-burst ABR. Results: Comparisons between the different frequencies showed lower thresholds in those frequencies below 2 kHz, independently of their age in months. The ARHL was detected by each one of the three auditory measurement systems, but with lower thresholds with the ASSR test. Finally, auditory clicks showed better correlations with 8 and 10 kHz elicited by ASSR, which was different to what was expected, based on human studies.
Keywords: Sprague-Dawley rats, ABR, ASSR, auditory clicks
Introduction Age-related hearing loss (ARHL), or presbycusis, is characterized by a reduction in hearing sensitivity and the ability to understand speech in noisy environments. Although the mechanisms underlying ARHL are not completely understood, it is generally accepted that there are at least two components to the onset of presbycusis, which implies degeneration of both the cochlea and the auditory portion of the central nervous system [1–4]. Both human [5,6] and animal studies [7,8] suggest that ARHL involves complex interaction patterns with respect to pure-tone threshold shifts and the underlying cochlear pathology.
Conventional behavioral pure-tone audiometry remains the gold standard for quantifying and describing hearing loss in subjects who are able to respond and cooperate. It is necessary to use alternative methods when assessing various difficult populations such as prelocutive children with neurological retardation, subjects who require medical or legal auditory evaluation, and experimental animals. The difficulty of testing these populations makes it necessary to use objective measures that do not rely on subjective responses. One of these techniques is auditory evoked potentials (AEPs), in which brainstem responses to acoustic stimuli are recorded. One of the most common AEPs
Correspondence: Ricardo Sanz Fernández, Department of Otolaryngology, Hospital Universitario de Getafe, Autovía de Toledo, km 12,500, 28905 Getafe, Madrid, Spain, Tel: +34 916 83 93 60. Fax: +34 916839748. E-mail: [email protected]
(Received 12 June 2014; accepted 4 August 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2015 Informa Healthcare DOI: 10.3109/00016489.2014.953203
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R. Sanz-Fernández et al.
used to evaluate auditory function in the brainstem is the auditory brainstem response (ABR). Rodents have served as excellent models in hearing research [9], and the use of ABR to detect threshold shifts is widely reported in the medical literature [10,11]. Click and tone-burst ABRs are commonly used to determine auditory sensitivity. ABRs conducted with click stimuli have high reproducibility and waveform stability. As a result, the acoustic click is one of the most common stimuli in clinical studies. This stimulus has markedly well defined timing and is ideal for evoking a synchronized neural population response. Unfortunately, according to many authors, it is a broadband stimulus in terms of its spectral composition [12]. Upon their introduction, it became clear that ABRs evoked by clicks would be unable to provide frequency-specific information because of spectral splatter. In fact, ABR testing records simultaneous firing from a large pool of neural units that respond most effectively to a stimulus characterized by an abrupt or rapid onset and a broad frequency bandwidth. Therefore, this type of test can assess hearing in the range of 2000–4000 Hz. The inadequacy of ABR for determining thresholds over 90 dB HL is another limitation of its applicability toward evaluating severe to profound hearing loss. In contrast to ABRs evoked with transient broadband clicks or tone bursts, the auditory steady-state response (ASSR) is evoked using continuous tones modulated either in amplitude or in amplitude and frequency. The use of continuous sinusoids allows for stimuli with narrower spectra than those of tone bursts, improving the frequency-specific threshold determination. Furthermore, the ASSR technique allows the clinician to present up to four different stimuli to both ears simultaneously, with objective responses determined using reliable statistical techniques. The ASSR technique has several potential advantages over tone-burst and click ABRs. Performing response detection in the frequency domain based on statistical tests ensures that the ASSRs are detected objectively. The method does not depend on subjective visual examination of the waveforms or response patterns. Reports on the accuracy of ASSR thresholds compared to behavioral and ABR thresholds have demonstrated good correlations across various age groups, although significant differences exist between studies. The variability can largely be accounted for by the degree of hearing loss, as ASSR thresholds approximate behavioral thresholds better as the severity of the sensorineural hearing loss increases. This phenomenon has been attributed to physiological recruitment.
