Automated threshold detection for auditory brainstem responses: comparison with visual estimation in a stem cell transplantation study
© Bogaerts et al; licensee BioMed Central Ltd. 2009
Received: 15 April 2009
Accepted: 26 August 2009
Published: 26 August 2009
Auditory brainstem responses (ABRs) are used to study auditory acuity in animal-based medical research. ABRs are evoked by acoustic stimuli, and consist of an electrical signal resulting from summated activity in the auditory nerve and brainstem nuclei. ABR analysis determines the sound intensity at which a neural response first appears (hearing threshold). Traditionally, threshold has been assessed by visual estimation of a series of ABRs evoked by different sound intensities. Here we develop an automated threshold detection method that eliminates the variability and subjectivity associated with visual estimation.
The automated method is a robust computational procedure that detects the sound level at which the peak amplitude of the evoked ABR signal first exceeds four times the standard deviation of the baseline noise. Implementation of the procedure was achieved by evoking ABRs in response to click and tone stimuli, under normal and experimental conditions (adult stem cell transplantation into cochlea). Automated detection revealed that the threshold shift from pre- to post-surgery hearing levels was similar in mice receiving stem cell transplantation or sham injection for click and tone stimuli. Visual estimation by independent observers corroborated these results but revealed variability in ABR threshold shifts and significance levels for stem cell-transplanted and sham-injected animals.
In summary, the automated detection method avoids the subjectivity of visual analysis and offers a rapid, easily accessible http://axograph.com/source/abr.html approach to measure hearing threshold levels in auditory brainstem response.
The auditory brainstem response (ABR) is a voltage response evoked by acoustic stimuli as sound is processed along the auditory pathway. It consists of electrical signals resulting from the sum of sound-evoked activity along the auditory nerve and brainstem nuclei. ABR analysis determines the sound intensity at which a neural response first appears (hearing threshold) . Previous studies in rats in mice have shown that ABR thresholds do not indicate absolute behavioural hearing thresholds [2, 3]. However, ABR audiometry has been used extensively in animal hearing research for examining gene therapy [4–10], cell-replacement therapies [11–14], and noise-induced hearing loss [15–20].
The ABR offers an objective measurement of auditory signal processing. The objectivity is diminished by conventional visual inspection of the ABR threshold level. Subjectivity and variability are introduced when the investigator has to decide when a complex, multi-component response first becomes distinguishable from background noise . Methodologies have been developed to address the subjective component of threshold detection by including criteria about the shape, pattern, or absolute amplitude of the response, yet these still require a visual decision about the presence of a signal. Eliminating subjectivity in auditory threshold determination would improve the sensitivity and reliability of this important audiometric technique.
While visual estimation remains the conventional technique for ABR threshold detection, a need for automated statistical methods for detection is highly recognised. Several methods have been developed based on the techniques of Fsp analysis [22–25], cross correlation [26–28] and feature vectors [29–32]. Fsp analysis requires calculation of a variance ratio in the ABR waveform followed by application of the F-statistic to this ratio. Cross correlation measures the degree of similarity between a sliding template and an averaged waveform. Feature vectors quantify selected components of the response's time course. Fsp has been incorporated into available software (Compumedics Ltd) yet the other methods lack comparable implementation.
Here we develop a simple, fully automated auditory threshold detection method to address the subjectivity and variability associated with visual estimation of ABRs. This method is based on the signal-to-noise ratio and the software has been made readily available . The algorithm is calibrated by comparison with visual estimation, implemented via investigation of stem cell transplantation, and compared against variability obtained with visual estimation.
Results and discussion
The automated detection method is based on a statistical foundation. Assuming a normal distribution of baseline noise data, 99.98% of noise values will lie within ± 4 SD of the mean. Any amplitude values occurring outside this range allow rejection of the null hypothesis (no ABR signal) at the p < 0.0002 level. This confidence level is valid when a single data point in the ABR signal is examined. If we search for a peak amplitude across a time window containing 100 data points, then a peak signal > 4 SD is significantly different from baseline noise at the p < 0.02 level, thus confirming the presence of an ABR signal and providing a statistically reliable estimate of the hearing threshold level.
