Blink patterns and kinematics of eyelid motion in ophthalmologically normal horses

Lori J. Best Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Vasilios Alexiades Department of Mathematics, College of Arts and Sciences, University of Tennessee, Knoxville, TN 37996.

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Diane V. H. Hendrix Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Thomas Chen Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Daniel A. Ward Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Abstract

OBJECTIVE To describe qualitative blinking patterns and determine quantitative kinematic variables of eyelid motion in ophthalmologically normal horses.

ANIMALS 10 adult mares.

PROCEDURES High-resolution videography was used to film blinking behavior. Videotapes were analyzed for mean blink rate, number of complete versus incomplete blinks, number of unilateral versus bilateral blinks, and subjective descriptions of blinking patterns. One complete blink for each horse was analyzed with image-analysis software to determine the area of corneal coverage as a function of time during the blink and to calculate eyelid velocity and acceleration during the blink.

RESULTS Mean ± SD blink rate was 18.9 ± 5.5 blinks/min. Blinks were categorized as minimal incomplete (29.7 ± 15.6%), moderate incomplete (33.5 ± 5.9%), complete (30.8 ± 13.1%), and complete squeeze (6.0 ± 2.8%); 22.6 ± 9.0% of the blinks were unilateral, and 77.3 ± 9.1% were bilateral. Mean area of exposed cornea at blink initiation was 5.89 ± 1.02 cm2. Mean blink duration was 0.478 seconds. Eyelid closure was approximately twice as rapid as eyelid opening (0.162 and 0.316 seconds, respectively). Deduced maximum velocity of eyelid closure and opening was −16.5 and 7.40 cm/s, respectively. Deduced maximum acceleration of eyelid closure and opening was −406.0 and −49.7 cm/s2, respectively.

CONCLUSIONS AND CLINICAL RELEVANCE Kinematic variables of ophthalmologically normal horses were similar to values reported for humans. Horses had a greater percentage of complete squeeze blinks, which could increase tear film stability. Blinking kinematics can be assessed as potential causes of idiopathic keratopathies in horses.

Abstract

OBJECTIVE To describe qualitative blinking patterns and determine quantitative kinematic variables of eyelid motion in ophthalmologically normal horses.

ANIMALS 10 adult mares.

PROCEDURES High-resolution videography was used to film blinking behavior. Videotapes were analyzed for mean blink rate, number of complete versus incomplete blinks, number of unilateral versus bilateral blinks, and subjective descriptions of blinking patterns. One complete blink for each horse was analyzed with image-analysis software to determine the area of corneal coverage as a function of time during the blink and to calculate eyelid velocity and acceleration during the blink.

RESULTS Mean ± SD blink rate was 18.9 ± 5.5 blinks/min. Blinks were categorized as minimal incomplete (29.7 ± 15.6%), moderate incomplete (33.5 ± 5.9%), complete (30.8 ± 13.1%), and complete squeeze (6.0 ± 2.8%); 22.6 ± 9.0% of the blinks were unilateral, and 77.3 ± 9.1% were bilateral. Mean area of exposed cornea at blink initiation was 5.89 ± 1.02 cm2. Mean blink duration was 0.478 seconds. Eyelid closure was approximately twice as rapid as eyelid opening (0.162 and 0.316 seconds, respectively). Deduced maximum velocity of eyelid closure and opening was −16.5 and 7.40 cm/s, respectively. Deduced maximum acceleration of eyelid closure and opening was −406.0 and −49.7 cm/s2, respectively.

CONCLUSIONS AND CLINICAL RELEVANCE Kinematic variables of ophthalmologically normal horses were similar to values reported for humans. Horses had a greater percentage of complete squeeze blinks, which could increase tear film stability. Blinking kinematics can be assessed as potential causes of idiopathic keratopathies in horses.