For these reasons, click-evoked ABR has been suggested as a useful, time-efficient physiological cross-check for ASSR results, although behavioral audiometry remains the gold standard. The aim of this study was to evaluate ARHL in our animal model by comparing three measurements: click ABRs, tone-burst ABRs, and ASSR. Material and methods Animals Healthy female Sprague-Dawley rats were tested in this study. All animals were obtained from and maintained at the University Hospital of Getafe’s animal care facility until use. The animals were housed in plastic cages with water and food ad libitum and maintained in a 12 h light/dark cycle. Rats with skin lesions, splenomegaly, macroscopically visible tumors, and inner ear infections were excluded. Five groups were created based on their age in months: 3, 6, 12, 18, and 24 months old. No rats died or were excluded during the study period. There were 10 Sprague-Dawley rats per group, with a total of 50 animals. Every measurement was obtained for both the right and left ears, yielding 100 ears examined for each measurement taken. The 3-month-old group was considered the baseline against which the other four groups were compared. Rats were anesthetized with an intraperitoneal injection of ketamine 90 mg/kg and diazepam 5 mg/ kg. All procedures involving the use and care of the animals were reviewed and approved by the University Hospital of Getafe’s Animal Care and Use Committee. ASSR and ABR measurements ASSR and ABR tests were performed on all rats in the study before euthanization. Rats were anesthetized as described above. An ER3 insert earphone was placed directly into the external auditory canal. Subcutaneous electrodes were placed over the vertex (active) and in the pinna of each ear (reference). Ground electrodes were placed over the neck muscles. ASSRs were recorded using an evoked potential averaging system (Intelligent Hearing System SmartEP, Miami, FL, USA) in an electrically shielded, double-walled, sound-treated booth in response to tone bursts at 0.5, 1, 2, 4, 8, 10, 12, 14, and 16 kHz with a 10 ms plateau and 1 ms rise/fall time. Intensities were expressed in decibels sound pressure level (db SPL) peak equivalent. Intensity series were
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Auditory evoked potentials in ARHL in rats recorded, and an ASSR threshold was defined as the lowest intensity capable of eliciting a replicable, visually detectable response. The evoked responses were amplified with a gain of 100.00 K and bandpass filtered from 30 to 300 Hz. A total of 250 sweeps were averaged at each stimulus level. The line filter was on at 50 Hz, with an artifact rejection region of –511 to 512 ms. Using the same hardware previously described, 100 ms click and tone-burst ABRs were also recorded. Each tone burst (exact Blackman envelope) and click was 1 ms in duration and had a 0.5 ms rise/fall time with no plateau. Tone bursts were presented at a rate of 27.7/s and were bandpass filtered from 30 to 1500 Hz. Clicks were presented at a rate of 19.3/s and bandpass filtered from 30 to 3000 Hz. The evoked responses in the rats were amplified with a gain of 100.00, with artifact rejection between 15 and 20 mV. A total of 250 sweeps were averaged at each stimulus level. The level of the signal was decreased in 10 dB steps from 90 dB SPL to 5 dB SPL to acquire data. Nine frequencies were tested: 0.5, 1, 2, 4, 8, 10, 12, 14, and 16 kHz. Every test (ASSR and click and tone-burst ABRs) conducted on a single animal was performed during the same session and by the same investigator.
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significantly lower thresholds were observed in 0.5 and 1 kHz (p < 0.05). These auditory profiles of lower thresholds in the frequencies below 2 kHz were observed in every group studied, independent of the age of the rats.
Tone-burst ABR When the auditory thresholds obtained in each group were compared to those in the 3-month-old group, we found a significant decrease in hearing that affected more frequencies as the groups were older (Figure 1). In the 6-month-old group, a significant increase of the thresholds was observed exclusively at 4 kHz. Significant differences were observed at 2, 4, 8, and 16 kHz when the 12-month-old group was compared with the initial group. Similarly, when the 18-monthold rats were compared across methods, additional increases were observed at 10 and 12 kHz. Finally, the oldest group showed significant differences in every frequency studied except for 0.5 kHz. Table I shows the p values of every group compared.
Click ABR Statistics analysis Data are presented as the mean ± standard deviation (SD). The analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Although the data showed normality according to the Kolmogorov–Smirnoff test, the small sample size and discontinuous data distribution led us to use a non-parametric test. The Mann–Whitney test was used to compare the differences between groups (control versus treated or measures over time). Differences with a p value < 0.05 were considered to be statistically significant. The concordance among the auditory measurement systems was analyzed using an intra-class correlation coefficient. Ethical considerations The study was approved by the Clinical Research Committee of the University Hospital of Getafe (Exp. PI09/02472, July 14, 2004).
Although ARHL was first recognized in the 6-monthold group, the first significant differences appeared in the 24-month-old group (Figure 2).
ASSR Similar to the observations for tone-burst ABRs, a progressive and significant decrease in hearing threshold was observed as the rats grew older. As shown in Figure 3, no relevant changes were observed in the 6- month-old group, except at the frequency of 4 kHz. In the 12-month-old group, a significant decrease in the thresholds was present exclusively at 16 kHz. When the 18-month-old group was compared with the baseline group, significant differences were observed in the frequency range from 4 to 10 kHz and at 16 kHz. Finally, the 24-month-old group showed significant differences at every frequency except 0.5 kHz.
Results
Comparison between ASSR and tone-burst ABR
Auditory profile
When the thresholds registered using both techniques were compared in every group, significantly lower values (p £ 0.01) were obtained in every frequency studied, using ASSRs.
When we compared every frequency elicited by all of the auditory measurement systems (0.5–16 kHz),
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R. Sanz-Fernández et al. Frequency (kHz ) 0.5
1
2
4
8
10
12
14
16
0 10 3m
20
6m 30
*
dB
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40
*
12 m
*
*
*
18 m 24 m
* *
50
*
60 70 80 90 100
Figure 1. Age-related hearing loss elicited by the tone-burst auditory brainstem response. Mean and standard deviation bars of every frequency are presented. Asterisks are placed where there is significance (p < 0.05) when the 24-month-old group was compared to the initial group. m, months.
Click ABR vs tone-burst ABR vs ASSR The concordance among the auditory measurement systems using intra-class correlations indicated higher
concordance rates at 8 and 10 kHz between clicks and ASSR. Although significant correlations were observed in every comparison made, the correlations were higher at the abovementioned frequencies (Table II).
