Carbon monoxide as a cellular protective agent in a swine model of cardiac arrest protocol

John C. Greenwood; Ryan W. Morgan; Benjamin S. Abella; Frances S. Shofer; Wesley B. Baker; Alistair Lewis; Tiffany S. Ko; Rodrigo M. Forti; Arjun G. Yodh; Shih-Han Kao; Samuel S. Shin; Todd J. Kilbaugh; David H. Jang

doi:10.1371/journal.pone.0302653

Peer Review History

Original SubmissionAugust 3, 2023
2 Nov 2023 Decision Letter - Miquel Vall-llosera Camps, Editor PONE-D-23-24722The feasibility of carbon monoxide as a cellular protective agent in a swine model of cardiac arrestPLOS ONE Dear Dr. Jang, 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. I would like to sincerely apologise for the delay you have incurred with your submission. It has been exceptionally difficult to secure reviewers to evaluate your study. We have now received two completed reviews; the comments are available below. Reviewer#1 has raised significant scientific concerns about the study that need to be addressed in a revision. About Reviewer#2 comments, I'd like to note here that Study Protocols are in scope for PLOS ONE (https://journals.plos.org/plosone/s/what-we-publish#loc-study-protocols). Please revise the manuscript to address all the reviewer's comments in a point-by-point response in order to ensure it is meeting the journal's publication criteria. Please note that the revised manuscript will need to undergo further review, we thus cannot at this point anticipate the outcome of the evaluation process. Please submit your revised manuscript by Dec 09 2023 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. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. 2. Please amend your current title to include the word "protocol". 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. 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Please amend either the title on the online submission form (via Edit Submission) or the title in the manuscript so that they are identical. 7. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. 8. Please upload a copy of Figure 1, to which you refer in your text. If the figure is no longer to be included as part of the submission please remove all reference to it within the text. 9. 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. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions? The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses? The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory. Reviewer #1: Partly Reviewer #2: Yes ****** 3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable? Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors described where all data underlying the findings will be made available when the study is complete? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics. You may also provide optional suggestions and comments to authors that they might find helpful in planning their study. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Thank you for giving me an opportunity to review this manuscript. This manuscript describes a study protocol to investigate the feasibility of CO as a cellular protective agent in a pig model of cardiac arrest. Overall, the manuscript clearly presents the rationale and method of the study. I have only a few minor suggestions listed as follows. Abstract Abstract, Line 9. Please revise the term ‘post-cardiac patients’. (patients with cardiac arrest or patients resuscitated from cardiac arrest?) Introduction Introduction, Line 7. It may be better to move the sentence “In one of our studies, we found that swine exposed to 2000 ppm for over an hour reliable COHb of around 50%.3” from the introduction section to the method or discussion section. Introduction. Please spell out all abbreviations including MI and CI in the first appearance. Please check all abbreviations throughout the manuscript. Introduction. A more detailed description of why CO is a more ideal agent than NO or H2S is needed. It is necessary to highlight the necessity of this study in relation to previous studies that investigated the effect of CO administration after arrest. Materials and methods The double quotation marks in the first sentence in the animals and overall study design section are incomplete. Please provide a more detailed description on the CI monitoring. The “up to 12 min” is unnecessarily repeated in the first sentence of the cardiac arrest and carbon monoxide experimental protocol section. Please provide the rationale for choosing 8 min duration of untreated cardiac arrest. Please provide the reason why epinephrine is chosen as a medication to titrate CI. Epinephrine is well known to affect cerebral perfusion adversely. There are two sentences describing the timing of randomization: “All subject animals will be randomized to one of three groups prior to arrival.” and “After successful resuscitation, the animals will be randomly divided into three groups:”. It would be helpful if you clearly described the timing of commencement of CO administration. It would be nice if the authors could share their previous experiences of CO administration, such as hemodynamic changes after CO administration. How many hours after ROSC does the experimental protocol end and euthanasia occur? Please define the primary outcome of this study. Figure legend Please spell out all abbreviations within the Sources of Funding section. Please revise the figure to include the timing of each intervention. Reviewer #2: The proposed study is highly relevant with a very well described method. It will allow a very comprehensive evaluation of the effect of CO after cardiac arrest. The submission of the design of experimental study should be encouraged. However, I do not know whether method paper could comply to Plos One Editorial policy, since the journal guidelines state that research articles "must present the results of original research". In order to include original findings, the article should at least present preliminary original data with a natural history of the model in control conditions. ****** 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 ******** [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.0302653.r001
