Comparative analysis of spontaneous blinking and the corneal reflex

Ocular surface health, the cognitive status, psychological health or human neurological disorders, among others, can be assessed by studying eye blinking, which can be differentiated in spontaneous, reflex and voluntary. Its diagnostic potential has provided a great number of works that evaluate their characteristics and variations depending on the subject's condition (sex, tiredness, health, …). The objective of this study was to analyse the differences in blinking kinematics of spontaneous and reflex blinks, distinguishing between direct and consensual reflexes, using a self-developed, non-invasive and image processing-based method. A video-oculography system is proposed using an air jet driven by a syringe to induce reflex and a high-speed camera to record the blinking of both eyes. The light intensity diffused by the eye changes during blinking and peaks when the eyelid closes. Sixty-second sequences were recorded of 25 subjects blinking. Intensity curves were off-line fitted to an exponentially modified Gaussian (EMG) function, whose σ, μ and τ parameters were analysed. A two-way analysis of variance (ANOVA) of these parameters was conducted to test the influence of the subject, the eye and blink type. In the closing phase, direct and consensual corneal reflexes are faster than spontaneous blinking, but there was no significant difference between them, nor between right and left eyes. In the opening phase, the direct corneal reflex was the slowest and significant differences appeared between right and left eyes.


Comments: -
On page 3, line 48: Can you please provide more details about the difference between direct and consensual reflex? - The authors used the amount of light reflected from the ROI to register eye blinking. I believe this technique is highly affected by ambient light and the size of the ROI. Can the authors try different techniques like eye alignment? -On page 5, line 37: Did the author fix the ambient light intensity in all the recordings? Please, mention the light intensity value in lux (or lumens).
-What is the distance between the air tubes and the eyes? -Are the syringes controlled manually? Does, the piece of paper used to detect the stimulus onset affect the air-flow? -Why didn't the authors record more than two sequences (or longer sequences)? I believe the final number of blinks in each group is not sufficient to draw reliable conclusions.

Decision letter (RSOS-201016.R0)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Dr Espinosa
The Editors assigned to your paper RSOS-201016 "Comparative analysis of spontaneous blinking and the corneal reflex" have now received comments from reviewers and would like you to revise the paper in accordance with the reviewer comments and any comments from the Editors. Please note this decision does not guarantee eventual acceptance.
We invite you to respond to the comments supplied below and revise your manuscript. Below the referees' and Editors' comments (where applicable) we provide additional requirements. Final acceptance of your manuscript is dependent on these requirements being met. We provide guidance below to help you prepare your revision.
We do not generally allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available, we may invite new reviewers.
Please submit your revised manuscript and required files (see below) no later than 21 days from today's (ie 20-Oct-2020) date. Note: the ScholarOne system will 'lock' if submission of the revision is attempted 21 or more days after the deadline. If you do not think you will be able to meet this deadline please contact the editorial office immediately.
Please note article processing charges apply to papers accepted for publication in Royal Society Open Science (https://royalsocietypublishing.org/rsos/charges). Charges will also apply to papers transferred to the journal from other Royal Society Publishing journals, as well as papers submitted as part of our collaboration with the Royal Society of Chemistry (https://royalsocietypublishing.org/rsos/chemistry). Fee waivers are available but must be requested when you submit your revision (https://royalsocietypublishing.org/rsos/waivers). The reviewers raise a number of critical comments in relation to you work. You need to carefully address their comments in a revision, and provide a point-by-point response to them. Please bear in mind that you will not generally be permitted to revise your paper further.

