Comparing animal well-being between bile duct ligation models

Guanglin Tang; Wiebke-Felicitas Nierath; Emily Leitner; Wentao Xie; Denis Revskij; Nico Seume; Xianbin Zhang; Luise Ehlers; Brigitte Vollmar; Dietmar Zechner

doi:10.1371/journal.pone.0303786

Peer Review History

Original SubmissionSeptember 17, 2023
23 Nov 2023 Decision Letter - Muhammad Salman Bashir, Editor PONE-D-23-30237Comparing animal well-being between bile duct ligation modelsPLOS ONE Dear Dr. Zechner, 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 Jan 07 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, Muhammad Salman Bashir, M.S.C 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. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free. Upon resubmission, please provide the following: The name of the colleague or the details of the professional service that edited your manuscript A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a supporting information file) A clean copy of the edited manuscript (uploaded as the new manuscript file) 3. Thank you for stating the following financial disclosure: “GT, WFN, WX, EL, DZ and BV were supported by the Deutsche Forschungsgemeinschaft (DFG research group FOR 2591, ZE 712/1-1, ZE 712/1-2, VO 450/15-1 and VO 450/15-2). LE and DR received funding through the research project “EnErGie” by the European Social Fund (ESF; reference: ESF/14-BM-A55-007/18) and the Ministry of Education, Science, and Culture of Mecklenburg-Vorpommern.” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: “The authors are grateful for the perfect technical assistance from Berit Blendow and Dorothea Frenz. This study was supported by the Deutsche Forschungsgemeinschaft (DFG research group FOR 2591, ZE 712/1-1, ZE 712/1-2, VO 450/15-1 and VO 450/15-2). LE and DR received funding through the research project “EnErGie” by the European Social Fund (ESF; reference: ESF/14-BM-A55-007/18) and the Ministry of Education, Science, and Culture of Mecklenburg-Vorpommern.” We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “GT, WFN, WX, EL, DZ and BV were supported by the Deutsche Forschungsgemeinschaft (DFG research group FOR 2591, ZE 712/1-1, ZE 712/1-2, VO 450/15-1 and VO 450/15-2). LE and DR received funding through the research project “EnErGie” by the European Social Fund (ESF; reference: ESF/14-BM-A55-007/18) and the Ministry of Education, Science, and Culture of Mecklenburg-Vorpommern.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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: In the manuscript "Comparing Animal Well-being between Bile Duct Ligation Models", Guanglin Tang et al. compared the differences between cBDL and pBDL mice. They found that pBDL animals had a significantly higher survival rate and their well-being was significantly improved when compared to cBDL animals. After 14 days of ligation, liver enzymes such as aspartate and alanine aminotransferase, alkaline phosphatase, and glutamate dehydrogenase were significantly elevated after cBDL, but only glutamate dehydrogenase activity was increased after pBDL. However, several concerns regarding the research must be clarified. (1) The authors should provide serum levels of TBA (total bile acids), TBIL (total bilirubin), IBIL (indirect bilirubin), and DBIL (direct bilirubin). These markers are commonly used to assess the degree of cholestasis in animals. (2) The authors should provide inflammatory and fibrotic markers from cBDL and pBDL mice. Inflammation and fibrosis are common pathophysiological processes in biliary obstruction, and analysis of these markers will further support the differences between the two models. (3) The authors should provide data on the levels of Mrp2, Mrp3, Mrp4, Bsep, Ntcp, Cyp7a1, Cyp8b1, and Cyp27a1 mRNA in total liver tissue. This is important because these genes encode enzymes involved in bile acid synthesis and transport, and their expression levels can provide insights into the extent of cholestasis and liver injury in the two models. By comparing the expression levels of these genes between cBDL and pBDL mice, the authors can further validate their hypothesis that pBDL results in less severe liver damage compared to cBDL. Therefore, it is essential to include this information in the manuscript to strengthen the study's conclusions. (4) This study may have some value in certain aspects, but overall, it lacks innovation. Reviewer #2: The common bile duct ligation (cBDL) is the most used animal model to mimic hepatic diseases such as obstructive cholestasis, primary biliary or sclerosing cholangitis and acute liver injury. Recently, it has been introduced a modification in this model, just the left hepatic bile duct is ligated creating a partial bile duct ligation (pBDL). Within the left lobe pBDL causes similar conditions of cBDL like necrosis, inflammation and fibrosis. The Authors aimed to compare animal wellbeing between cBDL and pBDL mice, demostrating that mice after pBDL show less distress and a better survival than mice after cBDL. The manuscipt is interesting but some points need to be improved: - Regarding the morphological study of the heaptic tissue, in the comparison of liver necrosis and fibrosis (Figure 6), it is important to add pictures and data of cBDL and unligated lobe or control tissue. In general, they should insert the study even of the right lobe to exclude any type of damage in that side compared the left