Preservation of neurons in an AD 79 vitrified human brain

Pierpaolo Petrone; Guido Giordano; Elena Vezzoli; Alessandra Pensa; Giuseppe Castaldo; Vincenzo Graziano; Francesco Sirano; Emanuele Capasso; Giuseppe Quaremba; Alessandro Vona; Maria Giuseppina Miano; Sergio Savino; Massimo Niola

doi:10.1371/journal.pone.0240017

Peer Review History

Original SubmissionAugust 5, 2020
9 Sep 2020 Decision Letter - Fernando de Castro, Editor PONE-D-20-24415 Preservation of Neurons in an AD 79 Vitrified Human Brain PLOS ONE Dear Dr. Petrone, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has a remarkable merit and just need slights changes to better describe parts of the samples as suggested by expert Reviewer #2. Therefore, we invite you to submit a revised version of the manuscript that addresses these very minor points raised during the review process and if I consider myself as clear enough, it would not be necessary any further round of revision. Please ensure that your decision is justified on PLOS ONE’s publication criteria because your work can be considered as unique (at least unique up to date) and its impact is out of question. Please submit your revised manuscript by Oct 24 2020 11:59PM. 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Please ensure that you refer to Tables 1 & 2 in your text; if accepted, production will need this reference to link the reader to the Table. 4. Please include a caption for all of your figures [Note: HTML markup is below. Please do not edit.] 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: Partly ******** 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: This is an excellent analysis of the preservation of neural tissue by vitrification from a young male from Herculano. Authors provides detailed characterization of neurons and axons using SEM analysis. Reviewer #2: This contribution is probably the most surprising article I have had the opportunity to review. Indeed, it is a difficult task to provide the authors with a well-argued opinion. Having a chance of inspecting a 2000-year-old vitrified CNS specimen of course is a very rare event. Here the authors took advantage of natural vitrification process enabling –for the first time if I am right- the accurate identification of neuronal structures at an unprecedented level of resolution. Taken together, this can be viewed as a “neuroarcheological” report. My only concern is related to the method conducted for identifying neuronal processes from any other CNS structure like small blood vessels. As indicated in page 5, it seems that the main criteria is that identified tubular structures are smaller in diameter that blood vessels in the CNS. Although this is overall fine to me, I am wondering to what extent this will suffice. For instance, the structure shown in Figure 4 is here taken as a good example of non-myelinated axon arising from a spinal cord neuron. Although I am obviously not too much familiar with the conducted analytical procedure, I guess this could also be considered as a blood vessel of small caliber. In summary, I guess this undoubtedly is a very rare example of neuroarcheology taken from a very rare event and of course of great interest from potential readers, likely representing a wide audience. Nevertheless and bearing in mind my background in neuromorphology, I guess the authors should make a greater effort in better explaining the criteria used for ensuring the neuronal origin of some of the observed structures. ****** 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.0240017.r001
Revision 1
16 Sep 2020 Author Response Rebuttal letter – Ref.: PONE-D-20-24415 Title: Preservation of Neurons in an AD 79 Vitrified Human Brain Response to the comments from the editor and reviewers We wholeheartedly agree with comments from the editor and reviewers therefore we made a revision of the critical issues. In particular, following the suggestions of reviewer #2, we have treated in more depth and clarified the neuronal origin of the structure shown in figure 4, in order to avoid any possible misinterpretation concerning non-myelinated axons vs small blood vessels. As also suggested, we ensured to refer to Tables 1 & 2 in the text, and a caption for all of the figures has been added at the end of the manuscript. The four additional citations have also been added and renumbered within the text and bibliography as well. We have also improved the description of the databases used for the expression study, both in “Results” and “Materials and Methods” sections. Reviewer #1: This is an excellent analysis of the preservation of neural tissue by vitrification from a young male from Herculano. Authors provides detailed characterization of neurons and axons using SEM analysis. We thank the reviewer for his/her positive and insightful comments on the manuscript. Reviewer #2: This contribution is probably the most surprising article I have had the opportunity to review. Indeed, it is a difficult task to provide the authors with a well-argued opinion. Having a chance of inspecting a 2000-year-old vitrified CNS specimen of course is a very rare event. Here the authors took advantage of natural vitrification process enabling ? for the first time if I am