An automated detection method based on the signal-to-noise ratio has previously been reported [22, 23, 25]. This method requires calculation of a parameter, Fsp, which is the variance of the amplitude values across an specified time window of the averaged response (VAR(S)), divided by the variance of the amplitude of a single time point across several hundred sweeps (VAR(SP)). This deterministic approach assumes that the evoked ABR voltage waveform is constant from trial to trial yet neural population signals typically fluctuate in amplitude from trial to trial due to changes in the number of neurons contributing to the response. Such ABR amplitude fluctuations will cause VAR(SP) to be systematically overestimated. In contrast, assumption of biological variability has been included in our algorithm.
This relatively simple and intuitive threshold detection method was developed as a plug-in module for AxoGraph X, a data analysis application (AxoGraph Scientific). The automated analysis module, with source code, is freely available with an application license . The module imports a graph containing the family of averaged voltage responses recorded at different sound intensity levels, then automatically outputs a plot of the signal-to-noise ratio versus sound intensity, with the ABR threshold level indicated.
Automated method detects accurate and consistent hearing threshold levels
For click and tone stimuli, automated detection produced a similar threshold value as visual estimation for the same series of ABR traces in normal-hearing mice (Figure 2). Summary data indicated that automated detection provided a mean threshold value of 33 ± 2 dB (n = 21) for click stimuli that was not significantly different than the mean threshold value predicted by visual estimation (31 ± 2 dB; n = 21; Figure 2A). This was also consistent in response to tone stimuli where automated detection showed a mean threshold value of 32 ± 3 dB (n = 18) while visual estimation predicted a mean threshold value of 29 ± 2 dB; n = 18; Figure 2A). Individual data revealed that automated detection produced identical estimates of ABR threshold compared to visual estimation in 48% of mice (n = 10/21) for click stimuli and in 50% of mice (n = 9/18) for tone stimuli. In the non-identical data, the threshold estimates for automated detection differed from visual estimation by a mean of 6 ± 0.6 dBs (n = 11) for click stimuli and by 9 ± 3 dBs (n = 9) for tone stimuli.
Two independent observers estimated significantly different threshold levels for the same series of ABRs (Figure 2B). The absolute difference in threshold values between observers was statistically different than zero for click stimuli (1.7 ± 0.7 dB; n = 21; p < 0.05) and for tone stimuli (3.9 ± 0.9 dB; n = 18; p < 0.001). Together, the results confirm that a signal-to-noise ratio value of four (SD ± 4) predicts equivalent and consistent threshold levels in comparison to visual estimation and that variability in threshold detection is associated with visual estimation.
Automated detection method is used to investigate vestibular stem cell transplantation
The mean ABR threshold shift between pre- and post-surgery hearing levels for mice transplanted with vestibular cells was 34 ± 6 dB (n = 5) in response to click stimuli. This threshold shift was not significantly different than for mice receiving a sham injection (48 ± 5 dB; n = 6). For pure tone stimuli, the mean ABR threshold shift was also not significantly different for stem cell-transplanted (33 ± 9 dB; n = 5) versus sham-injected animals (41 ± 3 dB; n = 6; Figure 3B).
Automated detection avoids variability associated with visual estimation
A simple, fully automated auditory threshold detection method was developed to address the subjectivity and variability associated with visual estimation of ABRs. The threshold values provided by automated detection were similar to those provided by visual estimation, indicating the automated method predicts valid threshold levels. The automated detection method was implemented in experimental conditions and revealed no difference between stem cell-transplanted and sham-injected mice. Visual estimation by independent observers corroborated these results but revealed variability in ABR threshold shifts and significance levels for stem cell-transplanted and sham-injected animals. In summary, the automated detection method developed here offers an accessible, accurate, and reproducible approach for measuring hearing threshold levels in auditory brainstem responses.
