The precorneal tear film as a fluid shell: the effect of blinking and saccades on tear film distribution and dynamics.

Clinical Science J. DANIEL NELSON, MD, EDITOR

The Precorneal Tear Film as a Fluid Shell: The Effect of Blinking and Saccades on Tear Film Distribution and Dynamics NORIHIKO YOKOI, MD, PHD, 1 ANTHONY J. BRON, BSC, MB, DO, FRCS, FRCOPHTH, 2 GEORGI AS. GEORGIEV, PHD3

ABSTRACT We conducted a series of experiments to elucidate the behavior of the human precorneal tear film (PCTF) during blinking and horizontal and vertical saccades. Methodology included video-interferometry with subsequent image cross-correlation (tear film lipid layer [TFLL]) and video-microscopy (mucoaqueous subphase [MAS]). We observed that the TFLL interference pattern deteriorates rapidly with successive blinks and degrades slowly with repeated horizontal saccades during blink suppression when dark arcs of thinning appear in the fluorescein-stained PCTF. Furthermore, after full downgaze and a return to the primary position, a transient horizontal bright band appears, deep to the spreading TFLL. It may be followed by local disturbances in the interference pattern. Two horizontal dark bands form in the stained PCTF after the return saccade. PCTF disruption may occur below the lower band during blink suppression. We concluded that shearing during horizontal saccades is insufficient to disturb the tear film

Accepted for publication January 2014. From the 1Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, John Radcliffe Hospital, Oxford, UK, and 3 Department of Biochemistry, Faculty of Biology, University of Sofia, Sofia, Bulgaria. This study was supported in part by a Grant-in-Aid for Scientific Research (C) (25462728) from the Ministry of Education, Culture, Sports, Science and Technology in Japan. The authors have no commercial or proprietary interests in any concept or product discussed in this article. Single-copy reprint requests to Norihiko Yokoi, MD, PhD (address below). Corresponding author: Norihiko Yokoi, MD, PhD, Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto-city 602-8566, Japan. Tel: þ81-75-251-5578. Fax: þ81-75-251-5663. E-mail address: [email protected] © 2014 Elsevier Inc. All rights reserved. The Ocular Surface ISSN: 15420124. Yokoi N, Bron AJ, Georgiev GA. The precorneal tear film as a fluid shell: The effect of blinking and saccades on tear film distribution and dynamics. 2014;-(-):1-15.

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structure greatly. The MAS and TFLL move together as a fluid shell. The dark arcs/bands are caused by meniscusinduced thinning, imprinted onto the PCTF at the lid margin. Their stability during blink suppression suggests that the MAS has gel-like properties. The horizontal bright bands are probably due to transient corneal indentation in downgaze. In downgaze, the disturbance of the TFLL and MAS below the dark bands is possibly due to shearing across the MAS in the return phase. This could cause desiccating stress in everyday activities, such as working at a computer. KEY WORDS blinking, dry eye, mucin, precorneal tear film, tear film dynamics, tear film lipid layer, tear film structure

I. INTRODUCTION A. Structure of the Tear Film uch of our understanding of tear film dynamics comes from studies of tear film behavior during the blink and the blink interval.1-4 A complex pattern of events occurs as the preocular film is deposited and re-formed from blink to blink. Wolff’s original description of the precorneal tear film as a 3-layered structure with a superficial lipid layer, an aqueous layer, and a deep mucin layer5 remains a convenient approximation, although this concept has been modified from time to time. Holly suggested that tear mucin (referring to goblet cell mucin) was present in the tear film as a coascervate, separated into two phases. A deep phase, intimately associated with the epithelium, contained most of the macromolecules and was of high viscosity, while the superficial phase contained mucin in dilute solution, directly associated with the lipid layer.6 Today, this view is no longer tenable, as a coacervate denotes an aggregate of molecules held together by hydrophobic interactions,7 which is unlikely to be the situation in the tear film. Holly also proposed that gel mucin (goblet cell mucin), by lowering the surface tension of the tears, was responsible for the wettability of the ocular surface epithelium, which was otherwise hydrophobic in its native state.6 It has since

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PRECORNEAL TEAR FILM AS FLUID SHELL / Yokoi, et al

OUTLINE I. Introduction A. Structure of the Tear Film B. Spreading and Stability of the Lipid Layer II. Experiments Conducted A. Recruitment and Experimental Conditions B. Experimental Procedures 1. Horizontal Saccades a. Effect on the Tear Film Lipid Layer 1) Nasal Paradigm [n ¼ 6] 2) Nasal and Temporal Paradigm [n ¼ 8] 3) Cross-Correlation Analysis b. Effect on the Mucoaqueous Subphase 2. Downgaze, Followed by a Return Saccade to the Primary Position a. Effect on Lipid Layer Integrity b. Effect on the Mucoaqueous Subphase III. Results A. Horizontal Saccades 1. Nasal Paradigm a. General Observations b. Lipid Layer Degradation 2. Nasal/Temporal Paradigm 3. Fluorescein-Stained Tear Film During Horizontal, Nasal and Temporal Saccades: Dark Arcs B. Downgaze Paradigm 1. The Lipid Layer: Bright Bands 2. The Aqueous Sub phase: Dark Bands and Coating Events IV. Discussion A. Tear Film Lipid Layer 1. Compression and Expansion with Blinking 2. Stability During Horizontal Saccades 3. Influence of Downgaze and Return Saccades B. Mucoaqueous Subphase V. Summary and Conclusions

been shown that the apical plasma membranes of the surface epithelial cells are intrinsically wettable,8 due to the presence of a glycocalyx. This is formed by membrane spanning mucins (MUC 1,4 and 16) whose exodomains project into the tear film. They are expressed to a varying degree by the apical membranes of the surface epithelial cells9 together with other molecules such as galectin-3.10,11 Cher speaks of a mucoaqueous layer whose deepest aspect contains a hydrated mucin gel anchored to the surface cells of the epithelium and therefore, by implication, although not stated, to the surface glycocalyx.12 It has also been suggested that there is no distinct segregation of gel mucin within the aqueous subphase.13-15 On the basis of the studies presented here, we conclude that secretory mucin forms a distinct but dynamic gel layer over the ocular surface, based on weak non-covalent 2


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interactions, hydrogen bonds, Van der Waals attractions and steric interactions, between the polymer molecules. The gel would be expected to disintegrate with each blink and be restored at the end of the blink, when no shear was applied.14,16 B. Spreading and Stability of the Lipid Layer Various authors have considered the manner in which the aqueous layer (or mucoaqeuous subphase [MAS]) of the tear film spreads or is deposited during the blink. The tear film lipid layer (TFLL) is a viscoelastic film whose spreading characteristics have been determined, either directly by interferometry2,4 or indirectly with high-speed video-photography of particles embedded in this layer.17 The rate of spreading of the lipid layer is far slower than the speed of the blink. The blink is completed in 200-300 ms, while the lipid layer, spreading with an initial velocity (measured in 5 normal subjects) of between 3.49e9.83 mm/second (Figure 1), slows asymptotically to stabilize at about 1 second or more after the blink. Following this, the colored interference pattern remains almost unchanged in appearance for the remainder of the blink interval, apart from a small, continued upward drift. In normal subjects, this stability of lipid layer structure has been shown to persist for short periods, from blink to blink.18-20 Thus, it has been observed that, in a series of consecutive blinks, the gross features of the complex, colored, interference patterns are retained over several blinks, (e.g., 2-6 blinks or more) although undergoing a variable degree of degradation with each successive blink. At some point, there is an abrupt and complete change in pattern at the end of the series. The colored fringes of the interference pattern represent regional variations in thickness of the lipid film, related to the molecular organization

