The non-benzodiazepine anxiolytic etifoxine limits mechanical allodynia and anxiety-like symptoms in a mouse model of streptozotocin-induced diabetic neuropathy

Géraldine Gazzo; Marlene Salgado Ferrer; Pierrick Poisbeau

doi:10.1371/journal.pone.0248092

Peer Review History

Original SubmissionFebruary 17, 2021
26 May 2021 Decision Letter - Rosanna Di Paola, Editor PONE-D-21-05383 The non-benzodiazepine anxiolytic etifoxine limits the sensory-affective expression of pain following streptozotocin-induced diabetic neuropathy PLOS ONE Dear Dr. 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 10 days. 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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Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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 manuscript investigates the effects of the non-benzodiazepine anxiolytic Etifoxine on mechanical allodynia and anxiety in a mouse model of painful diabetic neuropathy induced by streptozotocin (STZ). The Authors demonstrates that Etifoxine prevents and reverses mechanical allodynia and anxiety in the STZ mice. The results are intriguing and could lead to a novel and effective treatment for painful diabetic neuropathy, a prevalent and significant health issue. The paper is well written and the logic of the experiments is easy to follows. Major points: 1) The Authors should describe whether the experimental set up included male and female mice and if the experimental design allowed for disaggregation of data so that results obtained from males and females can be analyzed separately and compared. These days it is not acceptable to just perform experiments on male animals. It has been shown that males and females have extremely different pain responses driven by different factors in some instances. 2) Blinding is not mentioned in the behavior studies. Given that blinding is critical for these experiments, authors should describe whether and how blinding was included in the experimental design. In addition, there is not mention of randomization methods in the experimental design. These factors weaken the ability of the Authors to reproduce the work. 3) The STZ model is a model of type 1 diabetes. Most commonly, however, painful neuropathy is associated with type 2 diabetes. In order for the study to be more impactful, the Authors should confirm their findings in models of painful diabetic neuropathy associated with type 2 diabetes, such as the High-Fat-Diet mouse model or db-db obese mice. Minor points 1) The Authors should rework the title. It is not clear what the Authors mean for “sensory-affective expression of pain”. The Authors in the study are measuring measure static tactile reflexes; a potential measure related to tactile allodynia in addition to anxiety. 2) The Authors could present the preventive experiment first and then conclude the paper with the reversal experiment. The reversal is even more relevant from a translational perspective. 3) The Authors should not use the term: “curative”. Indeed, the Authors are not providing evidence of disease modification, such as reversal of small fiber degeneration etc. Reviewer #2: Interesting manuscript, even if the topic cannot be regarded as very original. The title is too generic. Please, specify in the title that the study has been carried out in rats or at least specificy streptozotocin-induced diabetic neuropathy "model". The Introduction is just a little bit long because the authors should draw readers' attention to simply general overview of PDN and few specific statements about EFX. Materials&methods is well-written and statistical analysis is accurate. Results, supported by statistics, are easily "visualized" owing to well-constructed figures and well-detailed legends. The Discussion is to shorten because too redundant. Authors should more focus on the effect of etifoxine against PDN, the topic of their study, and less wander off. I would suggest authors to give some more details about microglia activation and neuropathic chronic states due to metabolic dysfunction, trying to better clarify the role of EFX. Reference list is good and updated. ****** 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 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.0248092.r001
Revision 1
5 Jul 2021 Author Response Dear Editor of Plos one, Please find below our detailed answers to the reviewers comments. We thank them for their constructive comments, which help us revise the manuscript. We do hope that our answers will clarify the concerns raised after their reading. Below are the point-by-point answers preceded by some editorial changes we were asked to do. Answer to Editor’s requests: 1. Style of the journal: We have checked all parts in order to respect the recommended style. 2. Sacrifice method was added to the “animals” paragraph of the materials and methods section. 3. As there is non ethical or legal restrictions on sharing, all data are freely available in the seafile depository server of the University of Strasbourg as recommended by our research organization for open access. These data are now also accessible to the public under the following DOI number : 10.17605/OSF.IO/9GP68 4. We have transferred the funding bodies informations to the Financial Disclosure section. 5. A competing interest statement is included in the submission. It indicates that the financial support by Biocodex laboratories does not alter adherence to PLOS ONE policies on sharing data and materials. This is why data could be filed in an open source depository. Moreover, the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Answer to Reviewers' comments: Reviewer #1: The manuscript investigates the effects of the non-benzodiazepine anxiolytic Etifoxine on mechanical allodynia and anxiety in a mouse model of painful diabetic neuropathy induced by streptozotocin (STZ). The Authors demonstrates that Etifoxine prevents and reverses mechanical allodynia and anxiety in the STZ mice. The results are intriguing and could lead to a novel and effective treatment for painful diabetic neuropathy, a prevalent and significant health issue. The paper is well written and the logic of the experiments is easy to follows. Major points: 1) The Authors should describe whether the experimental set up included male and female mice and if the experimental design allowed for disaggregation of data so that results obtained from males and females can be analyzed separately and compared. These days it is not acceptable to