Table I. p values in comparison of the auditory thresholds elicited by all of the auditory measurement systems. Age
500 Hz
1000 Hz
2000 Hz
4000 Hz
8000 Hz
10 kHz
12 kHz
14 kHz
16 kHz
Tone-burst auditory brainstem response 6 months
0.853
0.143
0.105
0.011
0.143
0.247
0.393
0.529
0.436
12 months
0.796
0.105
0.075
0.035
0.015
0.023
0.123
0.739
0.123
18 months
0.035
0.011
0.075
0.015
0.005
0.007
0.035
0.143
0.035
24 months
0.684
0.003
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
0.001
< 0.001
Auditory steady-state response 6 months
0.971
0.219
0.796
0.105
0.436
0.684
0.579
0.218
0.739
12 months
0.739
0.631
1.000
0.143
0.190
0.247
0.190
0.971
0.043
18 months
0.315
0.075
0.016
0.005
0.007
0.089
0.436
0.481
0.004
24 months
0.043
0.005
0.001
< 0.001
0.001
0.009
0.029
0.009
< 0.001
Click auditory brainstem response 6 months
0.579
12 months
0.481
18 months
0.143
24 months
< 0.001
The auditory thresholds obtained in each group were compared to those in the 3-month-old group. Statistical significance was accepted when p £ 0.01 (values shown in bold type).
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Auditory evoked potentials in ARHL in rats 3m
6m
12 m
18 m
24 m
(ASSR and tone-burst and click ABRs) showed improved results at higher frequencies (>4 kHz) compared with those obtained at lower ones (0.5, 1, and 2 kHz). This auditory profile persisted in every age group studied and in various animal groups, always showing improved auditory thresholds at higher frequencies regardless of whether and to what degree ARHL was present. This finding has been reported previously by several authors [13–15] in various studies that repeatedly indicated the highest level of normal auditory sensitivity at approximately 12–24 kHz. A second conclusion can be inferred regarding ARHL. In the present study, every auditory measurement system detected progressive hearing loss, which was more pronounced when older groups were compared with younger ones. This deterioration was more accurate at acute frequencies. Schuknecht developed the knowledge of degenerative changes in the lateral wall and stria vascularis as a major contributor to hearing loss associated with advancing age [16], a condition known as metabolic presbycusis, strial presbycusis, or pure presbycusis [17]. Various animal models have been used to investigate ARHL. The most frequently studied model is the
0 10 20 30 40
60 70 Figure 2. Age-related hearing loss elicited by the click auditory brainstem response. Mean and standard deviation bars of every age group are presented. m, months.
Discussion Several conclusions can be drawn from this study. First, in our animal model, the thresholds calculated using each of the auditory measurement systems
Frequency (kHz ) 0.5
1
2
4
8
10
12
14
16
0 10 3m
20
6m
* 30
* *
40
*
* *
12 m 18 m 24 m
* dB
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50
50 60
* *
70 80 90 100 Figure 3. Age-related hearing loss elicited by the auditory steady-state response. Mean and standard deviation bars of every frequency are presented. Asterisks are placed where there is significance (p < 0.05) when the 24-month-old group was compared to the initial group. m, months.
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R. Sanz-Fernández et al.
Table II. Intra-class correlation with the concordance rates of auditory brainstem response (ABR) elicited with auditory clicks and auditory steady-state response (ASSR). ASSR vs click ABR
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Frequency
ICC
95% CI
p value
500 Hz
0.294
–0.198–0.458
0.143
1000 Hz
0.733
0.063–0.820
< 0.001
2000 Hz
0.758
0.640–0.837
< 0.001
4000 Hz
0.763
0.747–0.840
< 0.001
8000 Hz
0.834
0.753–0.888
< 0.001
10 kHz
0.799
0.702–0.865
< 0.001
12 kHz
0.772
9.662–0.847
< 0.001
14 kHz
0.744
0.620–828
< 0.001
16 kHz
0.779
0.671–0.851
< 0.001
CI, confidence interval; ICC, intra-class correlation.