The cornea requires a healthy tear film to protect and nourish the ocular surface and to provide optical clarity for optimal vision. An accurate and precise blinking mechanism is essential for the distribution and drainage of the tear film and to provide corneal protection.1 Numerous techniques have been used in humans to evaluate eyelid motion during normal blinking and to specifically analyze such quantitative kinematic variables as blink velocity and acceleration.2–6 Deviations in kinematic values can be both a cause or an effect of corneal disease. For example, abnormalities in blink kinematics of humans are believed to contribute to the development of keratopathy associated with Graves disease6 and superior limbic keratoconjunctivitis.7 Conversely, there are increases in the frequency of incomplete blinking, compared with the frequency of complete blinking, secondary to dry eye disease,4,6,8,9 and humans with tear film abnormalities may compensate by blinking with a higher percentage of complete blinks and therefore spend a greater amount of time with the affected eyelids closed.2,10 Thus, evaluation of blinking kinematics may assist in determining the underlying causes of a number of keratopathies in humans.

Horses are particularly prone to corneal disease owing in part to the anatomic placement of their eyes, which leaves them comparatively exposed, with corneal disorders ranging from relatively benign to vision- or globe-threatening. However, the authors are not aware of any published studies regarding blinking physiology of horses. The purpose of the study reported here was to determine values for SEBR and characterize blink patterns of horses in a controlled environment. We intended to use high-speed videography and image-analysis software to generate a mathematical description of the normal blink mechanism and to determine maximum velocity and acceleration of the superior eyelid of horses. We hypothesized that a relatively greater percentage of blinks would be complete versus incomplete to adequately spread the tear film over the large prominent cornea in equids. We further hypothesized that the maximum velocity and acceleration of a blink would be greater than in humans to allow for a complete blink over a larger cornea without increasing the total time the eyelids are closed.

Materials and Methods

Animals

Ten adult horses from the University of Tennessee teaching herd were included in the study. Horses were free of systemic disease and were not receiving medical treatment at the time of the study. All the horses were females (mean ± SD age, 16.3 ± 3.7 years) and were housed in an outdoor paddock. There were 6 American Quarter Horses, 3 Tennessee Walking Horses, and 1 mixed-breed horse. A complete ophthalmic examination, which included Schirmer tear testing, applanation tonometry, slit-lamp biomicroscopy, and indirect ophthalmoscopy, was performed on each horse. Horses with ocular abnormalities, including an abnormal eyelid conformation, were excluded from the study. All experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and visual research and were approved by the University of Tennessee Institutional Animal Care and Use Committee.

Videography

Videography of each horse was performed during 2 weeks in February 2014. Videography was performed between 9:20 am and 5:00 pm. Horses were placed in standard stocks without sedation or head restraint. Horses that displayed anxiety when placed in the stocks were excluded from the study. The stocks were located in an indoor facility with a temperature ranging from 20° to 21.1°C and relative humiditya between 20% and 46%.

A ruler was taped to the skin at a location 3 cm ventral to the right eye to provide a reference for image analysis measurements. The ruler was placed approximately 15 minutes before initiation of videography to allow horses to become acclimated to its presence; horses also acclimated to the presence of the cameras, lights, and personnel during that 15-minute period. Videography was performed before data collection to reduce the impact of stress and anticipatory blinking on spontaneous blink activity.

Videography was performed with 2 camcordersb (1,920 × 1,080 pixels; 1080 interlaced). Both cameras were positioned so that they were level (horizontal) at the eye height of each horse; both cameras recorded simultaneously. The primary camera was positioned in line with the pupil-optic nerve axis of the right eye, and the secondary camera was positioned perpendicular to the first camera (Figure 1). Lights were positioned in the room behind the cameras to provide sufficient illumination for the video recordings. To minimize stress to the horses, the cameras and lights were positioned before each horse was brought into the room and placed in the stocks. Digital images were stored on secure-digital memory cardsc and transferred as .mov files to a digital editing systemd for video and image analysis.

Figure 1—
Figure 1—

Schematic diagram of the videography setup depicting the relationship among the horse, the primary camera that recorded only the right eye, the secondary camera that recorded both eyes, and the lights.

Citation: American Journal of Veterinary Research 79, 6; 10.2460/ajvr.79.6.650

Recording was conducted for a minimum of 5 minutes for each horse. Frame rate for the recordings was 30 frames/s. Video recordings from the primary camera were analyzed to determine blink rate and blink kinematic variables and to enable a subjective description of blinks; only recordings of the right eye were used for these analyses. The secondary camera was positioned to record both eyes simultaneously; therefore, recordings from that camera were analyzed to determine blink rate and the ratio of unilateral to bilateral blinks.

