PONE-D-22-25555Round-window delivery of lithium chloride protects cochlear synapses from noise-induced excitotoxic trauma by inhibiting NMDA receptorPLOS ONE

Dear Dr. Jung,

Thank you for submitting your manuscript to PLOS ONE. The manuscript has been reviewed, reviews attached, and after careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript is of interest, but the reviewers have raised a number of concerns that need to be addressed.  Some of these listed are relatively minor and I recommend you address them but there are a number of more significant concerns about the statistical analyses and the lack of a control group, which both need to be considered and addressed in any revision before we can consider accepting the paper for publication.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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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?

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

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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: This is a well written paper on the application of a LiCl treatment for the protection of the hair cell-auditory neuron synapse following a noise exposure in rats. The results would be of interest to researchers and clinicians in the field of hearing research.

I have a few specific comments that should be addressed:

The abstract mentions synaptic regeneration, but later talks about reducing synaptic loss. In this paper, it is more likely to be the latter that is occurring, since the treatment is given 24 hours after noise exposure. Please check the manuscript to make sure there are no other inconsistencies with respect to protection/regeneration.

The introduction was appropriately reference and introduced all relevant topics.

In this sentence in the methods: “The control group was not exposed to noise or surgery to access normal cochlear synaptic counts.” Do you mean assess here?

It’s not clear from the methods whether the lithium is injected through the RWM or into the niche. Please amend the text to clarify.

Figure 1 is a nice summary of the experimental protocol. Thanks for including this.

In this sentence in the results: “As quantitatively shown in Fig. 3C and D, the mean synaptic count per IHC was reduced”, do you mean Fig 4C and D?

And here as well: “Synaptic rescue can be seen in the noise + 2 mM LiCl group both one week (Fig. 3A) and two weeks (Fig. 3B) after the treatment.” I think you mean Fig 4A and B

In figure 4, there are some differences in the brightness of the green stain between images, particularly in Fig 4B noise + vehicle which seems dimmer than the ones on either side of it. It’s hard to see the synaptic puncta. Also in Fig 4A noise + vehicle and noise + 2 mM LiCl. Could these images be adjusted to be of similar intensity?

In figure 5 there are 2 bands in the NR2B image. Are they both relevant to the protein? Do you expect to see a reduction in NR2B compared to control? In the results section you only compare to noise-induced increase in total NR2Band pNR2B, but what would you find if you compared to control?

The first sentence in the discussion seems a little off topic, mentioning a therapeutic window in which to regenerate “peripheral axons”, whereas this study is looking at synapses.

In the discussion – Could you please include discussion on the timing of the experimental protocol, and the clinical applicability of delivering a therapeutic within 1 day of a noise exposure, particularly a noise exposure that only causes synaptopathy, as opposed to one that causes a significant threshold shift, and particularly where the treatment requires deliver of the agent in a clinical setting (as opposed to taking an oral medication for example). If synaptopathy in the human population occurs because of moderate noise exposure over a long period of time, how applicable is the animal model used in this study? How would the patient identify that a damaging noise exposure has occurred?

Depending on the requirements of the journal, a conclusion would be nice to have.

Reviewer #2: This manuscript investigated the effects of round-window delivery of lithium on noise-induced cochlear synaptopathy and hearing loss in a rat model. They also investigated the changes in NMDA receptor expression as potential underlying mechanisms. The experimental design is straightforward, although including a control + vehicle group would add more confidence for some of the conclusions (see specific comments below). The methods were appropriate and well described except the statistical analysis (see specific comments below). The results section could be improved with a more appropriate statistical analysis.

Since there is no page numbers and line numbers for the downloaded manuscript, the page numbers mentioned in the comments below is referred to the page number of the entire PDF file , which does not corresponding to the actual page numbers of the manuscript.

1. Title: The animal species needs to be included in the title

2. Page 9, Abstract:

a. Please provide the dose (or dose range) of lithium chloride and clarify whether it was a single treatment or multiple treatment

b. Throughout the whole abstract, there is no mentioning of what animals was used.

3. Page 10: “it could contribute to difficulties understanding …” should be “…difficulties in understanding…”.

4. Page 14, first line: Will the ABR testing at 3 days after treatment not only assess the “effects of the surgical manipulations”, but also the treatment effects?

5. Page 14, line 6: n = 2 for groups 1 and 2?

6. Page 15, line 1: What does “imaged at frequencies of 8, 12, 16 and 32 kHz” mean? Please clarify how the frequency location was determined.

7. Page 15, Western blot analysis: The statement “To assess NR2B activity” is not appropriate, as Western blot can only look at the protein expression of the receptor, but not the activity of the receptor.

8. Page 16, line 15: “followed by autoradiography”. “Autoradiography” is referred to the photochemical technique used to record the spatial distribution of radiolabeled compounds within a specimen or an object, which shouldn’t be used to describe the imaging analysis procedure for Western blot using ChemiDoc.

9. Pages 16 – 17: “To compare the ABR thresholds, synapse counts, and western blot result between experimental groups.” This is not a complete sentence.

10. Page 17: The sentence “parametric data were analyzed using the repeated measures one-way analysis of variance (ANOVA) was used with the Greenhouse-Geisser correction” is not grammatically correct.

11. Statistical analysis:

a. For the ABR data, you have 5 groups (control, noise-only, noise + vehicle, noise + 1mM and noise + 2mM), 4 time pints (baseline, 3 days, 1 week and 2 weeks) and 4 frequencies (8, 12, 16 and 32). A 3-way ANOVA (group x time x frequency) with repeated measures or a linear mixed model analysis should be used. If you want to analyse each frequency separately, you should use a two-way ANOVA (group x time) with repeated measures or a linear mixed model analysis. An one-way ANOVA is not appropriate for this data. In addition, why the baseline data was not included in the analysis?

b. For the wave I amplitude data (i.e., figure 3), no statistical method was mentioned. Assume that a one-way ANOVA was used, but looking at the data, at least a two-way ANOVA (time x stimulus level) with repeated measures should be used for each group instead, but a 3- way analysis would be even better.

