PONE-D-19-30197

A Spectral-Domain Optical Coherence Tomographic Analysis of RDH5-/- Mice Retina

PLOS ONE

Dear Professor Nakazawa,

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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Knut Stieger, D.V.M. Ph.D.

Academic Editor

PLOS ONE

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https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210439

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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: No

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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 manuscript entitled “A Spectral-Domain Optical Coherence Tomographic Analysis of RDH5-/- Mice Retina” Misuru Nakazawa and colleagues have described a detailed morphological phenotype of a RDH5-/- deficient mouse model for fundus albipunctatus (FA), a human hereditary retinal dystrophy. Based on the first description of the mouse mutant for the 11-cis-retinol dehydrogenase gene (11-cis-RoDH-/- mouse) by Driessen et al., 2000, the authors have investigated the retinal phenotype by using non-invasive diagnostic tools, e.g., OCT, SLO and ERG. Results were confirmed by histological and statistical analyses.

This manuscript offers a broad spectrum of retinal techniques for the investigation of a mild phenotype. The study is related to great diligence of work and is addressed to several interesting aspects in using this model for an improved understanding of FA. The authors have discussed important findings:

1) First description of a detailed morphological data set which reveal a mild phenotype caused by the gene disruption in RDH5.

2) Due to the morphological mild phenotype (decrease in longitudinal in the thickness of the retinal layers), the ERG recordings of mutants are inconspicuously compared to wild-type controls.

3) The whitish spots which are revealed in fundus images can not be depicted in any OCT-scans caused by limitation of the OCT-technique.

Comments and suggested improvements:

Style:

The notation of protein and gene should name according to the guidelines set by the International Committee on Standardized Genetic Nomenclature for Mice (https://www.jax.org/jax-mice-and-services/customer-support/technical-support/genetics-and-nomenclature) both for C57BL6 (e.g. C57BL/6N) and for the mutant.

In the discussion part the sentence “This may be due to spices-related differences...” should correct to “This may be due to species-related differences...”

Results:

Fig 2: The wavelength and the orientation of the OCT-scan (horizontal or vertical orientation) should be completed in the legend and/or in the images. The scans show predominantly a wavelike retinal structure. Is this phenomenon based on properties of the device or may due to any phenotype characteristics?

Fig 3: Please correct the arrow color in C.

Fig 5: Please adapt the position of the scale bars as well the size of images on a consistent standard.

Fig 7: Please adapt and correct the magnification properties in panel A-E. It appears that panel C shows a higher magnification. Panel F is not included in the Fig but described in the legend. Please correct.

Fig 8: It might be helpful for a better understanding of the ERG recordings if the months of investigation and the species (mutant vs control) are included into the figures.

Results and discussion:

The authors have declared no statistical significant differences in the ERG recordings between mutants and controls at PM3 and 5 (Fig 8). But, it might be that the b-wave part in mutations is smaller than in the controls at both time points of investigations by a careful consideration. Therefore, I recommend an overlay of the ERG recordings to proof this suggestion. Furthermore, I recommend to present a separate illustration of the ERG recordings under scotopic (dark adapted), rod-only, as well under scotopic (light adapted), cone-dominated, conditions (please see the publication: Tanimoto et al. 2009, Vision tests in the mouse: Functional phenotyping with electroretinography. Front Biosci (Landmark Ed). Additionally, in 2017, Kuehlewein et al. has described a cone dysfunction in patients with FA.

Reviewer #2: The authors investigated retinal structure and function in RDH5-/- mice. In general, it is a very good work, except for one topic (see below).

There are some remarks the authors should take into the consideration.

M&M:

The authors called the layers of photoreceptor inner segments and outer segments “photoreceptor layer C”, although the photoreceptor cell bodies stretch to the outer rim of the outer plexiform layer, with their nuclei constituting the outer nuclear layer. Maybe the authors could call their layer C “photoreceptor segments layer” or so?

Results:

The authors write: “No qualitative differences were observed in the retina between RDH5-/- and C57BL6 mice. In addition, this tendency was consistent even in the midperipheral areas (Fig 3).” Do they mean Fig. 2 instead of Fig. 3?

