Dear Dr. Liu,

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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We look forward to receiving your revised manuscript.

Kind regards,

Nafisa M. Jadavji, PhD, MSc, BSc

Academic Editor

PLOS ONE

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Additional Editor Comments:

Dear Author,

The reviewers have listed several concerns about your submission. If you choose to resubmit, I urge to address each concern in your revised manuscript and respond.

Sincerely,

Nafisa

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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?

Reviewer #1: Partly

Reviewer #2: No

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

Reviewer #1: No

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

Reviewer #1: No

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: No

Reviewer #2: No

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Reviewer #1: This is a good study. Provided the author addresses all the gaps and clarifies certain information in this manuscript:

Introduction:

In general, it's acceptable.

Methodology

The language in this submission requires improvement, as some sentences are unclear or awkwardly phrased. Additionally, the sentence arrangement could be better organised for coherence and readability. In the statistical analysis section, the explanation of the validity for OPLS-DA was missing, as were the values of the selected VIP and the criteria used for comparisons. Information about the software used for statistical analysis was also not provided.While comparisons between groups (intergroup comparisons) were mentioned, it is unclear whether intragroup comparisons were performed. This should be clarified. No references were provided for some of the major methods.

The total number of rats was stated as 30, with 15 SHR and 15 WKY. However, in Section 1.3, it is mentioned that there are three groups: WKY (10 rats), model (10 rats), and SHR (10 rats). Does the model group comprise 5 SHR and 5 WKY?

Results and discussion

More discussion is needed and references. It is suggested to provide a diagram to highlight which metabolites are shared and different among those three groups.

Conclusion:

Didn't highlight the major findings in the study—too simple and general.

Others:

Kindly provide the limitations of the study. Formatting was not consistent throughout the manuscript (example: 1.5.2).

Please find the comments in the attached documents too.

Reviewer #2: The paper studies the molecule mechanism of vascular cognitive impairment using model rats by various methods, such as metabolomics, proteomics, Morris water maze and pathological slides. The paper is not well written in good language, which makes it difficult to understand. Moreover, the statistical and computational analysis were not clearly described, and the results are not well analyzed. Please see the following comments:

Section 1.8. P<0.05 threshold should be adjusted because of the multiple testing problem to reduce false discovery rate.

Figure 2. What does the error bar mean? If the error bars mean confidence interval, since many of the overlap with each other, the difference between different groups should not be significant. Hence, the significant levels (***, **) are questionable.

What softwares were used to annotate the metabolites? The authors should report the metabolite annotation level of the analysis.

What softwares were used to annotate protein?

Figure 5A and 6A. what is the total number of metabolites shown? The abundance table of them should be provided as supplementary material. It seems there are more than 437 and 449 significant metabolites.

Figure 5C and 6C. They look very confusing. Please describe more.

There are a lot of typo and grammar issues. e.g.

- Section 1.1 Is there is typo here? 15 -> 15 rats?

- page 7 last paragraph. "After the operation ...". Please rewrite the sentence with correct grammar

- Section 1.8. "Univariate statistical analysis ...". Please rewrite this sentence with correct grammar

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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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.

Attachments

Attachment

Submitted filename: PONE-D-24-59603_reviewer.pdf

Dear Dr. Liu,

We thank-you for making revisions to your manuscript and hope to read the revised manuscript soon. 

Please submit your revised manuscript by Aug 23 2025 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.

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,

Nafisa M. Jadavji, PhD, MSc, BSc

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comments:

Dear Authors,

Thank-you for taking the time to make revisions to your manuscript. Some minor revisions are still required. We look forward to reading your revised manuscript.

Sincerely,

Nafisa

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

Reviewer #5: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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

The PLOS Data policy

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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Reviewer #3:  The revised manuscript is well-structured, and the methodology and results are clearly presented. No major corrections appear necessary at this stage.

However, I would like to raise one conceptual point for clarification and potential revision. The authors have focused on pathological changes in the hippocampus, but vascular cognitive impairment (VCI) differs fundamentally from Alzheimer’s disease in its pathophysiology. VCI is typically associated with accumulated asymptomatic ischemic events, leading to white matter degeneration and diffuse global brain decline, rather than localized hippocampal dysfunction.

Therefore, while it is acceptable that hippocampal pathology was examined in this study, it would be helpful for the authors to clarify that:

Only the hippocampus was assessed histologically,

The behavioral evaluation mainly reflects cognitive performance and does not encompass depressive or affective domains,

The findings should be interpreted within these limitations.

Moreover, while the metabolomic and proteomic analyses are well conducted, many of the identified molecular changes—particularly those involving neurotransmitters and amino acid metabolism—are broadly linked to aging or psychiatric disorders. Some brief discussion on this point would strengthen the clinical relevance of the findings.

In summary, I recommend only minor textual revisions, particularly to the limitations and discussion sections, with the goal of making the implications more clinically accessible for practicing physicians. Overall, the manuscript is of high quality and should be considered favorably for publication after these small adjustments.

