Temporal dynamics of pro-inflammatory cytokines and serum corticosterone following acute sleep fragmentation in male mice

Van Thuan Nguyen; Cameron J. Fields; Noah T. Ashley

doi:10.1371/journal.pone.0288889

Peer Review History

Original SubmissionJuly 5, 2023
12 Oct 2023 Decision Letter - Dragan Hrncic, Editor PONE-D-23-20980Temporal Dynamics of Pro-inflammatory Responses and Glucocorticoid Release from Acute Sleep Fragmentation in Male MicePLOS ONE Dear Dr. Ashley, 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. ============================== ACADEMIC EDITOR: - please do follow directions from our reviewer to improve your study- could some of your results be explained by alteration in hormonal status affected by sleep fragmentation. Extend discussion to neuroinflammation and explain potential limitations and clinical benefits of your results more deeply. ============================== Please submit your revised manuscript by Nov 26 2023 11:59PM. 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If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 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 ****** 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 ******** 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 straightforward study that examines serum corticosterone and serum IL-6, as well as Il-1b mRNA and TNF-a mRNA) after experimental acute sleep fragmentation in male mice. The methods appear generally sound. However, I have some suggestions for improvement. Major comments - Title. I would suggest “Temporal dynamics of corticosterone and pro-inflammatory cytokines following acute sleep fragmentation in male mice” (or something similar). - General comment. Fewer abbreviations would make the text easier to read (e.g. can remove “SF” and “OSA”). Why not use “control” or “CON” instead of “NSF”? - Methods. Why were only male mice used? This should be explained. - Methods. What was the body mass of the animals? - Methods. Please provide more information on the water, diet, and bedding. This is an important part of the ARRIVE guidelines. - Methods. For peripheral tissues, was the whole organ collected and used for analysis? How much tissue was collected for each brain region? How much tissue was used to measure gene expression? - Methods. Why not measure serum IL-1b and TNF-a too? Why not measure IL-6 mRNA too? I did not understand the rationale for what was measured. This should be clearer. - Results. Figures for relative mRNA expression: Differing lowercase and uppercase letters are difficult to differentiate between 0, 1 and 2 hours. - Discussion. I am not sure acute sleep fragmentation reveals much about the effects of chronic apnea. This is worth a short paragraph in the Discussion. Minor comments - Figure 1. Consider indicating when the lights turn on (ZT0) and off (ZT12). Each rectangle during the duration of SF indicates 1 hour, but the two rectangles for the acclimation period represent two days (48 hours). Could clarify this in the legend. - Figure 2B. The color and shape for NSF group (light blue circle) is different from the rest of the figures (dark blue triangle). - Figure 4 legend. Typo: “PCF: prefrontal cortex” - Results (line 270). Typo: “IL-1” - Discussion (lines 347-348). This sentence is a bit confusing. Consider rewording the sentence. ******** 6. 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 ******** [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. https://doi.org/10.1371/journal.pone.0288889.r001
Revision 1
3 Nov 2023 Author Response We note that Figure 1 in your submission contain copyrighted images. Thank you. We have made a new original figure to replace Figure 1. ACADEMIC EDITOR: - please do follow directions from our reviewer to improve your study We believe that we have satisfactorily addressed the reviewer’s comments below. - could some of your results be explained by alteration in hormonal status affected by sleep fragmentation. This raises an interesting point. It is possible that sleep fragmentation could alter other hormones (besides glucocorticoids) that could affect inflammatory responses, e.g., androgens. We discuss this possibility in the discussion. Extend discussion to neuroinflammation and explain potential limitations and clinical benefits of your results more deeply. We have provided more info on neuroinflammation and also discuss potential limitations and clinical benefits in the discussion. Reviewer #1: This is a straightforward study that examines serum corticosterone and serum IL-6, as well as Il-1b mRNA and TNF-a mRNA) after experimental acute sleep fragmentation in male mice. The methods appear generally sound. However, I have some suggestions for improvement. Major comments - Title. I would suggest “Temporal dynamics of corticosterone and pro-inflammatory cytokines following acute sleep fragmentation in male mice” (or something similar). Thank you. We have made this change and have added “serum” in front of corticosterone. - General comment. Fewer abbreviations would make the text easier to read (e.g. can remove “SF” and “OSA”). Why not use “control” or “CON” instead of “NSF”? Thank you. We have cut down on the abbreviations in the text as recommended. We have spelled out OSA. We also have replaced “NSF” with “CON” throughout texts and figures. However, we believe