right- the accurate identification of neuronal structures at an unprecedented level of resolution. Taken together, this can be viewed as a "neuroarcheological" report. My only concern is related to the method conducted for identifying neuronal processes from any other CNS structure like small blood vessels. As indicated in page 5, it seems that the main criteria is that identified tubular structures are smaller in diameter that blood vessels in the CNS. Although this is overall fine to me, I am wondering to what extent this will suffice. For instance, the structure shown in Figure 4 is here taken as a good example of non-myelinated axon arising from a spinal cord neuron. Although I am obviously not too much familiar with the conducted analytical procedure, I guess this could also be considered as a blood vessel of small caliber. In summary, I guess this undoubtedly is a very rare example of neuroarcheology taken from a very rare event and of course of great interest from potential readers, likely representing a wide audience. Nevertheless and bearing in mind my background in neuromorphology, I guess the authors should make a greater effort in better explaining the criteria used for ensuring the neuronal origin of some of the observed structures. We thank the reviewer for his/her suggestion. We have now included two references (Müller et al., 2008; Schmid et al., 2019) in the main text showing that the smallest capillaries in the human brain have diameters ranging from 8 to 1 µm and a wall thickness of about 1 µm (Figure 1). Figure 1 . The typical diameters/wall thicknesses of blood vessel for humans and mice are given in micrometers. The smallest capillaries have diameters of a very few micrometers and a wall thickness of about one micrometer (from Müller et al., 2008). In Figure 4 of our manuscript the mean diameter of a non-myelinated axon is 452·50 ∓ 20·50 nm, that is significantly inferior to the minimal dimension of the diameter of a capillary as reported in literature. Moreover, we can add two other important morphological details that can confirm our description. The first one is that this structure originates in fact from the changing curvature of the cellular membrane and it extends for several microns away from the cell body. To be clearer, here below we show a comparison between our SEM image (left) and a TEM image (right) of the axon initial segment (red arrows) of a cerebral cortical pyramidal neuron (Bender et al, 2012) that shows the same section that we found in our sample. Starting with this description we can assume that in our sample we have found a section of axon initial segment that is a non-myelinated axon (Palay et al., 1968). The last point that validates our description is that the wall of this structure is around 70 nm, that is far below the size of the wall thickness of the capillary ranging around 1 / 0.5 µm (Müller et al., 2008). Therefore, we have added the following sentences (page 6, lines 166-175 of Revised Manuscript with Track Changes): "… diameter (nm, mean ∓ SD) (452·50 ∓ 20·50) (Fig 4A). Several evidence seems to confirm the neuronal origin of such structure: i. its diameter is significantly inferior to the minimal diameter of a capillary, the smallest one of which in the human brain have diameters ranging from 8 to 1 µm, and a wall thickness of about 1 µm [38,39]; ii. it originates from the cellular membrane and it extends for several microns away from the cell body; iii. its high similarity with the axon initial segment of a cerebral cortical pyramidal neuron [40]; iiii. the thickness of its wall (ca 70 nm) is far below the size of the wall thickness of a capillary (ca 1 to 0.5 µm) [38]. On this basis, we can assume that in our sample we have found a section of an axon initial segment which is a non-myelinated axon [41]". Additional Bibliography 38. Müller B, Lang S, Dominietto M, Rudin M, Schulz G, Deyhle H et al. High-resolution tomographic imaging of microvessels. In Developments in X-Ray Tomography VI, Stuart R. Stock (ed), Proc. of SPIE Vol. 7078, 70780B, 2008; 0277-786X/08/$18 · doi: 10.1117/12.794157. 39. Schmid F, Barrett MJ, Jenny P, Weber B. Vascular density and distribution in neocortex. Neuroimage. 2019; 197:792-805. 40. Bender KJ, Trussell LO. The physiology of the axon initial segment. Ann Rev Neurosci. 2012; 35:249-265. 41. Palay SL, Sotelo C, Peters A, Orkand PM. The axon hillock and the initial segment. J Cell Biology. 1968; 38(1):193-201. Attachments Attachment Submitted filename: Respons to Reviewers.docx https://doi.org/10.1371/journal.pone.0240017.r002
18 Sep 2020 Decision Letter - Fernando de Castro, Editor Preservation of Neurons in an AD 79 Vitrified Human Brain PONE-D-20-24415R1 Dear Dr. Petrone, 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, Fernando de Castro Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0240017.r003
Formally Accepted
24 Sep 2020 Acceptance Letter - Fernando de Castro, Editor PONE-D-20-24415R1 Preservation of Neurons in an AD 79 Vitrified Human Brain Dear Dr. Petrone: 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. Fernando de Castro Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0240017.r004

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