Biochemical evaluation of tears

After videography was completed, osmolality and electrolyte composition of tears were measured as described elsewhere.11 Tears were collected in capillary tubes at the medial canthus, and tear samples obtained from each horse were stored at −62°C until analysis. Tear samples were allowed to thaw completely prior to analysis. Osmolality was measured with a vapor-pressure osmometer.e Immediately after the osmolality was measured, sodium, potassium, magnesium, and calcium concentrations were determined by use of an automated biochemical analyzer.f The SEBR and blink characteristics of each horse were compared to the osmolality and electrolyte composition, and correlations between SEBR and biochemical variables were calculated.

Image analysis

Video recordings were reviewed. One complete blink for each horse in which the horse's head did not move immediately before or after the blink and in which the cornea remained perpendicular to the primary camera throughout the blink was selected for detailed analysis. Each frame was analyzed separately from the other frames. The first obvious downward movement of the superior eyelid was marked as the starting point for each blink, and the blink endpoint was the point after which there was no additional widening of the palpebral fissure. Fifteen frames were adequate to capture a complete blink for each horse.

Each frame was downloaded into an image-analysis system.g A computer cursor was used to outline the palpebral fissure, thus defining the fissure as an ROI (Figure 2). The image-analysis system then calculated the area within the ROI of each frame via reference to the ruler located 3 cm ventral to the right eye. In some instances, frames were too blurred to allow accurate placement of the cursor and creation of the ROI; such frames were eliminated from the analysis. For each horse, areas were expressed as the percentage of fully exposed area prior to initiation of the blink. Data for the percentage of fully exposed area versus time were used to calculate a mean value for the 10 horses at each of 15 time points and fitted by use of the method of least squares to a cubic function of time.

Figure 2—
Figure 2—

Photograph of the right eye of a horse. An ROI is outlined (red line) prior to commencement of a complete blink. The area of the ROI was calculated by use of an image-analysis system. Notice the ruler (scale in centimeters) used to calibrate the ROI data was affixed to the horse's face at a point 3 cm ventral to the right eye.

Citation: American Journal of Veterinary Research 79, 6; 10.2460/ajvr.79.6.650

Images then were used to assess the position of the superior eyelid as a function of time during a complete blink. Midpoint of the superior eyelid was identified and traced to its corresponding midpoint on the inferior eyelid following eyelid closure. Position of the midpoint of the superior eyelid was defined as the distance from the corresponding midpoint of the inferior eyelid (as measured by use of the image-analysis system), and the position of the superior eyelid midpoint in each frame was plotted as a function of time. These plots were fitted to cubic functions of time by use of the method of least squares, and the first and second derivatives of these plot fittings were used to create plots of velocity versus time and acceleration versus time, respectively. Maximum and minimum velocities and accelerations during eyelid closure and opening were derived from equations of the plot fittings. Movement of the superior eyelid in a ventral (downward) direction was defined as the negative direction, and movement of the superior eyelid in a dorsal (upward) direction was defined as the positive direction.

Statistical analysis

Kinematic variables were reported as mean ± SD. Osmolality and electrolyte composition of tear film were correlated with SEBR (Pearson correlation coefficient) by use of commercially available statistical software.h Correlations were considered significant at values of P < 0.05.

Results

Fifteen horses were originally evaluated for inclusion in the study. Five did not remain motionless for a sufficient amount of time to enable us to obtain interpretable video recordings, and they were therefore not included in the analyses. The remaining 10 horses were tolerant of the stocks and stood extremely still during videography.

Evaluation of video recordings revealed that the inferior eyelid did not move appreciably with most blinks, with corneal coverage provided almost exclusively by the superior eyelid. Evaluation of the recordings revealed 4 distinctive patterns of blinking as determined on the basis on the amount of the cornea that was covered and forcefulness of the blinks. Blinks were categorized as minimal incomplete blinks (< 50% of the cornea was covered by the superior eyelid), moderate incomplete blinks (50% to 95% of the cornea was covered), complete blinks (> 95% of the cornea was covered), and complete squeeze blinks (a complete blink with forceful contraction of the orbicularis oculi muscles [Supplementary Video S1, available at http://avmajournals.avma.org/doi/suppl/10.2460/ajvr.79.6.650]). Overall mean ± SD blink rate was 18.9 ± 5.5 blinks/min, which consisted of 29.7 ± 15.6% minimal incomplete blinks, 33.5 ± 5.9% moderate incomplete blinks, 30.8 ± 13.1% complete blinks, and 6.0 ± 2.8% complete squeeze blinks. Blinks were unilateral (22.6 ± 9.1%; right eye, 11.9 ± 9.9%; left eye, 10.7 ± 3.9%) and bilateral (77.3 ± 9.2%).