Revision 1
18 Dec 2023 Author Response Response to Reviewers Reviewer #1: Thank you for giving me an opportunity to review this manuscript. This manuscript describes a study protocol to investigate the feasibility of CO as a cellular protective agent in a pig model of cardiac arrest. Overall, the manuscript clearly presents the rationale and method of the study. I have only a few minor suggestions listed as follows. Abstract 1. Abstract, Line 9. Please revise the term ‘post-cardiac patients’. (patients with cardiac arrest or patients resuscitated from cardiac arrest?) Response: We reviewed the manuscript and updated the term for post cardiac arrest patients to “post-arrest patients” which is the proper term. We revised both the abstract and the main manuscript. 2. Introduction, Line 7. It may be better to move the sentence “In one of our studies, we found that swine exposed to 2000 ppm for over an hour reliable COHb of around 50%.3” from the introduction section to the method or discussion section. Response: We move this sentence from the introduction to the methods as recommended by reviewer 1. We also restructured the introduction to focus more on cardiac arrest as well as the gaps related to the need for more therapies as opposed to starting with carbon monoxide poisoning that we feel flows better. 3. Introduction. Please spell out all abbreviations including MI and CI in the first appearance. Please check all abbreviations throughout the manuscript. Response: We spell out all abbreviations such as MI (myocardial infarction) and CI (cardiac index) in the manuscript in tracked changes. 4. Introduction. A more detailed description of why CO is a more ideal agent than NO or H2S is needed. Response: We have already described the differences between CO, NO and H2S in the discussion section that flows better here as opposed to placing in the introduction. Discussion Paragraph 5 “Other agents with similar properties to CO have been investigated, including nitric oxide (NO) and hydrogen sulfide (H2S). H2S is also a toxic gas with overlapping properties to CO that also can be found in the gas industry and sewage handling. While H2S has been shown to have cardioprotective benefits, it is considerably more toxic than CO with a very low therapeutic index that makes clinical implementation challenging. Another gas that also has been used is NO that is produced endogenously with important biological functions that primarily involve vasodilation and angiogenesis. NO has clinically been used for the treatment of ischemic heart disease but also has been used post-arrest to reduce IR injury. Despite the potential benefits of NO, NO is reactive at higher doses that can cause increased ROS production leading to DNA damage and can also cause methemoglobinemia (MetHb). MetHb can worsen ischemia as the oxidized iron in the affected hemoglobin can no longer accept or deliver oxygen to tissue. Taken together, while H2S, NO and CO are all potentially poisonous, CO is not considered as reactive as NO and has considerably more robust experimental and clinical data supporting use of CO over H2S and NO with a more favorable safety profile.” 5. It is necessary to highlight the necessity of this study in relation to previous studies that investigated the effect of CO administration after arrest. Response: We revised to better discuss the necessity of this study as there is a paucity of work in this area despite both in vitro and small animal work that support the of CO as a therapeutic in cardiac arrest. Discussion Paragraph 3 “While there are some small animal models in this area, there is a paucity of large animal models that recapitulate what can occur in patients. Our prior work with swine demonstrates a much more translational model that better captures the physiology and biomolecular findings in patients. Leveraging our swine models of both CO exposure and cardiac arrest, positive findings from this study would allow more rapid clinical translation in the future.” Materials and methods 6. The double quotation marks in the first sentence in the animals and overall study design section are incomplete. Response: This has been fixed as recommended. 7. Please provide a more detailed description on the CI monitoring. Response: We revised the methods to provide a more detailed description of cardiac index or CI monitoring. Perioperative procedures and monitoring section “Continuous aortic pressure (MAP), central venous pressure (CVP), cardiac output (CO) and cardiac index (CI) will be monitored with CI is calculated by dividing CO by body surface area.” 8. The “up to 12 min” is unnecessarily repeated in the first sentence of the cardiac arrest and carbon monoxide experimental protocol section. Response: This was corrected 9. Please provide the rationale for choosing 8 min duration of untreated cardiac arrest. Response: Extensive preclinical and clinical research has examined the time-sensitive progression of resuscitation physiology. We first considered the 3 phase model of cardiac arrest which was previously described by Weisfeldt & Becker (JAMA 2002), where immediate defibrillation within a 5-minute arrest period did not benefit from CPR (Neimann et al, Circulation, 1992). For our proposed experiment, we wanted to mimic a real world scenario of sudden cardiac arrest followed by bystander CPR, while also maximizing our chances for return of spontaneous circulation. A number of clinical and preclinical studies have identified that ROSC rates can be optimized in the 5-10 minute no-flow window by including CPR + Defibrillation (Yakaitis et al., Crit Care, 1980; Menegazzi et al, Ann of Emerg Med, 1993; Yu et al, Circulation, 2002). Extending beyond the 10 minute no-flow time appears to decrease chances of ROSC, and the severity of brain injury (outcome of interest) will likely outweigh any benefit from the proposed intervention. 