C57BL/6J (B6) mouse. These mice exhibit early and progressive hearing loss, and the broad degeneration throughout the cochlea and auditory cortex overlaps each of the previously mentioned major types of ARHL proposed by Schuknecht [17]. This strain has been considered to be a model of human sensory presbycusis [16]. Sprague-Dawley rat strains are also often used as animal models to study the mechanisms underlying ARHL. The first signs of scattered hair cell loss are detected in the early life of Sprague-Dawley rats and progress with age [18]. Inner and outer hair cell loss has been observed in 2-month-old rats. A pronounced spiral ganglion nerve loss with aging has also been described [19]. These histological findings exhibit good correlations with our electrophysiological evidence, supporting a deterioration of the thresholds elicited by ABR and ASSR. Certain differences were observed among toneburst and click ABRs and ASSR. All of the thresholds elicited using the ASSR method were lower than those obtained using ABR, regardless of the stimulus type (click or tone burst). Although ASSR and tone-burst ABR use different stimuli to elicit the response, the frequency ranges studied with both methods were similar. The improved detection of auditory thresholds together with the possibility of a broader frequency range makes ASSR a more suitable test to study auditory responses in animal models. Because the differences between ABR and ASSR were always independent of the age group, we were able to extend our preference for ASSR to studying ARHL. In our animal model, the click ABR correlated moderately well with every frequency studied using the ASSR system. Nevertheless, the strongest
correlations were found at the 8 and 10 kHz frequencies, which was unexpected. Because the estimated levels typically tended to correlate reasonably well with the audiogram from approximately 2–4 kHz according to the ABR tests, we expected to find stronger correlations in those at the 2 and 4 kHz frequencies. The click is relatively flat in a coupler. However, due to the resonances of the outer and middle ear, there will likely be increased energy in the 1.5 and 3 kHz regions. The click truly stimulates all frequencies up to nearly f = 1/duration of the DC pulse driving the transducer and within the frequency response of the transducer and the filter effects of the auditory periphery. The latter two factors impart some frequency-specific nuance to the effective stimulus; nuances of the ABR further favor the 2–4 kHz region during an unmasked, unfiltered click response [20]. One possible explanation for this displacement of the auditory click energy toward 8 and 10 kHz could be the anatomic differences between humans and our animal model. Real ear measurements in humans reveal differences from couplers and can reveal the nuances of each individual. These differences can be more evident in subjects with outer and/or middle ear pathologies and during comparisons between adults and very young children, infants, or newborns. In all species, including humans, the external ear and ear canal modify the acoustic spectrum of sounds reaching the tympanic membrane. This spectral modification plays a particular role in sound localization mechanisms, and the pinnae of many mammalian species have distinct designs, and in some cases mobility, to enhance the spectral cues that provide information on sound sources. The environment of the middle ear cavity clearly influences the signal that reaches the cochlea in terms of both the amplitude and the spectral content. Pressure changes within the middle ear can attenuate signals and change the spectral transfer function of the middle ear. However, the reality is that the click is a broadband stimulus that simulates most audible frequencies in the cochlea. The broadband character of the click and the differences in the Sprague-Dawley rat middle ear may explain the observed variations in the click properties compared with those observed in humans. Conclusions These results support the idea that the ASSR is a more accurate test for studying ARHL in Sprague-Dawley rats. Differences in the rat middle ear may explain the variations of the click properties, with a displacement
Auditory evoked potentials in ARHL in rats of the energy toward 8 and 10 kHz compared with humans.
Acknowledgment This research was funded by FIS Exp. PI09/02472, July 14, 2004.
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Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References [1] Torre P, Lasky RE, Fowler CG. Aging and middle ear function in rhesus monkeys (Macaca mulatta). Audiology 2000;39:300–4. [2] Walton JP, Simon H, Frisina RD. Age-related alterations in the neural coding of envelope periodicities. J Neurophysiol 2002;88:565–78. [3] Simon H, Frisina RD, Walton JP. Age reduces response latency of mouse inferior colliculus neurons to AM sounds. J Acoust Soc Am 2004;116:469–77. [4] Gates GA, Mills JH. Presbycusis. Lancet 2005;366:1111–20. [5] Gates GA, Schmid P, Kujawa SG, Nam B, D’Agostino R. Longitudinal threshold changes in older men with audiometric notches. Hear Res 2000;141:220–8. [6] Rosenhall U. The influence of ageing on noise-induced hearing loss. Noise Health 2003;5:47–53. [7] Mills DM. Interpretation of distortion product otoacoustic emission measurements. I. Two stimulus tones. J Acoust Soc Am 1997;102:413–29. [8] Kujawa SG, Liberman MC. Adding insult to injury: cochlear nerve degeneration after “temporary” noise-induced hearing loss. J Neurosci 2009;29:14077–85.
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[9] Jaumann M, Dettling J, Gubelt M, Zimmermann U, Gerling A, Paquet-Durand F, et al. cGMP-Prkg1 signaling and Pde5 inhibition shelter cochlear hair cells and hearing function. Nat Med 2012;18:252–9. [10] Au A, Stuyt JG, Chen D, Alagramam K. Ups and downs of viagra: revisiting ototoxicity in the mouse model. PLoS One 2013;8:e79226. [11] Nevado J, Sanz R, Sánchez-Rodríguez C, García-Berrocal JR, Martín-Sanz E, González-García JA, et al. Ginkgo biloba extract (EGb761) protects against aging-related caspasemediated apoptosis in rat cochlea. Acta Otolaryngol 2010; 130:1101–12. [12] Jones SM, Subramanian G, Avniel W, Guo Y, Burkard RF, Jones TA. Stimulus and recording variables and their effects on mammalian vestibular evoked potentials. J Neurosci Methods 2002;118:23–31. [13] Ehret G. Development of absolute auditory thresholds in the house mouse (Mus musculus). J Am Audiol Soc 1976;1: 179–84. [14] Borg E. Auditory thresholds in rats of different age and strain. A behavioral and electrophysiological study. Hear Res 1982; 8:101–15. [15] Overbeck GW1, Church MW. Effects of tone burst frequency and intensity on the auditory brainstem response (ABR) from albino and pigmented rats. Hear Res 1992; 59:129–37. [16] Fetoni AR1, Picciotti PM, Paludetti G, Troiani D. Pathogenesis of presbycusis in animal models: a review. Exp Gerontol 2011;46:413–25. [17] Schuknecht HF, Gacek MR. Cochlear pathology in presbycusis. Ann Otol Rhinol Laryngol 1993;102:1–16. [18] Keithley EM, Ryan AF, Feldman ML. Cochlear degeneration in aged rats of four strains. Hear Res 1992;59:171–8. [19] Keithley EM, Feldman ML. Hair cell counts in an agegraded series of rat cochleas. Hear Res 1982;8:249–62. [20] Schwartz DM, Morris MD, Spydell JD, Ten Brink C, Grim MA, Schwartz JA. Influence of click polarity on the brain-stem auditory evoked response (BAER) revisited. Electroencephalogr Clin Neurophysiol 1990; 77:445–57.