12. Page 17, Results: The statement of “a temporary ~15 dB SPL threshold shift and a permanent ~15 dB SPL threshold shift” is not appropriate, as “temporary” means lasting for only a limited period of time; not permanent, but the threshold shift was “permanent” in your case. So, please rewrite this sentence.

13. Figure 2: The baseline ABR for all the groups should be included in order to see changes before and after noise exposure/drug treatment.

14. Page 17, Results: “The surgery for the round-window delivery of vehicle caused temporary increases in the 8 kHz and 12 kHz thresholds for only one week after the surgery, as observed by comparing the noise-only and noise + vehicle groups”. Without a control + vehicle group, the conclusion that “the surgery for the round-window delivery of vehicle caused temporary increases in the 8 kHz and 12 kHz thresholds” cannot be made. As shown in Fig 2, there seems no difference in the threshold between 3 days and 1 week for the noise + vehicle group (there seems slight improvement).

15. Page 18, Results: “All experimental groups had statistically significant differences before and after noise exposure as determined by one-way ANOVA with the Greenhouse-Geisser correction for all levels from 65 -80 dB SPL”. This statement is not backed up by the one-way ANOVA analysis, as you were talking about two factors here, i.e., time and levels. It is not clear how the one-way ANOVA could be run to tell you the differences for the two factors. As commented on the statistical analysis, a two-way ANOVA or 3-way ANOVA/linear mixed model should be used.

16. Figure 3 legend: what does the grey area mean? The exposed intensity? Please clearify.

17. Page 19, Results: For the synapses data, it is also better to do a 2-way ANOVA or linear mixed model analysis for similar reasons mentioned above. In addition, n = 2 seems a very small sample size for valid statistical analysis.

18. Figure 5: please add sample size for each group. From the western blot image, there seems only one sample in the control group and 2 samples in the other two groups. Please clarify.

19. The Supplementary figure seems not necessary.

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Reviewer #1: Yes: Rachael T Richardson

Reviewer #2: Yes: Yiwen Zheng

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Attachments

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Submitted filename: review comments.docx

PONE-D-22-25555R1Round-window delivery of lithium chloride regenerates rat cochlear synapses from noise-induced excitotoxic trauma by inhibiting NMDA receptorPLOS ONE

Dear Dr. Jung,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: Please address the minor changes recommended by both reviewers, including the comparison between control and experimental groups.

==============================

Please submit your revised manuscript by Apr 10 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Alan G. Cheng, M.D.

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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: (No Response)

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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: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

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: Yes

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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: No

Reviewer #2: Yes

**********

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: Thank you for the changes you made to the manuscript.

I have a couple of minor revisions that I think need to be addressed prior to publication.

Suggested change to the title from "Round-window delivery of lithium chloride regenerates rat cochlear synapses from noise-induced excitotoxic trauma by inhibiting NMDA receptor" to one of the following:

"Round-window delivery of lithium chloride regenerates cochlear synapses damaged by noise-induced excitotoxic trauma via inhibition of the NMDA receptor in the rat"

OR

"Regeneration of cochlear synapses in the rat following noise-induced excitotoxic trauma by round-window delivery of lithium chloride to inhibit the NMDA receptor" or something similar, because I don't think "regenerates rat cochlear synapses from noise-induced excitotoxic trauma" makes sense.

Line 208: "the two-way repeated measures one-way analysis of variance" Was it one way or two way?

Line 224 "in the noise + vehicle group had returned to that in the noise-only group." change to ...of the noise-only group.

Line 302 "There is a robust literature on potential therapeutic agent to prevent or regenerate noise-induced synaptic loss in the cochlea [7, 23-26]. " Change to "There is a robust literature on the delivery of potential therapeutic agents to prevent or regenerate noise-induced synaptic loss in the cochlea [7, 23-26].

Suggested change to Line 371 ""Fourthly, many key questions remain for clinical application. There is likely a significant clinical population with loss of cochlea synapse that could be addressed by a treatment analogous to that used here in the noise-exposed rat. Although the local delivery of lithium to the round window was effective in regenerating cochlear synapses 24 hours after the noise exposure, a key question would be how long this time window could be extended for effective synaptogenesis in a clinical setting."

Reviewer #2: The authors have addressed most of my comments to my satisfaction. However, for the ABR results, although the authors added the control group to Figure 2, the control group was still excluded from the statistical analysis. Without statistically analyse the difference between control and other experimental groups, especially, between control and noise + drug groups, a piece of very important information is missing. That is whether or not the drug treatment would have any therapeutic value/potential, i.e., to make the hearing threshold return to the control level. Therefore, control group should be included in the analysis and the results need to be discussed accordingly. In addition, in lines 208 – 208, please clarify what does “the two-way repeated measures one-way analysis of variance (ANOVA)” mean?

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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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Rachael Richardson

Reviewer #2: Yes: Yiwen Zheng

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Round-window delivery of lithium chloride regenerates cochlear synapses damaged by noise-induced excitotoxic trauma via inhibition of the NMDA receptor in the rat

PONE-D-22-25555R2

Dear Dr. Jung,

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,

Alan G. Cheng, M.D.

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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Yes

**********

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: Yes

**********

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: (No Response)

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Rachael Richardson

Reviewer #2: Yes: Yiwen Zheng

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