The SEBR and percentage of complete blinks were evaluated for correlations with electrolyte concentrations and osmolality of the tear film. We did not collect a sufficient tear volume to assess osmolality for 1 horse and electrolyte composition for 3 horses. A direct correlation was found between SEBR and sodium concentration (134.75 ± 10 mmol/L [n = 7]; r = 0.845; P = 0.02), and an indirect relationship was found between SEBR and potassium concentration (16.3 ± 5.77 mmol/L [7]; r = −0.968; P = 0.01). There was a direct relationship (r = 0.983; P = 0.01) between SEBR and the sodium-to-potassium ratio (Figure 3). There was not a significant correlation between SEBR and magnesium, calcium, or chloride concentrations (n = 7) or osmolality (9).

Figure 3—
Figure 3—

Plot of the SEBR versus the tear sodium concentration-to-potassium concentration ratio for 7 horses. Each circle represents results for 1 horse. Notice the strong positive correlation.

Citation: American Journal of Veterinary Research 79, 6; 10.2460/ajvr.79.6.650

Mean ± SD area of exposed cornea at the initiation of each blink (as determined by use of the image-analysis system) was 5.89 ± 1.02 cm2. The percentage of corneal coverage versus time was plotted (Figure 4). Data were best described by the following equation: area(t) = 1.14 – (11.5•t) + (45.4•t2) – (47.3•t3), where area is the area of the exposed ocular surface (in centimeters squared), and t is time (in seconds).

Figure 4—
Figure 4—

Mean ± SD percentage of the area of cornea exposed as a function of time for complete blinks of the superior eyelids of 10 horses. Notice that the line of best fit for the data was best described by a cubic equation. t = Time.

Citation: American Journal of Veterinary Research 79, 6; 10.2460/ajvr.79.6.650

The position of the superior eyelid during a complete blink was evaluated as a function of time by use of a single function to describe the entire blink (both eyelid closure and eyelid opening phases) as well as independent functions (1 for the eyelid closure phase and 1 for the eyelid opening phase). Use of the independent functions yielded the best fit of the data; thus, independent functions were used for the remainder of the analyses. Position of the midpoint of the superior eyelid was plotted against time during eyelid closure (Figure 5). Data were best described by the following equation: positionc(t) = 2.02 – (203•t2) + (834•t3), where positionc is the position of the midpoint of the superior eyelid with respect to the midpoint of the inferior eyelid (in centimeters). The first derivative of that equation yielded the following equation: velocityc(t) = (−406•t) + (2,502•t2), where velocityc is the superior eyelid velocity during closure. The time to eyelid closure, which was calculated by setting velocityc(t) = 0, was 0.162 seconds. The time at which maximum velocity was detected, which was calculated by setting the derivative of the velocityc(t) function = 0, was 0.0811 seconds, and maximum velocity was −16.5 cm/s (velocity was negative because movement of the superior eyelid was in a downward direction). Mean velocity during eyelid closure was calculated by use of the following equation: mean velocity = (1/0.162)∫00.162(dp/dt)dt, where dp/dt is change in position per change in unit time. Mean velocity during eyelid closure was −11.0 cm/s.

The second derivative of the equation positionc(t) = 2.02 – (203•t2) + (834•t3) yielded the following equation: accelerationc(t) = −406 + (5,004•t), where accelerationc represents acceleration of the eyelid during closure. Because this was a linear relationship, maximum acceleration during closure was equal to the value of acceleration(t) at one of the endpoints of the interval (ie, when t = 0 or t = 0.162), whichever was greater. Acceleration was approximately the same (albeit in different directions) at both endpoints (−406 cm/s2 at t = 0 seconds and 405 cm/s2 at t = 0.162 seconds [Figure 5]). Mean acceleration during eyelid closure was −0.676 cm/s2.