Reviewer comments to Author:
Reviewer: 1 Comments to the Author(s) In the paper entitled "Comparative analysis of spontaneous blinking and the corneal reflex" the authors investigated in 25 healthy subjects some dynamic characteristics of spontaneous blinking and reflex blinking, and their significant differences by using a video-oculography system, even if rationale is not established. They found that during the closing phase, direct and consensual responses of blink reflex are faster than spontaneous blinking, but there was no significant difference between them, nor between right and left eye. On the contrary, during the opening phase, the direct response was the slowest and significant differences appeared between right and left eye.
The statistical analysis is well performed, however some major concerns are present: 1. The rationale of research should be better explained in the Introduction and in the Abstract. 2. Since spontaneous blinking may be influenced by cognitive functions (Jongkees BJ and Colzato LS, 2016), I believe that the authors should have investigated these aspects in the participants enrolled in the study. 3. The authors enrolled 25 subjects aged 20-61 years (33 ± 14 years, 16 women and 9 men). Since blink reflex as well as spontaneous blinking may depend on age and sex (Peddireddy et al, 2006), the authors should have investigated any differences due to sex or age. 4. In the Methods the authors should give more information about the setting where subjects are studied (for example was it the same for all the participants? Was the lighting of the room artificial or not?). 5. To exclude subjects with increased spontaneous blinking the authors should have evaluate the spontaneous blink at rest. 6. I think the authors should better discuss their results.
Reviewer: 2 Comments to the Author(s) The paper investigates the blinking behaviour of two different types of blinking: spontaneous and corneal reflex (direct and consensual) in humans. The study used video recordings of eyes during blinking behaviour under natural and stimulation conditions (stimulation is performed randomly using air jets). Although the authors were motivated by the medical applications of characterizing the blinking behaviour, the data acquisition was performed from healthy subjects only. It would add more value to the paper if the data involved patients and control groups.
Comments: -On page 3, line 48: Can you please provide more details about the difference between direct and consensual reflex? -The authors used the amount of light reflected from the ROI to register eye blinking. I believe this technique is highly affected by ambient light and the size of the ROI. Can the authors try different techniques like eye alignment? -On page 5, line 37: Did the author fix the ambient light intensity in all the recordings? Please, mention the light intensity value in lux (or lumens).
-What is the distance between the air tubes and the eyes? -Are the syringes controlled manually? Does, the piece of paper used to detect the stimulus onset affect the air-flow? -Why didn't the authors record more than two sequences (or longer sequences)? I believe the final number of blinks in each group is not sufficient to draw reliable conclusions.

===PREPARING YOUR MANUSCRIPT===
Your revised paper should include the changes requested by the referees and Editors of your manuscript. You should provide two versions of this manuscript and both versions must be provided in an editable format: one version identifying all the changes that have been made (for instance, in coloured highlight, in bold text, or tracked changes); a 'clean' version of the new manuscript that incorporates the changes made, but does not highlight them. This version will be used for typesetting if your manuscript is accepted. Please ensure that any equations included in the paper are editable text and not embedded images.
Please ensure that you include an acknowledgements' section before your reference list/bibliography. This should acknowledge anyone who assisted with your work, but does not qualify as an author per the guidelines at https://royalsociety.org/journals/ethicspolicies/openness/.
While not essential, it will speed up the preparation of your manuscript proof if accepted if you format your references/bibliography in Vancouver style (please see https://royalsociety.org/journals/authors/author-guidelines/#formatting). You should include DOIs for as many of the references as possible.
If you have been asked to revise the written English in your submission as a condition of publication, you must do so, and you are expected to provide evidence that you have received language editing support. The journal would prefer that you use a professional language editing service and provide a certificate of editing, but a signed letter from a colleague who is a native speaker of English is acceptable. Note the journal has arranged a number of discounts for authors using professional language editing services (https://royalsociety.org/journals/authors/benefits/language-editing/).

===PREPARING YOUR REVISION IN SCHOLARONE===
To revise your manuscript, log into https://mc.manuscriptcentral.com/rsos and enter your Author Centre -this may be accessed by clicking on "Author" in the dark toolbar at the top of the page (just below the journal name). You will find your manuscript listed under "Manuscripts with Decisions". Under "Actions", click on "Create a Revision".
Attach your point-by-point response to referees and Editors at Step 1 'View and respond to decision letter'. This document should be uploaded in an editable file type (.doc or .docx are preferred). This is essential.
Please ensure that you include a summary of your paper at Step 2 'Type, Title, & Abstract'. This should be no more than 100 words to explain to a non-scientific audience the key findings of your research. This will be included in a weekly highlights email circulated by the Royal Society press office to national UK, international, and scientific news outlets to promote your work.

