PONE-D-21-05849

A simple and robust method for automating analysis of naïve and regenerating peripheral nerves

PLOS ONE

Dear Dr. Wong,

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.

The manuscript is appreciated by all reviewers, but minor revisions are requested.

Did the authors note any differences between embedding protocols? Resin vs. paraffin. This would be interesting to know as this has implications on time commitment to sample preparation.

Have the authors attempted to manually correct the ADS data to determine if this would further improve the accuracy? As ADS much faster than manual segmentation, it may be beneficial to include this step if this is not a time consuming process.

The discussion would be improved if this methodology is compared with other published reports. This is not to say the authors should implement other methodologies but compare other authors result with their own.

Can the authors comment on the efficacy of counting non-myelinated axons. This is the bigger challenge and is important when considering sensory nerve recovery.

The quality of the figures is quite low; please improve the resolution of the figures showing the data (Figures 1/2 appear fine).

Please, consider moving your motivation for using ADS to the introduction, rather than the discussion, so the reader understands up front why you are studying this software in particular.

Please, consider introduction of the g-ratio definition early.

Line 77: Period missing.

Line 175: Can you explain the ‘minferet’ command for users not familiar with imagej?

Line 196: Does the GIMP software automatically label these images? Any reason why the software was not compared using this software?

Line 362 a citation has not been included.

Line 363: Ref not inserted

Please, provide in the INTRODUCTION examples of recent works on peripheral nerve regeneration (see, for example doi: 10.1088/1758-5090/aaaf50).

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Gennady S. Cymbalyuk, Ph.D.

Academic Editor

PLOS ONE

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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: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: 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: 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 author's are to be congratulated on the quality of the manuscript presented and for addressing a key component that has limited peripheral nerve research. The application of ADS is novel as it is free and provides fast results and is of great interest to the research community. However, there are a few issues that need to be addressed prior to acceptance.

1) Did the authors note any differences between embedding protocols? Resin vs. paraffin. This would be interesting to know as this has implications on time commitment to sample preparation.

2) Have the authors attempted to manually correct the ADS data to determine if this would further improve the accuracy? As ADS much faster than manual segmentation, it may be beneficial to include this step if this is not a time consuming process

3) The discussion would be improved if this methodology is compared with other published reports. This is not to say the authors should implement other methodologies but compare other authors result with their own.

4) Can the authors comment on the efficacy of counting non-myelinated axons. This is the bigger challenge and is important when considering sensory nerve recovery.

5) In line 362 a citation has not been included.

Reviewer #2: Manual axon histomorphometry (AH) is time- and resource-intensive, which has inspired

many attempts at automation. Axon histomorphometry using ADS and manual analysis (gold-standard) was performed on light micrographs of naïve or regenerating rat median nerve cross-sections prepared with

either toluidine-resin or osmium-paraffin embedding protocols. The parameters of interest included axon count, axon diameter, myelin thickness, and g-ratio. In the current study, the authors aimed to validate the use of the newly developed deep learning program AxonDeepSeg (ADS) for automated axon histomorphometry (AH) and found that even without algorithm retraining, ADS was able to appropriately identify critical differences between naïve and regenerating nerves and work in different sample preparation methods. This was a carefully done counting procedure. However, in the INTRODUCTION the authors shall give several examples of recent works on peripheral nerve regeneration (see, for example doi: 10.1088/1758-5090/aaaf50)

Reviewer #3: Reviewer Comments

This manuscript presents results using an automated deep learning software, AxonDeepSeg, to automatically perform axon histomorphometry. The automated results are compared to the gold standard hand count method, and the results between the two are compared. The main outcomes are measuring axon size/diameter, myelin thickness, and the g-ratio. The results demonstrate that the automated method can detect differences between naïve and regenerating axons, in terms of axon density and g-ratio, although some differences between this method and the hand counts.

This article is straightforward and the results are well documented. Automated methods for axon histomorphometry are an important tool that need to be widely developed/deployed, and this article helps demonstrate the utility of one of the proposed solutions that is already available. The article could be made more impactful if several different automated methods were compared head-to-head in order to benchmark the currently available tools, but this manuscript still has validity by presenting a single software against manual counts. I have a few comments that should be addressed before publication, however, which are mostly minor.

Minor Comments

General: The quality of the figures is quite low; please improve the resolution of the figures showing the data (Figures 1/2 appear fine).

General: I would recommend moving your motivation for using ADS to the introduction, rather than the discussion, so the reader understands up front why you are studying this software in particular.

General: You may want to introduce the g-ratio definition early, I do not think it is explained before discussing in the results.

Line 77: Period missing.

Line 175: Can you explain the ‘minferet’ command for users not familiar with imagej?

Line 196: Does the GIMP software automatically label these images? Any reason why the software was not compared using this software?

Line 363: Ref not inserted

**********

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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: Jonatha I. Leckenby

Reviewer #2: No

Reviewer #3: No

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A simple and robust method for automating analysis of naïve and regenerating peripheral nerves

PONE-D-21-05849R1

Dear Dr. Wong,

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,

Gennady S. Cymbalyuk, Ph.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

Reviewer #3: 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

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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: The Authors present an improved revision of their manuscript. All my questions or comments have been addressed in a satisfactory manner and the manuscript is acceptable for publication.

Reviewer #2: The authors have satisfactorily addressed my concerns, the manuscript has been improved and could be published in its present form

Reviewer #3: The figures (3-7) still appear to be low quality. This may be an issue with the downloaded PDF, but I will let the authors and the editorial staff address this issue moving forward. All other issues have been addressed.

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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: Jonathan I. Leckenby, MBBS, PhD

Reviewer #2: No

Reviewer #3: Yes: Thomas Eggers