Development of a constant pressure perfused ex vivo model of the equine larynx

Sven Otto; Jule K. Michler; Stefan Dhein; Christoph K. W. Mülling

doi:10.1371/journal.pone.0251530

Peer Review History

Original SubmissionFebruary 8, 2021
9 Apr 2021 Decision Letter - Maurizio Mandalà, Editor PONE-D-21-04296 Development of a constant pressure perfused ex vivo model of the equine larynx PLOS ONE Dear Dr. Michler 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 10th April 2021. 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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We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 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: 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 ****** 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: This manuscript presents for the readership of Plose One interesting results of an experimental study on tissue survival versus hypoxia of postmortem excised equine laryngeal organs during the course of a 6-hour in vitro perfusion. This manuscript represents original research. A pilot study on this project was published as a case study in the congress proceedings of a BMT (Biomedical Engineering) congress in 2013 (see reference 42). The five larynxes were perfused with an oxygen-enriched, body-warm Tyrode saline solution at constant pressure via the superior laryngeal arteries. Venously, the irrigation solution ran into the laryngeal surrounding basin.Addition of vasoactive substances demonstrated vitality of blood vessels with preservation of vascular regulation of feeding arteries in both directions (vasoconstriction and vasodilatation) and their reversibility. The hypoxia marker lactate dehydrogenase (LDH), which can be determined in the perfusion circuit, was determined three times (at the beginning, after changing the perfusion fluid (after approximately 2 h), and at the end of the experiment). The first two measurements were still in the upper normal range. At the end, LDH levels increased statistically significantly, but did not reach the level of a non-perfused larynx. Thus, anaerobic metabolism occurred after the 2h value. As the authors correctly note, continuous LDH measurements would have been helpful to more accurately document the transition to anaerobic metabolism. In addition, tissue hypoxia was determined at the end of the experiment in a very elaborate manner using histological examinations of the only vocal fold abductor muscle of the equine larynx, the cricoarytenoideus dorsalis muscle (CAD), with the aid of an immunofluorescent analysis of hypoxia-inducible factor (HIF-1α) adapted specifically for this study.For this purpose, an additional positive control had to be established in the form of a cell culture. As a result, severe hypoxia damage of the muscle fibers after 6 h of perfusion could be excluded. This information is of great importance for the future use of such an in vitro model as a substitute for in vivo experiments, e.g. for electrical stimulation of vocal fold motility. A major future application of such an in vitro perfusion model of mammalian larynxes will be its use for surgery simulation and for testing new dynamic therapeutic approaches, such as functional electrical stimulation (FES). This experimental study demonstrated that electrical stimulation of still-vital nerve endings in CAD muscle at the end of the experiment could still elicit a muscle contraction comparable to the initial one, as measured by an intramuscular pressure sensor. In my eyes, this is the most significant statement of this work. It is therefore recommended that the authors emphasize this aspect more strongly in the discussion. The stimulation parameters used cannot trigger direct muscle fiber stimulation, thus after 6 h the nerve-muscle unit was still vital. For the future experiments mentioned by the authors to measure the stimulation current propagation in CAD depending on the location, configuration, and size of possible stimulation electrodes, it is of importance how long such a model remains vital under recurrent or continuous electrical stimulation. In my understanding, the supply of both oxygen and substrate nutrients to the tissue then plays a role. This should be added when discussing the change to a fresh blood perfusion model.In addition to current intensity, pulse rate, and pulse duration, the authors were asked to specify the time for which electrical stimulation was applied at the beginning and end of each experiment. For future experiments, it would be important from the point of view of future clinical questions to investigate tissue hypoxia as a function of stimulation duration and current input and to continue the experiment in each case until the model can no longer be used. From this, the maximum useful life of such a model could be determined. Overall, however, this does not diminish the importance of this work, which is a milestone of basic research towards a replacement model for in vivo animal experiments on laryngeal electrostimulation. All Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail.The conclusions, except for the additions to electrostimulation listed above, are adequately presented and supported by the data. The article is presented in an intelligible fashion and is written in standard English. The research meets all applicable standards for the ethics of experimentation and research integrity. The article adheres to the appropriate reporting guidelines and community standards for data availability and can be recommended for publication in Plos One with minor revisions. Reviewer #2: Interesting paper. It appears to be a well designed setup which will hopefully allow further investigation into RLN. Just a few comments. Page 5 line 58, Page 6 line 74- Please elaborate on your experimental