Mechanisms and therapeutic implications of RTA 408, an activator of Nrf2, in subarachnoid hemorrhage–induced delayed cerebral vasospasm and secondary brain injury

Tai-Hsin Tsai; Szu-Huai Lin; Chieh-Hsin Wu; Yi-Cheng Tsai; Sheau-Fang Yang; Chih-Lung Lin

doi:10.1371/journal.pone.0240122

Peer Review History

Original SubmissionJune 24, 2020
29 Jul 2020 Decision Letter - Jinglu Ai, Editor PONE-D-20-19417 Mechanisms and therapeutic implications of RTA 408, an activator of Nrf2, in subarachnoid hemorrhage–induced delayed cerebral vasospasm and secondary brain injury PLOS ONE Dear Dr. Lin, 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 August 28th. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Jinglu Ai, M.D., Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines: (1) Please state the source of the rats used in the study (2) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment) (3) Please include the method of euthanasia and clarify whether the rats were anaesthetised before they were euthanized by perfusion and fixatiion. Please provide the reagents used in the method of euthanasia. (4) Please describe the post-operative care received by the animals, including the frequency of monitoring and the specific clinical, physiological and behavioural criteria used to assess animal health and well-being. Thank you for your attention to these requests. 3. In the Methods section, please provide the product number and any lot numbers of the primary antibodies used in the Western blot analysis for your study. 4. In the Methods section, please provide the source, product number and any lot numbers of the RTA-408 used in the animal experiments for your study. 5. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses, including the specific name and version of the software used. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. 6. We noticed minor instances of text overlap with the following previous publication(s), which need to be addressed: (1) https://www.sciencedirect.com/science/article/abs/pii/S1878331712001313?via%3Dihub (2) https://thejns.org/view/journals/j-neurosurg/110/3/article-p457.xml The text that needs to be addressed involves the (1) Introduction and (2) Results section (pages 8-9). 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This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 9. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [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: The authors have examined the potential efficacy of RTA 408 in attenuating cerebral vasospasm and acute brain injury following SAH in a rodent model. There are some minor grammatical errors that should be corrected. How were the dosages of RTA 408 selected? Did the authors examine Nrf2 nuclear translocation? A limitations section should be provided. ****** 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.0240122.r001
Revision 1
8 Sep 2020 Author Response Dear reviewers: Thank you for the opportunity to revise our paper on ‘Mechanisms and therapeutic implications of RTA 408, an activator of Nrf2, in subarachnoid hemorrhage–induced delayed cerebral vasospasm and secondary brain injury.’ I have included the reviewer comments immediately and responded to them individually, indicating exactly how we addressed each concern or problem and describing the changes we have made. The revisions have been approved by all authors. The changes are marked in red in the paper as you requested, and the revised manuscript is attached to this email message. Reply to reviewer's comment Reviewer 1 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at Answer: The manuscript has meet PLOS ONE's style requirements as the PLOS ONE'S style templates. 2. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines Answer: We have been reported the additional details in Methods section regarding animal care such as the source of the rats, animal welfare, the method of euthanasia and postoperative care. Page 4-5 3. In the Methods section, please provide the product number and any lot numbers of the primary antibodies used in the Western blot analysis for your study. Answer: We have been provided the product numbers of the primary antibodies used in the Western blot analysis in this manuscript. Page 6 4. In the Methods section, please provide the source, product number and any lot numbers of the RTA-408 used in the animal experiments for your study. Answer: We have been provided the source, product number of the RTA 408 used in the animal experiments. Page 5 5. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses, including the specific name and version of the software used. Answer: We have been provided the specific name and version of the software regarding the specific name and version of the software in the Methods section. Page 6 6. We noticed minor instances of text overlap with the following previous publication(s), which need to be addressed involves the (1) Introduction and (2) Results section (pages 8-9). Answer: We also noticed the text overlap with the previous publications. Although the previous publications are our own works, we have been rephrased and quoted the duplicate text to avoid plagiarism or self-plagiarism. Page2-3, 7-8 7. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. Answer: Data from this study are available upon request and there are no restrictions on sharing data publicly. We will upload the data set necessary to replicate our study findings as Supporting Information files. 8. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. Answer: We will provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures. 9. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Answer: We have an ORCID iD for the corresponding author and that it is validated in Editorial Manager. Reviewer 2: 1. There are some minor grammatical errors that should be corrected. Answer: The grammatical errors of the article have been edited. 2. How were the dosages of RTA 408 selected? Answer: In this experiment, Group 3-5 received experimental SAH with RTA 408 treatment (Intraperitoneal injection) 30 min after the first induction of SAH for 7 days following the first hemorrhage. The dosages of RTA 408 are 0.5 mg/kg/day, 1 mg/kg/day and 1.5 mg/kg/day. The dosages of RTA 408 were selected according to the previous experiments.Peng Han, et al. RTA-408 Protects Kidney from Ischemia-Reperfusion Injury in Mice via Activating Nrf2 and Downstream GSH Biosynthesis Gene. Oxid Med Cell Longev. 24 December 2017. Page 4-5 3. Did the authors examine Nrf2 nuclear translocation? Answer： The authors do not examine Nrf2 nuclear translocation, because this experimental design is for the study of downstream protein changes in the Nrf2 pathway caused by RTA 408 treatment. Despite the changes of several Nrf2 downstream of protein levels after RTA-408 treatment, the authors did not directly sought out a clear mechanism for the neuroprotective effects of RTA-408. Therefore, additional intervention studies including Nrf2 nuclear translocation are still needed to verify the links between RTA408 and Nrf2 and the consequences of SAH. This problem was addressed in the limitation section. 4. A limitations section should be provided. Answer: A limitation section has been provided. Page 12 We hope the revised manuscript will better suit the PLOS ONE but are happy to consider further revisions, and we thank you for your continued interest in our research. Sincerely, Tai-Hsin Tsai, MD Department of Neurosurgery Kaohsiung Medical University Hospital https://doi.org/10.1371/journal.pone.0240122.r002
