PONE-D-23-36233Multivariate description and scoring of neuromotor changes in a mouse model of peripheral nerve injuryPLOS ONE

Dear Dr. Zhang,

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.

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Aliaa Rehan Youssef, PhD

Academic Editor

PLOS ONE

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"Research reported in this publication was supported by the National Institutes of Health under Award Numbers F30DK123985, and T32GM008152 (BAN); DOD Department of the Army: W81XWH2110862 (Zhang), McCormick Foundation/Northwestern Memorial Hospital (Zhang and Werthiem), Julius N. Frankel Foundation via Northwestern Memorial Foundation (Zhang, Han, and Wang); American Heart Association and CIHR (Koss); and R01LM013337 (Luo). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding sources had no roles in study design, collection, analysis, and interpretation of the data, in the writing of the report, nor in the decision to submit the article 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: No

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: I Don't Know

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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

Reviewer #3: No

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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: No

Reviewer #2: Yes

Reviewer #3: 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: The authors present a novel project that endeavors to use machine leaning and multivariate to develop a better assessment of neuromotor recovery after limb transplant or nerve resection in a mouse model. There is good information presented, but enthusiasm for the manuscript is reduced due to a number of potentially correctable factors.

1. It isn't clear if this is a project focused on improving assessment of nerve injury patients and VCA recipients, or a project seeking to implement novel machine learning techniques. The manuscript as presented doesn't completely do either.

2. There are multiple grammar/usage errors. For example, in the abstract on line 74, "Not all interventions can be experimented in humans". I assume the authors mean not all interventions can be studied in humans. The following sentences which state "methods for evaluating rodent gait lack the multivariate description applied to humans limiting their effectiveness. The authors then go on to say that multivariate evaluation of murine gait models is a novel clinical problem. This statement is not clear, and examples like this are common in the submitted manuscript.

3. There is no clear hypothesis that will be tested. This should be added to the Objective of the abstract. Was the hypothesis that multi-variate analysis is better than univariate analysis? Why was a hind limb transplant model used? Did the authors hypothesize that multivariate analysis would detect deficits better than univariate analysis in transplants than in nerve resection models? Was that the case? The only proof I saw was that more factors were identified by a more complex analysis. Does that empirically mean it is better?

4. The authors need a better description in the methods section of what machine learning, and training of classifier models is and what it means in relation to normal animals, nerve transection and hind limb transplant models.

5. Based on clinical experience to date, it is unlikely that clinical lower limb transplantation will become common. Results with lower limb prosthetics are just too good. The authors might want to relate how the proposed work might benefit upper limb transplantation.

6. Pay attention to grammar, another example, line 138 - "peripheral nervous injury" - should be peripheral nerve injury

7. Be careful about rationale for using male mice in methods section. The statement that male mice were used, because in the author's experience "they appear to be more tolerant to hindlimb transplant surgery" is a difficult statement to defend.

8. Figure 2 - comparing normal function to nerve resection or a transplant and looking for differences is not difficult. The authors state that only 44% had a p value below the 0.001 threshold. Why is this not higher? State clearly what the authors expected and why it is important to show this data.

9. The same lack of purpose is exhibited in table 1. Is the point that there are more features with multivariate analysis? The statement "Within each type of of analysis (univariate vs. multivariate), the two pathological states have high degree of overlap with key differences among both pathological states" is very confusing. What are the authors trying to say?

10. Please go through the rest of the manuscript and clearly state the reason for the analysis, and how it relates to the hypothesis that will be presented in the abstract.

11. In figure 5, Why do healthy animals have scores close to no function, (1 = fully functional, 0 = no function)? Figure 5 also highlights the fact hat nerve function after only two weeks of healing is very limited. Better nerve function would be seen at one month. Did the authors want animals with incomplete nerve recovery, and if so, what was the rationale?

Reviewer #2: COMMENTS TO THE AUTHORS

1. The introduction of the article is quite long and needs to be shortened.

2. The hypothesis of the article should be stated more clearly in the introduction.

3. There seems to be an inconsistency between the title of the study and the hypothesis of the study. While the study title states "Multivariate description and scoring of neuromotor changes in a mouse model of peripheral nerve injury", the study seems to test the validity and reliability of a video analysis program called DigiGait. Therefore, it seems necessary to change either the title or the hypothesis of the article or the content of the study.