Auditory Brainstem Responses (ABRs)
Auditory function was assessed by measuring ABR thresholds in CBA/CaH mice aged 4 to 6 postnatal weeks (n = 41) as previously described . ABR thresholds were recorded in two groups of animals: normal hearing mice (n = 30) and mice receiving a unilateral stem cell transplantation or sham injection into the left cochlea (n = 11). Briefly, mice were anaesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg), and ABRs were recorded differentially between subdermal platinum electrodes placed at the vertex and lateral to the left cheek with an electrode at the lower back serving as ground. Clicks (1 ms duration, 100 ms interstimulus interval) and tone pips (16 kHz; 1 ms rise/fall; 3 ms duration, 90 ms interstimulus interval) were delivered via an electrostatic insert speaker and ABRs were obtained by reducing the intensity in 5 dB steps beginning at 90 dB (Tucker Davis Technologies). The ABR signal was obtained by time locked averaging with a minimum of 512 averages. ABRs were band passed filtered above 300 Hz and below 1500 Hz. No stimulus artifact was observed. With ideal recording conditions, a baseline noise of 100–200 μV was achieved. ABRs were recorded with BioSig software (Tucker Davis Technologies) and converted to TIFF files for visual estimation in printed format or to ASCII files for automated computer analysis.
All experiments were performed with the approval of the Garvan Institute and St Vincent's Hospital Animal Ethics Committee, in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (National Health and Medical Research Council, 2004).
Stem cell transplantation
For vestibular primary cell culture, male and female CBA/CaH mice (n = 5; aged 10–15 postnatal days) were anesthetized with CO2 and decapitated. Inner ears were placed in chilled Dulbecco's modified Eagle medium (D-MEM) containing 9.6 mg/ml HEPES, and the utricular maculae and ampullary cristae carefully dissected. The outer margins of the sensory epithelia were then trimmed away and the tissues processed according to a method adapted from that of Oshima et al. . Tissues were incubated in 0.5 mg/ml thermolysin (Sigma) in D-MEM for 30 min at 37°C and then in 0.125% trypsin in Hank's Balanced Salt Solution for 20 min at 37°C. Tissues were washed in Advanced D-MEM/F-12 medium containing 20 mM glutamine and 10% fetal bovine serum and gently triturated. Dissociated cells were centrifuged at 400 × g and resuspended in 10 ml Advanced D-MEM/F-12 medium containing 20 mM glutamine, B-27 supplement minus vitamin A, N2 supplement, 20 ng/ml EGF, 20 ng/ml bFGF (both Millipore), 100 U/ml penicillin G and 100 μg/ml streptomycin. The cell suspension was then poured through a 70 μm cell strainer (BD Falcon) into plastic tissue culture dishes (BD Falcon) and cultured at 37°C with 5% CO2. Cells were collected after seven days in vitro for transplantation experiments after dissociation with TrypLE Express. All reagents from Invitrogen unless otherwise stated.
For transplant surgery, cochleostomies were performed on CBA/CaH mice aged 4 to 6 postnatal weeks (n = 11) as previously described . Briefly, minimal trauma surgery was performed on mice anaesthetised with 75 mg/kg ketamine and 15 mg/kg xylazine. A minimally invasive procedure was initiated by micro drilling through the bulla to access the inner ear and perform a lateral wall cochleostomy in the basal turn of the cochlea. Transplantations were made using a glass capillary needle (tip diameter of 100 μm) inserted into the cochleostomy. For stem cell injections (n = 6), one microliter of stem cells suspended in phosphate buffer was injected over one minute to deliver 2000–4000 cells. For sham injections (n = 5), identical techniques were followed except that phosphate buffer was substituted for the stem cell solution.
Statistics are quoted as mean ± standard error of the mean (SEM). Significant differences in mean threshold values were determined using a two-tailed unpaired t-test (Prism, GraphPad).
The authors SB, JMS, and SO have no competing interests; JDC is a software programmer at AxoGraph Scientific.
The authors are grateful to the Australian Deafness Research Foundation and the Fairfax Foundation for research funding.