setup. I am quite familiar with developing an ex vivo perfusion apparatus and was having a hard time figuring out exactly how the perfusion system works with your paper. What arteries were cannulated? Passive drainage (allowing the veins to drain to the tub below)? Was the larynx immersed in the solution? please be more specific Page 6 line 85- “A pressure transducer (Transducer Model SPR-524, Millar Instruments, Inc., Houston, Texas, USA) was inserted into the medial part of the CAD... The electrical impulses and the change of the intramuscular pressure during contraction were recorded by a PowerLab” Unfortunately a twitch or local contraction as noted on the pressure transducer does not always result in movement of the laryngeal musculature. Was the movement of the CAD noted visually or just via the transducer? ****** 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: Yes:** Prof. Andreas H. Mueller, M.D. 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. 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Revision 1
23 Apr 2021 Author Response Comments raised by Reviewer 1 (Prof. Müller) First, we would like to explicitely thank you for your detailed and appreciative review of our manuscript. 1. Rev. 1 asked for a revision of the discussion towards the CAD contraction at the beginning/end of the experiment. We rewrote this by adding the abducted vocal fold and the perfusion duration (lines 294-295). This is indeed a central finding of this study, but as it already has a front row seat in the discussion, we don´t want to overinterpret this as it is the first study to report such data in a modest number of ex vivo larynges and we tend to judge our data as critical as possible. 2. The next point Rev. 1 recommends is to add information to the outlook about fresh blood perfusion in hindsight of oxygen supply and substrate nutrients. We were happy to do this and rearranged the text accordingly (lines 364-373). 3. Rev. 1 then asked to specify the duration of the electrical stimulation at the beginning and at the end of the experiment which was 8 s (minimum). We supplemented the manuscript with this (material and methods section, line 138). We would like take the opportunity to comment on this factor of central importance to you: of course, one of the logical next steps would consist of stress testing the model by moving the vocal fold for a certain period of time/try different pulses etc. to determine the point where this model reaches its capacity. This will require quite a number of organs, an intelligent sub-grouping and will be complicated by the overall lacking data in this field for ex-vivo larynx perfusion towards the strength of the muscular answer, what is considered a weak answer (etcpp), where the cut-off is... Nevertheless, we absolutely agree that this is something highly desirable! We intendedly used this test to prove our approach as resulting in a functional model and learned during our experiments what a hallmark this relatively simple test presents. Comments raised by Reviewer 2 1. Rev.2 asked for a more detailed description of our set-up. We extended the caption of Fig 1 that explains the experimental set-up and inserted an additional schematic drawing of an equine larynx in order to better illustrate the catheterization. The corresponding caption reads now (lines 120-134): A: The perfusion system (technical drawing) consists of a closed circuit. Red color indicates the hot water circuit, blue represents the perfusion circuit, gassed with carbogen. For active perfusion, the arteries were catheterized, passive venous outflow drains into the perfusion tub. The gassing tubes (one in the supply tank 1 and one in the perfusion tub) are denoted with an asterisk. The perfusion flow was measured using a flowmeter (F). In the inset photograph, a dorsal view of the perfusion setup in situ can be observed. The larynx, immersed in perfusion solution, is placed on a larynx retainer to stabilize its position in the perfusion tub. Both cranial thyroid arteries were catheterized (intravenous catheter, 18 gauge). For pH-control, a pH-meter is added (marked with an arrow). B: Schematic drawing of an equine larynx, lateral aspect. Relevant structures are the cricoarytenoideus dorsalis muscle [CAD, a)], the common carotid artery [b) running parallel to the external jugular vein], the catheterized arterial branch [c), cranial thyroid artery] followed by its ramification into the ascending pharyngeal artery (d) and the laryngeal branch (e). The latter two vessels nourish the CAD. f) Recurrent laryngeal nerve reaches the larynx as caudal laryngeal nerve. Thank you very much for this helpful comment, we hope to have addressed it sufficiently. 2. The next comment by Rev. 2 addresses the electrical stimulation of the CAD. The contraction of the CAD can be seen by an abduction of the vocal fold. This was judged visually. We did not develop a comparable measurement approach for this and therefore it is stated descriptively in the text. To improve this passage, we refined the results accordingly (lines 225-226). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0251530.r002
28 Apr 2021 Decision Letter - Maurizio Mandalà, Editor Development of a constant pressure perfused ex vivo model of the equine larynx PONE-D-21-04296R1 Dear Dr. Michler, 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, Maurizio Mandalà, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0251530.r003
Formally Accepted
7 May 2021 Acceptance Letter - Maurizio Mandalà, Editor PONE-D-21-04296R1 Development of a constant pressure perfused ex vivo model of the equine larynx Dear Dr. Michler: 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. Maurizio Mandalà Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0251530.r004

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