Retinal vessel multifractals predict pial collateral status in patients with acute ischemic stroke

Adnan Khan; Patrick De Boever; Nele Gerrits; Naveed Akhtar; Maher Saqqur; Georgios Ponirakis; Hoda Gad; Ioannis N. Petropoulos; Ashfaq Shuaib; James E. Faber; Saadat Kamran; Rayaz A. Malik

doi:10.1371/journal.pone.0267837

Peer Review History

Original SubmissionSeptember 19, 2021
14 Dec 2021 Decision Letter - Aurel Popa-Wagner, Editor PONE-D-21-30340Retinal Vessel Multifractals Predict Pial Collateral Status in Patients with Acute Ischemic StrokePLOS ONE Dear Dr. Malik, 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 28 2022 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. 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We will update your Data Availability statement to reflect the information you provide in your cover letter. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? 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Reviewer #1: 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 study “Retinal Vessel Multifractals Predict Pial Collateral Status in Patients with Acute Ischemic Stroke” by Khan et al, represents a very thorough image analysis of the retinal blood vessels aiming to assess their morphology as a fast and accessible predictor tool for pial collateral circulation availability in stroke patients. Leptomeningeal collaterals of the pia vessels are small arterial connections of the terminal cortical branches of major cerebral arteries along the surface of the brain. These vessels are closed under normal conditions when blood flow from all major cerebral arteries is not affected, but are recruited when one major artery is occluded. Minor issues - The authors should clearly present the phisiopathological connection between the retinal vessels pattern and pial collateral circulation, and whether this is or not an independent risk factor. Many prior publications have demonstrated that retinal vessel morphology can be a diagnostic marker for early detection and monitoring of ischemic stroke, based on the fact that ophthalmic artery originates in the internal carotid artery, the same as for MCA, and thus both vessels might reflect a common pressure and motility change. Major issues - Binarized images were analysed utilising the Frac Lac plugin for ImageJ. This software applies the sliding box algorithm and looks only at the silhouettes of the defined Regions of Interest (ROI), and does not consider the inner holes of the ROIs. Were multifractal and lacunarity analyses enough to evaluate the complete filling patterns of the ROIs. The authors might also consider utilising a sliding box algorithm (for example Image ProPlus from Media Cybernetics) that considers the full patterns and not only the profile lines, this could improve the monofractal discriminating efficiency. - How did the main physiopathological denominators of a direct vascular pathology influenced the connection between retinal vessels, pial vessels, and stroke in these patients? Besides the presence/absence of hypertension, the analysis needs to consider diabetes, atheromatosis and vasculitis in these patients. These parameters should in fact be evaluated for both the control and the study group. - How did the retina blood vessels correlate with the area of the core of the infarct and penumbra, on CT scans? - Where the patients analysed during the ischemic thrombolysis window? Was thrombolysis performed on these patients? If yes, how was the recovery considering the retina vascular pattern as a predictor? Altogether, this is a very valuable study in the field, and I will fully endorse its publication after answering the above questions. ****** 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 [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.0267837.r001
Revision 1
12 Apr 2022 Author Response Dear Editor, We thank you and the reviewers for their constructive and valuable comments, which have helped us to improve the quality of our manuscript. A point-by-point response (blue) to you and the reviewers’ comments is provided below along with the revised manuscript. Responses to Editor Questions: 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 Answer: Thank you. We have followed the PLOS ONE’s style requirements and amended the documents. 2. Please describe in your methods section how capacity to provide consent was determined for the participants whom have suffered a stroke in this study. Please also state whether your ethics committee or IRB approved this consent procedure. If you did not assess capacity to consent please briefly outline why this was not necessary in this case. Answer: Thank you. Each participant was consented for the study. The study was approved from Weill Cornell medicine-Qatar and Hamad General Hospital IRBs. It has been stated on page 4, last paragraph. “This study adhered to the tenets of the declaration of Helsinki and was approved by the Institutional Review Board of Weill Cornell Medicine-Qatar (15–00021) and Hamad General Hospital (15304/15). Informed, written consent was obtained from all patients/guardians before participation in the study. 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Answer: Thank you. We have provided the correct grant number BMRP20038654. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: Supported by Qatar National Research Fund Grant BMRP20038654. The funders had no role in study design, data collection and analysis, decision to publish, or publish, or preparation of the manuscript. Please note that 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: The author(s) received no specific funding for this work. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Answer: Thank you. We would like to add following funding statement. “Supported by Qatar National Research Fund Grant BMRP20038654. The funders had no role in study design, data collection, analysis and interpretation, and decision to prepare the manuscript and the publication process.” 4. Please note that in order to use the direct billing option the corresponding author must be affiliated with the chosen institute. Please either amend your manuscript to change the affiliation or corresponding author, or email us at plosone@plos.org with a request to remove this option. Answer: Thank you. Billing will be paid by Weill Cornell Medicine-Qatar. 