Acute postoperative pain and dorsal root ganglia transcriptomic signatures following total knee arthroplasty (TKA) in rats: An experimental study

David E. Komatsu; Sardar M. Z. Uddin; Chris Gordon; Martha P. Kanjiya; Diane Bogdan; Justice Achonu; Adriana DiBua; Hira Iftikhar; Amanda Ackermann; Rohan J. Shah; Jason Shieh; Agnieszka B. Bialkowska; Martin Kaczocha

doi:10.1371/journal.pone.0278632

Peer Review History

Original SubmissionJune 13, 2022
26 Sep 2022 Decision Letter - Rosanna Di Paola, Editor PONE-D-22-16993Acute postoperative pain and dorsal root ganglia transcriptomic signatures following total knee arthroplasty in ratsPLOS 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 Nov 10 2022 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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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. [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: Partly Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The study was performed at a high level, but there are several technical issues that require correction for publication. To make a decision on further publication, it is necessary to revise the indicated sections and add mandatory papers with a second review: + A shorter and more capacious name that meets modern trends: "Acute postoperative pain and dorsal root ganglia transcriptomic signatures following TKA in rats: experimental study" + In the introduction and relevance, it is better not to refer to papers that are 15 years old, it is better to find references up to 5 years old. There are also recent studies regarding the level of pain after TKA, for example DOI: 10.1007/s00167-017-4713-5 + Where is the conclusion section? The results must be summarized. + Line 474 – What document is the link to? And then the URL: ... + A level of evidence needs to be added. Reviewer #2: As the authors point out, there is a need for better animal models of human health problems. Fig 1-3 concern the surgery and functional recovery profile of a rat model of knee replacement. Fig 6 reports RNA-seq data identifying upregulated transcripts and pathways in the acute postoperative period. My main concern is that the appropriate controls are missing from the figures seeking to demonstrate that clinically utilized perioperative analgesics can reduce the behavioral measures of recovery (Fig 4), and to investigate if the recovery from knee replacement in animals with preexisting OA is altered (Fig 5). The absence of these controls impacts on their validity. A vehicle control is needed in Fig 4 and an OA - sham group needs to be added to Fig 5. minor points: I prefer a graph reporting inflammatory mediator changes. Are the levels you measured over the recovery period what you expected based on previously published data? Fig 4E, why is the before morphine SFI elevated? is this significant cf to before index for the other two groups? Reconsider how to frame the abstract/introduction and start of discussion. I am not convinced that the severe post-TKA pain state observed in people with OA following TKA is the most compelling argument to use, and the data presented doesn't address the need for larger doses of analgesics at all. Take care to use the correct terminology when discussing the interpretation of the behavioral outputs measured. Changes in locomotion, rearing and gait are not "pain" per se. The argument that functional recovery is only partially improved by analgesia would be clearer if incapacitance, SFI, and stride length values from sham animals were somehow indicated in fig 4 Include more information about why DMM was chosen as a OA model and state how long it persists (is the incapacitance level reported in Fig 5C stable for the whole duration of the experimental protocol) The axis text in Fig 6C is blurred and can not be read in the version supplied. What transcripts were down-regulated in your study? Would you expect morphine/ketorolac treatment to fully restore incapacitance? In addition to being a pain associated behavior, what else does it capture? 436: .....may reflect reduced endogenous opioid tone leading to enhanced pain sensitivity - but see https://doi.org/10.1016/j.neuron.2021.04.011 and https://doi.org/10.1016/j.neuron.2021.03.012 ****** 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.0278632.r001
Revision 1
18 Oct 2022 Author Response ¬¬¬Reviewer 1 A shorter and more capacious name that meets modern trends: "Acute postoperative pain and dorsal root ganglia transcriptomic signatures following TKA in rats: experimental study" We have changed the title in accordance with the reviewer’s suggestion. In the introduction and relevance, it is better not to refer to papers that are 15 years old, it is better to find references up to 5 years old. There are also recent studies regarding the level of pain after TKA, for example DOI: 10.1007/s00167-017-4713-5 The older citations have been removed and newer citations added, including the suggested study on post TKA pain. Where is the conclusion section? The results must be summarized. A conclusion section has been added after the discussion. Line 474 – What document is the link to? And then the URL: ... The URL was to a Centers for Disease Control website. This has been removed and a similar manuscript citation has been added. A level of evidence needs to be added. The level of evidence is II and this has been added to the end of the abstract. Reviewer 2 My main concern is that the appropriate controls are missing from the figures seeking to demonstrate that clinically utilized perioperative analgesics can reduce the behavioral measures of recovery (Fig 4), and to investigate if the recovery from knee replacement in animals with preexisting OA is altered (Fig 5). The absence of these controls impacts on their validity. A vehicle control is needed in Fig 4 and an OA - sham group needs to be added to Fig 5. The reviewer makes a valid point; however, these analyses were designed as a within group pre- to post-drug response and not as a between group vehicle control study. Nevertheless, we did collect vehicle data for incapacitance, which expectedly showed no effect. These data have been added to the revised figure 5 (previously figure 4). Unfortunately, vehicle controls were not collected on the SFI and stride length analyses, so these could not be added. However, saline was used as a vehicle for all of the drugs employed and thus would not be expected to alter SFI or stride length. As for the OA-sham in Figure 6 (previously figure 5), the intent of this series of experiments was to determine if preexisting OA influenced postoperative pain and recovery following TKA surgery by performing a within group comparison to presurgical baseline values. We elected to use a sham+sham control to determine if repeated surgical procedures affected our outcomes, and it did not. We did not actually compare sham+sham to OA+sham. Moreover, this study was not designed to compare TKA surgery to the natural progression of OA to see if surgery alleviates long-term pain and function. However, this is an intriguing idea that we may explore in a subsequent study. Minor points: I