Figure 1. Spreading of the tear film lipid layer. The graph shows the velocity of the lipid layer in a normal subject, obtained by plotting the movement of a rectangular region of interest in the TFLL interference pattern over time [blue circles]. The initial velocity is 4.45 mm/sec. The behavior of the TFLL corresponds to that of a viscoelastic film as predicted by the superimposed Voigt model plot [red line] based on an initial velocity of 4.45 mm/sec.4

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PRECORNEAL TEAR FILM AS FLUID SHELL / Yokoi, et al of its lipid molecules. Therefore, the stability of pattern from blink to blink has been interpreted to imply retention of molecular organization during its concertina-like compression (sometimes called “pleating” [Mishima and Maurice21]) and expansion during each blink. From the discussion above, it can be inferred that during compression of the lipid layer in the downstroke of the blink, the lipid layer is sheared from the MAS so that the two layers become dissociated. A similar dissociation appears to occur in the upstroke of the blink, when an upward moving lipid front, driven by surface tension forces, can be followed, travelling over the aqueous subphase. Although the visible front of the interference pattern lags behind the movement of the upper lid, it may be that the spreading lipid layer keeps pace with the rising lid margin, but that its initial, polar component is too thin to be detected by interferometry. It occurred to us that further information about the properties of the tear film and interactions of these two phases could be obtained by examining their relationship during horizontal and vertical versions. In the former, the tear film would be impelled by forces transmitted from the anterior globe through the MAS to the lipid layer. In the case of vertical versions, movement of the globe across the lid mucosa would be expected to influence tear film behavior in a different way. On the basis of this, two novel paradigms were devised in which tear film behavior was observed following the performance of both horizontal and vertical saccades. It was argued that this would be of particular interest, given the stereotyped nature and speed of saccades and therefore their likely reproducibility.22

Table 1.

II. EXPERIMENTS CONDUCTED A. Recruitment and Experimental Conditions Approval was obtained from the Institutional Review Board of Kyoto Prefectural University of Medicine, Kyoto, Japan, before the study was initiated. The research was conducted in compliance with the Declaration of Helsinki, and informed consent was obtained from all subjects after explanation of the nature of the study and possible consequences associated with participation in the study. Thirteen subjects with normal eyes were recruited (11 males and 2 females), with no history of eye disease or of contact lens wear (Table 1). Lids and anterior segments were normal on slit-lamp examination, and subjects were free from topical or systemic medication at the time of study. Fluorescein staining was absent in all subjects. Studies were conducted in a quiet room, in dim illumination, in the absence of drafts, at a temperature of about 25 C. Subjects were allowed to relax in the room for at least 15 minutes before starting any procedure. Two kinds of study were conducted, on the left eye only, to examine the effect of saccades on the TFLL or on the MAS. For studies of the TFLL, each subject was seated comfortably at a DR-1TM video-interferometer with their chin on the chin rest and blinking spontaneously. Videos were recorded at 21 fps. For studies of the aqueous subphase, subjects were similarly seated, at a digital slit-lamp (Rodenstock RO5000), and the eye examined at a magnification of x12 after the instillation of fluorescein. A single drop of unpreserved saline (BSS) was placed on a fluoresceinimpregnated tip of a FLUORES Ocular Examination Test Paper (Showayakuhinkako Co. Japan) and shaken off briskly

Table of all subjects indicating experimental paradigms

Subject

Initial

Age years

Sex

Expt.

1

MB

36

M

J

2

TK

29

M

J

3

JM

29

M

J

4

KY

26

F

J

5

KK

34

M

J

6

SK

43

M

J

7

AK

45

F

{y

8

HK

33

M

{y

9

JHL

35

M

{y

10

KA

24

M

{

11

MM

34

F

{y

12

MS

30

M

{y

13

SJK

34

M

{y


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Experimental Paradigms

J ¼ Nasal followed by return saccades only. Tear film lipid layer studied. { ¼ Nasal and return saccaades followed by temporal and return saccades. Tear film lipid layer and aqueous subphase studied. y ¼ Downgaze and return saccade. Tear film lipid layer and aqueous subphase.

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PRECORNEAL TEAR FILM AS FLUID SHELL / Yokoi, et al to reduce instilled volume. The tip was then tapped briefly onto the lower tarsal conjunctiva of the left eye, and the subject was asked to blink lightly several times in order to mix the fluorescein with the tears. Tear film behavior was recorded continuously at a frame rate of 30 frames/sec, under blue light, using the cobalt blue exciter filter of the slitlamp. B. Experimental Procedures 1. Horizontal Saccades a. Effect on the Tear Film Lipid Layer With the eyes in the primary position, the interferometric pattern of the TFLL could be viewed in the pupil zone against the fundus reflex, which allowed images of the spreading lipid layer to be captured in the blink interval. The frame rate of the video-interferometer was not sufficient to capture TFLL pattern changes during saccades; for this reason, a paradigm was adopted which allowed pattern changes to be recorded at the end of each of a series of return-saccades to the primary position, after deviation to one side or the other. In response to a verbal signal, the subject stopped blinking and performed a series of saccades, under instruction. In series [a], this was performed to the nasal side and back to the primary position, during a tolerable period of blink suppression. In series [b], a saccade was first performed to the nasal side and back to the primary position, and then after a pause, to the temporal side, with a return to the primary position. This sequence was then repeated. The following sequence was maintained: 1) Nasal Paradigm [n ¼ 6]. With the eyes in the primary position, a baseline interference pattern was captured, at least 2 seconds after a blink. At this point the TFLL had spread across the aqueous phase of the precorneal tear film and had stabilized.17 The subject then performed a saccade (termed here the primary saccade) to a target about 60 to the nasal side, returning after a pause, on verbal command, to the primary position. This is termed the return saccade. An image of the TFLL pattern was captured at the end of each return saccade. The maximum time from the onset of a nasal

saccade to the end of a return saccade was around 1.7 seconds. This sequence was repeated by each subject as many times as could be comfortably achieved in the extended blink interval, the longest sequence (with 4 return saccades, in subject 1) taking 10.3 seconds. In each subject a series of spontaneous blinks, sometimes followed by a light, voluntary blink, preceded the performance of saccades. A baseline image of the TFLL pattern (the baseline pattern) could then be acquired in the primary position at least 2 seconds after the last blink, which could be compared with TFLL patterns captured after a variable number of return saccades (return saccade patterns, 1, 2, 3, etc.). Each image acquired on the return saccade reflected the impact of one nasal, primary saccade and one return saccade, on the integrity of the TFLL. 2) Nasal and Temporal Paradigm [n ¼ 8]. As noted, in these subjects, a saccade was first performed to the nasal side and back to the primary position, and then after a pause, to the temporal side, with a return to the primary position. This sequence was repeated within an extended blink interval and videos of the entire sequence were recorded. 3) Cross-Correlation Analysis. In subject 1, who was able to perform a series of 4 return saccades from the nasal position, it was possible to analyze the degradation of the interference pattern between each sequence of return saccades, in the following way. The videos used to capture the primary and return saccade patterns were separated into individual frames. The frames between each saccade were analyzed, and a region of interest was identified within the TFLL pattern for comparison of the primary pattern with those following the return saccades. The selected patterns are presented in Figure 2. The cross-correlation between grayscale images was performed with the Image CorrelationJ IO plugin (http://www. gcsca.net/IJ/ImageCorrelationJ.html) to the ImageJ software (Chinga and Syverud 2007). A cross-correlation coefficient (Pearson correlation coefficient, r) is estimated between the aligned images. The implemented statistics secure a complete assessment of the pattern in the images, including the mean grey level, standard deviation, skewness and kurtosis of the grey level (basis weight) distribution.