- Jewett DL, Williston JS: Auditory-evoked far fields averaged from the scalp of humans. Brain. 1971, 94 (4): 681-696. 10.1093/brain/94.4.681.View ArticlePubMedGoogle Scholar
- Heffner HE, Heffner RS: Audition. Handbook of Research Methods in Experiemtnal Psychology. Edited by: Davis SF. 2003, Malden, MA: BlackwellGoogle Scholar
- Heffner HE, Koay G, Heffner RS: Comparison of behavioral and auditory brainstem response measures of threshold shift in rats exposed to loud sound. J Acoust Soc Am. 2008, 124 (2): 1093-1104. 10.1121/1.2949518.PubMed CentralView ArticlePubMedGoogle Scholar
- Ishimoto S, Kawamoto K, Kanzaki S, Raphael Y: Gene transfer into supporting cells of the organ of Corti. Hear Res. 2002, 173 (1–2): 187-197. 10.1016/S0378-5955(02)00579-8.View ArticlePubMedGoogle Scholar
- Kawamoto K, Ishimoto S, Minoda R, Brough DE, Raphael Y: Math1 gene transfer generates new cochlear hair cells in mature guinea pigs in vivo. J Neurosci. 2003, 23 (11): 4395-4400.PubMedGoogle Scholar
- Kawamoto K, Kanzaki S, Yagi M, Stover T, Prieskorn DM, Dolan DF, Miller JM, Raphael Y: Gene-based therapy for inner ear disease. Noise Health. 2001, 3 (11): 37-47.PubMedGoogle Scholar
- Kawamoto K, Sha SH, Minoda R, Izumikawa M, Kuriyama H, Schacht J, Raphael Y: Antioxidant gene therapy can protect hearing and hair cells from ototoxicity. Mol Ther. 2004, 9 (2): 173-181. 10.1016/j.ymthe.2003.11.020.View ArticlePubMedGoogle Scholar
- Kawamoto K, Yagi M, Stover T, Kanzaki S, Raphael Y: Hearing and hair cells are protected by adenoviral gene therapy with TGF-beta1 and GDNF. Mol Ther. 2003, 7 (4): 484-492. 10.1016/S1525-0016(03)00058-3.View ArticlePubMedGoogle Scholar
- Praetorius M, Baker K, Weich CM, Plinkert PK, Staecker H: Hearing preservation after inner ear gene therapy: the effect of vector and surgical approach. ORL J Otorhinolaryngol Relat Spec. 2003, 65 (4): 211-214.View ArticlePubMedGoogle Scholar
- Stover T, Yagi M, Raphael Y: Cochlear gene transfer: round window versus cochleostomy inoculation. Hear Res. 1999, 136 (1–2): 124-130. 10.1016/S0378-5955(99)00115-X.View ArticlePubMedGoogle Scholar
- Hakuba N, Hata R, Morizane I, Feng G, Shimizu Y, Fujita K, Yoshida T, Sakanaka M, Gyo K: Neural stem cells suppress the hearing threshold shift caused by cochlear ischemia. Neuroreport. 2005, 16 (14): 1545-1549.PubMedGoogle Scholar
- Hildebrand MS, Dahl HH, Hardman J, Coleman B, Shepherd RK, de Silva MG: Survival of partially differentiated mouse embryonic stem cells in the scala media of the guinea pig cochlea. J Assoc Res Otolaryngol. 2005, 6 (4): 341-354.PubMed CentralView ArticlePubMedGoogle Scholar
- Iguchi F, Nakagawa T, Tateya I, Endo T, Kim TS, Dong Y, Kita T, Kojima K, Naito Y, Omori K, et al.: Surgical techniques for cell transplantation into the mouse cochlea. Acta Otolaryngol Suppl. 2004, 551: 43-47.PubMedGoogle Scholar
- Okano T, Nakagawa T, Endo T, Kim TS, Kita T, Tamura T, Matsumoto M, Ohno T, Sakamoto T, Iguchi F, et al.: Engraftment of embryonic stem cell-derived neurons into the cochlear modiolus. Neuroreport. 2005, 16 (17): 1919-1922. 10.1097/01.wnr.0000187628.38010.5b.View ArticlePubMedGoogle Scholar