4. It is not understood why isotransplantation was preferred over autotransplantation in the study. However, autotransplantation is the first choice in limb amputations in the clinic.

5. In addition, histopathologic examination methods of neural recovery were not included in the study. Therefore, the study does not include detailed recovery data including the histopathological and biochemical examinations of the subjects who underwent transsection and limb transplantation.

6. Many other neuromotor analysis methods (such as swimming, sciatic function test, and climbing) were also not included in the study. In addition, target organ (such as gastrocnemius muscle) involvement levels were not included in the study. Therefore, detailed analysis of neuromotor recovery was not performed.

7. In addition, there are already studies similar to this study in the literature as follows:

a. Ganguly A, McEwen C, Troy EL, Colburn RW, Caggiano AO, Schallert TJ, Parry TJ. Recovery of sensorimotor function following sciatic nerve injury across multiple rat strains. J Neurosci Methods. 2017 Jan 1;275:25-32. doi: 10.1016/j.jneumeth.2016.10.018. Epub 2016 Oct 29. PMID: 27984099.

Reviewer #3: Thank you so much for providing me to review this manuscript. It seems that its aim is to use multivariate statistics to characterize and distinguish among etiologies of gait deficit in animals.

Below are some of the comments and suggestions for the manuscript:

• The first sentence in the abstract (lines 72-73) is not clear. “Quantitative scoring enabling comparison of neuromotor recovery in peripheral nerve injury models is critical to understanding and gauging the extent of injury and repair.” Just add commas to the sentence to be more readable and to separate the introductory phrase from the main clause. “Quantitative scoring, enabling comparison of neuromotor recovery in peripheral nerve injury models, is critical to understanding and gauging the extent of injury and repair.”

• Lines 97-98 are not enough for the conclusion. You may need to add briefly the contributions and advantages for your proposed solution.

• The introduction was written in a clear way, and the authors presented several works in the literature. It is better to add at least a recent reference from the last year.

• You should add references to the two approaches used in Lines 227-229.

• In line 254, you mentioned that the same animal was not included in both the training and test sets within a respective fold. How did you ensure that using randomly 10-fold cross validation?

• Authors should describe the dataset in more details. How many samples in the dataset? What is the number of samples of each class (healthy, surgery)? What is the number of mice that participated in the experiment? These are not mentioned in the methodology section.

• Authors should describe in more detail the data that is processed in the classifiers. How many input characteristics are there?

• What are the hyperparameters that were used in each classifier (e.g. C and gamma in SVM, number of trees in Random Forests…etc.)?

• Did you perform hyperparameter tuning to select the best parameters for each model? This may improve the performance.

• It is better to add the confusion matrix of the results and appropriate evaluation metrics.

• Maybe it is worth testing other ensemble-based classifiers (e.g. XgBoost or stacking classifiers) to improve the performance.

I hope that these comments are useful for you going forwards.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-23-36233R1Multivariate description of gait changes in a mouse model of peripheral nerve injury and traumaPLOS ONE

Dear Dr. Zhang,

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 minor points raised during the review process.

Please submit your revised manuscript by Oct 26 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,

Annesha Sil, Ph.D.

Associate 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 #4: 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 #4: Yes

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

Reviewer #4: Yes

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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 #4: 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 #4: Yes

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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 #4: Well written study addressing a notable gap in current PNI research and incorporation of machine learning to further minimize observational bias. The authors propose that a multivariate analysis of gait could uncover significant connections between spatiotemporal aspects of gait that are biologically pertinent to PNI. They further propose that these relationships will enable a more precise identification of distinct gait patterns compared to using only univariate analysis.

The authors have addressed all previous comments and concerns raised by the reviewers. Yet, concern still remains in generalizing the authors hypothesis to current gait research protocols within the scientific community. This issue may benefit from further discussion.

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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 #4: No

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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.

Multivariate description of gait changes in a mouse model of peripheral nerve injury and trauma

PONE-D-23-36233R2

Dear Dr. Zhang,

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.

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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,

Antal Nógrádi, M.D., Ph.D., D.Sc.

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 #4: 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 #4: Yes

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

Reviewer #4: 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 #4: 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 #4: 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 #4: All of the reviewers concerns have been addressed by the authors. I have no further unaddressed comments

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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 #4: No

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