Figure 2a-e. TFLL patterns acquired from subject 1 during the performance of a sequence of nasal saccades followed by a return to the primary position. Patterns are selected from a series of 10 consecutive frames, which remained grossly constant over time. a: TFLL pattern at baseline, stabilized >2 sec, following the blink and prior to the primary saccade. b-e: Patterns after a sequence of 4 return saccades. The black rectangle in panel a denotes the area over which the image cross-correlation was measured. The area was selected to avoid the eyelashes, but measurements over a larger selection area followed the same quantitative trend.

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PRECORNEAL TEAR FILM AS FLUID SHELL / Yokoi, et al b. Effect on the Mucoaqueous Subphase The tear film was stained with fluorescein in the manner described, and the subject was seated at the slit-lamp, blinking spontaneously. At some point, the subject was asked to suppress blinking and perform a series of horizontal saccades, through about 60 , first to the nasal side and then to the temporal side, starting from the primary position and pausing in each extreme gaze position, and in the primary position after each return saccade. Movements of the precorneal tear film were recorded continuously in the midzone of the palpebral aperture. The period of blink suppression was between 15 and 22 sec in some subjects. This sequence was repeated several times during an extended blink interval and was well tolerated by the subjects. After each set of primary and return saccades, fixation was held briefly in the primary position so that the behavior of the stained tear film could be observed and recorded. 2. Downgaze, Followed by a Return Saccade to the Primary Position a. Effect on Lipid Layer Integrity The following paradigm was adopted in 6 subjects (Table 1). After a period of blinking, the TFLL pattern of the left eye was recorded in the primary position. Subjects were then asked to look fully downward in the sagittal plane (i.e., they performed a downward saccade) and hold gaze downward for a few seconds. This caused a marked narrowing of the palpebral aperture, which varied in extent from subject to subject. They then performed a return saccade to the primary position, at which point the TFLL pattern was recaptured. This sequence was repeated several times by each subject. With vertical excursions, the upper lid is said to follow the movement of the globe precisely, and therefore no major displacement of the tear film is predicted in relation to the upper lid. However, with the downward saccade performed here, followed by the return saccade, the lower parts of the cornea and globe glide over the lower tarsal conjunctiva, generating friction between the apposed surfaces. b. Effect on the Mucoaqueous Subphase The paradigm followed was identical to that used in the study of the TFLL. After the tear film was stained with fluorescein, the subjects performed a series of downward saccades, each followed by a return to the primary position. Tear film behavior was recorded at the slit-lamp, as before, with the Rodenstock slit lamp. III. RESULTS The field of view of the DR1-1 is a circle of about 7-8 mm, delimited by the pupil margin. By contrast, the stained film was imaged across the full extent of the cornea in the horizontal plane (approximately 12 mm) and a shorter distance in the vertical plane, depending on the degree of overlap of the limbus by the lid margins. For this reason, some features imaged in the periphery of the stained film (such as dark arcs [see below]) were not apparent during interferometry; e.g., dark arcs are not imaged in the interference studies. THE OCULAR SURFACE /

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A. Horizontal Saccades 1. Nasal Paradigm a. General Observations A variable number of return saccade patterns were observed in each subject, depending on their individual capacity to suppress blinking during these maneuvers. In all subjects, a satisfactory baseline interference pattern was obtained, but in one subject (subject 6), the image quality of the return-saccade patterns was low and did not permit comparison.

b. Lipid Layer Degradation In subject 1, where four return saccade patterns were recorded (Figure 2 a-e), it can be seen visually that there is a remarkable similarity between consecutive TFLL patterns, although with a gradual degradation of the pattern from one return saccade to the next, with a slight drift of the pattern to the temporal side. This similarity between baseline and return saccade patterns was also seen in subjects 2-5, where two return saccade images could be recorded, although, here again, a variable degradation of the return saccade pattern is also seen (Figure 3 a-i). In subject 2, where the saccade sequence was repeated twice with separate post-blink baselines, in each case the return saccade patterns strongly resembled the respective baseline patterns (Figure 4 a-f). The interference patterns from subject 1 (Figure 2) were analyzed further, using two types of image cross-correlation analysis, as follows: (i) comparison of the TFLL stationary pattern realized after each saccade with the pre-saccade pattern, and (ii) comparison between two consecutive stationary patterns. Measurements were made within the area defined by the box in Figure 2a. Typical output of image cross-correlation comparison by the Image CorrelationJ plugin is shown in Figure 5. The results are summarized in Table 2. As can be seen, the correlation between the consecutive patterns is quite high, but the correlation of each pattern with the initial pattern decreases with saccade number, indicating a steady change in pattern with repeat saccades. For comparison, a similar analysis of TFLL patterns was made in a single subject (12), after a series of spontaneous blinks (Table 3), comparing patterns after the first, third, and fourth blinks (image 1 [baseline] and images 3 and 4). Correlation coefficients were obtained between the baseline blink of (i) post-blink TFLL stationary pattern with TFLL pattern after the first opening of the eye (image 1), and of (ii) consecutive stationary patterns. In this subject, it can be seen that the similarity between patterns deteriorates more rapidly than that between saccade patterns (Table 3 and Figure 6). This is to be expected as the blink applies strong dilational and shear deformation and causes major restructuring in TFLL. 2. Nasal/Temporal Paradigm The TFLL pattern response to a series of nasal followed by temporal saccades was identical to that found with nasal plus return saccades alone. In general, the pattern established at the end of a series of blinks was grossly maintained during the series of nasal and temporal saccades, although with a -

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Figure 3a-i. TFLL patterns. Left eye: Primary and return saccade patterns in subjects 3, 4, and 5.

progressive degradation of pattern with each step, as observed with nasal saccades alone. 3. Fluorescein-Stained Tear Film During Horizontal, Nasal and Temporal Saccades: Dark Arcs In each subject, the fluorescent precorneal film appeared to be segregated from the rest of the preocular tears, behaving like a single, cap-like body of fluid, which moved with the eye during horizontal versions. Additionally, in the blink interval, the overall fluorescence intensity of the film varied from subject to subject and further, from blink

to blink. In one subject, following a nasal saccade, it was noted that this body of fluid made a small movement to the nasal side at the end of the return saccade. In others, in the downgaze paradigm, a slight upward shift was noted immediately following downgaze and a downward shift was noted immediately following the return saccade. In all subjects, to a varying degree, following an excursion to either side, curved, dark arcs of thinning appeared in the fluorescent film when viewed in the primary position. The curvature of the arcs corresponded to that of the upper or lower lid margin when the globe rested briefly in gaze to one side or the

Figure 4a-f. Subject 2. Baseline and return saccade patterns after 2 separate blinks. a-c: baseline and return saccade patterns after first blink. d-f: after second blink.