11 Sep 2020 Decision Letter - Jinglu Ai, Editor PONE-D-20-19417R1 Mechanisms and therapeutic implications of RTA 408, an activator of Nrf2, in subarachnoid hemorrhage–induced delayed cerebral vasospasm and secondary brain injury PLOS ONE Dear Dr. Lin, 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. ============================== Additional Editor Comments (if provided): Thank you for the revision. Before I accept your revised manuscript. I would like you to revise all your figures to make them look more professional. E.g., Figure 1, the y-axis label is not centered to the axis, and the labeling of the three doses of the drug is way too long. You could simply label them as 0.5 mg, 1 mg and 1.5 mg, and on top of them use a bracket to include all three, and label the bracket with text, SAH+RTA408. This is the same for the chart labeling as well, and for all the figures. After the modification, and save some spaces, you may want to increase the font size for all the labeling on the charts. In the PDF file, the figures, especially the charts in the figures are of very bad quality, some are even not readable, especially the last figure with 12 charts. I am not sure whether the journal office has a better version of the figures. It is not publishable as presented in the current PDF version. Thank you, Jinglu Ai ============================== Please submit your revised manuscript by Sept 21st. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Jinglu Ai, M.D., Ph.D. Academic Editor PLOS ONE [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.0240122.r003
Revision 2
17 Sep 2020 Author Response Reply to reviewer's comment Additional Editor Comments: 1.Please revise all your figures to make them look more professional and I have uploaded a copy o Figure 5. Answer : I have revised all figures to make them look more professional. Reviewer 1 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at Answer: The manuscript has meet PLOS ONE's style requirements as the PLOS ONE'S style templates. 2. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines Answer: We have been reported the additional details in Methods section regarding animal care such as the source of the rats, animal welfare, the method of euthanasia and postoperative care. 3. In the Methods section, please provide the product number and any lot numbers of the primary antibodies used in the Western blot analysis for your study. Answer: We have been provided the product numbers of the primary antibodies used in the Western blot analysis in this manuscript. 4. In the Methods section, please provide the source, product number and any lot numbers of the RTA-408 used in the animal experiments for your study. Answer: We have been provided the source, product number of the RTA 408 used in the animal experiments. 5. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses, including the specific name and version of the software used. Answer: We have been provided the specific name and version of the software regarding the specific name and version of the software in the Methods section. 6. We noticed minor instances of text overlap with the following previous publication(s), which need to be addressed involves the (1) Introduction and (2) Results section (pages 8-9). Answer: We also noticed the text overlap with the previous publications. Although the previous publications are our own works, we have been rephrased and quoted the duplicate text to avoid plagiarism or self-plagiarism. 7. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. Answer: Data from this study are available upon request and there are no restrictions on sharing data publicly. We will upload the data set necessary to replicate our study findings as Supporting Information files. 8. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. Answer: We will provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures. 9. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Answer: We have an ORCID iD for the corresponding author and that it is validated in Editorial Manager. Reviewer 2: 1. There are some minor grammatical errors that should be corrected. Answer: The grammatical errors of the article have been edited and the editing certificate is attached. 2. How were the dosages of RTA 408 selected? Answer: In this experiment, Group 3-5 received experimental SAH with RTA 408 treatment (Intraperitoneal injection) 30 min after the first induction of SAH for 7 days following the first hemorrhage. The dosages of RTA 408 are 0.5 mg/kg/day, 1 mg/kg/day and 1.5 mg/kg/day. The dosages of RTA 408 were selected according to the previous experiments. Peng Han, et al. RTA-408 Protects Kidney from Ischemia-Reperfusion Injury in Mice via Activating Nrf2 and Downstream GSH Biosynthesis Gene. Oxid Med Cell Longev. 24 December 2017. 3. Did the authors examine Nrf2 nuclear translocation? Answer： The authors do not examine Nrf2 nuclear translocation, because this experimental design is for the study of downstream protein changes in the Nrf2 pathway caused by RTA 408 treatment. Despite the changes of several Nrf2 downstream of protein levels after RTA-408 treatment, the authors did not directly sought out a clear mechanism for the neuroprotective effects of RTA-408. Therefore, additional intervention studies including Nrf2 nuclear translocation are still needed to verify the links between RTA408 and Nrf2 and the consequences of SAH. This problem was addressed in the limitation section. 4. A limitations section should be provided. Answer: A limitation section has been provided. Attachments Attachment Submitted filename: Response to reviewers comment.docx https://doi.org/10.1371/journal.pone.0240122.r004
21 Sep 2020 Decision Letter - Jinglu Ai, Editor Mechanisms and therapeutic implications of RTA 408, an activator of Nrf2, in subarachnoid hemorrhage–induced delayed cerebral vasospasm and secondary brain injury PONE-D-20-19417R2 Dear Dr. Lin, 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, Jinglu Ai, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0240122.r005
Formally Accepted
25 Sep 2020 Acceptance Letter - Jinglu Ai, Editor PONE-D-20-19417R2 Mechanisms and therapeutic implications of RTA 408, an activator of Nrf2, in subarachnoid hemorrhage–induced delayed cerebral vasospasm and secondary brain injury Dear Dr. Lin: 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 Dr. Jinglu Ai Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0240122.r006

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