Otoacoustic emissions (OAE)

Under normal circumstances, mammals’ ears generate endogenous sounds from within the inner ear. These sounds, referred to as otoacoustic emissions (OAEs), are physiological and spontaneously generated. Naturally produced OAEs are of different intensities, depend on the situation and vary among individuals.

The production of OAEs originates from a physical mechanism involving the interaction between the tectorial membrane and the outer hair cells of the Organ of Corti, which serves as the ear’s mechano-electric transducer. This mechanism occurs when a sound wave (stimulus) propagates through the auditory canals, causing deformation of the Organ of Corti and displacement of the tectorial membrane over the hair cells. In response to the sound stimulus, the vibration of the outer hair cells generates an OAE, which then travels antidromically towards the eardrum, ultimately resulting in a detectable sound in the external auditory canal.

Typically, OAEs are subtle and not easily detectable in individuals with normal hearing due to the presence of concurrent sounds within the ear and the mitigating effects of reflective phenomena.

Cochlear outer hair cells (OHCs)

Cochlear outer hair cells (OHCs) are responsible for the sensitivity, dynamic range, and frequency-resolving capacity of the mammalian auditory system. They serve as energy sources for sound amplification. The OHC amplification function is nonlinear, meaning that soft or quiet sounds are amplified to a greater extent than loud sounds. This nonlinearity allows for a broad range of sound pressures to be reduced into a much narrower range of hair displacements.

OHCs are highly sensitive and have been identified as toxicity targets of certain drugs. For instance, aminoglycosides induced ototoxicity involves the irreversible destruction of OHCs, primarily in the basal turn of the cochlea.

Since OHCs generate otoacoustic emissions, OAE assessment provides a direct measure of their viability. Therefore, evaluating OAEs is crucial for characterizing the ototoxicity profile of drugs or assessing their protective effects in models of injury.

Cochlear outer hair cells (OHCs) are responsible for the sensitivity, dynamic range, and frequency-resolving capacity of the mammalian auditory system. They serve as energy sources for sound amplification. The OHC amplification function is nonlinear, meaning that soft or quiet sounds are amplified to a greater extent than loud sounds. This nonlinearity allows for a broad range of sound pressures to be reduced into a much narrower range of hair displacements.

OHCs are highly sensitive and have been identified as toxicity targets of certain drugs. For instance, aminoglycosides induced ototoxicity involves the irreversible destruction of OHCs, primarily in the basal turn of the cochlea.

Since OHCs generate otoacoustic emissions (OAEs), OAE assessment provides a direct measure of their viability. Therefore, evaluating OAEs is crucial for characterizing the ototoxicity profile of drugs or assessing their protective effects in models of injury.

Principles of OAE assessment

It is possible to consistently generate specific sounds that, when delivered to the ear, result in measurable otoacoustic emissions. Two commonly used methods for eliciting OAEs are Transient Evoked Otoacoustic Emissions (TEOAEs) and Distortion Product Otoacoustic Emissions (DPOAEs).

Both TEOAEs and DPOAEs involve generating standardized sound stimuli, delivering them into the external auditory canal, and recording the resulting OAEs, which are the sounds produced by the OHCs in the inner ear as a response. TEOAEs utilize click sounds as the stimulus, while DPOAEs employ pure tones. DPOAEs are often preferred as pure tones trigger OAEs that are easier to interpret.

The DPOAE measuring technique involves delivering two pure tones with known frequencies F1 and F2 (primary frequencies) to the external ear canal. To obtain accurate DPOAE responses, it is important that this pair of primary frequencies used as stimuli are relatively close to each other, typically with an F2/F1 ratio around 1.2. When these two pure tones are simultaneously delivered into the ear canal, they generate two waves that propagate asynchronously within the cochlea’s tympanic and vestibular scales.

These waves cause deformations in the tonotopic regions near F1 and F2, resulting in a physical interference effect within the cochlear segment located between the tonotopic cells for F1 and F2. This physical interference maximizes the stimulation of the OHCs corresponding to the tonotopic region of 2xF1-F2. Additionally, other areas in the cochlea may experience some stimulation due to wave motion, although to a lesser extent.

The presence or absence of a DPOAE response at a certain level of stimulation, and the magnitude or amplitude of this response, directly indicate the viability of the OHCs in the assessed region of the cochlea. In other words, the DPOAE response provides a measure of the health and functioning of the outer hair cells in that specific area.

The below diagram provides a visual depiction of the DPOAE measuring technique.

Assessing the viability and function of OHCs should cover the entire cochlea. This means that several pairs of primary frequencies (or pure tones) at a constant F2/F1 ratio are sequentially delivered into the ear canal to elicit DPOAE responses in different regions of the cochlea. The frequency pairs typically range from ~4kHz to ~32kHz. High frequencies are sensed in the base of the cochlea and are therefore used to assess OHC function in these proximal cochlear regions. Conversely, low frequencies are sensed closer to the apex and are therefore used to assess OHC function in these distal regions.