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PRECORNEAL TEAR FILM AS FLUID SHELL / Yokoi, et al erased during an eye movement or a blink, these arcs remained stable in morphology and were not filled in by the adjoining tear film during continued blink suppression.

Figure 5. Typical output of image cross-correlation comparison by the Image CorrelationJ plugin. The algorithm estimates a statistic, a crosscorrelation coefficient (Pearson correlation coefficient, r), within each local region having a predefined size, on two aligned images. The crosscorrelation map provides the evaluation of the local correlation between the compared images at each position.

other. Thus, for instance, after a saccade to the temporal side, a dark arc would be formed over the lower lateral cornea, corresponding in shape to the contour of the lower lateral lid margin (Figure 7 a-h). Each excursion of the eye would create a fresh arc, either below or, less commonly, above, which would superimpose upon a previous arc. Careful observation indicated that these arcs were imprints of meniscus-induced thinning generated while the cornea was displaced nasally or temporally from the straight-ahead position. This conclusion was confirmed in 9 additional subjects with normal ocular surfaces, in whom horizontal head rotation was performed while maintaining forward gaze. In each case, the manner of formation of the arcs could be observed directly during these slow, smooth movements (Figure 8 a-f). Additionally it was observed, that passage of the lower part of the cornea behind lower tarsal plate during these horizontal versions can re-coat the apposed cornea with a subaqueous layer, which can then obliterate a segment of a preformed dark arc (Figure 7 e,f). Importantly, unless

Table 2.

B. Downgaze Paradigm 1. The Lipid Layer: Bright Bands In most subjects, the TFLL spread upward, rapidly or more slowly (subjects 7,13), as the eye returned to the primary position, much as it does following the upstroke of the blink In a few subjects, however, it moved downward (e.g., subject 12). In one subject (13), movement was up with some return saccades but down with others. In each subject, with the eye restored to the primary position, a broad, horizontal, curved bright band with refractile margins, was immediately revealed against the red reflex, which appeared to lie deep to the spreading TFLL (Figure 9 a-d[e.g., subjects 7, 13]). These bands faded rapidly over time. In some subjects, curious phenomena were observed in the region of the refractile band with continued forward gaze. In one subject, a series of parallel vertical spikes evolved over time, extending into the refractile band (subject 9 [Figure 10]). In another, there was an increased downward concavity of the band, with the appearance of crenations along the upper margin. In others still, there was a disjointed pattern, or the evolution of a pattern resembling a Marangoni effect. (Figure 11c). 2. The Aqueous Sub phase: Dark Bands and Coating Events A strong resemblance in location was observed between events seen in interference mode and those observed in the stained film. Although the TFLL and fluorescein-stained tear film studies were not performed simultaneously, similarities in their spatio-temporal features suggest that key events in the TFLL and aqueous layers were coincident.

Correlation coefficients obtained after cross-correlation of (i) post-saccade TFLL stationary pattern with pre-saccade TFLL pattern, and of (ii) consecutive stationary patterns Change in TFLL pattern from baseline to each return saccade R

R2

Pattern prior saccade 1 to pattern after saccade 1

0.93

0.86

Pre-saccade pattern to pattern after saccade 2

0.91

0.83


0.88

0.77


0.87

0.76

Comparison

Change between consecutive TFLL patterns Pre-saccade pattern to pattern after saccade 1

0.93

0.86

Pattern after saccade 1 to pattern after saccade 2

0.97

0.94


0.94

0.89


0.89

0.79


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Table 3.

Correlation coefficients obtained after crosscorrelations between a baseline pattern after an initial blink and that following a 3rd and 4th blink R

R2

Image 1 to image 2

0.53

0.28

Image 1 to image 3

0.65

0.42

Image 2 to image 3

0.58

0.38

Comparison

In all subjects, on return to the primary position, one or two horizontal dark bands were observed in the fluorescent film, interpreted as thinning of the aqueous layer generated by zones of meniscus-induced thinning in relation to the upper and lower lids, while the eyes were held momentarily in downgaze (Figure 12). When only one was present, it related to the position of the lower lid, and when two were present, the lower of these zones was always darker and broader than the upper, implying greater thinning in relation to the lower lid meniscus. In most subjects, the dark bands retained their position, sometimes broadening or showing an increased density as long as blinking was suppressed. However, in subject 12, with sustained blink suppression after the return saccade, a steady disruption of the stained film occurred below the lower dark band, appearing as if this part of the tear film was draining away downward (Figure 12 c-f). In some subjects, with sustained forward gaze after the return saccade, additional dark bands were superimposed onto the fluorescent film by partial blinks (Figure 12 e,f; Figure 13). In yet another subject, vertically disposed, finger-like patterns evolved after the return saccade in both the DR-1TM images and the stained tear film, after the return saccade, resembling ‘Marangoni’ patterns (Figure 14). In all cases, dark bands and related patterns could be cleared by a single blink. In one subject who had less narrowing of the palpebral aperture in downgaze than the others, it was possible to observe that the tear film overlying the upper cornea during downgaze moved upward slowly, as a fluorescent cap, immediately after the downward saccade. IV. DISCUSSION A. Tear Film Lipid Layer 1. Compression and Expansion with Blinking In a previous study we reported that during blinking, the gross TFLL pattern can be relatively constant over a series of

blinks, although showing a small scale, stepwise degradation over succeeding blinks.18 At some point, a further blink leads to a total change in pattern, and the sequence is repeated. These phenomena have been confirmed by others.19,20 In the present report, we analyzed a sequence of TFLL images after a series of blinks in one subject (subject 12) and confirmed these observations quantitatively. We have interpreted this stepwise degradation of the interference pattern to be due to a reorganization of lipid molecules in the TFLL and/or a limited exchange of lipid between the film and meibomian reservoirs at the lid margins. A further possibility would be some loss of lipid to the MAS.23,24 We consider that the catastrophic change that occurs from time to time is due to a more extensive mixing between the lipid film and the marginal reservoir, although, here again, a major internal reorganization, without exchange, is a possibility, although less likely. During blinking, when the eyes are stationary, the TFLL is driven chiefly by movements of the upper lid. Using the specific paradigms explored in the current study, we were able to study responses of the lipid layer to rotational forces impelled by horizontal saccades or to interactions between the globe and the lower lid, during vertical saccades. Our observations of the TFLL were confined to the precorneal film and shed no direct light on the behavior of the rest of the preocular film, overlying the bulbar conjunctiva. 2. Stability During Horizontal Saccades Observation of the tear film after the primary and the first return saccade in the horizontal meridian is physiologically relevant, as such events occur frequently in natural viewing conditions, entailing a change of gaze direction. Such saccades are, however, usually coupled with a blink,25 so that tear film structure would be normally expected to be re-set after a change in gaze. Comparison of primary saccade patterns with those generated by later return saccades (second, third, fourth, etc.) relate to less physiological events, but do, however, provide further information about the properties of the tear film. In order to interpret the findings of this study, it should be emphasized that in the blink interval, the TFLL, a viscoelastic film,4,26-28 extends in continuity across the aqueous subphase of the tear film from the upper to the lower meibomian reservoir and does not pass behind the lids at any time. The disposition of the aqueous layer is more complex. In the upstroke of the blink, fluid for the formation of the menisci and tear film is derived chiefly from fluid residing behind the upper lid, presumably supplemented by fluid Figure 6. Subject 12. DR-1TM images showing the TFLL images >2 sec after the 1st (baseline), 3rd and 4th consecutive blinks, observed during spontaneous blinking. Their general similarity is shown, but also their serial degradation from blink to blink.