The reviewer has the impression that OCT scans shown in Figs. 3B and 3D do not correspond to the horizontal lines visible in Fig. 3A and 3C. Moreover, it would be nice to have arrows indicating the sites of the hyperreflective spots also in Figs. 3B and 3D.

In Figure 5, the scale bars are strange, and also the general appearance of the pictures. Were the pictures of the histological sections collected from different sources?

Discussion:

In the sentence “This may be due to spices-related difference between mice and humans.” May be a typo.

The biggest problem the reviewer sees in the presented manuscript are the electroretinography (ERG) data that are provided in supplement S4. Firstly, the number of animals (n=4) is by far too small to make any meaningful statements. In general, it is estimated that there may occur a deviation of 10-15% between single ERG measurements. In order to achieve sufficient statistical power to check for differences between experimental groups, approximately 10 animals per group are necessary.

As the next, it is very questionable to perform a normality test with a group of only n=4. It simply does not make any sense! Anyhow, Shapiro-Wilk normality test is not recommended in many cases, as the D'Agostino & Pearson omnibus normality test is better.

The deviations inside each group are very big, which raises the question if the ERG measurements were performed properly, and if these ERG data are useful at all. The reviewer has a long history in ERG and VEP measurements in rats and mice, and he finds the big deviations inside the groups very surprising, in particular with respect to the first mouse in the two PM5 groups. Moreover, the big decline of amplitudes in wild-type mice from PM3 to PM5 is surprising and was never observed by the reviewer in his own work. In contrast, almost no amplitude decline was seen in the RDH5-/- mice, which is suprising.

Consequently, the authors are advised to thouroughly check the ERG data and perfom the measurements in a higher number of animals, or to not touch the topic of ERG in their manuscript at all.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-19-30197R1

A Spectral-Domain Optical Coherence Tomographic Analysis of Rdh5-/- Mice Retina

PLOS ONE

Dear Professor Nakazawa,

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 address the final points raised by reviewer 1. Furthermore, please elaborate more on the point that without the functional readout (ie. ERG), the dataset contains sufficient amount of new data to be published in PONE. 

We would appreciate receiving your revised manuscript by Mar 30 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Knut Stieger, D.V.M. Ph.D.

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: 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: 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: 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

**********

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: Misuru Nakazawa and colleagues have revised the manuscript entitled “A Spectral-Domain Optical Coherence Tomographic Analysis of RDH5-/- Mice Retina” by following the comments of both reviewers. The major revision which was made by the authors was the deletion of the obtained ERG data at all. The rationale for that decision was the awareness that the ERG data are insufficiently in terms of animal numbers and protocols used for the experiments to allow any interpretation of the RDH5 murine mutant functional phenotype.

Comments:

For two requested changes for the following aspects, the edit could not found in the revised version:

1) In the discussion part the sentence “This may be due to spices-related differences...” should correct to “This may be due to species-related differences...”

2) Fig 3: Please correct the arrow colour in C.

Reviewer #2: The comments of the reviewer have been addressed. It is good that the topic of electroretinography was omitted as long as there are no valid data available.

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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.

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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: 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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-19-30197R2

A Spectral-Domain Optical Coherence Tomographic Analysis of Rdh5-/- Mice Retina

PLOS ONE

Dear Professor Nakazawa,

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.

Dear Authors, 

you did not address my point on elaborating on the issue of why do you think that a pure morphological study describing the retinal changes without discussing the potential effect on the retinal function is sufficient for publication in PONE. I would like to encourage you to do so.

We would appreciate receiving your revised manuscript by Apr 11 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Knut Stieger, D.V.M. Ph.D.

Academic Editor

PLOS ONE

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

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

A Spectral-Domain Optical Coherence Tomographic Analysis of Rdh5-/- Mice Retina

PONE-D-19-30197R3

Dear Dr. Nakazawa,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Knut Stieger, D.V.M. Ph.D.

Academic Editor

PLOS ONE

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