ORIGINAL ARTICLE
Acta Otolaryngol Downloaded from informahealthcare.com by University of Louisville on 01/19/15 For personal use only.
Utility of auditory steady-state and brainstem responses in age-related hearing loss in rats
RICARDO SANZ-FERNÁNDEZ1, CAROLINA SÁNCHEZ-RODRIGUEZ2, JOSÉ JUAN GRANIZO3, ENRIQUE DURIO-CALERO1 & EDUARDO MARTÍN-SANZ1 1
Department of Otolaryngology, University Hospital of Getafe, Madrid,Spain, 2Fundación para la Investigación Biomédica, University Hospital of Getafe, Madrid, and 3Clinical Epidemiology Unit, Hospital Infanta Cristina, Parla, Madrid, Spain
Abstract Conclusions: The results support the idea that auditory steady-state response (ASSR) is a more accurate test for studying age-related hearing loss (ARHL) in Sprague-Dawley rats. Differences in the rat middle ear may explain the variations of the click properties, with a displacement of the energy toward the 8 and 10 kHz frequencies compared with humans. Objectives: The purpose of this study was to evaluate ARHL in older and younger Sprague-Dawley rats using auditory clicks and tone burst with auditory brainstem response (ABR), in addition to ASSR. Methods: This was a prospective cohort study with 50 animals divided into 5 groups based on their age in months. A total of 100 registers were elicited from each one of the 3 auditory measurements systems in an electrically shielded, double-walled, sound-treated cabin. Nine frequencies, from 0.5 to 16 kHz were analyzed with the auditory steady-state response and compared with the results elicited by the clicks and tone-burst ABR. Results: Comparisons between the different frequencies showed lower thresholds in those frequencies below 2 kHz, independently of their age in months. The ARHL was detected by each one of the three auditory measurement systems, but with lower thresholds with the ASSR test. Finally, auditory clicks showed better correlations with 8 and 10 kHz elicited by ASSR, which was different to what was expected, based on human studies.
Keywords: Sprague-Dawley rats, ABR, ASSR, auditory clicks
Introduction Age-related hearing loss (ARHL), or presbycusis, is characterized by a reduction in hearing sensitivity and the ability to understand speech in noisy environments. Although the mechanisms underlying ARHL are not completely understood, it is generally accepted that there are at least two components to the onset of presbycusis, which implies degeneration of both the cochlea and the auditory portion of the central nervous system [1–4]. Both human [5,6] and animal studies [7,8] suggest that ARHL involves complex interaction patterns with respect to pure-tone threshold shifts and the underlying cochlear pathology.
Conventional behavioral pure-tone audiometry remains the gold standard for quantifying and describing hearing loss in subjects who are able to respond and cooperate. It is necessary to use alternative methods when assessing various difficult populations such as prelocutive children with neurological retardation, subjects who require medical or legal auditory evaluation, and experimental animals. The difficulty of testing these populations makes it necessary to use objective measures that do not rely on subjective responses. One of these techniques is auditory evoked potentials (AEPs), in which brainstem responses to acoustic stimuli are recorded. One of the most common AEPs
Correspondence: Ricardo Sanz Fernández, Department of Otolaryngology, Hospital Universitario de Getafe, Autovía de Toledo, km 12,500, 28905 Getafe, Madrid, Spain, Tel: +34 916 83 93 60. Fax: +34 916839748. E-mail: [email protected]
(Received 12 June 2014; accepted 4 August 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2015 Informa Healthcare DOI: 10.3109/00016489.2014.953203
Acta Otolaryngol Downloaded from informahealthcare.com by University of Louisville on 01/19/15 For personal use only.