- Davis RR, Newlander JK, Ling X, Cortopassi GA, Krieg EF, Erway LC: Genetic basis for susceptibility to noise-induced hearing loss in mice. Hear Res. 2001, 155 (1–2): 82-90. 10.1016/S0378-5955(01)00250-7.View ArticlePubMedGoogle Scholar
- Kujawa SG, Liberman MC: Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth. J Neurosci. 2006, 26 (7): 2115-2123. 10.1523/JNEUROSCI.4985-05.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Ou HC, Bohne BA, Harding GW: Noise damage in the C57BL/CBA mouse cochlea. Hear Res. 2000, 145 (1–2): 111-122. 10.1016/S0378-5955(00)00081-2.View ArticlePubMedGoogle Scholar
- Vlajkovic SM, Housley GD, Munoz DJ, Robson SC, Sevigny J, Wang CJ, Thorne PR: Noise exposure induces up-regulation of ecto-nucleoside triphosphate diphosphohydrolases 1 and 2 in rat cochlea. Neuroscience. 2004, 126 (3): 763-773. 10.1016/j.neuroscience.2004.04.023.View ArticlePubMedGoogle Scholar
- Wang Y, Hirose K, Liberman MC: Dynamics of noise-induced cellular injury and repair in the mouse cochlea. J Assoc Res Otolaryngol. 2002, 3 (3): 248-268. 10.1007/s101620020028.PubMed CentralView ArticlePubMedGoogle Scholar
- Willott JF, VandenBosche J, Shimizu T, Ding DL, Salvi R: Effects of exposing gonadectomized and intact C57BL/6J mice to a high-frequency augmented acoustic environment: Auditory brainstem response thresholds and cytocochleograms. Hear Res. 2006, 221 (1–2): 73-81. 10.1016/j.heares.2006.07.016.PubMed CentralView ArticlePubMedGoogle Scholar
- Vidler M, Parker D: Auditory brainstem response threshold estimation: subjective threshold estimation by experienced clinicians in a computer simulation of a clinical test. Int J Audiol. 2004, 43: 417-429. 10.1080/14992020400050053.View ArticlePubMedGoogle Scholar
- Elberling C, Don M: Quality estimation of averaged auditory brainstem responses. Scan Audiol. 1984, 13 (3): 187-197. 10.3109/14992028409043059.Google Scholar
- Elberling C, Don M: Threshold characteristics of the human auditory brain stem response. J Acoust Soc Am. 1987, 81 (1): 115-121. 10.1121/1.395019.View ArticlePubMedGoogle Scholar
- Hyde M, Sininger YS, Don M: Objective detection and analysis of auditory brainstem response: an historical perspective. Ear Hear. 1998, 19 (1): 97-113.Google Scholar
- Sininger YS: Auditory brain stem response for objective measures of hearing. Ear Hear. 1993, 14 (1): 23-30. 10.1097/00003446-199302000-00004.View ArticlePubMedGoogle Scholar
- Cone-Wesson BK, Hill KG, Liu GB: Auditory brainstem response in tammar wallaby (Macropus eugenii). Hear Res. 1997, 105 (1–2): 119-129. 10.1016/S0378-5955(96)00199-2.View ArticlePubMedGoogle Scholar
- Davey R, McCullagh P, Lightbody G, McAllister G: Auditory brainstem response classification: a hybrid model using time and frequency features. Artif Intell Med. 2007, 40 (1): 1-14. 10.1016/j.artmed.2006.07.001.View ArticlePubMedGoogle Scholar
- Ozdamar O, Delgado RE, Eilers RE, Urbano RC: Automated electrophysiologic hearing testing using a threshold-seeking algorithm. J Am Acad Audiol. 1994, 5 (2): 77-88.PubMedGoogle Scholar
- Acir N, Ozdamar O, Guzelis C: Automatic classification of auditory brainstem responses using SVM-based feature selection algorithm for threshold detection. Eng Appl Artif Intel. 2006, 19: 209-218. 10.1016/j.engappai.2005.08.004.View ArticleGoogle Scholar