The same process was repeated for eyelid opening. Position of the midpoint of the superior eyelid was plotted against time (Figure 5). Data were best described by the following equation: positiono(t) = −0.815 + (4.7•t) + (15•t2) – (27.8•t3), where positiono is the position of the midpoint of the superior eyelid with respect to the midpoint of the inferior eyelid (in centimeters). The first derivative of that equation yielded the following equation: velocityo(t) = 4.7 + (30•t) – (83.4•t2), where velocityo(t) is the superior eyelid velocity during opening. Maximum velocity during eyelid opening was 7.40 cm/s and was detected at 0.180 seconds. Mean velocity during eyelid opening was 5.07 cm/s. Time to the end of the blink, which was calculated by setting velocityo(t) = 0, was 0.478 seconds. Duration of the eyelid opening phase of the blink was 0.478 – 0.162 = 0.316 seconds. The second derivative of that equation yielded the following equation: accelerationo(t) = 30 – (166.8•t), where accelerationo(t) is the acceleration during eyelid opening. Maximum acceleration of eyelid opening was −49.7 cm/s2 at the time of complete reopening of the eyelid (t = 0.478 seconds). Mean acceleration during the eyelid opening phase of the blink was −23.4 cm/s2.

Figure 5—
Figure 5—

Position (A and B), velocity (C and D), and acceleration (E and F) of the superior eyelid as a function of time for eyelid closure (A, C, and E) and eyelid opening (B, D, and F). Notice that the scale on the x-axis differs between panels A and B, C and D, and E and F and that the scale on the y-axis differs between panels C and D and E and F. Eyelid closure occurred at a mean of 0.162 seconds, and eyelid opening occurred at a mean of 0.478 seconds; thus, opening required approximately twice as much time as closure. Maximum closing velocity was −16.5 cm/s and occurred at 0.0811 seconds (ie, when the slope of the curve was 0). Maximum velocity of eyelid opening was 7.40 cm/s and was attained at 0.180 seconds (approx 0.018 seconds after the initiation of eyelid opening). Acceleration during eyelid closure was greatest at the beginning and end of the closure phase (> 400 cm/s2 at both points), whereas maximum acceleration of eyelid opening was −49.7 cm/s2 and occurred at the time of complete reopening of the eyelid (0.478 seconds). For velocity and acceleration, movement of the superior eyelid in a ventral (downward) direction was defined as the negative direction, and movement of the superior eyelid in a dorsal (upward) direction was defined as the positive direction. Results for panels A and B represent mean ± SD. The line of best fit was determined for all data; all results were for analyses of complete blinks of the superior eyelids (n = 10 horses). See Figure 4 for remainder of key.

Citation: American Journal of Veterinary Research 79, 6; 10.2460/ajvr.79.6.650

Velocities and accelerations were obtained (Table 1). Values were determined by use of the lines of best fit at the midpoint of each time interval.

Table 1—

Change in the position of the superior eyelid during opening and closing phases of a complete blink for 10 horses.

Blink phaseTime interval (s)Starting and ending positions for interval (cm)Velocity during interval (cm/s)Acceleration during interval (cm/s2)
Closing0–0.0332.020 and 1.990–6.07–323
 0.033–0.0671.990 and 1.480–14.0–156
 0.067–0.1001.480 and 0.666–16.511.1
 0.100–0.1330.666 and 0.443–13.3178
 0.133–0.1670.443 and 0.245–4.61345
Opening0.167–0.2000.245 and 0.5757.40–0.582
 0.200–0.2330.575 and 0.7477.28–6.14
 0.233–0.2670.747 and 1.0706.99–11.7
 0.267–0.3001.070 and 1.2406.50–17.3
 0.300–0.3331.240 and 1.2305.84–22.8
 0.333–0.3671.230 and 1.6604.98–28.4
 0.367–0.4001.660 and 1.6203.94–33.9
 0.400–0.4331.620 and 1.8802.72–39.5
 0.433–0.5331.870 and 1.740−0.28NA

