PONE-D-23-27605Identification of compounds that cause axonal dieback without cytotoxicity in dorsal root ganglia explants and intervertebral disc cells with potential to treat pain via denervationPLOS ONE

Dear Dr. Wachs,

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. Both reviewers view your manuscript positively. However, Reviewer 2, in particular, makes detailed criticisms that you are requested to address as fully as possible in your revised manuscript.

Please submit your revised manuscript by Jan 04 2024 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,

Israel Silman

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Note from Emily Chenette, Editor in Chief of PLOS ONE, and Iain Hrynaszkiewicz, Director of Open Research Solutions at PLOS: Did you know that depositing data in a repository is associated with up to a 25% citation advantage (https://doi.org/10.1371/journal.pone.0230416)? If you’ve not already done so, consider depositing your raw data in a repository to ensure your work is read, appreciated and cited by the largest possible audience. You’ll also earn an Accessible Data icon on your published paper if you deposit your data in any participating repository (https://plos.org/open-science/open-data/#accessible-data).

3. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files.

4. Thank you for stating the following financial disclosure:

“RAW received the NSF CAREER award (NSF CAREER1846857) from the National Science Foundation (NSF) which provided funds to conduct this research. RAW, FSL, UNN received funding by NSF CAREER1846857. EJM received funding from NSF Research Experiences for Undergraduates (REU) program EEC 2050587 as a part of this work.”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

5. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“We would like to thank the National Science Foundation (NSF) for providing funds to conduct this research (NSF CAREER1846857). We would also like to acknowledge the NSF Research Experiences for Undergraduates (REU) program EEC 2050587 for supporting part of this work.”

We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“RAW received the NSF CAREER award (NSF CAREER1846857) from the National Science Foundation (NSF) which provided funds to conduct this research. RAW, FSL, UNN received funding by NSF CAREER1846857. EJM received funding from NSF Research Experiences for Undergraduates (REU) program EEC 2050587 as a part of this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

6. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

7. Please include a separate caption for each figure in your manuscript.

8. We note that Figure 1 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

1. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

2. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

9. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

10. We notice that your supplementary figures are uploaded with the file type 'Figure'. Please amend the file type to 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list.

[Note: HTML markup is below. Please do not edit.]

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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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: Yes

**********

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: The work describes the effects of pyridoxine (Pyr), vincristine sulfate (Vcr) and ionomycin (Imy) on DRG explant axonal dieback and cytotoxicity. The experiments and results have been described very clearly.

I have only two comments:

-As the Authors stated, the used DRG explant model has the limitation that both the neuronal cell bodies and the axons have been exposed to the tested compounds. I wonder whether this model is physiologically

relevant to study axonal dieback. While this first screening was important, the Authors should include an experiment where the compounds are tested on the axonal compartment only, to make their findings more physiologically relevant.

-All the analysis have been done on bright-field images. It would be meaningful complementing with fluorescence analysis, using axonal markers, to have a better understanding of the axon morphology (axon blebbing, axon retraction).

Reviewer #2: The manuscript investigates potential application of pyridoxine (Pyr) and vincristine sulfate (Vcr) to cause local denervation and alleviate pain induced by aberrant nerve growth in tissue. The author utilized rat DRG explant hydrogel culture platform for axonal dieback compound screening under four drugs: capsaicin (Caps), Pyr, Vcr, and ionomycin (Imy). Axonal length was measured before and after drug treatment to calculate axon length ratio. To assess cytotoxicity of the drugs, DRG lactate dehydrogenase (LDH) activity was measured post drug treatment. Drug toxicity to human nucleus pulposus (NP) cells was also studied in vitro through cell viability kits. Results showed that Pyr and Vcr was able to induce reduction in axonal length at high concentration while Imy produced slower axonal dieback. Imy, Vcr and Pyr had only minimal neurotoxicity. Further, neither Pyr nor Vcr triggered NP cell death or affected cellular metabolic activity. Overall, the research is exciting and brought up new axon dieback candidates to treat pain without induced cytotoxicity to intervertebral disc cells, but there are some concerns need to be addressed:

1. 3.79 mM Caps was used as positive control for the study, but there lacks detailed description how the concentration was calculated. Although 8% Caps is used in commercial patch, it’s better to investigate the effects of different concentration on DRG explant to strengthen the advantages of Pyr and Vcr over Caps.

2. The image quality of DRG outgrowth (Figure 2) is low and it’s hard to visualize the axons.

3. Could the author specify how many axons were included for each DRG explant piece for axonal length (ratio) measurement? Meanwhile, it’s unclear if the axonal length referred to the maximum axonal length or to the average length of several selected axons. If only several axons were selected, could the author clarify the criteria of selection? How did the author make sure the exact same axon was captured before and after drug treatment to calculate the axonal length change (ratio)?

4. The author may consider doing immunostaining of the neurites for higher resolution images and more precise quantification. For example, TUJ staining will allow the visualization of neurite outgrowth and Sholl analysis in ImageJ for average axonal length measurement.

5. Instead of having two separate figures, the reviewer would recommend showing the dose-dependent drug effect on axonal length and cytotoxicity in one figure, i.e., combining the Figure 2 and supplementary Figure 2 for clearer interpretation.

6. Could the author explain why they normalized the LDH activity of DRG explant to lysed DRGs? In addition, the term “neurotoxicity” has to be used carefully since DRG consists of various types of cells.

7. The AlamarBlue reduction per cell plot in Figure 4 is a bit confusing. Could the author explain why the replicates in PBS control group had the same value? Some groups seem to have significant higher values compared to control (for example 1mM Pyr vs PBS in Donor1) while not highlighted in the plot. In addition to the 2-way ANOVA test, multiple comparison against control is something the author should consider performing here.

**********

6. 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: No

Reviewer #2: Yes: Mikhail Berezin

**********

[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.

Identification of compounds that cause axonal dieback without cytotoxicity in dorsal root ganglia explants and intervertebral disc cells with potential to treat pain via denervation

PONE-D-23-27605R1

Dear Dr. Wachs,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Israel Silman

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: (No Response)

Reviewer #2: All comments have been addressed

**********

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: Partly

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: (No Response)

**********

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: Thank you for the video and the high magnification pictures. They are much clearer.

Regarding my previous concern about the necessity of testing the compounds on the axon compartment, I do still believe it would be an important addition. With all the limitations of a 2D assay, it will add a valuable information and it will not be a duplication of the experiment but a valuable supplement. In fact, the Authors themselves write " There is a need to characterize the axonal degeneration effects when applying these drugs locally on nerve fiber endings to determine their potential efficacy to alleviate pain by local denervation without off-target effects".

Regarding the neurotoxicity vs cytotoxicity distinction: it would be important to perform the cytotoxicity assay on DRG neuron lysates. There are protocols to enrich the preparation for DRG neurons (for example: Tsantoulas et al., PLOSOne, 2013 https://doi.org/10.1371/journal.pone.0080722).

Reviewer #2: (No Response)

**********

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: No

Reviewer #2: Yes: Mikhail Berezin

**********