5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Answer: Thank you. We have provided the data availability statement on page 8, 1st paragraph. “The data used for statistical analysis in this study is available at repository with URL https://doi.org/10.6084/m9.figshare.16574498.v1 .” Reviewers' comments: Reviewer's Responses to Questions Reviewer: Reviewer #1: The study “Retinal Vessel Multifractals Predict Pial Collateral Status in Patients with Acute Ischemic Stroke” by Khan et al, represents a very thorough image analysis of the retinal blood vessels aiming to assess their morphology as a fast and accessible predictor tool for pial collateral circulation availability in stroke patients. Leptomeningeal collaterals of the pia vessels are small arterial connections of the terminal cortical branches of major cerebral arteries along the surface of the brain. These vessels are closed under normal conditions when blood flow from all major cerebral arteries is not affected, but are recruited when one major artery is occluded. We thank the reviewer for his/her positive evaluation of our study. Minor issues The authors should clearly present the phisiopathological connection between the retinal vessels pattern and pial collateral circulation, and whether this is or not an independent risk factor. Many prior publications have demonstrated that retinal vessel morphology can be a diagnostic marker for early detection and monitoring of ischemic stroke, based on the fact that ophthalmic artery originates in the internal carotid artery, the same as for MCA, and thus both vessels might reflect a common pressure and motility change. Answer: Thank you for suggesting that a common anatomical source for the origin of the ophthalmic and middle cerebral arteries may explain the comparable changes in the structure of the retinal and pial collaterals vessels. Given that standard retinal vessel metrics did not differ between ischemic stroke patients with good and poor pial collaterals, while retinal vessel complexity did . We have suggested (page 13) that hypoxia may lead to altered vessel complexity due to alterations in vasculogenesis and angiogenesis. Major issues Binarized images were analysed utilising the Frac Lac plugin for ImageJ. This software applies the sliding box algorithm and looks only at the silhouettes of the defined Regions of Interest (ROI), and does not consider the inner holes of the ROIs. Were multifractal and lacunarity analyses enough to evaluate the complete filling patterns of the ROIs. The authors might also consider utilising a sliding box algorithm (for example Image ProPlus from Media Cybernetics) that considers the full patterns and not only the profile lines, this could improve the monofractal discriminating efficiency. Answer: We thank the reviewer for the suggestion to consider using ImageProPlus software. However, this software was not available to us and we believe that the use of another pipeline would complicate the interpretation of essentially a proof of concept study because of different image pre-processing and segmentation pipelines. Nevertheless, Df, lacunarity and additional multifractal parameters (Fig.1) were quantified on the MONA REVA segmented images using open-source FracLac. The fractal dimension Df is the most widely used metric for quantifying vascular complexity. We have also used lacunarity, a proxy for heterogeneity and additional multifractality metrics to identify patterns of complexity that cannot be identified with the single Df. We have now clarified on page 5 (last paragraph), page 6 (1st, 2nd and 3rd paragraphs) of the revised manuscript the complementary role of the metrics we have assessed. In the revised manuscript on page 13 we have referred to the issue of using multiple different outcomes, especially in a proof of concept translational study: “A recent systematic review has confirmed an overall reduction in retinal vessel fractals in patients with stroke and dementia [52]. However, they showed variability in fractal outcomes in different studies attributed to differences in populations studied, the equipment used to capture retinal images, and segmentation tools used to define and calculate the fractals [53].” To further clarify this we have added the following sentence in the revised manuscript on page 13: “Other image capture and analysis techniques may help to further differentiate patients with poor and good pial collaterals.” How did the main physiopathological denominators of a direct vascular pathology influenced the connection between retinal vessels, pial vessels, and stroke in these patients? Besides the presence/absence of hypertension, the analysis needs to consider diabetes, atheromatosis and vasculitis in these patients. These parameters should in fact be evaluated for both the control and the study group. Answer: Thank you. We excluded patients with vasculitis as stated on page 4, 1st paragraph. Moreover, the systolic blood pressure and HbA1c did not differ between patients with ischemic stroke and good compared to poor pial collaterals (Table 1). We have also updated our material and method section, and stated the numbers with diabetes and hypertension on page 4, 3rd Paragraph. “In the ischemic stroke group, seven had diabetes and twenty had hypertension, whilst none of the healthy control participants had diabetes or hypertension.” How did the retina blood vessels correlate with the area of the core of the infarct and penumbra, on CT scans? Unfortunately, we did not have the image analysis software to measure the area of the core infarct volume and penumbra. Going forward we will try and incorporate these metrics. Where the patients analysed during the ischemic thrombolysis window? Was thrombolysis performed on these patients? If yes, how was the recovery considering the retina vascular pattern as a predictor? Thank you. Retinal imaging was performed outside the ischemic thrombolysis window. Thrombolysis was performed on 19 patients. We have measured the structural retinal metrics which we believe would not change after thrombolysis. https://doi.org/10.1371/journal.pone.0267837.r002
18 Apr 2022 Decision Letter - Aurel Popa-Wagner, Editor Retinal Vessel Multifractals Predict Pial Collateral Status in Patients with Acute Ischemic Stroke PONE-D-21-30340R1 Dear Dr. Malik, 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, Aurel Popa-Wagner Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0267837.r003
Formally Accepted
26 Apr 2022 Acceptance Letter - Aurel Popa-Wagner, Editor PONE-D-21-30340R1 Retinal Vessel Multifractals Predict Pial Collateral Status in Patients with Acute Ischemic Stroke Dear Dr. Malik: 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 Professor Aurel Popa-Wagner Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0267837.r004

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