PONE-D-24-20012Intraoperative phrenic nerve stimulation to prevent diaphragm fiber weakness during thoracic surgeryPLOS ONE

Dear Dr. Ferreira,

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.

Please submit your revised manuscript by Oct 06 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,

Steven E. Wolf, MD

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. Thank you for stating the following financial disclosure: 

"NIH R01 AR072328, HL130318, HL121500"

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.

3. Please expand the acronym “NIH R01” (as indicated in your financial disclosure) so that it states the name of your funders in full.

This information should be included in your cover letter; we will change the online submission form on your behalf.

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

"We thank the Division of Cardiothoracic Surgery research coordinators with their regulatory assistance. We are grateful for the participation of the patients. The study was funded by NIH R01 AR072328. LFF was supported by HL130318 and CAC was supported by HL121500."

We note that you have provided funding information that is not 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: 

"NIH R01 AR072328, HL130318, HL121500"

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

5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.   

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

[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: No

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: No

**********

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: This is a report of a small cohort study. There are some deficiencies in the description of the statistical considerations of the study:

1. Sample size discussion is inadequate. What is the primary outcome of the study? What test statistic is being used? It appears that random effects models are being used in the analysis, yet there is no discussion of assumptions on the model that are necessary for computing sample size. Referencing a prior study is insufficient, as this study needs to stand alone. The Discussion should also include a sentence on whether the assumptions used in the sample size computation (variability etc.) were actually realized in the clinical trial.

2. Random effects models have assumptions that require validation of those assumptions. What diagnostics did you use to ensure the models are apt?

3. p<0.05 to declare significance is fine for the primary outcome, but there are other tests being done and there does not appear to be any adjustment for multiple testing. Of course the other tests may be exploratory, in which case p<0.05 really has no meaning.

Reviewer #2: GENERAL COMMENTS

Bresciani et al assessed the effects of unilateral phrenic nerve stimulation during cardiothoracic surgery on diaphragm muscle fiber function. This is huge work, with a very complex population and very complicated collection of diaphragm biopsies. I provided point-by-point comments below. Some of them are already described by the authors as limitations. However, I consider them to be very important points that need to be considered while reviewing/considering the manuscript for publication. I hope these comments help you improve the quality of the manuscript.

There some differences between the clinical trials registration and the results reported here.

ABSTRACT

Please, clarify details of the stimulation (e.g., duration of each stimulation and etc)?

MAIN TEXT

It is not clear why the focus of this study was patients with normal lung function/inspiratory muscle strength. The question would be much more relevant in high-risk populations, such as patients with lung diseases.

The study used unilateral phrenic nerve stimulation and considered the other side as an internal control. Unilateral phrenic nerve stimulation can sometimes cause both hemidiaphragms to contract. This phenomenon occurs due to the presence of cross-innervation or central connections between the motor neurons controlling the diaphragm. When one phrenic nerve is stimulated, it can lead to a bilateral response, causing both hemidiaphragms to contract, although the effect is typically stronger on the side directly stimulated it imposes a limitation on what is considered the “control hemidiaphragm” for the study. This may underestimate the effect of the intervention, which could affect the conclusions that, for instance, only one type of fiber was found to be different.

Please, clarify whether or not neuromuscular block was used at any point in any patients for the surgical procedure.

If the protocol was extended to include patients with lung transplant, why reporting partial data only here? and from patients with preserved MIP? The results from patients post-lung transplant would be much more interesting from a clinical perspective.

Line 164. I am not sure I understand the parameters for the stimulation correctly. Please, clarify: each stimulation was continuously applied for 1 minute? If that is correct, I do not understand the rationale for these settings. Each inspiratory effort last approximately 0.8 to 1.2 seconds. I would expect a stimulation targeted to protect the diaphragm to have such a physiological range. If the stimulation lasted one minute, this does not resemble the way the diaphragm works normally. Or did the stimulation last 1.5 ms? If this is the case, this is the complete opposite and similarly does not resemble how the diaphragm works normally. Please, clarify the parameters in the text. I suggest this is addressed in the limitations section and please describe the rationale for this choice.

Sample size was based on prior work about mitochondrial function. The effect size or the magnitude of the difference between the two groups used for the sample size calculation is not described. Moreover, mitochondrial function is not a primary outcome in the study. Please, clarify the rationale for this approach and why not calculate the sample size based on differences expected for the primary outcomes of the study?

Calculated sample size was n=20 but 25 patients were included. Please, justify. Then, data is available from 19 patients, which is less than the calculated sample size.

Looking at the first clinical trial registration (https://clinicaltrials.gov/study/NCT03303040?tab=history&a=1#version-content-panel), estimated enrollment was 54 and not all outcomes described in the clinical trials registration are reported here. Please, clarify.

How was the intensity of the stimulation set since it was not the same for all? Based on the strength of the diaphragm contraction? The effect of the stimulation intensity and the effort generated by the diaphragm during each contraction caused by the stimulation could significantly affect the study results.

The authors discuss that “However, synchronization of phrenic stimulation and MV-delivered lung inflation is not necessary (or physiologically relevant) during open-chest thoracic surgery. The clinical environment did not permit us to make quantification of force generation, but we could clearly observe vigorous contractions of the stimulated side. The optimal parameters and timing of phrenic stimulation remains unresolved.” However, studies have shown that, during mechanical ventilation, the timing (i.e., synchronous or dyssynchronous) type of contraction (e.g., concentric vs eccentric) and the effort generated by the diaphragm (e.g., large vs small effort) all can affect whether it will develop injury or not. These factors can influence diaphragm function independently or in combination.

**********

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: No

**********

[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.

Intraoperative phrenic nerve stimulation to prevent diaphragm fiber weakness during thoracic surgery

PONE-D-24-20012R1

Dear Dr. Ferreira,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Steven E. Wolf, MD

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

**********

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: (No Response)

**********

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

Reviewer #1: (No Response)

**********

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: (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: (No Response)

**********

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)

**********

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

**********