Peer Review History
Original SubmissionMay 31, 2023 |
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PONE-D-23-16864An ex vivo rat trachea model reveals abnormal airway physiology and a gland secretion defect in cystic fibrosisPLOS ONE Dear Dr. Rowe, 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 Aug 19 2023 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:
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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 Reviewer #3: Yes ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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 Reviewer #3: 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 Reviewer #3: 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 “An ex vivo rat trachea model reveals abnormal airway physiology and a gland secretion defect in cystic fibrosis” describes development of an in vitro method to maintain excised tracheas for at least three weeks. This enables access to the tissue for pharmacological manipulation for longer times than fresh tissue while maintaining the cystic fibrosis phenotype of CFTR-/- rat tracheas. The major advantage with this approach compared to traditional ALI cultures is that this preparation includes submucosal glands. This enables the study of submucosal gland derived bundled strands and how they contribute to mucociliary clearance. As stated in the title, the preparation can also be used to study how mucus is altered in cystic fibrosis, where the bundled strands contribute to the pathology. The results presented are of great interest not only for scientists studying cystic fibrosis, but also other fields where the tracheal epithelium is studied. The results are in context of previous literature and the references are fair. The conclusions are supported by the results and the methods are described in enough detail for reproduction. The manuscript is very clear in organization and language. There are only some minor concerns which need to be addressed: 1: In line 287, it says: however the difference in MCT between CFTR−/− trachea (0.1 ± 0.1 mm/min) compared to WT (0.9 ± 0.7 mm/min; P<0.01; Figure 3E) was exaggerated after carbachol stimulation (Videos 5-6, Figure 3B,E). Quantification of MCT for each trachea showed a significant deficit in MCT after stimulation in CFTR−/− trachea (0.1 ± 0.1 mm/min) compared to WT (0.9 ± 0.7 mm/min; P<0.01; Figure 3E). The numbers given are the same for unstimulated and stimulated tracheas, but in the figure it is clear that the numbers are different. There must be a mistake somewhere. 2: In line 513, the text reads: Using PAS, we show that ex vivo tracheae provide roughly 4.7X the amount of mucin, the primary constituent of mucus, compared to fresh tracheae (Figure 6D-E), ruling out the possibility that the increase in pooled ASL and mucus protein from ex vivo tracheae is an artifact of an accumulation of fluid or cellular debris. This conclusion is most likely correct, but there is no explanation given for why there would be more mucus in the cultured tracheas. Was the number of goblet cells increased during culture? Was the number of mucus-producing cells in the glands increased? Is the explanation instead that mucus is collecting on the cultured trachea that would be removed by mucociliary clearance in the live animal? This deserves some further discussion. Reviewer #2: Harris et al. have set out a rat trachea model to study the abnormalities of the CF respiratory tract ex vivo. The paper describes a convincing, very carefully detailed method to isolate and maintain tracheae for ex vivo experimentation and drug testing. Main concerns: 1. Methods, Lines 180-185: This part can sound a bit confusing for someone who is not well acquainted with the topic. Have the whole tracheae undergone fixation and paraffinization for histopathology or just a part of each trachea? Also, step 4 says that µOCT was performed in freshly excised tracheae, which, I suppose, had not yet been processed for histological analysis. Kindly clarify the order in which these analyses – histopathology and imaging – were performed. 2. Results: When downloaded, some figures have dark backgrounds, which make them difficult to see. Please make sure that all figures are visible in case the paper is sent for another review round. 3. Lines 293-295: The word “trend” does not sound as the most appropriate. In general, trend is used to describe a change over time or, if much, a pattern after repeated measurements with different sample. In the present study, only a few animals have been used, which is in accordance with ethical protocols, so the amount may not be representative enough to point out any trend. 4. Figure 2: Were the experiments in Figure 2 conducted with CFTR-/- tissue or with WT tissue? If conducted with WT tissue, how long were the CFTR-/- tracheae viable? 5. Discussion: Please elaborate on the added value, difference and importance of this model compared to precision cut lung slices. 6. Please elaborate (discussion) on the contribution of trachea impairment in CF compared to the rest of the lung. Minor concerns: 1. This is merely technicality, but, overall, the authors mix the Results section with data interpretation, which, in turn, should be in the Discussion section. I would advise the authors to order the results in terms of what the authors consider most important, only with the description of the results. Likewise, the authors can interpret these findings in the Discussion section by following the same order in the result. This can make it easier to the reader to follow the authors’ train of thought. 2. Please avoid repetitive statements in the M&M (e.g., first sentence of 2.1 and 2.2). 3. The quality of the representative image of Figure S2 of the WT animal is not comparable to the CFTR-/- animal and should be replaced. 4. Include full names of abbreviations at the end of every figure legend as figure should be able to stand alone without reading the main text. 5. Please specify whether the ducts analyzed for Figure 4 were analyzed in the same trachea region (proximal vs. distal). 6. Line 325: small typing error “previously” should read “previous” 7. For Figure 4: please provide a supplemental figure showing clear ducts in both WT and CFTR-/- tracheae after the washing protocol. Reviewer #3: Introduction Lines 60- 61, page 9 – the statement “and not easily amenable to genetic modification or longitudinal studies” should be softened. There are several genetically modified pigs and ferrets available. The demand for genetically modified models in these species may be less, but that doesn’t mean they are not amenable to genetic modification. Similarly, there several longitudinal studies in ferrets and pigs. It is recommended that this statement be softened or re-worded. Methods 2.4 – is the washing procedure adequate to remove mucus from the CFTR-/- rat airway? For example, mucus is likely more adherent in CFTR-/- rat airways. 2.5 – has the equation listed been validated? Meaning, have the expected concentrations of drugs using that equation been measured in the media and/or in the tissues? 6.0 PAS Slot Blot – Is it assumed that PAS is only detecting secreted mucins and not other glycoproteins or glycolipids? Additionally, PAS since only detects neutral mucins, are conclusions draw about secreted mucins accurate (since some literature suggests that mucins in CF may be more acidic in form, and would be detected by Alcian blue preferentially. Results Were sex differences noted? The images in Figure 2 for ex vivo trachea give impression there is more vacuoles in the trachea – is that a true observation or just due to the image selected? Discussion Nicely written. A recommendation is to include a limitations paragraph. For example, one limitation is that ex vivo preps do not allow for studying the interaction of the epithelia with the immune cells, unless immune cells are added. Additionally, though cholinergic responses remained intact, there will be a loss of extrinsic neural innervation, which is important for surface epithelia and submucosal gland function. ********** 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 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.] 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. |
Revision 1 |
An ex vivo rat trachea model reveals abnormal airway physiology and a gland secretion defect in cystic fibrosis PONE-D-23-16864R1 Dear Dr. Steven M. Rowe, 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, Abdelwahab Omri, Pharm B, Ph.D, Laurentian University, Canada Academic Editor PLOS ONE |
Formally Accepted |
PONE-D-23-16864R1 An ex vivo rat trachea model reveals abnormal airway physiology and a gland secretion defect in cystic fibrosis Dear Dr. Rowe: 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. Abdelwahab Omri Academic Editor PLOS ONE |
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