Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model

Robert L. Bell; Ginger S. Withers; Frans A. Kuypers; Wolfgang Stehr; Aditi Bhargava

doi:10.1371/journal.pone.0246412

Peer Review History

Original SubmissionJanuary 14, 2021
22 Mar 2021 Decision Letter - Arda Yildirim, Editor PONE-D-21-01479 Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model PLOS ONE Dear Dr. Bhargava, 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. The paper could have an important technical contribution in the area. I agree with all reviewers think that mainly the manuscript have several technical deficiencies. There are a lot of careless mistakes in the manuscript and the whole manuscript shall be reviewed by the authors carefully. The article requires some technical revisions and a thorough proof-read for grammatical errors. Please submit your revised manuscript by May 02 2021 11:59PM. 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At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines: (a) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment) (b) Please include the method of euthanasia (c) Please provide additional information about the experimental endpoints used in the model. Please provide the frequency of monitoring and specific criteria used to determine when animals should be euthanized. In addition, please state whether any animals died before meeting the criteria for euthanasia. Thank you for your attention to these requests. 4. Please ensure you have discussed any potential limitations of your study in the Discussion. 5. Thank you for stating the following financial disclosure: 'DK070878 to AB ' At this time, please address the following queries: Please clarify, naming in full, the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” If any authors received a salary from any of your funders, please state which authors and which funders. If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 6. Please include a caption for figure 7. Additional Editor Comments: For your guidance, you can check the reviewers' comments. Thank you for giving us the opportunity to consider your work. [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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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: The study on Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model was an interesting study with novel ideas and conclusions on the role of CRF showing promising pharmacological role in treatment or prevention of necrotising enterocolitis in the neonates. However, there are some questions and concerns that need clarification. 1. What determined the quantity of formula feed given to each neonates? Is the quantity given not excessive, thereby responsible for the observed inflammatory damages in the bowel of the neonates. 2. What other markers of oxidative stress did you determine in the course of the study and the probable roles of antioxidants instead of CRF on the oxidative stress and inflammation in the affected neonates. There are specific observations in some of the sections that may require your correction. Abstract 1. Line 50: Stressed neonates, how were they stressed. Please indicate in one or two words. 2. Lines 60-61: The conclusion was not explicit. Too shallow because much of the findings were not captured. Introduction Lines 136-138: The hypothesis mentioned in the justification for the study is not entirely the same as the ones mentioned in the abstract. Please adjust it! Materials and methods 1. Line 195: Evaluation of specific oxidative stress markers from post mitochondrial fractions of homogenized intestinal tissue of ought to have been added. Like lipid peroxidation, glutathione levels and antioxidant enzymes such as superoxide dismutase. 2. Line 197: Names or initial of researchers are not necessary here, why not put them in the acknowledgment if they are not part of the authors, or authors' contributions. 3. Mention the parameters evaluated in the antibodies, Western blot and immunoflourescence analysis. Results 1. Line 337: AST, render in full and put the abbreviation in bracket. Discussion 1. Line 401-402: Please cite appropriate reference. Reviewer #2: The manuscript entitled as 'Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model' is well designed l study. However, minor revision is needed, than it can be published in this journal. Suggestions are indicated on the manuscript. ****** 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: Yes:** Dr O. I Azeez 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.] 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-21-01479_reviewer-1.pdf Attachment Submitted filename: 1-PONE-D-21-01479_reviewer.pdf https://doi.org/10.1371/journal.pone.0246412.r001
Revision 1
29 Apr 2021 Author Response Arda Yildirim, Ph.D. Academic Editor PLOS ONE RE: PONE-D-21-01479 Dear Dr. Yildirim, Please find attached a revised version of our manuscript entitled “Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model”. Changes in text are in blue. Below, we provide a point-by-point responses to reviewers. Reviewer #1: The study on Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model was an interesting study with novel ideas and conclusions on the role of CRF showing promising pharmacological role in treatment or prevention of necrotising enterocolitis in the neonates. However, there are some questions and concerns that need clarification. 1. What determined the quantity of formula feed given to each neonates? Is the quantity given not excessive, thereby responsible for the observed inflammatory damages in the bowel of the neonates. Response. The quantity of formula feed was informed by established neonatal rat models of necrotizing enterocolitis and approved by our IACUC committee’s veterinarian. Overfeeding is a valid concern and was addressed during study design. It is notable that formula feeding—and perhaps overfeeding—is associated with the development of NEC in humans. In our experiments, formula feeding alone (i.e. without exposure to physiologic stress or corticotropin releasing factor) was associated with some inflammatory changes, but was not clearly associated with the development of overt NEC, thus formula-feeding alone is not associated with the observed effects. These changes were ameliorated with the administration of the CRF antagonist, Astressin. 