- Keohane BM, Mason SM, Baguley DM: Clinical evaluation of the vector algorithm for neonatal hearing screening using automated auditory brainstem response. J Laryngol and otology. 2004, 118 (2): 112-116.View ArticleGoogle Scholar
- Mason SM: Automated system for screening hearing using the auditory brainstem response. British journal of audiology. 1988, 22 (3): 211-213. 10.3109/03005368809076454.View ArticlePubMedGoogle Scholar
- Sanchez R, Riquenes A, Perez-Abalo M: Automatic detection of auditory brainstem responses using feature vectors. International journal of bio-medical computing. 1995, 39 (3): 287-297. 10.1016/0020-7101(95)01110-Z.View ArticlePubMedGoogle Scholar
- Automated ABR threshold detection. [http://axograph.com/source/abr.html]
- Galbraith G, Waschek J, Armstrong B, Edmond J, Lopez I, Liu W, Kurtz I: Murine auditory brainstem evoked response: putative two-channel differentiation of peripheral and central neural pathways. J Neurosci Methods. 2006, 153 (2): 214-220. 10.1016/j.jneumeth.2005.10.017.View ArticlePubMedGoogle Scholar
- Henry KR: Auditory brainstem volume-conducted responses: origins in the laboratory mouse. Journal of the American Auditory Society. 1979, 4 (5): 173-178.PubMedGoogle Scholar
- Zhou X, Jen PH, Seburn KL, Frankel WN, Zheng QY: Auditory brainstem responses in 10 inbred strains of mice. Brain Res. 2006, 1091 (1): 16-26. 10.1016/j.brainres.2006.01.107.PubMed CentralView ArticlePubMedGoogle Scholar
- Hildebrand MS, Newton SS, Gubbels SP, Sheffield AM, Kochhar A, de Silva MG, Dahl HH, Rose SD, Behlke MA, Smith RJ: Advances in molecular and cellular therapies for hearing loss. Mol Ther. 2008, 16 (2): 224-236. 10.1038/sj.mt.6300351.View ArticlePubMedGoogle Scholar
- Sekiya T, Kojima K, Matsumoto M, Holley MC, Ito J: Rebuilding lost hearing using cell transplantation. Neurosurgery. 2007, 60 (3): 417-433. 10.1227/01.NEU.0000249189.46033.42.View ArticlePubMedGoogle Scholar
- Pandit S, Cohen M, Sullivan JM, Bogaerts S, Oleskevich S: Stem cell therapy for noise-induced hearing loss in mice. Proc Aust Neuroscience Soc. 2009, 19: 99.Google Scholar
- Li H, Liu H, Heller S: Pluripotent stem cells from the adult mouse inner ear. Nat Med. 2003, 9 (10): 1293-1299. 10.1038/nm925.View ArticlePubMedGoogle Scholar
- Martinez-Monedero R, Yi E, Oshima K, Glowatzki E, Edge AS: Differentiation of inner ear stem cells to functional sensory neurons. Developmental neurobiology. 2008, 68 (5): 669-684. 10.1002/dneu.20616.View ArticlePubMedGoogle Scholar
- Oshima K, Grimm CM, Corrales CE, Senn P, Martinez Monedero R, Geleoc GS, Edge A, Holt JR, Heller S: Differential distribution of stem cells in the auditory and vestibular organs of the inner ear. J Assoc Res Otolaryngol. 2007, 8 (1): 18-31. 10.1007/s10162-006-0058-3.PubMed CentralView ArticlePubMedGoogle Scholar
- Bogaerts S, Douglas S, Corlette T, Pau H, Saunders D, McKay S, Oleskevich S: Microsurgical access for cell injection into the mammalian cochlea. J Neurosci Methods. 2008, 168 (1): 156-163. 10.1016/j.jneumeth.2007.09.016.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.