prefer a graph reporting inflammatory mediator changes. Are the levels you measured over the recovery period what you expected based on previously published data? Fig 4E, why is the before morphine SFI elevated? is this significant cf to before index for the other two groups? We have changed table 1 to the new figure 4. As for the apparent before morphine elevation, we believe that this merely represents variability observed in this model. This also supports our decision to use a within group pre-post design so that such differences do not adversely impact our results. We did compare the before levels between all groups and they did not significantly differ. We identified a reference to IL-6 elevation similar to what we observed for a slightly different rat knee surgical model and have added that to the discussion. TNFa is elevated in many OA models, but we could find no references to it after surgical procedures in rats. Reconsider how to frame the abstract/introduction and start of discussion. I am not convinced that the severe post-TKA pain state observed in people with OA following TKA is the most compelling argument to use, and the data presented doesn't address the need for larger doses of analgesics at all. We have made minor modifications to these sections; however clinical data support the assertion that pain is higher in TKA patient than other surgeries and this is one of the reasons for the development of this model. We have removed the reference to the clinical use of larger doses of analgesics. Take care to use the correct terminology when discussing the interpretation of the behavioral outputs measured. Changes in locomotion, rearing and gait are not "pain" per se. We agree that these are surrogates of pain and have modified our manuscript accordingly. The argument that functional recovery is only partially improved by analgesia would be clearer if incapacitance, SFI, and stride length values from sham animals were somehow indicated in fig 4 The graphs in figure 5 (previously fig 4) now have lines indicating mean sham values added to them. Include more information about why DMM was chosen as a OA model and state how long it persists (is the incapacitance level reported in Fig 5C stable for the whole duration of the experimental protocol) Justification for the DMM model has been added to the Methods section under DMM surgery and is supported by a new reference. We did not continue to measure incapacitance levels beyond 4 weeks for this experiment. However, this model has been studied out to 12 weeks and shows no further changes in incapacitance after 4 weeks (PMID: 27856294, 34136155). The axis text in Fig 6C is blurred and can not be read in the version supplied. What transcripts were down-regulated in your study? The font sizes of axes have been increased and now appears clearer. In addition, downregulated transcripts have been added to the volcano plot. These are also listed in Table 1 (formerly Table 2). Would you expect morphine/ketorolac treatment to fully restore incapacitance? In addition to being a pain associated behavior, what else does it capture? Our goal in combining morphine/ketorolac was to model commonly used perioperative analgesics. We hypothesized that the combination would be more efficacious than either drug alone but did not expect them to fully restore incapacitance. Incapacitance may also capture differences in proprioception and nerve dysfunction leading to differential weight bearing. 436: .....may reflect reduced endogenous opioid tone leading to enhanced pain sensitivity - but see https://doi.org/10.1016/j.neuron.2021.04.011 and https://doi.org/10.1016/j.neuron.2021.03.012 We are indeed aware of this study and agree that in contrast to the enhanced opioid tone observed upon NaV1.7 deletion, we do not know whether endogenous opioids contribute to pain in our model. Nevertheless, our transcriptomic data offer a tantalizing suggestion of altered opioid function and therefore we highlighted this possibility. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0278632.r002
14 Nov 2022 Decision Letter - Antal Nógrádi, Editor PONE-D-22-16993R1Acute postoperative pain and dorsal root ganglia transcriptomic signatures following total knee arthroplasty (TKA) in rats: An experimental studyPLOS ONE Dear Dr. Kaczocha, 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. Both reviewers have raised minor points. The correction of these points would further improve the manuscript. Please amend the paper according to their suggestions. Please submit your revised manuscript by Dec 29 2022 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. 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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. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Good job on paper! The only thing for the correct presentation in the reference databases and the correct presentation of the work is to categorize the abstract (aim, materials&methods, results, etc.). Reviewer #2: Thanks to the authors for responding to concerns raised and clarifying some aspects in the text. Fig 6 - especially 6C is still very difficult to read and needs to be improved for publication. Should Orthop Surg Res. 2021 Dec 20;16(1):726. doi: 10.1186/s13018-021-02871-y. be referenced in the introduction? ****** 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.0278632.r003
Revision 2
15 Nov 2022 Author Response ¬¬¬Reviewer 1 Good job on paper! The only thing for the correct presentation in the reference databases and the correct presentation of the work is to categorize the abstract (aim, materials&methods, results, etc.). We thank the reviewer for this suggestion. We would like to note that our Abstract is formatted in accordance with journal guidelines (i.e., not categorized). Reviewer 2 Thanks to the authors for responding to concerns raised and clarifying some aspects in the text. Fig 6 - especially 6C is still very difficult to read and needs to be improved for publication. Should Orthop Surg Res. 2021 Dec 20;16(1):726. doi: 10.1186/s13018-021-02871-y. be referenced in the introduction? In accordance with the reviewer’s suggestion, we have enlarged the graphs presented in panel C. We have also included the aforementioned reference (now #17) in the introduction. https://doi.org/10.1371/journal.pone.0278632.r004
21 Nov 2022 Decision Letter - Antal Nógrádi, Editor Acute postoperative pain and dorsal root ganglia transcriptomic signatures following total knee arthroplasty (TKA) in rats: An experimental study PONE-D-22-16993R2 Dear Dr. Kaczocha, 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, Antal Nógrádi, M.D., Ph.D., D.Sc. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0278632.r005
Formally Accepted
23 Nov 2022 Acceptance Letter - Antal Nógrádi, Editor PONE-D-22-16993R2 Acute postoperative pain and dorsal root ganglia transcriptomic signatures following total knee arthroplasty (TKA) in rats: An experimental study Dear Dr. Kaczocha: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Antal Nógrádi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0278632.r006

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