one. - In the same Figure 6, the images of sirius red are very out of focus, requiring higher contrast to better appreciate the differences between the various samples. - The Discussion section needs to be rewrite, in the present form is full of repetitive concepts. The Authors should semplify it by discussing and speculating the points presented in a gradual and progressive way. - It is not completely clear the reason why the pBDL animal model could be used to study local features of the mentioned hepatic diseases. Please, clarify better this point in the Discussion section. - When they observed that the necrosis in pBDL + pAL animal model is caused by interrupting of arterial blood supply and, at the same time, induce cholestasis, they should insert the important role of the Peribiliary Plexus (PBP) which supply bile ducts originating from the hepatic artery. - Since the crucial point of the study is the ligation of the left hepatic bile duct, it would be really important to add pictures of the surgical procedure. - Several typos are present along the text, please revise it carefully. ****** 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.0303786.r001
Revision 1
22 Apr 2024 Author Response Reply to reviewer 1 Thank you very much for all of your valuable suggestions to improve the manuscript. We have followed all of your recommendations and incorporated a significant amount of additional data. This process has expanded the number of figures and supplemental figures from 9 in the original manuscript to 20 in the current version. In order to effectively convey the main messages of our study, we restructured the results section of our manuscript into three blocks. We now compare CBDL to pBDL in Fig 1 and S1 Fig. We compare cBDL to the v-pBDL animal model in SFig 2-4 and Fig 2-4. And we compare the v-pBDL to the pBDL+pAL animal model (both ligated as well as unligated liver lobes) in SFig 5-9 and Fig 5-9. I hope that these additional experiments are now sufficient for acceptance as a publication in PLOS ONE. For a point to point reply please see text below. Point 1 from Reviewer 1: The authors should provide serum levels of TBA (total bile acids), TBIL (total bilirubin), IBIL (indirect bilirubin), and DBIL (direct bilirubin). These markers are commonly used to assess the degree of cholestasis in animals. Answer: For comparing cBDL to the v-pBDL animal model we now present TBA (total bile acids) and TBIL (total bilirubin) in figure 3E and figure 3F and IBIL (indirect bilirubin), and DBIL (direct bilirubin) in supplemental figure 4. For comparing v-pBDL to pBDL+pAL we now present TBA (total bile acids) and TBIL (total bilirubin) in figure 7E and figure 7F and IBIL (indirect bilirubin), and DBIL (direct bilirubin) in supplemental figure 9. Point 2 from Reviewer 1: The authors should provide inflammatory and fibrotic markers from cBDL and pBDL mice. Inflammation and fibrosis are common pathophysiological processes in biliary obstruction, and analysis of these markers will further support the differences between the two models. Answer: For comparing cBDL to the v-pBDL animal model we now present fibrotic (collagen I deposition) and inflammatory markers (CAE+ cells and TNFa as well as IFNg expression) in figure 2E-L, and supplementary figure 3. For comparing the ligated liver lobes of v-pBDL to pBDL+pAL nice we now present fibrotic (collagen I deposition) and inflammatory markers (CAE+ cells and TNFa as well as IFNg expression) in figure 6D-I, and supplementary figure 7. For comparing the unligated liver lobes of v-pBDL to pBDL+pAL mice we now present fibrotic (collagen I deposition) and inflammatory markers (CAE+ cells and TNFa as well as IFNg expression) in supplemental figure 6D-I, and supplementary figure 8. Point 3 from Reviewer 1: The authors should provide data on the levels of Mrp2, Mrp3, Mrp4, Bsep, Ntcp, Cyp7a1, Cyp8b1, and Cyp27a1 mRNA in total liver tissue. This is important because these genes encode enzymes involved in bile acid synthesis and transport, and their expression levels can provide insights into the extent of cholestasis and liver injury in the two models. By comparing the expression levels of these genes between cBDL and pBDL mice, the authors can further validate their hypothesis that pBDL results in less severe liver damage compared to cBDL. Therefore, it is essential to include this information in the manuscript to strengthen the study's conclusions. Answer: For comparing cBDL to the v-pBDL animal model we now present data on Mrp2, Mrp3, Mrp4, Bsep, Ntcp, Cyp7a1, Cyp8b1, and Cyp27a1 mRNA in total liver tissue in figure 4A-H. For comparing the ligated liver lobes of v-pBDL to pBDL+pAL nice we now present data on the expression of these genes in figure 8A-H. For comparing the unligated liver lobes of v-pBDL to pBDL+pAL mice we now present the expression of these genes in supplemental figure 9A-H. Point 4 from Reviewer 1: This study may have some value in certain aspects, but overall, it lacks innovation. Answer: We had already the following completely novel aspects in the original publication: We could demonstrate that pBDL causes significant less distress than cBDL and leads to a higher survival rate of the animals. In addition, we described for the first time pathological consequences when ligating the left bile duct with the corresponding left hepatic artery. In the revised manuscript we added a more detailed