PONE-D-20-01760

MicroRNA-9 Mediated the Protective Effect of Ferulic Acid on Hypoxic-Ischemic Brain Injury in Neonatal Rats

PLOS ONE

Dear Ms Yao,

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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

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

Reviewer #1: 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

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Reviewer #1: 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 paper authored by Keli Yao, and colleagues investigates the neuroprotective effects of Ferulic acid (FA) on learning and memory improvement in rats subjected to neonatal hypoxic-ischemic brain damage (NHIBD). They hypothesized that neuroprotective effects of FA are likely the result of the regulation of miR-9 expression through increased release of BDNF. In particular, researchers demonstrated that the intraperitoneally administration of Sodium ferulic (50 mg/kg) into the rats starting from to postnatal day twenty-one, one time per day for 5 days, induced the reduction hippocampal damage and prevented impairment in spatial learning and memory ability, evaluated by using Morris water maze test. In addition, they showed that the expression levels of miR-9 was significantly increased in the hippocampus of hypoxic-ischemic rats compared to sham rats. Moreover, they demonstrated that there was a significant reduction of the expression levels of miR-9 after treating the rats with SF daily for 5 days and then compared them with the levels in the sham group. In the same way, in vitro model, they observed in PC12 cells cultured under serum-oxygen deprivation conditions for 2 hours, that miR-9 release was significantly reduced after treatment with SF. Furthermore, they showed that the inhibition of miR-9, by using of LNA-miR-9, protected the hippocampus and ameliorated the spatial learning ability of rats subjected to NHIBD. They also observed, in the rat hippocampus and PC12 cells, that the treatment with SF and LNA-miR-9 determined the reduction of expression levels of AchE mRNA and protein, and induced the increase of expression levels of PSD-95 mRNA and protein. In the last part of work, the authors demonstrated that expression levels of BDNF protein significantly decreased in the hippocampus of the rats in HIBD group compared with those in the sham group after hypoxic-ischemic damage, conversely, in HIBD+SF and HIBD+LNA-miR-9 groups, increased BDNF protein expression had been observed.

Although the manuscript technically sounds, experiments have been performed with rigor, through appropriate controls, replication and sample size and the data produced support the conclusions, some points should be improved to reinforce the significance of the paper.

1. In the “discussion” section, it is necessary to explain, why they choose the dose of Sodium ferulic (50 mg/kg) for the intraperitoneally administration into the rats, since the mentioned papers used different dosage protocols.

2. In the “discussion” section, it is necessary to explain why they choose to start FA 21 days postnatally. It would be appropriate to understand whether the administration of FA starting from the phases immediately subsequent the induction of the damage, may also determine a greater neuroprotective effect in animals.

3. Authors should show the effect of FA administration in the cortex, since cortex is one of the main damaged areas after an hypoxic-ischemic damage.

4. In ”methods” section, it is appropriate to indicate the total number of rats were used in the paper.

5. In ”methods” section, it is appropriate to indicate the total number of slices for each mouse used to perform the H-E staining, also reporting the bregma coordinate.

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Reviewer #1: No

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MicroRNA-9 Mediated the Protective Effect of Ferulic Acid on Hypoxic-Ischemic Brain Damage in Neonatal Rats

PONE-D-20-01760R1

Dear Dr. Yao,

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.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Giuseppe Pignataro, MD, PhD

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: (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

**********

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

Reviewer #1: 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

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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

**********

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 manuscript technically sounds, experiments have been performed with rigor, through appropriate controls, replication and sample size and the data produced support the conclusions.

The authors answered to comments and made the necessary changes to improve the manuscript.

I accepted the revision of the paper by Keli Yao and colleagues.

However, I would like to precise that, although the paper has been generally improved and is now suitable for publication, it is necessary to introduce these modifies before accepting it for publication:

1) In the revised paper, in the “introduction” section, in the sentence “In conclusion, we aimed to explore neuroprotective effect of FA on HIBD patients and …” (this sentence have been highlighted by authors by using red text to indicate the changes made). It is necessary that the authors correct the word “patients” with rats, because the study has been performed in rats and not in patients.

2) In the revised paper, in the “methods” section, in the sentence “The rats were divided into six groups randomly and treated in the following cohorts: In HIBD group, HIBD+SF group, HIBD+LNA group and HIBD+PBS group, the HIBD model was established by the classic method of Rice-Vannucci (this sentence have been highlighted by authors by using red text to indicate the changes made). It is necessary that the authors, indicate that it has been performed a few modification, because they reported in the next sentence that: ”The left carotid artery of 7-day-old SD rats was permanently unilateral

ligated and removed,…”(this sentence was already present in the first version of paper).

3) In the revised paper, in the “methods” section, It is necessary that the authors, indicate the gender of rats used in the procedure.

4) In the revised paper, in the “discussion” section, in the sentence “We established HIBD model used on P7 rats, because P7 rats correspond to human neonatal” (this sentence have been highlighted by authors by using red text to indicate the changes made). It is necessary that the authors improve the second part of this affirmation and add a reference.

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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: No