10. Please provide the reason why epinephrine is chosen as a medication to titrate CI. Epinephrine is well known to affect cerebral perfusion adversely. Response: The post-cardiac arrest period is well known to result in reperfusion vasoplegia that would make it very difficult to initiate inodilator therapies (dobutamine, milrinone, etc.) without causing hemodynamic collapse. It is true that during ACLS, preclinical studies have identified that bolus dose (30 mcg/kg) epinephrine can reduce cerebral perfusion (Ristago, Crit Care Med, 2007), but the data is mixed, but our lab has found that lower dose (20 mcg/kg) may actually increase cerebral perfusion (Mavroudis et al, Crit Care, 2020). Post-arrest titration of an epinephrine effusion will allow us to augment cardiac output, while also avoiding excessive vasoplegia. It is unclear if epinephrine infusions, at lower continuous doses, negatively impact cerebral perfusion. 11. There are two sentences describing the timing of randomization: “All subject animals will be randomized to one of three groups prior to arrival.” and “After successful resuscitation, the animals will be randomly divided into three groups:” Response: We revised to the following below Material and methods under cardiac arrest and carbon monoxide experimental protocol “After successful resuscitation, the animals will be randomly divided into four groups: (1) CPR: Animals in this group will only receive CPR without CO upon ROSC; (2) CPR and CO: Cardiac arrest followed by CO treatment; (3) Sham: The same operation but no cardiac arrest or CO being given; (4) CO alone: Will be the same as Control except CO will be administrated to assess possible adverse effects of CO alone [Figure 1].” 12. It would be helpful if you clearly described the timing of commencement of CO administration. Response: We revised both the methods and Figure 1 to better describe the timing of each intervention so it is clear when CPR is induced followed by when CPR and CO administration occurs. For CO administration, an hour after achieving ROSC, CO (100 ppm) will be administered for 1 hour followed by 1 hour of re-oxygenation for a total post ROSC time of 3 hours. Figure 1: Experimental Protocol and flow diagram of study The following flow diagram will serve as a general framework for this proposed study with corresponding times: The perioperative period will include the induction of anesthesia to allow for the described procedures such as placement of central lines, cMD catheter, etc that typically takes about 2 hr. The induction of cardiac arrest will take about 4 seconds with cardiac arrest being allowed to continue for 8 min before the initiation of CPR with the first defibrillation at 10 min. If no ROSC is achieved, resuscitation will continue for an additional 10 min for a total of 20 min of CPR. Once ROSC is achieved, CO treatment with 100 ppm will be administrated for 1 hr followed by 1 hr of re-oxygenation for a total of 3 hr post-ROSC. 13. It would be nice if the authors could share their previous experiences of CO administration, such as hemodynamic changes after CO administration. Response: We revised the paper to cite our work both in the area of CO poisoning and cardiac arrest that demonstrates our expertise in this area. “While patients typically manifest symptoms with higher concentrations of CO, our lab has demonstrated cellular dysfunction in our swine model of acute CO poisoning using a relative lower dose (400 ppm) without overt clinical symptoms” 14. How many hours after ROSC does the experimental protocol end and euthanasia occur? Please define the primary outcome of this study. Response: We revised the methods and Figure 1 to better describe the timing of each key event in our procedure. Material and Methods Figure 1: Experimental Protocol and flow diagram of study “The following flow diagram will serve as a general framework for this proposed study with corresponding times: The perioperative period will include the induction of anesthesia to allow for the described procedures such as placement of central lines, cMD catheter, etc that typically takes about 2 hr. The induction of cardiac arrest will take about 4 seconds with cardiac arrest being allowed to continue for 8 min before the initiation of CPR with the first defibrillation at 10 min. If no ROSC is achieved, resuscitation will continue for an additional 10 min for a total of 20 min of CPR. Once ROSC is achieved, CO treatment with 100 ppm will be administrated for 1 hr followed by 1 hr of re-oxygenation for a total of 3 hr post-ROSC.” Figure legend 15. Please spell out all abbreviations within the Sources of Funding section. Response: We added the full funding information as recommended 1. National Institute of Environmental Health Sciences R21ES031243 (Jang) 2. National Heart, Lung, and Blood Institute R01HL141386 (Kilbaugh) 3. National Heart, Lung, and Blood Institute R56HL158696 (Jang) 4. National Heart, Lung, and Blood Institute R01HL166592 (Jang, Baker) 5. Children’s Hospital of Philadelphia Frontier Program (Baker, Ko, Forti, Kilbaugh) 6. National Institute of Neurologial Disorders and Stroke R01NS113945 (Baker, Lewis, Forti, Kilbaugh) 7. U.S. Department of Defense DoD W81XWH-22-1-0887/8 (Baker, Ko, Forti, Kilbaugh) 8. Toyota Way Forward Fund (Baker, Ko, Forti, Kilbaugh) 16. Please revise the figure to include the timing of each intervention. Response: We revised the figure caption to include the timing of each intervention. Reviewer #2: The proposed study is highly relevant with a very well described method. It will allow a very comprehensive evaluation of the effect of CO after cardiac arrest. 