Molecular mechanisms of splenectomy-induced hepatocyte proliferation

Andrey V. Elchaninov; Timur Kh. Fatkhudinov; Polina A. Vishnyakova; Maria P. Nikitina; Anastasiya V. Lokhonina; Andrey V. Makarov; Irina V. Arutyunyan; Evgeniya Y. Kananykhina; Anastasiya S. Poltavets; Kirill R. Butov; Igor I. Baranov; Dmitry V. Goldshtein; Galina B. Bolshakova; Valeria V. Glinkina; Gennady T. Sukhikh

doi:10.1371/journal.pone.0233767

Peer Review History

Original SubmissionMarch 31, 2020
14 Apr 2020 Decision Letter - Matias A Avila, Editor PONE-D-20-09227 Molecular mechanisms of splenectomy-induced hepatocyte proliferation PLOS ONE Dear Dr. Elchaninov, 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 all issues raised by the reviewer. We would appreciate receiving your revised manuscript by May 29 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No ****** 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: In this study Andrey Elchaninov and colleagues investigated the outcomes and molecular mechanisms of splenectomy-induced hepatocyte proliferation. The findings are of interest, however lacking sufficient novelty and the general organisation of the paper is not satisfactory to my point of view. Several points should be addressed: 1. All operated animals received Meloxicam (1.0 mg/kg/day) and additionally gentamicin (3.0 mg/kg/day). Did animals from the sham group received the same treatment? 2. Information about the age of rats should be included. 3. Figure 1 - Liver/body weight ratio has to be converted into the form of a percentage (%) - The liver/body weight ratio before operation (0h time point) has to be presented for both groups - Did authors check the hepatosomatic ratio at later time points? 10-14 days after splenectomy? 4. Figure 2 - The quality of H&E and especially Ki67 figures is very low. Moreover, the chosen areas in the liver sections are not representative. Such as, only the control and 24 h H&E images include the hepatic area with central vein and therefore can’t be compared to the rest of images. -The ki67positive cells are not visible. -The quantification of the images should be presented as diagram. 5. The general structure of the paper has to be changed. Figure 2 has to be reorganised. Cell cycle related qPCR has to be included into figure 2. Moreover, authors should perform PCNA and cyclin D Western blots. 6. Figure 3. I don’t recommend to use CK18 as general hepatocytes marker. Instead, the authors can use HNF4α staining for hepatocytes. Another option: authors can perform IHC Ki67 staining. With detail analysis of IHC Ki67 images the proliferating hepatocytes (relatively big, brown round nuclei) can be easily distinguished from nonparenchymal cells. 7. The quality of CD68 IF staining is very low. May be include F4/80 staining can be another option. 8. Please include 0h (before operation) controls in all qPCR 9. The English in the presented manuscript is not of publication quality and require major improvement ****** 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0233767.r001
Revision 1
4 May 2020 Author Response Dear reviewers, Thank you for the detailed and in-depth analysis of the submitted manuscript. We have tried to answer all your questions in detail and to the point. 1. All operated animals received Meloxicam (1.0 mg/kg/day) and additionally gentamicin (3.0 mg/kg/day). Did animals from the sham group received the same treatment? Yes; we specify this in the revision. 2. Information about the age of rats should be included. The corresponding section has been corrected. 3. Figure 1 - Liver/body weight ratio has to be converted into the form of a percentage (%) - The liver/body weight ratio before operation (0h time point) has to be presented for both groups - Did authors check the hepatosomatic ratio at later time points? 10-14 days after splenectomy? The corresponding figure has been corrected. The ratio of liver mass and body weight of experimental rats at later time points (10-14 days after the surgery) was not examined. The observed hepatocyte proliferation was transient, as indicated by the lack of Ki67-positive hepatocytes after day 1, and the ratio of liver mass to body weight for the operated rats remained constant, similarly with the control animals. 4. Figure 2 - The quality of H&E and especially Ki67 figures is very low. Moreover, the chosen areas in the liver sections are not representative. Such as, only the control and 24 h H&E images include the hepatic area with central vein and therefore can’t be compared to the rest of images. -The ki67positive cells are not visible. -The quantification of the images should be presented as diagram. All original illustrations (charts and microphotographs) were uploaded to the Editorial Manager in high quality with a resolution of 300 dpi. The original microphotographs clearly showed the specific staining, as well as characteristic features of liver microanatomy. Apparently, a decrease in the image quality occurred upon subsequent conversion of the image for the Reviewer’s copy of the manuscript. In the revised manuscript, the photographs of H&E stained sections of the liver are replaced with more representative images. 5. The general structure of the paper has to be changed. Figure 2 has to be reorganised. Cell cycle related qPCR has to be included into figure 2. Moreover, authors should perform PCNA and cyclin D Western blots. Figure 2 has been corrected. The western blot analysis was performed for cyclins D1 and E. At the same time, we prefer Ki67 as a proliferation marker for its specificity, since it has been reported to produce fewer false positive signals compared to PCNA [https://www.ncbi.nlm.nih.gov/pubmed/14623936]. 6. Figure 3. I don’t recommend to use CK18 as general hepatocytes marker. Instead, the authors can use HNF4α staining for hepatocytes. Another option: authors can perform IHC Ki67 staining. With detail analysis of IHC Ki67 images the proliferating hepatocytes (relatively big, brown round nuclei) can be easily distinguished from nonparenchymal cells. CK18 is a well-established hepatocyte marker [https://www.ncbi.nlm.nih.gov/pubmed/21420381]; for this reason, we preserved the original illustrations supplementing them with microphotographs showing the combined expression of HNF4α and Ki67 in hepatocytes. 7. The quality of CD68 IF staining is very low. May be include F4/80 staining can be another option. The corresponding figure has been corrected. We selected more representative microphotographs that appropriately illustrate CD68 expression in the liver. F4/80 is a well-established macrophage marker indeed, although it has been predominantly characterized for mice and partially for humans, and not for rats [https://www.ncbi.nlm.nih.gov/pubmed/28455709]. 8. Please include 0h (before operation) controls in all qPCR The corresponding section and figures have been corrected. 9. The English in the presented manuscript is not of publication quality and require major improvement The proofreading and text editing have been performed. Yours faithfully, Dr. Andrey Elchaninov Corresponding Author Dr Andrey Elchaninov, Laboratory of Regenerative Medicine, National Medical Research Center for Obstetrics, Gynecology and Perinatology Named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russia, 4 Oparina Street, Moscow 117997, Russia E-mail: elchandrey@yandex.ru; phone: +7 (916) 8885292 Attachments Attachment Submitted filename: Reply280420.docx https://doi.org/10.1371/journal.pone.0233767.r002
13 May 2020 Decision Letter - Matias A Avila, Editor Molecular mechanisms of splenectomy-induced hepatocyte proliferation PONE-D-20-09227R1 Dear Dr. Elchaninov, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Matias A Avila, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0233767.r003
Formally Accepted
15 May 2020 Acceptance Letter - Matias A Avila, Editor PONE-D-20-09227R1 Molecular mechanisms of splenectomy-induced hepatocyte proliferation Dear Dr. Elchaninov: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Matias A Avila Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0233767.r004

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