PONE-D-22-04666

Cognitive and behavioral effects of whole brain conventional or high dose rate (FLASH) proton irradiation in a neonatal Sprague Dawley rat model

PLOS ONE

Dear Dr. Williams,

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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Please address the following issues raised by the reviewers

- Reviewer 1: Provide additional explanation for the limited protective effect of FLASH , which incorporates findings from other groups. The results of the ELISA and Western blot analysis should also be mentioned in the discussion.

- Reviewer 2: Please provide a rationale for the order of the behavioural tasks particularly in regards to the ASR and TSR being close to the start of the testing battery

- Reviewer 3:

1) While the days on which the behavioral tests are described in each section, it would be helpful to have a timeline figure that shows the organization of these days over time. This gives the reader a better idea of the differences in time needed for each behavioral test.

2) The authors should comment on the potential for numerous behavioral tests to interact with or cause changes in future behavioral performance. For example, they noted that the behavioral tests were run from assumed least stressful to most stressful, but it is possible that learning one test could alter learning of subsequent behavioral tests. Given that only one test was repeated and the results were different from the first assessment (5 Gy FLASH), would differences in performances of the other groups potentially be reversed if the rats were re-tested?

3) There were limited effects on the neuronal markers chosen (DRD1, DRD2, DAT, TH, NMDA-NR1). The authors should comment on the lack of measurements typical of other FLASH studies, such as neuroinflammation and neurogenesis and what role these processes might play in the differences between these groups.

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We look forward to receiving your revised manuscript.

Kind regards,

Joohyung Lee, PhD

Academic Editor

PLOS ONE

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This research was supported by a Research Agreement from Varian, a Siemens Healthineers company. Additional funding support included research support from the TQL Foundation, Cincinnati, OH and Fund the Cure Next Door 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: 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: Yes

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: This is a well designed, careful neurotoxicity investigation of whole brain irradiation using 11 days old rats comparing the neurognitive and behavioral effects and concentration of neurochemical markers after conventional and FLASH proton single dose delivery at two dose levels (5 and 8 Gy).

The authors revealed important findings on the vulnerability of the young rat brain, identified underlying pathomechanism of cognitive and behavioural changes, but found only modes protective effect of the FLASH irradiation, and only at the 5 Gy dose level. They pointed out in the manuscrip correcty the limitations of the experiment, but these limitations are not explaine completely the lack of more pronounced protective effect of the FLASH. The reasons for that and the more evident results of other groups should be discussed in more details. As well as the results of the ELISA and Western blot analysis should be mentioned in the discussion.

If some senteces will be added on these points to the discussion I fully support the publication of this valuable manuscript.

Reviewer #2: Fantastic study and in-depth analysis of many crucial cognitive domains as a part of your behavioural suite, well done. The statistical analysis was also great to show the level of detail, and made all relevant comparisons for not only your hypotheses, but the validity of the measures. One thing that came to mind was the order of the behavioural tasks - I was intrigued to see ASR and TSR so close to the start of the testing battery. My belief was that exposure to the acoustic startle stimuli in the ASR (not sure about TSR) changes the underlying neurobiology of the dopamine system, so that it is different post-exposure to if the animals are naiive to this task. In this way, ASR is typically always measured as the last task? Overall, great quality paper for submission number 1.

Reviewer #3: The current manuscript details an experiment comparing conventionally delivered doses of protons (5 and 8Gy) and the same doses delivered using FLASH dose rates. Overall, the manuscript is well-written and provides a number of behavioral assays covering multiple neurobehavioral domains. I have a few minor comments.

1) While the days on which the behavioral tests are described in each section, it would be helpful to have a timeline figure that shows the organization of these days over time. This gives the reader a better idea of the differences in time needed for each behavioral test.

2) The authors should comment on the potential for numerous behavioral tests to interact with or cause changes in future behavioral performance. For example, they noted that the behavioral tests were run from assumed least stressful to most stressful, but it is possible that learning one test could alter learning of subsequent behavioral tests. Given that only one test was repeated and the results were different from the first assessment (5 Gy FLASH), would differences in performances of the other groups potentially be reversed if the rats were re-tested?

3) There were limited effects on the neuronal markers chosen (DRD1, DRD2, DAT, TH, NMDA-NR1). The authors should comment on the lack of measurements typical of other FLASH studies, such as neuroinflammation and neurogenesis and what role these processes might play in the differences between these groups.

********** 

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Cognitive and behavioral effects of whole brain conventional or high dose rate (FLASH) proton irradiation in a neonatal Sprague Dawley rat model

PONE-D-22-04666R1

Dear Dr. Williams,

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,

Joohyung Lee, 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 #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 #2: Yes

Reviewer #3: Yes

**********

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

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 #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 #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 #2: All comments from all reviewers have been addressed well, strengthening the overall quality of the paper. Well done.

Reviewer #3: (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

Reviewer #3: No

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