1. The submission of the design of experimental study should be encouraged. However, I do not know whether method paper could comply to Plos One Editorial policy, since the journal guidelines state that research articles "must present the results of original research". In order to include original findings, the article should at least present preliminary original data with a natural history of the model in control conditions. Response: As editor mentioned, this submission is appropriate for the protocol section. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0302653.r002
8 Mar 2024 Decision Letter - Meijing Wang, Editor PONE-D-23-24722R1Carbon monoxide as a cellular protective agent in a swine model of cardiac arrest protocolPLOS ONE Dear Dr. Jang, 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 Academic Editor is satisfied with scientific evaluation of your manuscript, and with the revisions you have made. Before we can can proceed with publication, we ask you to address the following concern: During internal evaluation of your manuscript, editorial staff noted significant text overlap between your manuscript and previous publications by one or more of the authors of this submission, specifically with the following publications: 1) "Alteration in Cerebral Metabolism in a Rodent Model of Acute Sub-lethal Cyanide Poisoning" - https://doi.org/10.1007/s13181-022-00928-w2) "Preliminary Research: Application of Non-Invasive Measure of Cytochrome c Oxidase Redox States and Mitochondrial Function in a Porcine Model of Carbon Monoxide Poisoning" - https://doi.org/10.1007/s13181-022-00892-53) "Cerebral mitochondrial dysfunction associated with deep hypothermic circulatory arrest in neonatal swine" - https://doi.org/10.1093/ejcts/ezx467 The Academic Editor has been consulted about this overlap, and they have not raised any concerns regarding duplication of work between these previous publications and this protocol submission, and they are satisfied that this protocol submission does warrant separate publication. However, while publication 3) above is published under an open access license, publications 1) and 2) are subject to copyright terms that prevent us from being able to publish your manuscript with the overlapping text in its current form. We apologize that this concern was not brought to your attention at an earlier stage in the publication process. In order to proceed, we ask you to revise your manuscript to reduce the text overlap with publications 1) and 2) linked above. Please also ensure, if you have not done so already, that the the previous publications are clearly referenced in the manuscript text as the sources for the methodologies included in this protocol. This is necessary for publication 3) as well. We appreciate your attention to these requests. Please submit your revised manuscript by Apr 22 2024 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. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: 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, Hugh Cowley Senior Editor PLOS ONE on behalf of Meijing Wang, MD Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions? The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field. Reviewer #2: Yes ******** 2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses? The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory. Reviewer #2: Yes ****** 3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable? Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible. Reviewer #2: Yes ****** 4. Have the authors described where all data underlying the findings will be made available when the study is complete? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 ****** 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 ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics. You may also provide optional suggestions and comments to authors that they might find helpful in planning their study. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: This is perfect if it can go to a protocol section. -------------------------------------------------------------------- ****** 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 ******** [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.0302653.r003
Revision 2
25 Mar 2024 Author Response Thank-you for the comments related to overlap for the two papers list below (There was not concern for the third paper list above and we were not asked to address any overlap related to the third paper): 1) Reference 47-"Alteration in Cerebral Metabolism in a Rodent Model of Acute Sub-lethal Cyanide Poisoning" -https://doi.org/10.1007/s13181-022-00928-w 2) Reference 29- "Preliminary Research: Application of Non-Invasive Measure of Cytochrome c Oxidase Redox States and Mitochondrial Function in a Porcine Model of Carbon Monoxide Poisoning" - https://doi.org/10.1007/s13181-022-00892-5 Response: We reviewed our manuscript as well as the two papers above (published by our group). For ease for the editor, we make comments regarding changes in the manuscript related to overlap of the two list papers. As also recommended by the reviewer, the list papers are already cited