36
R. Sanz-Fernández et al.
used to evaluate auditory function in the brainstem is the auditory brainstem response (ABR). Rodents have served as excellent models in hearing research [9], and the use of ABR to detect threshold shifts is widely reported in the medical literature [10,11]. Click and tone-burst ABRs are commonly used to determine auditory sensitivity. ABRs conducted with click stimuli have high reproducibility and waveform stability. As a result, the acoustic click is one of the most common stimuli in clinical studies. This stimulus has markedly well defined timing and is ideal for evoking a synchronized neural population response. Unfortunately, according to many authors, it is a broadband stimulus in terms of its spectral composition [12]. Upon their introduction, it became clear that ABRs evoked by clicks would be unable to provide frequency-specific information because of spectral splatter. In fact, ABR testing records simultaneous firing from a large pool of neural units that respond most effectively to a stimulus characterized by an abrupt or rapid onset and a broad frequency bandwidth. Therefore, this type of test can assess hearing in the range of 2000–4000 Hz. The inadequacy of ABR for determining thresholds over 90 dB HL is another limitation of its applicability toward evaluating severe to profound hearing loss. In contrast to ABRs evoked with transient broadband clicks or tone bursts, the auditory steady-state response (ASSR) is evoked using continuous tones modulated either in amplitude or in amplitude and frequency. The use of continuous sinusoids allows for stimuli with narrower spectra than those of tone bursts, improving the frequency-specific threshold determination. Furthermore, the ASSR technique allows the clinician to present up to four different stimuli to both ears simultaneously, with objective responses determined using reliable statistical techniques. The ASSR technique has several potential advantages over tone-burst and click ABRs. Performing response detection in the frequency domain based on statistical tests ensures that the ASSRs are detected objectively. The method does not depend on subjective visual examination of the waveforms or response patterns. Reports on the accuracy of ASSR thresholds compared to behavioral and ABR thresholds have demonstrated good correlations across various age groups, although significant differences exist between studies. The variability can largely be accounted for by the degree of hearing loss, as ASSR thresholds approximate behavioral thresholds better as the severity of the sensorineural hearing loss increases. This phenomenon has been attributed to physiological recruitment.
For these reasons, click-evoked ABR has been suggested as a useful, time-efficient physiological cross-check for ASSR results, although behavioral audiometry remains the gold standard. The aim of this study was to evaluate ARHL in our animal model by comparing three measurements: click ABRs, tone-burst ABRs, and ASSR. Material and methods Animals Healthy female Sprague-Dawley rats were tested in this study. All animals were obtained from and maintained at the University Hospital of Getafe’s animal care facility until use. The animals were housed in plastic cages with water and food ad libitum and maintained in a 12 h light/dark cycle. Rats with skin lesions, splenomegaly, macroscopically visible tumors, and inner ear infections were excluded. Five groups were created based on their age in months: 3, 6, 12, 18, and 24 months old. No rats died or were excluded during the study period. There were 10 Sprague-Dawley rats per group, with a total of 50 animals. Every measurement was obtained for both the right and left ears, yielding 100 ears examined for each measurement taken. The 3-month-old group was considered the baseline against which the other four groups were compared. Rats were anesthetized with an intraperitoneal injection of ketamine 90 mg/kg and diazepam 5 mg/ kg. All procedures involving the use and care of the animals were reviewed and approved by the University Hospital of Getafe’s Animal Care and Use Committee. ASSR and ABR measurements ASSR and ABR tests were performed on all rats in the study before euthanization. Rats were anesthetized as described above. An ER3 insert earphone was placed directly into the external auditory canal. Subcutaneous electrodes were placed over the vertex (active) and in the pinna of each ear (reference). Ground electrodes were placed over the neck muscles. ASSRs were recorded using an evoked potential averaging system (Intelligent Hearing System SmartEP, Miami, FL, USA) in an electrically shielded, double-walled, sound-treated booth in response to tone bursts at 0.5, 1, 2, 4, 8, 10, 12, 14, and 16 kHz with a 10 ms plateau and 1 ms rise/fall time. Intensities were expressed in decibels sound pressure level (db SPL) peak equivalent. Intensity series were
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Auditory evoked potentials in ARHL in rats recorded, and an ASSR threshold was defined as the lowest intensity capable of eliciting a replicable, visually detectable response. The evoked responses were amplified with a gain of 100.00 K and bandpass filtered from 30 to 300 Hz. A total of 250 sweeps were averaged at each stimulus level. The line filter was on at 50 Hz, with an artifact rejection region of –511 to 512 ms. Using the same hardware previously described, 100 ms click and tone-burst ABRs were also recorded. Each tone burst (exact Blackman envelope) and click was 1 ms in duration and had a 0.5 ms rise/fall time with no plateau. Tone bursts were presented at a rate of 27.7/s and were bandpass filtered from 30 to 1500 Hz. Clicks were presented at a rate of 19.3/s and bandpass filtered from 30 to 3000 Hz. The evoked responses in the rats were amplified with a gain of 100.00, with artifact rejection between 15 and 20 mV. A total of 250 sweeps were averaged at each stimulus level. The level of the signal was decreased in 10 dB steps from 90 dB SPL to 5 dB SPL to acquire data. Nine frequencies were tested: 0.5, 1, 2, 4, 8, 10, 12, 14, and 16 kHz. Every test (ASSR and click and tone-burst ABRs) conducted on a single animal was performed during the same session and by the same investigator.
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significantly lower thresholds were observed in 0.5 and 1 kHz (p < 0.05). These auditory profiles of lower thresholds in the frequencies below 2 kHz were observed in every group studied, independent of the age of the rats.