comparison between the three animal models, cBDL, pBDL and pBDL+pAL (the pBDL+pAL model is completely novel) offering among other things gene expression regulation of bile acid synthesizing enzymes and bile acid transporter proteins (this is to our knowledge completely novel for the pBDL and pBDL+pAL animal models). Reply to reviewer 2 Thank you very much for all of your valuable suggestions to improve the manuscript. We have followed all of your recommendations and incorporated a significant amount of additional data. This process has expanded the number of figures and supplemental figures from 9 in the original manuscript to 20 in the current version. In order to effectively convey the main messages of our study, we restructured the results section of our manuscript into three blocks. We now compare CBDL to pBDL in Fig 1 and S1 Fig. We compare cBDL to the v-pBDL animal model in SFig 2-4 and Fig 2-4. And we compare the v-pBDL to the pBDL+pAL animal model (both ligated as well as unligated liver lobes) in SFig 5-9 and Fig 5-9. I hope that these additional experiments are now sufficient for acceptance as a publication in PLOS ONE. For a point to point reply please see text below. Point 1 from Reviewer 2: Regarding the morphological study of the hepatic tissue, in the comparison of liver necrosis and fibrosis (Figure 6), it is important to add pictures and data of cBDL and unligated lobe or control tissue. In general, they should insert the study even of the right lobe to exclude any type of damage in that side compared the left one. Answer: For comparing cBDL to the v-pBDL animal model we now present pictures and data on liver necrosis, fibrosis and CAE staining in figure 2A-L. For comparing the ligated liver lobes of v-pBDL to pBDL+pAL nice we now present pictures and data on liver necrosis, fibrosis and CAE staining in figure 6A-I. For comparing the unligated liver lobes of v-pBDL to pBDL+pAL mice we now present pictures and data on liver necrosis, fibrosis and CAE staining in supplemental figure 6A-I. Point 2 from Reviewer 2: In the same Figure 6, the images of sirius red are very out of focus, requiring higher contrast to better appreciate the differences between the various samples. Answer: Those images, in my opinion were in focus (however a limitation might be that my eyesight has decreased over time), but the histology was hard to judge, because we did not counterstain the tissue with hematoxylin. In order to make it easier to judge the tissue, we did a collagen I immunohistochemistry and counterstained the tissue with hematoxylin. These images are now presented in figure 2E-G (comparing healthy control liver to liver sections from cBDL and v-pBDL mice), figure 6D-E (comparing the ligated liver lobes of v-pBDL to pBDL+pAL) and , supplemental figure 6D-E (comparing the unligated liver lobes of v-pBDL to pBDL+pAL). Point 3 from Reviewer 2: The Discussion section needs to be rewrite, in the present form is full of repetitive concepts. The Authors should semplify it by discussing and speculating the points presented in a gradual and progressive way. Answer: We shortened, restructured and rewrote the discussion section (line 350-403 and line 444-457). However, we also added a section about the expression of genes involved in in bile acid synthesis and transport, because analyzing those genes was requested by another reviewer (line 404-443). Point 4 from Reviewer 2: It is not completely clear the reason why the pBDL animal model could be used to study local features of the mentioned hepatic diseases. Please, clarify better this point in the Discussion section. Answer: we now clarified (by shortening and restructuring the text) the point that the same local pathophysiological features as observed in the mentioned hepatic diseases can be studied after pBDL (see line 378-386) Point 5 from Reviewer 2: When they observed that the necrosis in pBDL + pAL animal model is caused by interrupting of arterial blood supply and, at the same time, induce cholestasis, they should insert the important role of the Peribiliary Plexus (PBP) which supply bile ducts originating from the hepatic artery. Answer: we inserted the important role of the peripiliary venous plexus when we discussing pBDL + pAL mice (see line 395-399) Point 6 from Reviewer 2: Since the crucial point of the study is the ligation of the left hepatic bile duct, it would be really important to add pictures of the surgical procedure. Answer: We added pictures as supplemental figure 10 (S10 Fig). Point 7 from Reviewer 2: Several typos are present along the text, please revise it carefully. Answer: We corrected the typos throughout the manuscript. Attachments Attachment Submitted filename: Response to Reviewers-2024-03-31-fin.docx https://doi.org/10.1371/journal.pone.0303786.r002
1 May 2024 Decision Letter - Muhammad Salman Bashir, Editor Comparing animal well-being between bile duct ligation models PONE-D-23-30237R1 Dear Dr. Dietmar, 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, Muhammad Salman Bashir, M.S.C Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0303786.r003
Formally Accepted
28 May 2024 Acceptance Letter - Muhammad Salman Bashir, Editor PONE-D-23-30237R1 PLOS ONE Dear Dr. Zechner, 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. Muhammad Salman Bashir Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0303786.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .