PONE-D-20-27494

Differentiating axonal loss and demyelination in chronic MS lesions: a novel approach using single streamline diffusivity analysis.

PLOS ONE

Dear Dr. Klistorner,

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 expert reviewers found important concerns to be addressed, mainly regarding description of Methods and protocols, as well as in the quality and organization of figures.

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We look forward to receiving your revised manuscript.

Kind regards,

Fernando de Castro

Academic Editor

PLOS ONE

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'AK-National Multiple Sclerosis Society (NMSS), Novartis Save Neuron Grant, Sydney Eye Hospital foundation grant and Sydney University Medical foundation.

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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: 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

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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

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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: In this study authors developed a new method for evaluating axonal damage inside MS lesion on brain MRI in 30 MS cases by using a single streamline diffusivity analysis. They found a significant increase of both axial and radial diffusivity and AD showed a very high correlation with T1 hypointensity. This article is a new addition to the contribution of the team to the application of DWI to the understanding on MS lesions and quantification of axonal loss. The team is very well experienced and recognized and this new method is highly valuable for improving our understanding about how MS damage the brain and for testing new neuroprotective and remyelinating therapies.

Comments

1. Methods: indicates the number of voxels that 100 mm3 represent in this specific scanner. Explain how TractSeg algorithm solved the interruption of the tract within the lesions to provide the reconstruction of the tract. Authors explained that they use reference fibbers, but the algorithm should make use of a statistical or probability analysis to approach the fibbers within the lesion. Or it was always required a 100% match with the reference fibber? Indicate how many fibbers on average were present in the DWI voxels.

2. Can you provide the DTI protocol? I was unable to find it in the supplementary material

3. Figures are of low resolution (specially the insets with the graphs). Please, provide high quality figures

4. Figure 5 and 6: provide legends to the X and Y axis as indicated in the figure legend

Reviewer #2: The authors attempt to develop a single-streamlined approach to analyze diffusivity within chronic MS lesions. While the method seems potentially promising there are too many issues with the presentation and interpretation of the data to know if this method is sound.

Major issues:

• In the second last paragraph of the introduction, the authors state, “based on an assumption of uniform mean diffusivity of the brain white matter,” Why is that assumption valid?

• The authors state in the methods section about the MR parameters that “Specific parameters are presented in Supplementary material.” I do not see these parameters in the supplemental material. The administration of Gd can change metrics so I also expected to see the order in which the images were collected and that is not there either.

• Axis labels on the graphs in Figure 2 are necessary. What is the x-axis? What do the blue and the orange lines mean? The titles of the graph should be the y-axis labels. Is difference AD the delta-AD written in the manuscript? The authors should use consistent notation. In Figure 4 they seem to call this AD and RD asymmetry. Consistency is necessary. Labelling also applies to Figure 3-7.

• The bar graph in Figure 3 is also very confusing. Do the bars not represent Delta-RD and Delta-AD? If they do, then the legend needs to be changed. If they do not, a much better explanation of what is in the graph needs to be made. Using blue and orange is also VERY confusing. In the other graphs in Figure 3 and in the graphs in Figure 2, blue is meant to represent the lesion while orange is meant to represent the non-lesion tracks. Yet in the bar graph in Figure 3, blue and orange mean something else. The colors need to be changed for consistency. I am also unclear if I understand things correctly. Do only 2 voxels worth of data go into the lines in 2-7? Are data from a whole lesion core (line 1) really comparable with data from a single voxel ring around the lesion? It seems like the statistics would be poor for the analysis outside the lesion core. The lesion rim likely suffers inhomogeneously from partial voluming effects. Is that considered in the analysis?

• The author’s explanation of their new change in the way data are analyzed normalizing the differences as done in Figure 2 of the supplement is interesting but not convincing. What other factors can result in a change in RD and AD but not MD and have the authors checked to see if those effects are seen in their work and how they would affect their work. For example, some work has been done indicating axon diameter changes in different area of the brain. I would think that could result in a consistent MD but a change in AD and RD. From what the authors have written, they appear to have assumed that the changes they see are from crossing fibers and not from some other means. I need to be convinced they are not from other means or that these other means are irrelevant.

• Figure 4 switches the color scheme for Figure e. a-d had AD dark and RD light and Figure e has RD dark and AD light. Consistency is needed.

• Table 1: what are the uncertainties in the numbers in the table?

Minor issues:

• Very minor English mistakes can be found throughout the manuscript (for example “this represent”. A quick read though to fix these mistake should be done.

• Page 9 of the document, paragraph 4, the authors refer to “normal” cohorts. I believe the disease societies are asking scientists to stop using these terms and rather use terms such as “non-MS” cohorts.

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Reviewer #1: Yes: Pablo Villoslada

Reviewer #2: No

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PONE-D-20-27494R1

Differentiating axonal loss and demyelination in chronic MS lesions: a novel approach using single streamline diffusivity analysis.

PLOS ONE

Dear Dr. Klistorner,

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 very minor changes suggested by expert reviewer #2 are related to some overstatements in the text, as well as apparent confessions, between the title of figures and the axes in plots. Please, revise them and send them back to us for their final approval.

Please submit your revised manuscript by Jan 22 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Fernando de Castro

Academic Editor

PLOS ONE

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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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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: 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: Authors have addressed properly all my comments and doubts. The figures are now of good quality and the labels have been added to the axis

Reviewer #2: The authors have made substantive changes to the manuscript so that their work has become much clearer. I have two issues, which I believe can easily be corrected, with the manuscript as it stands.

1. The authors have a whole bunch of metrics which have been shown in their studies, or in others, to correlate with various pathologies. Yet, the authors do not do the correlations themselves with the people in this study. The manuscript itself does an excellent job not overstating what was done. The abstract, on the other hand, does overstate what was done which can be dangerous as the abstract is often the only part of a manuscript that is read by researchers who are looking for some trick to describe their own data. Thus, I suggest changing the overreaching statement “Our result demonstrates close association between an increase of AD and the degree of axonal loss and support the notion that the increase of RD in chronic MS lesions is driven by the combination of two factors: axonal destruction and loss of myelin sheaths in survived axons. While the effect of demyelination dominates in cases of mild axonal loss, neurodegeneration is a primary cause of increased RD in more destructive lesions. These finding highlight the importance of selecting appropriate patient cohorts for clinical trials of pro-remyelinating and neuroprotective therapeutics.” to something more accurate. The summary the authors wrote at the end of the manuscript has a nice statement that could be used instead, “Evaluation of lesions in a sizable cohort of MS patients using the proposed method supports the use of �AD as a marker of axonal loss; and the notion that demyelination and axonal loss independently contribute to the increase of RD in chronic MS lesions.”

2. Once again the plots, while much more clear, are not in the standard format for plots. The authors seem to be confusing the title of the plot with what should be on the y-axis. This is true for almost every plot in the figures presented. To make things clear, I will explain what I mean using just the top graph of Figure 2b. There should be no title. The y-axis should be labelled AD (µm2/ms). The x-axis is labelled sufficiently with streamline.

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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: Pablo Villoslada

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.

Differentiating axonal loss and demyelination in chronic MS lesions: a novel approach using single streamline diffusivity analysis.

PONE-D-20-27494R2

Dear Dr. Klistorner,

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,

Fernando de Castro

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 #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 #2: (No Response)

**********

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

Reviewer #2: (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 #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 #2: (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 #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 #2: No