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Figure 7a-h. Subject 12. Fluoresceinstained tear film of the left eye. a: Nasal saccade. b: A ‘dark arc’ is seen below and to the nasal side after the return saccade to the primary position. c: Temporal saccade. d: A lower temporal dark arc has been added to the return image, which superimposes on the previous arc. Note too that a coating event has obliterated the temporal limb of the first arc. e-f: Additional arcs and coating events are seen as further, consecutive saccades are performed, to the nasal/temporal/nasal/ and temporal sides.

delivered by the lacrimal glands to the upper and lateral fornices. Within about 100 ms of the upstroke, the preocular fluid segregates into the tear film and menisci,29,30 separated by a zone of marked thinning or absence of fluid, referred to as the black line in a fluorescein-stained film.29,31 Formation of the black lines is attributed to the negative hydrostatic pressure within each meniscus, which draws fluid from the preocular film until the two compartments separate. In the downstroke of the blink, most of the preocular fluid flows behind the descending upper lid and mixes with fluid in the retro-tarsal and to some extent upper and lateral fornical compartments.32 From this it can be deduced, in the context of the experiments performed here, that movements of the lipid layer will be constrained by their continuity with the lipid in the meibomian reservoirs, while those of the MAS, because of its continuity with fluid in the tarsoconjunctival compartments, will be less restricted. Considering the TFLL, a striking observation in the current study was that, during horizontal versions in the absence of blinking, the TFLL interference pattern after the first return saccade was almost identical to the pattern recorded at baseline. The most likely explanation is that THE OCULAR SURFACE /

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during the primary and return saccades, the TFLL and aqueous subphase of the precorneal film move together as a single unit, forming a continuous, fluid shell attached to the corneal surface. The absence of any inertial movements of the film after return to the primary position from a nasal or temporal saccade gives some indication of the strength of the interaction between these two layers. This contrasts with the separation of the TFLL from the aqueous layer that occurs in the downstroke of the blink, when the lipid layer is stripped from the subphase.18 This difference in behavior most likely relates to the different forces acting on the lipid and aqueous layers in the two situations. In the downstroke of the blink, the solid, upper lid margin descends with a velocity of 17-20 cm/sec,33,34 applying a compressive force to the TFLL. In this case, the eyelid produces shear-flow between the lipid and aqueous layers of the tear film. Although the exact behavior of the tear fluid behind the upper eyelid at the downstroke is unclear,12,34,35 it is reasonable to assume that the velocity of the tear fluid layers decreases in the normal direction, from the lipid layer to the corneal surface. When the eyelid moves upward, the mucoaqueous tears are deposited over the exposed ocular surface. The -

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Figure 8a-f. Normal subject, left eye. Induction of dark arcs in the fluoresceinstained tear film. a: A full upward saccade is performed. b: An upper dark arc is seen when the eye resumes the primary position, corresponding to the position of the upper meniscus when the eye was in upgaze (arrow). c: Full downgaze. d: Two dark arcs are seen when the eye resumes the primary position. The lower arc corresponds to the location of the lower meniscus and the upper arc to the location of the upper meniscus, while the eye was in downgaze (arrows). e: Head rotation to the right with eyes fixing straight ahead. The cornea comes into relation with the outer canthus. f: When the head is rotated to the primary position, two arcs are seen, the upper one relating to the upper meniscus and the lower to the upper meniscus, while the cornea was located temporally (arrows). Images were captured using a blue exciter filter and a yellow barrier filter.

TFLL spreads on top of it and the spread velocity is proportional to the thickness of the aqueous layer,36 which “screens” the friction between TFLL, the upwardly dragged fluid, and the immobile corneal surface. Generally, in each subject, the fluorescent precorneal film behaved as a single body of fluid, which moved with the eye during horizontal versions. The term perched tear film, coined by Miller et al,29 would seem to be apposite here. We found also that the overall fluorescence intensity of the stained precorneal tear film varied from subject to subject, and this too probably relates to differences in the volume, and also concentration, of fluorescein instilled.37 We also observed a variation in fluorescence intensity across the stained film from blink to blink, implying variations in tear film thickness38 over the cornea. This was most likely due to variations in the coating efficiency of the upper lid during each blink. Coating can be assumed to occur in both the downstroke and the upstroke of the blink, resembling the application of paint by a paint roller. The velocity of horizontal saccades in our experiments, with an angular subtense of about 60 , would be in the

region of >500 /sec.39 In blinking, the tear film is located between two solid surfaces, the moving eyelid surface and the immobile surface of the globe. In contrast, during the horizontal saccade, in the absence of blinking, the TFLLcovered tear surface remains in contact with the air, i.e., there is no friction between the lipid-coated surface and a solid immobile surface. For this reason, when the cornea moves during the saccade, there is no reason for the TFLL, located at the air/tear surface, to move at a slower speed than the aqueous tear fluid, adherent to the corneal surface. Thus, all the layers of the tear film move together as a fluid shell attached to the corneal surface. For this reason, and also because there is no compression of the precorneal TFLL during the maneuver, the TFLL pattern remains relatively static and constant between the saccades. In order for a gradient in the velocities of the tear film layers to be generated, i.e., for shear flow to take place between the layers, it is necessary to (i) increase the thickness of TFLL and/or to (ii) decrease the viscosity and disrupt the gellike structure of the tears.40 In a few subjects, on an individual basis, slight shifts in position of the stained film were

Figure 9a-d. Subject 12: Vertical saccade paradigm. A selection of TFLL images after 4 separate return saccades, demonstrating the horizontal, refractile ‘bright bands’ against the orange background. In d, the spreading TFLL pattern is seen to overlie the bright band.

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Figure 10a-c. Subject 9: DR-1TM images. A series of vertical spikes, evolving over a period of 3.2 sec after a vertical return saccade, extending into a bright band, whose upper border becomes crenated.