Results represent the value calculated for the superior eyelid at the midpoint of each time interval. Velocity during eyelid closing was calculated as follows: velocity(t) = (−406•t) + (2,502•t2). Acceleration during eyelid closing was calculated as follows: acceleration(t) = −406 + (5,004•t). Velocity during eyelid opening was calculated as follows: velocity(t) = 4.7 + (30•t) – (83.4•t2). Acceleration during eyelid opening was calculated as follows: acceleration(t) = 30 – (166.8•t). For all equations, t = time. For velocity and acceleration, movement of the superior eyelid in a ventral (downward) direction was defined as the negative direction, and movement of the superior eyelid in a dorsal (upward) direction was defined as the positive direction.

NA = Not applicable.

Discussion

The purpose of the study reported here was to describe the qualitative and quantitative variables of the blinking mechanism in ophthalmologically normal horses by use of readily available video recording equipment and computer software. Qualitatively, blinks could be segregated into 4 distinct types (2 types of incomplete blinks and 2 types of complete blinks). Complete blinks spread the tear film over the entire corneal surface, assist in drainage of the tear film, and have been associated with improved tear film stability in humans.8,9 Additionally, forceful or prolonged complete blinks (complete squeeze blinks) may further enhance expression of meibum and thus increase tear film stability.12 We hypothesized that horses may compensate for a larger amount of exposed corneal surface with a higher percentage of complete blinks relative to incomplete blinks, compared with the blink pattern of humans. This was not the case because the overall complete blink rate of 36.8% for the horses of the present study was similar to the rate of 40.2% reported for clinically normal humans.10 However, horses of the present study did have twice as many forceful complete squeeze blinks (6.0%), compared with only 3.4% for humans10; this may account for the stability of the tear film over the larger equine cornea given that these prolonged blinks enhance the expression of meibum.12

Additional factors that may have contributed to the complete versus incomplete blinking patterns in horses, compared with patterns in humans, included species differences and environmental factors. Horses are a prey species, and the stress of video recording and restraint in stocks may have artificially increased the frequency of incomplete blinks to minimize visual deprivation associated with complete eyelid closure. Corneal and tear film pathological conditions related to disturbances in the frequency of incomplete versus complete blinking have been identified in humans involved in high-focus activities (eg, use of video terminal displays).13 Horses engaged in high-stress or high-focus activities may also be predisposed to subtle exposure keratopathies secondary to blink abnormalities. In fact, corneal pathological conditions in race horses may be related to a decrease in eye blink rate or an increase in incomplete blinking.14 Further studies are warranted to investigate the percentage of complete versus incomplete blinks in various settings and in horses with subtle keratopathies.

Quantitative variables evaluated in the present study included the SEBR and the kinematic variables of velocity and acceleration of blinking. The SEBR has been evaluated extensively in humans and is influenced by both exogenous factors (tear film quality, corneal sensitivity, and environment)2,15 and endogenous factors (central pacemaker).16–18 The SEBR in clinically normal humans is between 6.2 and 23.3 blinks/min and differs with age, time of day, and mental focus and activity.16–18 The SEBR has been reported for guinea pigs (3 to 4 blinks/20 min),19 rabbits (1.00 ± 0.41 blinks/3 min),20 and horses (14.97 blinks/min).21 In the study reported here, the mean ± SD SEBR (18.9 ± 5.5 blinks/min) of horses was slightly higher than that reported previously.21 Exogenous environmental factors and ocular examination findings were not reported by the authors of that other study21 and could explain the small difference between results of their study and the study reported here. We attempted to limit exogenous variables in the present study by performing video recording in an indoor environment with a controlled temperature and relative humidity and by examining only horses with no ophthalmologic abnormalities. Video recordings were obtained while nonsedated horses were restrained in a standing position in stocks. Although this was not a natural environment and likely influenced the SEBR, it represented a commonly used setting for clinical examinations, so the data reported here should be valid for comparison with results for clinical cases. Video recording of horses with abnormal blinking or corneal pathological conditions could easily be performed in a similar setting and the results compared with those in the present study. Given that tear film stability is 1 factor that determines the SEBR and that the SEBR for the horses of the present study was similar to that reported for humans, it is likely that the tear film is at least as stable in horses as it is in humans.