that the abbreviation for SF should remain because we discuss it so much in this study. In the discussion, we attempt to remove SF and replace it with “sleep fragmentation” to make it more palatable to readers. - Methods. Why were only male mice used? This should be explained. We only used male mice to control for variation in immune/inflammatory responses that are well known to occur between the sexes. Adult female mice undergo a 4-day estrus cycle that may complicate their inflammatory responses to sleep fragmentation. Nonetheless, we are currently evaluating the temporal responses of female mice to sleep fragmentation and hope to compare them to male mice at a later date. - Methods. What was the body mass of the animals? We now report the average body mass of male mice used in the paper. - Methods. Please provide more information on the water, diet, and bedding. This is an important part of the ARRIVE guidelines. Thank you. We now report more information on the type of water used, the diet of the mice, and the type of bedding used. - Methods. For peripheral tissues, was the whole organ collected and used for analysis? How much tissue was collected for each brain region? How much tissue was used to measure gene expression? For peripheral tissues, a sub-sample of the whole organ was collected because the RNA extraction only permits less than 30 mg of tissue to be homogenized and measured for gene expression. Between 5-30 mg of brain tissue was collected for each brain region. - Methods. Why not measure serum IL-1b and TNF-a too? Why not measure IL-6 mRNA too? I did not understand the rationale for what was measured. This should be clearer. Thanks for your comments. We were limited in the amount of serum from each mouse, so were not able to assess more than one serum cytokine. The sandwich ELISA that we used is most sensitive to IL-6, so we made the decision to assess this pro-inflammatory cytokine in the serum. We were worried that IL-1b or TNF-a in the serum could be non-detectable (from prior experience). We would have liked to have measured IL-6 mRNA as well, but most of the previous studies in our lab have focused mostly on IL-1-beta and TNF-alpha gene expression, and the Taqman reagents/primers/probes are so expensive, we could not afford to measure another gene for this study. - Results. Figures for relative mRNA expression: Differing lowercase and uppercase letters are difficult to differentiate between 0, 1 and 2 hours. Thank you. We see what you are referring to. We have attempted to move those around slightly to make them more discernible. - Discussion. I am not sure acute sleep fragmentation reveals much about the effects of chronic apnea. This is worth a short paragraph in the Discussion. Thanks for pointing this out. We now provide a discussion on how the results are relevant to the etiology of chronic inflammatory diseases, like obstructive sleep apnea. Minor comments - Figure 1. Consider indicating when the lights turn on (ZT0) and off (ZT12). Each rectangle during the duration of SF indicates 1 hour, but the two rectangles for the acclimation period represent two days (48 hours). Could clarify this in the legend. Thank you. We have now clarified this in the figure and the legend. - Figure 2B. The color and shape for NSF group (light blue circle) is different from the rest of the figures (dark blue triangle). Thanks. We have now fixed this. - Figure 4 legend. Typo: “PCF: prefrontal cortex” Thanks. This has been fixed - Results (line 270). Typo: “IL-1” Also fixed. - Discussion (lines 347-348). This sentence is a bit confusing. Consider rewording the sentence. We have now reworded the sentence. “IL-1β expression was significantly elevated in hypothalamus after 6 and 12 h of sleep fragmentation and in hippocampus after 24 h of sleep fragmentation; Fig 4, Table 1). A similar pattern for TNF-α gene expression was also observed occurring slightly later (Fig 4, Table 1). Acute sleep fragmentation did not affect TNF-α or IL-1β expression in prefrontal cortex (Fig 4, Table 1).” Attachments Attachment Submitted filename: Response to reviewers comments.docx https://doi.org/10.1371/journal.pone.0288889.r002
29 Nov 2023 Decision Letter - Dragan Hrncic, Editor Temporal dynamics of pro-inflammatory cytokines and serum corticosterone following acute sleep fragmentation in male mice PONE-D-23-20980R1 Dear Dr. Ashley, 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, Prof. Dr. Dragan Hrncic, MD, MSc, MBE, 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: All comments have been addressed ******** 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 ****** 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: My comments have been addressed. The authors have responded to each of my questions and comments carefully. ****** 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 ******** https://doi.org/10.1371/journal.pone.0288889.r003
Formally Accepted
5 Dec 2023 Acceptance Letter - Dragan Hrncic, Editor PONE-D-23-20980R1 Temporal dynamics of pro-inflammatory cytokines and serum corticosterone following acute sleep fragmentation in male mice Dear Dr. Ashley: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Dragan Hrncic Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0288889.r004

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