observed after horizontal or vertical saccades, suggesting a recentering after slight, en bloc displacements. We cannot exclude the possibility that such shifts, implying looseness of attachment to the cornea, related to larger fluid instillation volumes, resulting in a thicker tear film. In unpublished experiments (not reported here), we have induced such an effect transiently, immediately after the instillation of a full, saline-containing eye drop, when it was observed that the stained precorneal bloc of fluid was displaced by a saccade to either side and re-centered on returning to the primary position. The TFLL pattern is also less stable in response to repeat horizontal saccades in this situation. We considered, but rejected, an alternative explanation for the strong resemblance between the primary- and return-saccade patterns during horizontal versions. In this, we imagined the TFLL to behave as a thin but rigid shell, flexing without displacement during the saccade, while traversed by the subjacent mucoaqueous layer.41 However, the TFLL is a viscoelastic film, which, even allowing for an increase in viscosity due to evaporative cooling in the blink interval, would not achieve the degree of stiffness required to satisfy this requirement.42,43 If it is accepted that the lipid and aqueous layers of the precorneal film move together during the saccade, the same will not be true for the rest of the preocular tear film over its full extent. With the eye open in the primary position, the preocular tear film conforms to the exposed globe, delimited by the shape of the palpebral aperture. If the precorneal tear film moves en bloc with the cornea during a saccade, then the triangles of tear film overlying the nasal and temporal bulbar conjunctiva must behave differently. Considering a nasal saccade, which will decrease the area of exposed nasal triangle, the triangular patch of lipid film must either be compressed (much as is proposed for the TFLL during the downstroke of the blink) and/or be distributed into the marginal meibomian reservoirs. In either case, it will be dissociated, in part, from the MAS. On resuming the primary

position in a return saccade, these events would be reversed and the film expanded, (opening up the “concertina”) and/ or receive a supplement of lipid from the meibomian reservoirs. Conversely, the temporal TFLL triangle would be stretched during the nasal saccade. This would tend to thin the film and perhaps draw lipid from the reservoirs. The situation would be reversed in the return saccade. Similar arguments can be made for events during a temporal saccade. In subject 1, where the TFLL pattern could be followed after multiple nasal saccades, a degradation of the interference pattern was observed and quantified between serial return saccades. The change was small from blink to blink, but progressed with the number of return saccades recorded. A slight change in pattern from baseline to a return saccade was seen too, visually, in subjects 2-5, where at least two return saccades were tracked. This is taken to mean that, in these conditions, there is a gradual rearrangement of lipid molecules within the TFLL, perhaps with some admixture with meibum from the marginal reservoirs, or loss of lipid to the aqueous phase of the tear film.44 It should, however, be remembered that the situation observed here was somewhat artificial, as in normal circumstances, saccades are frequently coupled to blinks.25 3. Influence of Downgaze and Return Saccades In the downgaze and return saccade experiments, the TFLL was compressed in downgaze and spread over the MAS with the return saccade. In most cases, this was an upward spread, as occurs in the upstroke of the blink, but in a few instances, surprisingly, the TFLL spread downward. We have no explanation for this, but it may relate to the fact that the palpebral aperture does not close completely in full downgaze so that the TFLL is not fully compressed in this position. This may influence the direction of forces driving TFLL movement when the primary position is resumed.

Figure 11a-c. DR-1TM images. Variations in bright band patterns in different subjects at various times after the return saccade. a: irregular. b: disjointed. c: Marangoni effect.


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PRECORNEAL TEAR FILM AS FLUID SHELL / Yokoi, et al Figure 12a-f. Subject 12. Dark bands seen in the fluorescein-stained tear film, immediately after 2 separate, vertical return saccades (a and d), corresponding to the position of the upper and lower zones of meniscusinduced thinning while the eyes were in downgaze. a-c and d-f: The tear film drains rapidly downward, below the location of the lowest dark band, over 4.6 sec (upper panel) and 5.5 sec (lower panel). Note too, that a fine dark band has been added to the tear film above, by a partial blink (e and f).

B. Mucoaqueous Subphase In the stained MAS of the tear film studied in the horizontal gaze paradigm, dark arcs were present, usually below, at the end of the return saccade. We consider these to be zones of meniscus-induced thinning, imprinted on the MAS layer as the cornea rests, briefly, in gaze to one side or the other. With each new saccade, a fresh arc is deposited, conforming in shape to the contour of the relevant lid margin, which was superimposed on the previous arc. We observed further that the cornea below the new arc was coated with a fresh tear film, presumably applied while the lower edge of the cornea rested behind the lower lid. This fresh coat obliterates the tail of the previous arc on that side (Figure 7). A striking event in the downgaze paradigm was the consistent formation of one or two horizontal dark bands, one above the other in the stained tear film, seen on return to the primary position of gaze. The mechanism of their formation appears to be identical to that involved in the formation of the dark arcs during horizontal versions; that is, they represent imprints of the upper and lower

Figure 13. Subject 8. Multiple dark bands are added to the stained film by frequent partial blinking. The drop instillation volume was probably high in this subject.

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zones of meniscus-induced thinning, here, laid down when the eyes are in full downgaze. Since the lid travels a greater distance than the limbus to reach its downgaze position, it may be concluded that their movements are not synchronous. In most instances, these dark bands were highly stable, remaining in place and sometimes deepening in intensity (i.e., darkening) for seconds after the end of the return saccade. Their failure to fill in, in the absence of blinking, implies that the stained bloc of precorneal fluid has properties of a gel rather than those of a purely aqueous layer which may be assumed to be composed chiefly of goblet cell mucin. We conclude that secretory mucin forms a distinct but dynamic gel layer within the tear film, based on weak noncovalent interactions, hydrogen bonds/Van der Waals attraction/steric interactions, between the polymer molecules. The gel disintegrates with the downstroke of each blink, is restored in the upstroke, and remains stable in open eye conditions, when the tear film is stable and no shear is applied. This does not preclude the presence of a more superficial watery layer covering the surface of the gel, which, perhaps, is partly drawn off as the menisci are formed in the upstroke of the blink. In subject 12, the stained film below the lower dark band appeared to drain downward during continued blink suppression (Figure 12). The cause for this is not known. It is unlikely to be due to the effect of gravity, which is thought not to influence flow within the tear film.45 It is possible that fluid movement is driven by surface tension forces or that it is due to an exaggeratedly negative hydrostatic pressure in the lower tear meniscus, but the latter would imply access between the meniscus and the fluid layer, which is normally denied after formation of meniscus-induced thinning after a blink.29,31 A final possibility is that there was a localized increase in evaporative water loss over the tear film below the lower dark band, perhaps due to a disruption of the TFLL. In this case, the vertical, dark zones would be construed as due to tear

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Figure 14. Subject 11. Multiple Marangoni-like patterns have formed in the stained film after a vertical return saccade.

film thinning or to the occurrence of fluorescein quenching.46,47 While this cannot be excluded, it did not fit in with the appearance of flow suggested by video records. The observations presented here have important implications for our understanding of tear film dynamics. We normally consider that the MAS of the tear film is spread by the act of blinking. The horizontal saccade paradigm suggests that, when the cornea moves behind the lower lid, the part that is covered by the tarsal plate acquires a fresh mucoaqueous tear film coating. It follows that with downward and return movements of the eye, a similar event will occur, with fresh tear film spreading mainly over the lower but also over the upper cornea, spread by the movement of the cornea over the tarsal conjunctiva. This offers a means by which the goblet cells of the lower tarsal conjunctiva may directly contribute to the MAS of the tear film and may explain why the behavior of the stained film below the lower dark bands was potentially different from that above it, after a vertical return saccade. If both lids contribute to coating during the downgaze paradigm, then the stained tear film at the end of the return saccade is a composite structure, having been coated by the upper tarsal conjunctiva above the upper dark band, by the lower tarsal conjunctiva below the lower dark band, and retaining its original, post-blink coating, between the bands. The dimensions and general location of the lower of the dark bands were similar to those of the bright bands seen against the red reflex in the TFLL studies. Because many of the bright bands occur at roughly the same level of the cornea as the dark bands, we initially considered that they might represent the same phenomenon imaged by two separate systems. However, further scrutiny of subject videos confirmed that they were independent events. The key distinguishing feature is that the dark bands, representing imprints of the upper and lower lid zones of meniscus-induced thinning, persist in the absence of blinking, but are cleared immediately in a single blink by the newly spread tear film. Conversely, the bright bands may fade in the absence of blinking, but if they do not, they persist if a blink occurs. Therefore, it can be concluded that the tear film itself is not the source of the bright bands. THE OCULAR SURFACE /