A quantitative mathematical description of the complete blink of ophthalmologically normal horses was generated to allow for more objective comparison between ophthalmologically normal horses and horses suspected of having abnormalities in blinking kinematics. Mean ± SD exposed corneal surface was 5.89 ± 1.02 cm2 for the horses of the present study, compared with 1.76 cm2 for humans.22 We hypothesized that the maximum velocity and acceleration of the blink during the closure phase in horses would be greater than those in humans because of the need for horses to cover the large corneal area in a similar total blink time, thus preventing premature breakup of the tear film. However, the calculated maximum velocity of 16.5 cm/s for the horses of the present study was similar to that reported in clinically normal humans (7.58 to 20.7 cm/s), which made our hypothesis untenable.3,5,6,23–27 As an alternative hypothesis, adequate spread of tear film over the large equine cornea could still be accomplished if the mean velocity over the entire blink duration was high, despite a pedestrian maximum velocity. Unfortunately, we could not test this alternative hypothesis because mean closing and opening velocities (−11.0 cm/s and 5.07 cm/s, respectively, for the horses of the present study) have not been reported for humans. However, we believe that it is unlikely the mean velocities of horses would be greater than those of humans because the total blink duration for the horses of the present study (0.478 seconds) is substantially longer than that reported for humans (< 0.300 seconds).3,6,23,26,27 Given that horses have a larger exposed surface area, similar maximum blink velocity, and longer blink duration, compared with results for humans, we propose that the equine tear film has superior stability because of its biochemical constitution (particularly the high calcium concentration) and increased thickness, compared with those in humans.11,28 The comparatively prolonged blink duration in horses would appear to be counterintuitive, given that horses are a prey species and visual deprivation occurs during the eyelid closure phase.

Maximum closing and opening accelerations for the horses of the present study were −406.0 and 49.7 cm/s2, respectively. This closing acceleration is approximately 25% greater than that reported for humans.29 Greater acceleration allows for rapid attainment of maximum velocity, which minimizes the amount of time for corneal exposure and overall duration of blinking. However, because acceleration-versus-time relationships are obtained by use of the second derivative of position-versus-time relationships, deviations between actual data points and lines of best fit are magnified and experimental error is increased substantially; therefore, conclusions about acceleration would have been strengthened with the inclusion of additional data points. Increasing the frame speed to > 30 frames/s would have the largest impact toward improving accuracy of acceleration determination by increasing the number of data points during each blink.

A significant negative correlation was detected between the potassium concentration of the tear film and the SEBR, and a significant positive correlation was detected between the sodium concentration and the SEBR. These correlations may have been the result of a change in tear film or ocular surface dynamics related to electrolyte concentrations. Additionally, as the SEBR increases, the amount of time for corneal exposure to UV light decreases. This could be important in that high potassium concentrations in the tear film have been found to protect corneal epithelial cells against UVB-induced apoptosis.30 Alternatively, these correlations may have been coincidental, and a larger population of ophthalmologically normal horses would be required to confirm this hypothesis.

Limitations of the study reported here included a relatively small sample size, inclusion of only middle-aged and geriatric horses, and inclusion of only female horses. These limitations were unavoidable because of the population of the university teaching herd at the University of Tennessee. In humans, there are differences in the SEBR on the basis of age and gender. Blink rates are consistently significantly higher in women than in men and also higher in older women than in younger women.25 In addition, there are gender and age influences on eyelid displacement in humans, with larger eyelid movements in older men and younger women, compared with eyelid movements in older women and younger men.25 Older horses and horses with pituitary pars intermedia dysfunction have a lower corneal touch threshold and may subsequently have a diminished SEBR.31 Additionally, increasing the frame speed and accounting for the 3-D curvature of the cornea would increase accuracy when formulating a mathematical description of blinking. It is also important to mention that the kinematic data of the present study were applied only to spontaneous complete blinks and that kinematic variables would likely differ between voluntary and reflexive blinks.32

In the present study, blink characteristics and the SEBR were determined for a group of ophthalmologically normal horses. Blinking variables have been widely evaluated in humans and correlated with corneal disease but have not been assessed in horses. Given the clinical importance of corneal disease in horses, we strongly believe that blinking abnormalities should be evaluated as possible contributing factors in affected horses and especially should be assessed in horses with idiopathic keratopathies. The mathematical descriptions derived for the study reported here provided an objective method for use in assessing kinematic eyelid behavior, and the data collection methods used in this study could be easily adapted to clinical settings. We believe that with continued improvements in quality and simplicity of video acquisition, analysis of blinking kinematics could become a routine part of the ocular examination of horses with idiopathic keratopathies in the future.