Tone-burst ABR When the auditory thresholds obtained in each group were compared to those in the 3-month-old group, we found a significant decrease in hearing that affected more frequencies as the groups were older (Figure 1). In the 6-month-old group, a significant increase of the thresholds was observed exclusively at 4 kHz. Significant differences were observed at 2, 4, 8, and 16 kHz when the 12-month-old group was compared with the initial group. Similarly, when the 18-monthold rats were compared across methods, additional increases were observed at 10 and 12 kHz. Finally, the oldest group showed significant differences in every frequency studied except for 0.5 kHz. Table I shows the p values of every group compared.
Click ABR Statistics analysis Data are presented as the mean ± standard deviation (SD). The analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Although the data showed normality according to the Kolmogorov–Smirnoff test, the small sample size and discontinuous data distribution led us to use a non-parametric test. The Mann–Whitney test was used to compare the differences between groups (control versus treated or measures over time). Differences with a p value < 0.05 were considered to be statistically significant. The concordance among the auditory measurement systems was analyzed using an intra-class correlation coefficient. Ethical considerations The study was approved by the Clinical Research Committee of the University Hospital of Getafe (Exp. PI09/02472, July 14, 2004).
Although ARHL was first recognized in the 6-monthold group, the first significant differences appeared in the 24-month-old group (Figure 2).
ASSR Similar to the observations for tone-burst ABRs, a progressive and significant decrease in hearing threshold was observed as the rats grew older. As shown in Figure 3, no relevant changes were observed in the 6- month-old group, except at the frequency of 4 kHz. In the 12-month-old group, a significant decrease in the thresholds was present exclusively at 16 kHz. When the 18-month-old group was compared with the baseline group, significant differences were observed in the frequency range from 4 to 10 kHz and at 16 kHz. Finally, the 24-month-old group showed significant differences at every frequency except 0.5 kHz.
Results
Comparison between ASSR and tone-burst ABR
Auditory profile
When the thresholds registered using both techniques were compared in every group, significantly lower values (p £ 0.01) were obtained in every frequency studied, using ASSRs.
When we compared every frequency elicited by all of the auditory measurement systems (0.5–16 kHz),
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R. Sanz-Fernández et al. Frequency (kHz ) 0.5
1
2
4
8
10
12
14
16
0 10 3m
20
6m 30
*
dB
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40
*
12 m
*
*
*
18 m 24 m
* *
50
*
60 70 80 90 100
Figure 1. Age-related hearing loss elicited by the tone-burst auditory brainstem response. Mean and standard deviation bars of every frequency are presented. Asterisks are placed where there is significance (p < 0.05) when the 24-month-old group was compared to the initial group. m, months.
Click ABR vs tone-burst ABR vs ASSR The concordance among the auditory measurement systems using intra-class correlations indicated higher
concordance rates at 8 and 10 kHz between clicks and ASSR. Although significant correlations were observed in every comparison made, the correlations were higher at the abovementioned frequencies (Table II).
Table I. p values in comparison of the auditory thresholds elicited by all of the auditory measurement systems. Age
500 Hz
1000 Hz
2000 Hz
4000 Hz
8000 Hz
10 kHz
12 kHz
14 kHz
16 kHz
Tone-burst auditory brainstem response 6 months
0.853
0.143
0.105
0.011
0.143
0.247
0.393
0.529
0.436
12 months
0.796
0.105
0.075
0.035
0.015
0.023
0.123
0.739
0.123
18 months
0.035
0.011
0.075
0.015
0.005
0.007
0.035
0.143
0.035
24 months
0.684
0.003
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
0.001
< 0.001
Auditory steady-state response 6 months
0.971
0.219
0.796
0.105
0.436
0.684
0.579
0.218
0.739
12 months
0.739
0.631
1.000
0.143
0.190
0.247
0.190
0.971
0.043
18 months
0.315
0.075
0.016
0.005
0.007
0.089
0.436
0.481
0.004
24 months
0.043
0.005
0.001
< 0.001
0.001
0.009
0.029
0.009
< 0.001
Click auditory brainstem response 6 months
0.579
12 months
0.481
18 months
0.143
24 months
< 0.001
The auditory thresholds obtained in each group were compared to those in the 3-month-old group. Statistical significance was accepted when p £ 0.01 (values shown in bold type).
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Auditory evoked potentials in ARHL in rats 3m
6m
12 m
18 m
24 m
(ASSR and tone-burst and click ABRs) showed improved results at higher frequencies (>4 kHz) compared with those obtained at lower ones (0.5, 1, and 2 kHz). This auditory profile persisted in every age group studied and in various animal groups, always showing improved auditory thresholds at higher frequencies regardless of whether and to what degree ARHL was present. This finding has been reported previously by several authors [13–15] in various studies that repeatedly indicated the highest level of normal auditory sensitivity at approximately 12–24 kHz. A second conclusion can be inferred regarding ARHL. In the present study, every auditory measurement system detected progressive hearing loss, which was more pronounced when older groups were compared with younger ones. This deterioration was more accurate at acute frequencies. Schuknecht developed the knowledge of degenerative changes in the lateral wall and stria vascularis as a major contributor to hearing loss associated with advancing age [16], a condition known as metabolic presbycusis, strial presbycusis, or pure presbycusis [17]. Various animal models have been used to investigate ARHL. The most frequently studied model is the
0 10 20 30 40
60 70 Figure 2. Age-related hearing loss elicited by the click auditory brainstem response. Mean and standard deviation bars of every age group are presented. m, months.