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On the one hand, when the eye returns to the primary position, the TFLL can be seen to be moving vertically across the bright bands, lying anterior to them. On the other hand, since these bands persist despite blinking, their source is not in the aqueous subphase. We therefore conclude that the bright bands originate in the cornea. We hypothesize that they are due to indentation of the corneal surface by the lower lid margin while the eye is in downgaze. This is consistent with reports of transient corneal indentation when the eye is in downgaze. Significant regions of topographic disturbance have been observed in young individuals maintaining downgaze and may give rise to polyopia. Effects are greater for larger than for smaller angles of downgaze,39 and presumably a time component is involved. These have been attributed to the effects of lid margin pressure, particularly of the upper lid, on corneal shape48-50 and may well be the cause of the bright bands seen here. Here, however, we have inferred that the lower lid margin may be more important. If this is true, then a further point to note is that the moving TFLL does not ripple as it flows over the bright band, which suggests that any surface indentation is occupied by fluid whose surface is in the anterior plane of the tear film. Specifically, this excludes the possibility that the bright bands are the interferometric counterpart of the meniscus-induced thinning giving rise to the lower dark bands. With this in mind, it became apparent that phenomena such as the Marangoni figures and the curious sharp peaks seen on interferometry represent the interferometric counterpart of events occurring in the “aqueous” subphase of the tear film, unrelated to the bright band phenomenon. In confirmation of this, we have noted that, although these phenomena may be superimposed on the bright band region in some subjects, they may also be present and evolve from points either above or below the bands. This explains the finding that Marangoni and other effects are also seen in the stained film, in relation to the lower dark bands. V. SUMMARY AND CONCLUSIONS It is apparent that the behavior of the precorneal TFLL during blinking and saccades is complex. As described earlier18 and as has been confirmed by others,19,20 the TFLL undergoes compression and expansion in a series of blinks during the downstroke and upstroke of the blink, respectively. For this to occur, the TFLL must be stripped from the aqueous subphase in the downstroke and must re-associate with it at the end of the upstroke. Our current research indicates that it exists as a stable fluid shell in combination with MAS, during horizontal saccades, attached to the underlying cornea. In a simple sequence of vertical saccades involving downgaze, a pause, and then a return to the primary position, the TFLL and also the aqueous subphase may be disturbed below with continued blink suppression. The MAS, assumed here to correspond to the fluoresceinstained tear film, retains the imprint of meniscus-induced thinning during both horizontal saccades and vertical -

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Figure 15. Schematic diagram, proposing a hypothetical coating mechanism by the tarsal conjunctiva during vertical saccades. Left eye, a-c: The formation of a lower dark arc is shown after a temporal saccade (see text for details). Note application of a tear film coat by the lower tarsal cornea while the eye is in temporal gaze. d-f: It is hypothesized that, in the downgaze paradigm, with downgaze and the return saccade, lower and upper parts of the cornea are coated by the corresponding tarsal conjunctivae (f. iii., iv.), while the intervening tear film, demarcated by upper and lower dark bands (f. i., ii.), remains undisturbed.

saccades, seen as dark arcs and bands in the fluorescent film (Figure 15). Persistence of these and of the familiar dark bands associated with partial blinks, occurring during blink suppression, strongly suggests the behavior of a gel, so that the term mucoaqueous subphase is appropriate. These studies also provide preliminary evidence that a tear film coating is applied to the cornea by the upper and lower tarsal plates in the downgaze paradigm and presumably whenever the surface of the globe moves under the tarsal plates. The downgaze paradigm mimics events that might occur on an everyday basis when an individual working at a computer changes gaze repetitively from the computer screen to text located at a lower level and might be expected to lead to an increase in evaporative loss over the lower cornea. This would compound desiccating stress from the slowing of the blink rate known to occur when engaging in difficult mental tasks, reading, or working at a video terminal.3,51-53 It could also be expected to contribute to ocular surface damage when eye movements are restricted, as in endocrine exophthalmos and supranuclear palsies. REFERENCES 1. Doane MG. Blinking and the mechanics of the lacrimal drainage system. Ophthalmology 1981;88:844-51

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2. Goto E, Tseng SC. Kinetic analysis of tear interference images in aqueous tear deficiency dry eye before and after punctal occlusion. Invest Ophthalmol Vis Sci 2003;44:1897-905 3. Tsubota K, Nakamori K. Dry eyes and video display terminals. N Engl J Med 1993;328:584 4. Yokoi N, Yamada H, Mizukusa Y, et al. Rheology of spread in normal and aqueous tear-deficient dry eyes. Invest Ophthalmol Vis Sci 2008;49: 5319-24 5. Wolff E. The muco-cutaneous junction of the lid margin and the distribution of the tear fluid. Trans Ophthalmol Soc UK 1946;66: 291-308 6. Holly FJ, Lemp MA. Wettability and wetting of corneal epithelium. Exp Eye Res 1971;11:239-50 7. Bakan J. Microencapsulation. In: Lachman L, Liberman HS, Kanig, (eds). The Theory and Practice of Industrial Pharmacy. USA, Lea & Febiger, 1970. pp 412-30 8. Cope C, Dilly PN, Kaura R, et al. Wettability of the corneal surface: a reappraisal. Curr Eye Res 1986;5:777-85 9. Argüeso P, Gipson IK. Epithelial mucins of the ocular surface: structure, biosynthesis and function. Exp Eye Res 2001;73:281-9 10. Argüeso P, Tisdale A, Spurr-Michaud S, et al. Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye. Invest Ophthalmol Vis Sci 2006;47:113-9 11. Argüeso P, Guzman-Aranguez A, Mantelli F, et al. Association of cell surface mucins with galectin-3 contributes to the ocular surface epithelial barrier. J Biol Chem 2009;284:23037-45 12. Cher I. Another way to think of tears: blood, sweat, and. “dacruon”. Ocul Surf 2007;5:251-4