At
Step 3 'File upload' you should include the following files: --Your revised manuscript in editable file format (.doc, .docx, or .tex preferred). You should upload two versions: 1) One version identifying all the changes that have been made (for instance, in coloured highlight, in bold text, or tracked changes); 2) A 'clean' version of the new manuscript that incorporates the changes made, but does not highlight them. --If you are requesting a discretionary waiver for the article processing charge, the waiver form must be included at this step.
--If you are providing image files for potential cover images, please upload these at this step, and inform the editorial office you have done so. You must hold the copyright to any image provided.
--A copy of your point-by-point response to referees and Editors. This will expedite the preparation of your proof.

At
Step 6 'Details & comments', you should review and respond to the queries on the electronic submission form. In particular, we would ask that you do the following: --Ensure that your data access statement meets the requirements at https://royalsociety.org/journals/authors/author-guidelines/#data. You should ensure that you cite the dataset in your reference list. If you have deposited data etc in the Dryad repository, please include both the 'For publication' link and 'For review' link at this stage.
--If you are requesting an article processing charge waiver, you must select the relevant waiver option (if requesting a discretionary waiver, the form should have been uploaded at Step 3 'File upload' above).
--If you have uploaded ESM files, please ensure you follow the guidance at https://royalsociety.org/journals/authors/author-guidelines/#supplementary-material to include a suitable title and informative caption. An example of appropriate titling and captioning may be found at https://figshare.com/articles/Table_S2_from_Is_there_a_trade-off_between_peak_performance_and_performance_breadth_across_temperatures_for_aerobic_sc ope_in_teleost_fishes_/3843624.

At
Step 7 'Review & submit', you must view the PDF proof of the manuscript before you will be able to submit the revision. Note: if any parts of the electronic submission form have not been completed, these will be noted by red message boxes.

Author's Response to Decision Letter for (RSOS-201016.R0)
See Appendix A.

Comments to the Author(s) I thank the authors for the answers and I endorse the final version of the manuscript for publication
Review form: Reviewer 2

Recommendation?
Accept as is

Comments to the Author(s)
The authors have addressed all comments. No additional comments.

Decision letter (RSOS-201016.R1)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Espinosa, It is a pleasure to accept your manuscript entitled "Comparative analysis of spontaneous blinking and the corneal reflex" in its current form for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org) and the production office (openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact --if you are going to be away, please nominate a co-author (if available) to manage the proofing process, and ensure they are copied into your email to the journal.
Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Please see the Royal Society Publishing guidance on how you may share your accepted author manuscript at https://royalsociety.org/journals/ethics-policies/media-embargo/.
Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal.
Kind regards, Royal Society Open Science Editorial Office Royal Society Open Science openscience@royalsociety.org on behalf of Prof Pietro Cicuta (Subject Editor) openscience@royalsociety.org

Reviewer: 1
In the paper entitled "Comparative analysis of spontaneous blinking and the corneal reflex" the authors investigated in 25 healthy subjects some dynamic characteristics of spontaneous blinking and reflex blinking, and their significant differences by using a video-oculography system, even if rationale is not established. They found that during the closing phase, direct and consensual responses of blink reflex are faster than spontaneous blinking, but there was no significant difference between them, nor between right and left eye. On the contrary, during the opening phase, the direct response was the slowest and significant differences appeared between right and left eye.
The statistical analysis is well performed, however some major concerns are present:

