PONE-D-20-06026

Combined abdominal heterotopic heart and aorta transplant model in mice

PLOS ONE

Dear Dr. Wong,

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.

We would appreciate receiving your revised manuscript by May 16 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Robert Jeenchen Chen, MD, MPH

Academic Editor

PLOS ONE

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2.  Thank you for stating in your Funding Statement:

'This work is supported, in part, by the International Society for Heart and Lung

Transplantation (ISHLT) Joel D. Cooper Career Development Award (BWW).'

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

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Additional Editor Comments (if provided):

Please address the issues and concerns from the reviewers.

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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: 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: “Combined Abdominal Heterotopic Heart and Aorta Transplant Model in Mice” is a manuscript that presents a novel microsurgical technique intended to improve upon the heart and aorta/carotid graft. The presented technique improves on the existing one because it has shorter graft ischemic times and is centered in only one body cavity. This model and the existing model are important as it may be used to recapitulate the clinical process of aortic vasculopathy. The methodology of the surgery is meticulously explained, and the statistics are appropriately performed. The illustrations are of good quality and add to the overall work. The discussion content is well thought-out and provides context for the study. This paper is marred by poor grammar, and since PLOS one does not provide proofreading and copyediting support, I do not believe it is publishable in its current state. I also suggest the following minor revisions for this manuscript in order to consider it for publication:

It would be helpful to the reader to provide the true distributions of the times for the operation (ischemic time, harvesting time), instead of reporting the approximate times. The authors should present the median time and the distribution (this could be represented graphically as well).

The validation of the new surgical method should be more adequately described. The authors write, “we subsequently performed allografts using Balb/c donor aorta and hearts transplanted into C57Bl/6 recipients in the presence of double co-stimulatory blockade”. The number of replicates of this procedure should be detailed, and it should be noted that the histologic examination of the aorta and cardiac graphs (6a and 6b) are representative of these experiments, if that is in fact the case.

Please add citations for the following sentence:

• Chronic allograft rejection remains the leading cause of graft loss one-year post-transplantation.

PLOS one does not provide any copyediting and proofreading service. There are multiple grammatical errors found in this manuscript. I believe that it is not publishable in its current state. I would suggest proofreading the article again. Examples of the many grammatical errors found throughout the paper are listed below:

• The result has further heightened the need for a parenchymal organ in combination with aorta transplantation in exploring clinic(al) relevance of CAV.

• Our results demonstrate that this new model is characterized by the reliable reproducibility.

oShould this sentence include “the”?

• After 1 minute for the systemic heparinization, an aortotomy of abdominal aorta is made to decompress the blood circulatory system,

othis should be an aortotomy of “the” abdominal aorta

• The IVC and the right superior vena cava (SVC) were proximally ligated with 7-0 sutures, respectively.

oIn this case respectively does not make sense.

Reviewer #2: Dear authors,

Thank you for submitting your manuscript titled "Combined abdominal heterotopic heart and aorta transplant model in mice" to PLOS ONE. It was a very well written manuscript and I commend you on accomplishing this very difficult microsurgery. Your description of the operation, as well as the diagrams truly help the reader follow along the operation. I do have a couple questions :

1. Have you compared this technique to the "conventional" heterotopic heart transplantation in mice? As in, have you compared the histology, alloresponses, coronary inflammation, etc between the techniques. This technique seems more difficult, with more anastomoses. If everything is similar, is there a reason to do this technique?

2. Unlike other solid organs, the heart is a dynamic structure that can fail with improper preload and afterload management which is critical post-transplantation. This can affect immunosuppression. Since this technique creates an organ in series, it may not be representative of the pressures seen in a real transplant. This may not matter in a liver for example. Do you think that since the heart is not exposed the similar pressures as in an orthotopic heart transplant that this technique may not be the best to address CAV? Please address. I think this is a major limitation of such models.

3. Despite heparin, there could still be issues with thrombosis. How can you tell if the coronaries are patent in the transplanted heart? Is the mouse continued on anticoagulation?

4. Do the transplant hearts go into arrhythmia?

5. Please review grammar and syntax.

Thank you very much for a very interesting manuscript.

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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.

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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 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.

PONE-D-20-06026R1

Combined abdominal heterotopic heart and aorta transplant model in mice

PLOS ONE

Dear Dr. Wong,

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.

==============================

Before we accept the manuscript, please address the issue found by one reviewer:

At the end of the first paragraph of the results the following sentence is hard to understand: “Survival of both the syngeneic aorta and heart graft was greater than 90 days.” Is this referring to all of the mice that survived the procedure?

I do not believe that the graft survival should be presented as 6/6 in the second paragraph of the results (since you are referring to three cardiac graphs and three aortic graphs). I think it should be presented as 3/3 cardiac graphs and 3/3 aortic graphs.

==============================

We would appreciate receiving your revised manuscript by Jul 03 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.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Robert Jeenchen Chen, MD, MPH

Academic Editor

PLOS ONE

[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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: 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

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

**********

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

Reviewer #3: 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

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors of Combined abdominal heterotopic heart and aorta transplant model in mice present a much improved study detailing a new microsurgical technique aimed at studying allograft vasculopathy. My prior comments on their manuscript have been adequately addressed; the grammar is much improved and the distributions of the procedure times are adequately detailed for the reader. The article’s introduction and discussion have been largely re-written, and the article is now fit for publication. Below, I have two comments for the authors to consider.

At the end of the first paragraph of the results the following sentence is hard to understand: “Survival of both the syngeneic aorta and heart graft was greater than 90 days.” Is this referring to all of the mice that survived the procedure?

I do not believe that the graft survival should be presented as 6/6 in the second paragraph of the results (since you are referring to three cardiac graphs and three aortic graphs). I think it should be presented as 3/3 cardiac graphs and 3/3 aortic graphs.

Reviewer #2: Dear authors,

Thank you for submitting your manuscript titled "Combined abdominal heterotopic heart and aorta transplant model in mice" to PLOS ONE. I would like to also thank you for the time and effort spent on the revisions. It is evident that significant portions of this manuscript has been revised. Grammatical errors have been addressed and the overall ease of reading has improved. Furthermore, you have addressed all of the comments submitted by myself and my co-reviewer in a very thorough manner. Please ensure grammar and syntax are appropriate. I would like to accept this manuscript.

Thanks

Reviewer #3: The methodology of the surgery is meticulously explained with nice schemes and photos. I have no further comment to be addressed.

**********

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

Reviewer #3: 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.

Combined abdominal heterotopic heart and aorta transplant model in mice

PONE-D-20-06026R2

Dear Dr. Wong,

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,

Robert Jeenchen Chen, MD, MPH

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #3: 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 #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: N/A

**********

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 #3: 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 #3: 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: No Further Comments. The authors have sufficiently revised the manuscript. It is now fit for publication in Plos One.

Reviewer #3: Authors revised the manuscript in accordance with previous reviewers' comments.

I have no further comment to be addressed.

**********

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 #3: No