In the next section, we will present a practical example of how the pairs of DPOAE stimulating frequencies are defined in a typical setting.

Planning DPOAE assessment in rats (illustrative example)

The optimal F2/F1 ratio depends on the species and the measurement setting. For instance, a ratio of 1.22 has been reported to yield the strongest DPOAE in humans (1) and laboratory mammals such as rats, gerbils and guinea pigs (2–4).

In order to assess the function of the OHCs throughout the entire rat cochlea, from the base to the apex, pairs of primary frequencies (F1 and F2) are delivered. These frequencies should be equidistant from the target frequencies 4, 8, 12, 24, and 32 kHz while maintaining a constant F2/F1 ratio of 1.22. To meet these requirements, the values of F1 and F2 can be calculated using equations [1] and [2] respectively; where TF is the target frequency and *R*is the constant F2/F1 ratio.

$$ F1 = \frac{2 \times TF}{R} \hspace{0.3cm} [1] \hspace{1cm} F2 = F1 \times R \hspace{0.3cm} [2]$$

The application of equations [1] and [2] to the selected target frequencies (TF) yields the corresponding pairs of primary frequencies (*F1*and F2). The table below presents these frequencies, along with the expected DPOAE response frequency defined as 2xF1-F2.

{ #dpoae }

DPOAE recording procedure

At Ototox, we have extensive experience in recording DPOAE in various species, including both rodents and non-rodents. DPOAE measurements are conducted under sedation or anesthesia, with a specific protocol tailored to each species. This minimally invasive technique is quick, taking only a few minutes.

Before proceeding with the DPOAE recording, a comprehensive examination of each ear is conducted while the animal is under sedation. This examination, which includes both an external inspection and an otoscopic examination of the ear canal, aims to check for any ear effusion, cerumen, otitis, or abnormalities in the ear structure. It should be noted that the feasibility of the otoscopic examination is dependent on the size of the animal; for example, it may not be possible in smaller animals such as mice but can be performed in larger animals like dogs. The auditory functional assessment is typically performed in the right ear unless any condition or abnormality is identified during the examination, which may hinder an accurate evaluation.

For DPOAE recording, we utilize specialized equipment consisting of a “TDT RZ6 MULTI-I/O PROCESSOR” connected to a “MEDUSA-Z4 AMPLIFIER”, two calibrated “TD MF1 MULTI-FIELD MAGNETIC SPEAKERS” and a registration microphone “ETIMOTYC ERB10+”.

DPOAEs are recorded in a double closed-field setting using a conical probe that effectively seals the auditory canal. The probe has connections for each speaker and the microphone. The DPOAE responses are presented in a plot that displays the stimulation thresholds in dB SPL on the ordinate axis and the frequency of the DPOAE response (2F1-F2) on the abscissa axis. Any DPOAE response measured above the noise floor (with a positive signal-to-noise ratio above 3 dB/V) is considered present.

In cases where the responses in the assessed ear (usually the right ear) exhibit inconsistency, the left ear is also evaluated to rule out any monoaural interference resulting from ear pathologies.

Once the DPOAE recording is completed, the following parameters are provided:

• DPOAE response thresholds measured at the selected pairs of primary frequencies with the applicable constant F2/F1 ratio, expressed in dB SPL.
• DPOAE response amplitude at 90 dB SPL of F1 and F2 stimulation, expressed in dB/V.

Statistical tests are conducted on the obtained parameters as deemed appropriate for analysis and interpretation.

References

1. Harris, F. P.; Lonsbury-Martin, B. L.; Stagner, B. B.; Coats, A. C.; Martin, G. K. Acoustic Distortion Products in Humans: Systematic Changes in Amplitudes as a Function of F2/F1 Ratio. J. Acoust. Soc. Am. 1989, 85 (1), 220–229. https://doi.org/10.¹¹²¹⁄₁.397728.
2. Brown, A. M. Acoustic Distortion from Rodent Ears: A Comparison of Responses from Rats, Guinea Pigs and Gerbils. Hear. Res. 1987, 31 (1), 25–37. https://doi.org/10.¹⁰¹⁶⁄₀₃₇₈-5955(87)90211-5.
3. Khvoles, R.; Freeman, S.; Sohmer, H. Transient Evoked Otoacoustic Emissions Can Be Recorded in the Rat. Hear. Res. 1996, 97 (1–2), 120–126.
4. Mills, D. M.; Rubel, E. W. Variation of Distortion Product Otoacoustic Emissions with Furosemide Injection. Hear. Res. 1994, 77 (1–2), 183–199. https://doi.org/10.¹⁰¹⁶⁄₀₃₇₈-5955(94)90266-6.