just perform experiments on male animals. It has been shown that males and females have extremely different pain responses driven by different factors in some instances. We completely agree with this crucial point and deplore the lack of studies performed on both male and female rodent models, especially considering the mentioned differential responses to pain and mechanisms of chronicization in males and females. The present study however only is a brief research report meant as a proof of concept of the potential effect of Etifoxine following STZ-induced diabetic neuropathy. This statement is in no case an excuse, however, considering the reported increased resistance of female rodents to STZ-induced diabetes (reviewed in Deeds et al. 2011 - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3917305/), which could be due in part to the protective action of estrogens (Le May et al., 2006 - https://pubmed.ncbi.nlm.nih.gov/16754860/), we decided to pursue our brief proof of concept in highly-susceptible male mice only, as a first step in a broader study. 2) Blinding is not mentioned in the behavior studies. Given that blinding is critical for these experiments, authors should describe whether and how blinding was included in the experimental design. In addition, there is not mention of randomization methods in the experimental design. These factors weaken the ability of the Authors to reproduce the work. Following the reviewer’s suggestion, we have added information in this regard in the “pharmacological treatment” section of the materials and methods. Blinding could not be included in the STZ vs VEH group considering the STZ-induced weight loss that even a blind experimenter could recognize. However, attribution of EFX or VEH treatment between CTRL and STZ groups was randomized. As added in the materials and methods, “STZ and VEH animals were randomly assigned to the EFX or VEH group, after verification that the difference in mechanical nociceptive baseline was not significant in-between both conditions in the established treatment groups. Experimenters were then blind to the treatment condition when performing behavioral assays”. 3) The STZ model is a model of type 1 diabetes. Most commonly, however, painful neuropathy is associated with type 2 diabetes. In order for the study to be more impactful, the Authors should confirm their findings in models of painful diabetic neuropathy associated with type 2 diabetes, such as the High-Fat-Diet mouse model or db-db obese mice. We agree with the reviewer, and had performed preliminary experiments in db/db-/- mice. However, our behavioral testing procedures were not calibrated for severely obese animals and we failed to highlight any significant results in this model. Minor points 1) The Authors should rework the title. It is not clear what the Authors mean for “sensory-affective expression of pain”. The Authors in the study are measuring measure static tactile reflexes; a potential measure related to tactile allodynia in addition to anxiety. The title has been reworked and specified and now states “The non-benzodiazepine anxiolytic etifoxine limits mechanical allodynia and anxiety-like symptoms in a mouse model of streptozotocin-induced diabetic neuropathy”. 2) The Authors could present the preventive experiment first and then conclude the paper with the reversal experiment. The reversal is even more relevant from a translational perspective. We took this comment into account and presented the preventive experiment first. 3) The Authors should use the term “curative”. Indeed, the Authors are not providing evidence of disease modification, such as reversal of small fiber degeneration etc. We took this comment into account. Since we only meant “curative” as a timely adjective in opposition to “preventive”, we replaced the term “curative” with the term “post-symptomatic”. Reviewer #2: Interesting manuscript, even if the topic cannot be regarded as very original. The title is too generic. Please, specify in the title that the study has been carried out in rats or at least specificy streptozotocin-induced diabetic neuropathy "model". The Introduction is just a little bit long because the authors should draw readers' attention to simply general overview of PDN and few specific statements about EFX. Materials&methods is well-written and statistical analysis is accurate. Results, supported by statistics, are easily "visualized" owing to well-constructed figures and well-detailed legends. The Discussion is to shorten because too redundant. Authors should more focus on the effect of etifoxine against PDN, the topic of their study, and less wander off. I would suggest authors to give some more details about microglia activation and neuropathic chronic states due to metabolic dysfunction, trying to better clarify the role of EFX. Reference list is good and updated. The authors thank the reviewer for their comments. * Title has been modified to specify that the study was performed in a mouse model and now states “The non-benzodiazepine anxiolytic etifoxine limits mechanical allodynia and anxiety-like symptoms in a mouse model of streptozotocin-induced diabetic neuropathy”. * The introduction was not shortened as we feel that an overview of EFX’s general mechanism of action relative to the pathogenesis of PDN allows the reader to understand why we sought to evaluate the properties of EFX in a model of STZ-induced neuropathy. * The discussion was shortened following reviewer’s comment as we had no intention to appear as if we were wandering off. Attachments Attachment Submitted filename: R1-Gazzo_response to reviewers.docx https://doi.org/10.1371/journal.pone.0248092.r002
15 Jul 2021 Decision Letter - Rosanna Di Paola, Editor The non-benzodiazepine anxiolytic etifoxine limits mechanical allodynia and anxiety-like symptoms in a mouse model of streptozotocin-induced diabetic neuropathy PONE-D-21-05383R1 Dear Dr. 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, Rosanna Di Paola, MD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0248092.r003
Formally Accepted
27 Jul 2021 Acceptance Letter - Rosanna Di Paola, Editor PONE-D-21-05383R1 The non-benzodiazepine anxiolytic etifoxine limits mechanical allodynia and anxiety-like symptoms in a mouse model of streptozotocin-induced diabetic neuropathy Dear Dr. Poisbeau: 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. Rosanna Di Paola Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248092.r004

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