2. What other markers of oxidative stress did you determine in the course of the study and the probable roles of antioxidants instead of CRF on the oxidative stress and inflammation in the affected neonates. Response. We thank the reviewer for this interesting question. In this study, we did not ascertain the roles of antioxidants. Determining the roles of antioxidants will require exhaustive set of experiments which is beyond the scope of this paper. We further feel that addition of more data will distract from message and the role of CRF system in NEC. As suggested, this will be a great follow-up study. We have discussed the potential role of oxidative stress in our discussion. There are specific observations in some of the sections that may require your correction. Abstract 1. Line 50: Stressed neonates, how were they stressed. Please indicate in one or two words. Response. Lines 48-49 already describe the stressor used (acute hypoxia followed by cold exposure). 2. Lines 60-61: The conclusion was not explicit. Too shallow because much of the findings were not captured. Response. We are perplexed as conclusions are meant to be short and general. We feel that given the word limits for an abstract, our conclusion statement is appropriate. Our discussion contains more explicit and in-depth conclusion. Introduction Lines 136-138: The hypothesis mentioned in the justification for the study is not entirely the same as the ones mentioned in the abstract. Please adjust it! Response. We thank the reviewer for catching this discrepancy. We have modified the hypothesis in lines 136-138 to be congruent. Materials and methods 1. Line 195: Evaluation of specific oxidative stress markers from post mitochondrial fractions of homogenized intestinal tissue of ought to have been added. Like lipid peroxidation, glutathione levels and antioxidant enzymes such as superoxide dismutase. Response. We thank the reviewer for suggesting alternative mechanisms. Indeed, as the reviewer points out, preterm babies as well as NEC babies are exposed to more oxidative stress than full-term babies. We have added this to our discussion (Lines 383-387) and provided 3 new references (#s 64-66). However, the tease out which precise oxidative stress pathway intersects with the CRF system, will require exhaustive sets of new experiments along with use of additional pharmacological agents. This is beyond the scope of this study. 2. Line 197: Names or initial of researchers are not necessary here, why not put them in the acknowledgment if they are not part of the authors, or authors' contributions. Response. Initials of the researchers help with further identifying their roles but as suggested, we have removed the initials from the methods. 3. Mention the parameters evaluated in the antibodies, Western blot and immunoflourescence analysis. Response. All sources of antibodies and dilutions used are provided (Lines 224-231). We are unclear as to what other parameters are required? Results 1. Line 337: AST, render in full and put the abbreviation in bracket. Response. AST is first defined in line 144, under materials section. Discussion 1. Line 401-402: Please cite appropriate reference. Response. Reference has been provided. Reviewer #2: The manuscript entitled as 'Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model' is well designed l study. However, minor revision is needed, than it can be published in this journal. Suggestions are indicated on the manuscript. 1. Define FF (formula fed, line 46). 2. The companies or sources are provided in parenthesis and we have removed the sentences that created this confusion. All suggestions indicated on the manuscript have been incorporated. Editorial comments to be included in the manuscript: Details of animal welfare – shelter, food, water, environmental enrichment – - Adult animals were housed in temperature- and humidity-controlled cages that utilized a laminar flow HEPA filter system. They had access to ad lib food, water, and environmental enrichment. Adult animals and neonates housed with dams were monitored at least daily. Neonates in experimental conditions were housed with members of their treatment groups in a temperature (35ºC) and humidity (15%)- controlled incubator. Each of these animals was inspected, fed, and provided with bladder and bowel stimulation six times daily. Method of euthanasia – Carbon dioxide exposure followed by bilateral thoracotomy and cardiac venting was used for euthanizing adult rats. Neonates were anesthetized with isoflurane and euthanized via decapitation. Information about experimental endpoints used in the model Besides gavage-induced thoracic injury, other experimental endpoints included body conditioning score of 2 or less and rectal prolapse. Frequency of monitoring – at least once daily during the experiment - Specific criteria used to determine which animals should be euthanized – o Criteria for euthanasia of neonates included loss of vigorous mobility, lethargy, failure to respond to stimuli; as well as findings suggestive of injury or aspiration that occurred during gavage feeding. These included respiratory distress, regurgitation of feeds, and pharyngeal bleeding. - Any animals died before meeting criteria for euthanasia o No We hope that our manuscript is now acceptable for publication. Sincerely, Aditi Bhargava Attachments Attachment Submitted filename: ResponseToReviewer 4.24.docx https://doi.org/10.1371/journal.pone.0246412.r002
25 May 2021 Decision Letter - Arda Yildirim, Editor Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model PONE-D-21-01479R1 Dear Dr. Bhargava, 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, Arda Yildirim, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Thanks for your hard work. 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 ******** 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 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: (No Response) Reviewer #2: The manuscript is well designed and also all required suggestion/corrections performed. Now it is suitable to be publish in the journal. ****** 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 Reviewer #2: No https://doi.org/10.1371/journal.pone.0246412.r003
Formally Accepted
1 Jun 2021 Acceptance Letter - Arda Yildirim, Editor PONE-D-21-01479R1 Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model Dear Dr. Bhargava: 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 plosone@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 Prof. Dr. Arda Yildirim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0246412.r004

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