in the reference as seen above. Introduction/background 1. There is not overlap with the introduction/background as this paper focused on cardiac arrest and the application of carbon monoxide as a therapeutic which is unique and not discussed in the above papers. We did make small grammatical revisions to improve the paper but no major edits were made. Methods 1. This paper describes many common methods also found in the two listed paper. For example, we describe methods to measure respirometry in this manuscript which was also employed in the two list papers. While it is not considered plagiarism to describe the same methods, we did revise in this current manuscript so it is more brief and also reference the original papers. We specifically revised the following sections due to the overlap: a. Non-invasive optical monitoring of cerebral oxygenation and CIV redox rates b. Measurement of mitochondrial respiration in isolated brain mitochondria c. Western Blot d. ATP fluometry Discussion 1. There is not overlap with the discussion as this paper focused on cardiac arrest and the application of carbon monoxide as a therapeutic which is unique and not discussed in the above papers. We made edits to improve overall clarity of the manuscript and streamline the reliability. https://doi.org/10.1371/journal.pone.0302653.r004
4 Apr 2024 Decision Letter - Meijing Wang, Editor PONE-D-23-24722R2Carbon monoxide as a cellular protective agent in a swine model of cardiac arrest protocolPLOS ONE Dear Dr. Jang, 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. Please submit your revised manuscript by May 19 2024 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. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: 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, Meijing Wang, MD 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: The substantial revision to reduce the overlap with previous publications from the same group in Methods is appreciated. However, there is still minor concern. Some information is needed to help the readers better understand certain specific methods: 1) please provide one or two sentences to describe how CIV redox rates are determined; 2) how much tissue protein is used for Western Blot and for ATP fluorometry; and 3) The wavelengths for ATP fluorometry should be stated. [Note: HTML markup is below. Please do not edit.] [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.0302653.r005
Revision 3
6 Apr 2024 Author Response Thank-you for the comments. In our previous revisions we addressed the issue with overlap for the related to two of our publications listed below which satisfied the reviewer. 1) Reference 47-"Alteration in Cerebral Metabolism in a Rodent Model of Acute Sub-lethal Cyanide Poisoning" -https://doi.org/10.1007/s13181-022-00928-w 2) Reference 29- "Preliminary Research: Application of Non-Invasive Measure of Cytochrome c Oxidase Redox States and Mitochondrial Function in a Porcine Model of Carbon Monoxide Poisoning" - https://doi.org/10.1007/s13181-022-00892-5 We were asked to address additional editor comments listed below. “The substantial revision to reduce the overlap with previous publications from the same group in Methods is appreciated. However, there is still minor concern.” Some information is needed to help the readers better understand certain specific methods: 1) Please provide one or two sentences to describe how CIV redox rates are determined We added the following to the optics section: Given that each tissue chromophore has a unique wavelength-dependent absorption signature, it is feasible to isolate the CIV influence on the brain's absorption spectrum from other tissue chromophores. We specifically used the UCLn algorithm to compute changes in cerebral oxidized CIV concentration from the measured wavelength-dependent changes in optical attenuation signals between 780–1000 nm wavelengths. 2) How much tissue protein is used for Western Blot and for ATP fluorometry For western blot: Based on previous work we will use a protein concentration of 10 μl per well. All experiments will be performed in duplicates and the local background corrected density values. For ATP fluorometry: ATP concentrations will be obtained in snap frozen brain tissue samples (ratio of 10 mg of tissue to 100 µl of the assay buffer) using an ATP fluorometric assay kit (Sigma MAK190) with an excitation of 535 nm and emission of 587 nm obtained in a similar manner based on our previous publications [40, 47]. 3) The wavelengths for ATP fluorometry should be stated. We revised the ATP fluorometry to include the wavelengths based on our previous publications. The below is the revised paragraph. ATP concentrations will be obtained in snap frozen brain tissue samples (ratio of 10 mg of tissue to 100 µl of the assay buffer) using an ATP fluorometric assay kit (Sigma MAK190) with an excitation of 535 nm and emission of 587 nm obtained in a similar manner based on our previous publications [40, 47]. Attachments Attachment Submitted filename: PLOS Response Sheet.docx https://doi.org/10.1371/journal.pone.0302653.r006
10 Apr 2024 Decision Letter - Meijing Wang, Editor Carbon monoxide as a cellular protective agent in a swine model of cardiac arrest protocol PONE-D-23-24722R3 Dear Dr. Jang, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Meijing Wang, MD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0302653.r007
Formally Accepted
26 Apr 2024 Acceptance Letter - Meijing Wang, Editor PONE-D-23-24722R3 PLOS ONE Dear Dr. Jang, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 Dr. Meijing Wang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0302653.r008

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