Reviewer #4:  PONE-D-24-59603R1

Combined metabolomics and proteomics analysis of vascular cognitive impairment in

hypertensive rats induced by endothelial injury

This is the first time I have seen this manuscript, but the paper was previously revised with provided. The authors have made many previous changes to text and methods sections.

The authors have looked at biological mechanisms of vascular cognitive impairment in SHR rat, following microcurrent damage, using metabolomics and proteomics tools as well as behaviour. The WKY rats and a “model” group serve as controls. The authors do notice changes in behaviour, which they relate suggest make the model clinically relevant, and they go on to describe various protein and metabolite pathways that appear different these group. The manuscript is a reasonable, descriptive study on a model vascular cognitive impairment.

Comments

While the authors studied both SHR, WHY and a model group and obtain a large amount of data (three groups and two modalities) there is little attempt to integrate these data together (e.g multivariate analysis / factor analysis) or even more simply to interpret the data together as a whole. This feels like a lost opportunity and the data is instead presented in long form and it is hard to pull out the most interesting findings from the noise. If this cannot be integrated, please make sure all data is deposited for others to analyse further. The discussion section appears overly speculative; a pathway description is not required for every metabolite seen in the study and most will not be necessary or mechanistic for this condition.

The remaining problems reading relate to ease of understanding the text and the presentation of the data. Suggestions are provided and require only minor revision.

Minor comments

You use the term “model” group, but this is ambiguous, I think actually mean control or sham or equivalent Please amend in all text.

Figure 1 – surgical images can be useful but the two right hand images are not necessary (microcurrent device and the low magnification rat and frame) – so please remove them. The two right hand images are useful but please add label with arrows and refer to each image in the figure legend. Note, in your methods you write that “neck skin depilated and disinfected with iodine tincture”, but this does not appear to have happen in the images? Please clarify.

Figure 3 – please add the group labels to the images directly for clarity and readability

Results 2.1 – Please explain the purpose of the experiment before you describe the data at the beginning of the paragraph, so the reader has context for what is to follow. Two sentences are sufficient.

Results 2.2 – Again, please explain the context of the experiment at the beginning of the paragraph before describing the data. This is otherwise quite hard to follow. Two sentences are sufficient.

Figure 4 – please add group labels to the images for clarity.

Suggestions – I think in Figures 3 and 4 it is awkward to compare down the columns. Would it not be easier to compare groups from left to right instead? You could rotate/ transform the images into this format. I leave this to author discretion.

Results 2.4.1 – again please explain the context of the experiment as in 2.1 and 2.2 above.

P22 - Some phrases are confusing in naming. E.g “In this study, SHR rats aged 13 to 14 weeks were selected, and the samples were collected 1 week after modelling” – is this the microcurrent damage? It is confusing that you have the other group called “model”. And on page 27 you refer to another group of “SHR model”. Please clarify in the text in each instance.

Discussion Figure 3 – this figure does not seem useful and is also not quantified. If the data is already in the results section, then it should not be duplicated. The name as Figure 3 also does not make sense as there are 7 figures above. Please either remove if it is essential then move it to the results.

Discussion text. This is overly speculative with long description of pathways with only passing relevance to the data in the manuscript e.g p26 GABA. Please cut down the discussion to include only those hypotheses most strongly supported by the data in this manuscript and the wider literature.

In a previous comment the authors say “All relevant data are within the manuscript and its Supporting Information files” however this does not appear to be in the submission. Please provide all raw data for this manuscript

Reviewer #5:  The authors responded to the the comments with care and diligence, making substantial revisions that improved the clarity, rigor, and overall quality of the manuscript. Methodological details were clarified, additional analyses were added, and key sections were rewritten for precision and coherence. The manuscript is now suitable for publication and makes a valuable contribution to the field.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #3: Yes:  keiichi abe

Reviewer #4: No

Reviewer #5: 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

Combined metabolomics and proteomics analysis of vascular cognitive impairment in hypertensive rats induced by endothelial injury

PONE-D-24-59603R2

Dear Dr. Liu,

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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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,

Nafisa M. Jadavji, PhD, MSc, BSc

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewer #3:

Reviewer #5:

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

Reviewer #5: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: Yes

Reviewer #5: Yes

**********

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

Reviewer #3: Yes

Reviewer #5: Yes

**********

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

The PLOS Data policy

Reviewer #3: Yes

Reviewer #5: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

Reviewer #5: Yes

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Reviewer #3: Since the authors have explicitly stated that this work is a preliminary study, it seems more appropriate not to extend the discussion too broadly to the relevance of the proteome across multiple other diseases.

Taking this into consideration, I find that the authors have adequately addressed the reviewers’ comments overall, and the manuscript has been sufficiently improved.

Reviewer #5: As I commented in previous versions of the article, the paper can be accepted for publication in PLOS ONE

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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 #3: Yes:  Keiichi Abe

Reviewer #5: No

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