-->PONE-D-26-20660-->-->Bone-conducted ultrasonic auditory brainstem response thresholds in a mouse model of cisplatin-induced hearing loss-->-->PLOS One

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Reviewers' comments:

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: ・General assessment

This manuscript investigates auditory brainstem responses (ABRs) evoked by bone-conducted ultrasonic stimulation in a mouse model of cisplatin-induced hearing loss. The finding that ultrasonic-evoked ABR thresholds are relatively preserved compared to those within the conventional hearing range is intriguing and potentially important. It suggests that ultrasonic-evoked responses may not simply reflect cochlear function in a tonotopic manner.

Although I do not consider additional experiments to be necessary and the data appear technically sound, the manuscript would benefit from improved clarity in its conceptual framework and interpretation. These issues can be addressed through revision of the text. Therefore, I recommend minor revision.

・Major comments

1. Clarification of the study objective and positioning

The overall aim and positioning of the present study require further clarification.

As currently written, the manuscript can be interpreted in multiple ways:

(1) as a comparative study between conventional and ultrasonic ABR,

(2) as a clinically oriented attempt to explore alternative indicators for cisplatin-induced hearing loss, or

(3) as an investigation into the mechanisms of bone-conducted ultrasonic hearing.

While each of these interpretations is potentially valid, the manuscript does not clearly define which represents the primary objective. This ambiguity makes it difficult to fully appreciate the significance of the findings.

In particular, if the study is framed merely as a comparison between stimulus modalities, the rationale for employing a cisplatin-induced hearing loss model becomes less clear. Instead, the present design appears more suitable for examining how cochlear damage differentially affects these responses, thereby providing constraints on possible mechanisms.

Given the absence of morphological or cellular analyses, the study is best positioned as a functional and phenomenological investigation that constrains possible mechanisms rather than directly elucidating them.

I therefore encourage the authors to explicitly state the primary aim of the study and align the Introduction and Discussion accordingly.

2. Strengthening the Discussion by integrating existing knowledge

The authors state that the purpose of this study is to describe physiological findings rather than to elucidate mechanisms. While this is a reasonable scope, the Discussion would benefit from a more structured and integrative interpretation of the results.

In particular, it would be helpful to relate the present findings to:

(i) the known pathophysiology of cisplatin-induced cochlear damage (e.g., preferential basal turn involvement and outer hair cell vulnerability), and

(ii) current hypotheses regarding bone-conducted ultrasonic hearing (e.g., harmonic distortion vs alternative cochlear or mechanical mechanisms).

The observed dissociation between conventional ABR and ultrasonic-evoked ABR appears difficult to reconcile with the known tonotopic vulnerability of the cochlea. This point is highly interesting but not sufficiently explored.

Without requiring additional experiments, discussing how the present results align with or challenge these frameworks would clarify the significance of the findings and strengthen the manuscript.

・Minor comments

1. Some parts of the Results section repeat methodological details. These descriptions should be consolidated in the Methods section. Please also add references to the corresponding figures/tables at appropriate locations in the text.

2. In Figure 1C, the label “control” appears to refer to recordings obtained before cisplatin administration rather than a saline-treated control group. To avoid confusion, please revise the labeling (e.g., “before treatment”) and clearly distinguish it from saline controls.

3. The terms “conventional hearing range” and “bone-conducted ultrasonic stimulation (beyond the conventional hearing range)” are repeatedly used and relatively long. Defining appropriate abbreviations (e.g., CHR and BCUS) would improve readability.

Reviewer #2: This manuscript investigates the threshold shifts of auditory brainstem responses (ABRs) to stimuli within the conventional hearing range and to bone-conducted ultrasonic stimulation using a mouse model of cisplatin-induced hearing loss. The authors demonstrate that while conventional hearing thresholds significantly increased after cisplatin administration, the threshold elevations evoked by bone-conducted ultrasonic stimulation remained relatively small. This dissociation between conventional and ultrasonic hearing under cochlear dysfunction is highly intriguing and opens a novel perspective in auditory research. Overall, the study is well-conceived and potentially impactful. However, there are several methodological ambiguities and structural redundancies that need to be addressed before publication.

Specific Comments:

The method used to calibrate the bone-conducted ultrasonic stimulation is not sufficiently clear. Although the authors cite previous literature, the specific procedure in the context of this study remains ambiguous. Because the output force of a piezoelectric actuator is heavily dependent on the mechanical impedance of the object it is attached to, it is critical to explain how the calibration was accurately achieved using a hydrophone. Furthermore, since the stimulus intensity is expressed as an absolute value in decibels relative to a standard bone compression pressure of 2 mPa (dB re 2 mPa), the theoretical rationale and specific determination method for using this pressure-based reference in bone conduction should be explicitly detailed in the text.The method used to calibrate the bone-conducted ultrasonic stimulation is not sufficiently clear. Although the authors cite previous literature, the specific procedure in the context of this study remains ambiguous. Because the output force of a piezoelectric actuator is heavily dependent on the mechanical impedance of the object it is attached to, it is critical to explain how the calibration was accurately achieved using a hydrophone. Furthermore, since the stimulus intensity is expressed as an absolute value in decibels relative to a standard bone compression pressure of 2 mPa (dB re 2 mPa), the theoretical rationale and specific determination method for using this pressure-based reference in bone conduction should be explicitly detailed in the text.

There is noticeable redundancy between the "Materials and Methods" and "Results" sections, particularly regarding the descriptions of the experimental design and the cisplatin administration protocol (e.g., dosage and consecutive days). To improve readability and flow, the authors should streamline these sections and ensure that the "Results" section focuses primarily on findings rather than repeating the experimental procedures.

Regarding the electrophysiological measurements, what specific criteria (e.g., visual inspection guidelines or signal-to-noise ratio thresholds) were used to reliably determine the presence or absence of ABR wave III? In addition, please provide information or data regarding the intra-individual reproducibility and consistency of these wave measurements across trials.

The exclusion rate of mice at baseline due to pre-existing high-frequency hearing impairment appears relatively high (9 out of 32 mice, ~28%). Could the authors discuss the potential causes of this pre-existing impairment? Specifically, is there a possibility that environmental noise within the animal housing facility influenced the baseline hearing thresholds of these young mice?

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Reviewer #1: No

Reviewer #2: Yes:  Takuji Koike

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Bone-conducted ultrasonic auditory brainstem response thresholds in a mouse model of cisplatin-induced hearing loss

PONE-D-26-20660R1

Dear Dr. Nin,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Toru Miwa

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: (No Response)

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: (No Response)

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-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: (No Response)

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: (No Response)

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-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: I thank the authors for their careful and constructive responses to the review comments. The manuscript has been substantially improved. In particular, the Introduction and Discussion now more clearly define the study objective and appropriately position the work as a functional and phenomenological investigation rather than a mechanistic study. The discussion has also been strengthened by relating the findings to the known pathophysiology of cisplatin-induced cochlear damage and current hypotheses regarding bone-conducted ultrasonic hearing. The remaining revisions have been appropriately addressed, and I have no further major concerns. I believe the manuscript is suitable for publication in its current form.

Reviewer #2: (No Response)

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes:  Takuji KOIKE

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