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PRECORNEAL TEAR FILM AS FLUID SHELL / Yokoi, et al 13. Spurr-Michaud S, Argüeso P, Gipson I. Assay of mucins in human tear fluid. Exp Eye Res 2007;84:939-50 14. Gipson IK. Distribution of mucins at the ocular surface. Exp Eye Res 2004;78:379-88 15. Gipson IK. The ocular surface: the challenge to enable and protect vision: the Friedenwald lecture. Invest Ophthalmol Vis Sci 2007;48: 4391-8 16. Sharma A. Energetics of cornea1 epithelial cell-ocular mucus-tear film interactions: some surface-chemical pathways of cornea1 defense. Biophys Chem 1993;47:87-99 17. Owens H, Phillips J. Spreading of tears after a blink: velocity and stabilization time in healthy eyes. Cornea 2001;20:484-7 18. Bron AJ, Tiffany JM, Gouveia SM, et al. Functional aspects of the tear film lipid layer. Exp Eye Res 2004;78:347-60 19. Rolando M, Valente C, Barabino S. New test to quantify lipid layer behavior in healthy subjects and patients with keratoconjunctivitis sicca. Cornea 2008;27:866-70 20. Ring MH, Rabensteiner DF, Horwath-Winter J, et al. Introducing a new parameter for the assessment of the tear film lipid layer. Invest Ophthalmol Vis Sci 2012;53:6638-44 21. Mishima S, Maurice DM. The oily layer of the tear film and evaporation from the corneal surface. Exp Eye Res 1961;1:39-45 22. Leigh RJ, Zee DS. Neurology of Eye Movements. Oxford University Press, 2006 23. Holly FJ. Physical chemistry of the normal and disordered tear film. Trans Ophthalmol Soc U K 1985;104:374-80 24. Sharma A, Ruckenstein E. The role of lipid abnormalities, aqueous and mucus deficiencies in the tear film breakup, and implications for tear substitutes and contact lens tolerance. J Colloid Interface Sci 1986;111: 8-34 25. Rottach KG, Das VE, Wohlgemuth W, et al. Properties of horizontal saccades accompanied by blinks. J Neurophysiol 1998;79:2895-902 26. King-Smith PE, Fink BA, Nichols JJ, et al. The contribution of lipid layer movement to tear film thinning and breakup. Invest Ophthalmol Vis Sci 2009;50:2747-56 27. Georgiev GA, Yokoi N, Koev K, et al. Surface chemistry study of the interactions of benzalkonium chloride with films of meibum, corneal cells lipids, and whole tears. Invest Ophthalmol Vis Sci 2011;52: 4645-54 28. Georgiev GA, Yokoi N, Ivanova S, et al. Surface chemistry study of the interactions of pharmaceutical ingredients with human meibum films. Invest Ophthalmol Vis Sci 2012;53:4605-15 29. Miller KL, Polse KA, Radke CJ. Black-line formation and the “perched” human tear film. Curr Eye Res 2002;25:155-62 30. Gaffney EA, Tiffany JM, Yokoi N, et al. A mass and solute balance model for tear volume and osmolarity in the normal and the dry eye. Prog Retin Eye Res 2010;29:59-78 31. McDonald JE, Brubaker S. Meniscus-induced thinning of tear films. Am J Ophthalmol 1971;72:139-46 32. King-Smith PE, Fink BA, Hill RM, et al. The thickness of the tear film. Curr Eye Res 2004;29:357-68


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33. Doane MG. Interactions of eyelids and tears in corneal wetting and dynamics of the normal human eyeblink. Am J Ophthalmol 1980;89:507-16 34. Wong H, Fatt I, Radke CJ. Deposition and thinning of the human tear film. J Colloid Interface Sci 1996;184:44-51 35. Holly FJ, Holly TF. Advances in ocular tribology. Adv Exp Med Biol 1994;350:275-83 36. Berger RE, Corrsin S. A surface tension gradient mechanism for driving the pre-corneal tear film after a blink. J Biomechanics 1974;7:225-38 37. Johnson ME, Murphy PJ. The effect of instilled fluorescein solution volume on the values and repeatability of TBUT measurements. Cornea 2005;24:811-7 38. Benedetto DA, Clinch TE, Laibson PR. In vivo observation of tear dynamics using fluorophotometry. Arch Ophthalmol 1984;102:410-2 39. Smeets JB, Hooge IT. Nature of variability in saccades. J Neurophysiol 2003;90:12-20 40. Afsar-Siddiqui AB, Luckham PF, Matar OK. The spreading of surfactant solutions on thin liquid films. Adv Colloid Interface Sci 2003;106: 183-236 41. Bron AJ, Yokoi N, Georgiev GA, et al. The lipid layer forms a semi-rigid carapace over the tear film, following spreading in the interblink. 2011; ARVO abstract N 2096 42. Rosenfeld L, Cerretani C, Leiske DL, et al. Structural and rheological properties of meibomian lipid. Invest Ophthalmol Vis Sci 2013;54: 2720-32 43. Georgiev GA, Yokoi N, Ivanova S, et al. Surface chemistry study of the interactions of hyaluronic acid and benzalkonium chloride with meibomian and corneal cell lipids. Soft Matter 2013;9:10841-56 44. Holly FJ. Formation and rupture of the tear film. Exp Eye Res 1973;15: 515-25 45. Ehlers N. The precorneal film: biomicroscopical, histological and chemical investigatioons. Acta Ophthalmol 1965;81(suppl):1-136 46. Nichols JJ, King-Smith PE, Hinel EA, et al. The use of fluorescent quenching in studying the contribution of evaporation to tear thinning. Invest Ophthalmol Vis Sci 2012;53:5426-32 47. King-Smith PE, Ramamoorthy P, Braun RJ, et al. Tear film images and breakup analyzed using fluorescent quenching. Invest Ophthalmol Vis Sci 2013;54:6003-11 48. Shaw AJ, Collins MJ, Davis BA, et al. Corneal refractive changes due to short-term eyelid pressure in downward gaze. J Cataract Refract Surg 2008;34:1546-53 49. Ford JG, Davis RM, Reed JW, et al. Bilateral monocular diplopia associated with lid position during near work. Cornea 1997;16:525-30 50. Buehren T, Collins MJ, Carney L. Corneal aberrations and reading. Optom Vis Sci 2003;80:159-66 51. Tsubota K. Tear dynamics and dry eye. Prog Retin Eye Res 1998;17: 565-96 52. Tsubota K, Miyake M, Matsumoto Y, et al. Visual protective sheet can increase blink rate while playing a hand-held video game. Am J Ophthalmol 2002;133:704-5 53. Jansen ME, Begley CG, Himebaugh NH, et al. Effect of contact lens wear and a near task on tear film break-up. Optom Vis Sci 2010;87:350-7

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Effect of benzalkonium chloride on the stability of the precorneal tear film in rabbit and man.

Tear film dynamics on soft contact lenses.

Sleep deprivation reduces tear secretion and impairs the tear film.

The importance of the precorneal tear film for the quality of optical imaging.

A Controlled Study on the Correlation between Tear Film Volume and Tear Film Stability in Diabetic Patients.

A new model of central lid margin apposition and tear film mixing in spontaneous blinking.

Effect of topically applied pilocarpine on tear film pH.

Effect of hydrogel lens wear on tear film stability.

Effect of blinking on tear elimination as evaluated by dacryoscintigraphy.

The pH in the precorneal tear film and under a contact lens measured with a fluorescent probe.

[Clinical diagnostics for the tear film and the ocular surface].

The tear ferning test: a simple clinical technique to evaluate the ocular tear film.

Staining of the conjunctiva and conjunctival tear film.

Dynamics and function of the tear film in relation to the blink cycle.

Botulinum toxin type a injection for lateral canthal rhytids : effect on tear film stability and tear production.

Tear film profile in Graves' ophthalmopathy.

Tear film measurement by optical reflectometry technique.

Dog Tear Film Proteome In-Depth Analysis.

[Tear film diagnosis in contact lens wearers].

Computed tear film and osmolarity dynamics on an eye-shaped domain.

Basal values, intra-day and inter-day variations in tear film osmolarity and tear fluorescein clearance.

Maximum-likelihood estimation in Optical Coherence Tomography in the context of the tear film dynamics.

Eyelid Tattooing Induces Meibomian Gland Loss and Tear Film Instability.

Dry eye in rheumatoid arthritis: tear film osmolarity and inflammation.