The rationale of research should be better explained in the Introduction and in the Abstract.
We would like to thank Reviewer's suggestion. The reasons of research were not clearly stated in the manuscript.
Noninvasive measurement of blinking has been a research subject by the authors of the manuscript since 2010 [26]. The implementation of image processing algorithms allows that the contact-free recording of the annexes of the eye can be used to gather and analyze quantitative blink kinematic parameters precisely, with an accuracy comparable to, or even better than, the search coil method. This fact together with the central nervous system connection with blinking encouraged us to deep in its research during the last years. In 2015, we presented a high-resolution binocular video-oculography system aimed to the assessment of pupillary light reflex, with which we detected an early incomplete blink and an upward eye movement [Espinosa et al. "A high-resolution binocular video-oculography system: assessment of pupillary light reflex and detection of an early incomplete blink and an upward eye movement," Bimed. Eng. Online 2015]. Corneal reflex, like the pupillary one, can be elicited by a light stimulus but we realized the few works that had referred to the study of the direct and consensual corneal reflexes considering their importance for example as indicator of both neurological and optic nerve pathologies. We preliminary addressed the assessment of direct and consensual corneal reflex and spontaneous blink in SPIE. Result suggested there existed differences between both reflexes, but statistic was poor. In this work, we modified the experimental setup, changing light stimulus by air jet shot, we evaluated a larger population and performed a complete statistical analysis. The objective of this study was to analyze the differences in blinking kinematics of spontaneous and reflex blinks, distinguishing between direct and consensual reflexes, using for the first time a self-developed, non-invasive, and image processing-based method. (Jongkees BJ and Colzato LS, 2016), I believe that the authors should have investigated these aspects in the participants enrolled in the study. 3. The authors enrolled 25 subjects aged 20-61 years (33 ± 14 years, 16 women and 9 men). Since blink reflex as well as spontaneous blinking may depend on age and sex (Peddireddy et al, 2006), the authors should have investigated any differences due to sex or age.

Since spontaneous blinking may be influenced by cognitive functions
The study population includes men and women within a wide adult range and no specific cognitive function was considered when making the measurements. Our interest is not in determining differences between age or sex but between types of blinking (spontaneous and direct and consensual reflexes). For this purpose, we proposed a technique that allows to extract kinematic parameters from isolated blink curves that are directly related to the opening and closing phases, but no rate analysis was performed. The central dopamine function is closely associated with the spontaneous blink rate and Jongkees BJ and Colzato LS, 2016 provides a comprehensive review. On the other hand, Peddireddy et al, 2006 hypothesized that blink reflex may be influenced by the effects of aging and gender; and with that aim evaluated electromyographic responses of two groups of 15 men of different mean age and other two ones of women.
We do not doubt about the interest of applying our technique to such analyses, but both are out of the scope of the manuscript and do not affect the validity of our results.

4.
In the Methods the authors should give more information about the setting where subjects are studied (for example was it the same for all the participants? Was the lighting of the room artificial or not?).
Two LED lamps (3500 K) were used to illuminate the subjects' eyes with an intensity of 1300±100 lux. They were placed obliquely without interrupting the line of sight or dazzling the participants. We have included that information in the manuscript to better describe the experimental setup. Responding to the issues raised by the reviewer, the lighting was artificial, and the illumination was the same for all the participants.

To exclude subjects with increased spontaneous blinking the authors should have evaluate the spontaneous blink at rest.
We agree with reviewer 1 that one way to stablish a criterium to exclude subjects with increased spontaneous blinking is to evaluate the blink at rest. All subjects of the study where pre-examined in order to discard subjects with excessive blinking or with an excessive number of incomplete blinks. The participants in this study were selected from a group of people that already participated in previous related studies. Therefore, only those with normal blinks and with no tendency to incomplete blinks were selected. We recognize that this was not a proper selection, but also the sampling was not completely blind. The measurements were performed without asking the participants making any work (reading, following a target, …) but looking at a distance point. In any case, the blink rate is out of the scope of this work; we are interested in isolated blinks and the establishment of characteristic parameters.
We have added a sentence in the text recognizing that a pre-evaluation of the subjects was performed in order to avoid people with excessive number of blinks or incomplete blinks.

I think the authors should better discuss their results.
We appreciate the reviewer's suggestion and have extended the discussion of the results.