Discussion Several conclusions can be drawn from this study. First, in our animal model, the thresholds calculated using each of the auditory measurement systems
Frequency (kHz ) 0.5
1
2
4
8
10
12
14
16
0 10 3m
20
6m
* 30
* *
40
*
* *
12 m 18 m 24 m
* dB
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50
50 60
* *
70 80 90 100 Figure 3. Age-related hearing loss elicited by the auditory steady-state response. Mean and standard deviation bars of every frequency are presented. Asterisks are placed where there is significance (p < 0.05) when the 24-month-old group was compared to the initial group. m, months.
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R. Sanz-Fernández et al.
Table II. Intra-class correlation with the concordance rates of auditory brainstem response (ABR) elicited with auditory clicks and auditory steady-state response (ASSR). ASSR vs click ABR
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Frequency
ICC
95% CI
p value
500 Hz
0.294
–0.198–0.458
0.143
1000 Hz
0.733
0.063–0.820
< 0.001
2000 Hz
0.758
0.640–0.837
< 0.001
4000 Hz
0.763
0.747–0.840
< 0.001
8000 Hz
0.834
0.753–0.888
< 0.001
10 kHz
0.799
0.702–0.865
< 0.001
12 kHz
0.772
9.662–0.847
< 0.001
14 kHz
0.744
0.620–828
< 0.001
16 kHz
0.779
0.671–0.851
< 0.001
CI, confidence interval; ICC, intra-class correlation.
C57BL/6J (B6) mouse. These mice exhibit early and progressive hearing loss, and the broad degeneration throughout the cochlea and auditory cortex overlaps each of the previously mentioned major types of ARHL proposed by Schuknecht [17]. This strain has been considered to be a model of human sensory presbycusis [16]. Sprague-Dawley rat strains are also often used as animal models to study the mechanisms underlying ARHL. The first signs of scattered hair cell loss are detected in the early life of Sprague-Dawley rats and progress with age [18]. Inner and outer hair cell loss has been observed in 2-month-old rats. A pronounced spiral ganglion nerve loss with aging has also been described [19]. These histological findings exhibit good correlations with our electrophysiological evidence, supporting a deterioration of the thresholds elicited by ABR and ASSR. Certain differences were observed among toneburst and click ABRs and ASSR. All of the thresholds elicited using the ASSR method were lower than those obtained using ABR, regardless of the stimulus type (click or tone burst). Although ASSR and tone-burst ABR use different stimuli to elicit the response, the frequency ranges studied with both methods were similar. The improved detection of auditory thresholds together with the possibility of a broader frequency range makes ASSR a more suitable test to study auditory responses in animal models. Because the differences between ABR and ASSR were always independent of the age group, we were able to extend our preference for ASSR to studying ARHL. In our animal model, the click ABR correlated moderately well with every frequency studied using the ASSR system. Nevertheless, the strongest
correlations were found at the 8 and 10 kHz frequencies, which was unexpected. Because the estimated levels typically tended to correlate reasonably well with the audiogram from approximately 2–4 kHz according to the ABR tests, we expected to find stronger correlations in those at the 2 and 4 kHz frequencies. The click is relatively flat in a coupler. However, due to the resonances of the outer and middle ear, there will likely be increased energy in the 1.5 and 3 kHz regions. The click truly stimulates all frequencies up to nearly f = 1/duration of the DC pulse driving the transducer and within the frequency response of the transducer and the filter effects of the auditory periphery. The latter two factors impart some frequency-specific nuance to the effective stimulus; nuances of the ABR further favor the 2–4 kHz region during an unmasked, unfiltered click response [20]. One possible explanation for this displacement of the auditory click energy toward 8 and 10 kHz could be the anatomic differences between humans and our animal model. Real ear measurements in humans reveal differences from couplers and can reveal the nuances of each individual. These differences can be more evident in subjects with outer and/or middle ear pathologies and during comparisons between adults and very young children, infants, or newborns. In all species, including humans, the external ear and ear canal modify the acoustic spectrum of sounds reaching the tympanic membrane. This spectral modification plays a particular role in sound localization mechanisms, and the pinnae of many mammalian species have distinct designs, and in some cases mobility, to enhance the spectral cues that provide information on sound sources. The environment of the middle ear cavity clearly influences the signal that reaches the cochlea in terms of both the amplitude and the spectral content. Pressure changes within the middle ear can attenuate signals and change the spectral transfer function of the middle ear. However, the reality is that the click is a broadband stimulus that simulates most audible frequencies in the cochlea. The broadband character of the click and the differences in the Sprague-Dawley rat middle ear may explain the observed variations in the click properties compared with those observed in humans. Conclusions These results support the idea that the ASSR is a more accurate test for studying ARHL in Sprague-Dawley rats. Differences in the rat middle ear may explain the variations of the click properties, with a displacement
Auditory evoked potentials in ARHL in rats of the energy toward 8 and 10 kHz compared with humans.
Acknowledgment This research was funded by FIS Exp. PI09/02472, July 14, 2004.
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Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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