Acknowledgments

Supported by the University of Tennessee Companion Animal Fund.

ABBREVIATONS

ROI

Region of interest

SEBR

Spontaneous eyeblink rate

Footnotes

a.

Model 613 indoor humidity monitor, AcuRite, Lake Geneva, Wis.

b.

AG-HMC80 3MOS AVCCAM HD shoulder-mount camcorder, Panasonic, Osaka, Japan.

c.

Ultra Plus 32GB class 10 SDHC cards, SanDisk, Milpitas, Calif.

d.

Final cut pro digital editing system, Apple, Cupertino, Calif.

e.

Vapro vapor pressure osmometer, model 5520, Wescor, Logan, Utah.

f.

Cobas 501c, Roche Diagnostics, Indianapolis, Ind.

g.

Image J. National Institutes of Health, Bethesda, Md. Available at: imagej.nih.gov/ij/. Accessed Jan 9, 2018.

h.

SigmaPlot for Windows, version 13, Systat Software, San Jose, Calif.

References

  • 1. Griepentroy GJ, Lucarelli MJ. Functions of the orbit and adnexa. In: Levin LA, Nilsson SFE, ver Hoeve J, et al, eds. Adler's physiology of the eye. 11th ed. Edinburgh: Saunders/Elsevier, 2011;333349.

    • Search Google Scholar
    • Export Citation
  • 2. Tsubota K, Hata S, Okusawa Y, et al. Quantitative videographic analysis of blinking in normal subjects and patients with dry eye. Arch Ophthalmol 1996;114:715720.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Malbouisson JM, Messias A, Garcia DM, et al. Modeling upper eyelid kinematics during spontaneous and reflex blinks. J Neurosci Methods 2010;191:119125.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Cardona G, Garcia C, Seres C, et al. Blink rate, blink amplitude, and tear film integrity during dynamic visual display terminal tasks. Curr Eye Res 2011;36:190197.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Berke A, Mueller S. The kinetics of lid motion and its effects on the tear film. Adv Exp Med Biol 1998;438:417424.

  • 6. Garcia DM, Messias A, Costa LO, et al. Spontaneous blinking in patients with Graves’ upper eyelid retraction. Curr Eye Res 2010;35:459465.

  • 7. Cher I. Blink-related microtrauma: when the ocular surface harms itself. Clin Experiment Ophthalmol 2003;31:183190.

  • 8. Wan T, Jin X, Lin L, et al. Incomplete blinking may attribute to the development of meibomian gland dysfunction. Curr Eye Res 2016;41:179185.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Hirota M, Uozato H, Kawamorita T, et al. Effect of incomplete blinking on tear film stability. Optom Vis Sci 2013;90:650657.

  • 10. Ousler GW, Abelson MB, Johnston PR, et al. Blink patterns and lid-contact times in dry-eye and normal subjects. Clin Ophthalmol 2014;8:869874.

    • Search Google Scholar
    • Export Citation
  • 11. Best LJ, Hendrix DVH, Ward DA. Tear film osmolality and electrolyte composition in healthy horses. Am J Vet Res 2015;76:10661069.

  • 12. Korb DR, Baron DF, Herman JP, et al. Tear film lipid layer thickness as a function of blinking. Cornea 1994;13:354359.

  • 13. Fenga C, Aragona P, Cacciola A, et al. Meibomian gland dysfunction and ocular discomfort in video display terminal workers. Eye (Lond) 2008;22:9195.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Wada S, Hobo S, Niwa H. Ulcerative keratitis in Thoroughbred racehorses in Japan from 1997 to 2008. Vet Ophthalmol 2010;13:99105.

  • 15. Collins M, Seeto R, Campbell L, et al. Blinking and corneal sensitivity. Acta Ophthalmol (Copenh) 1989;67:525531.

  • 16. Doughty MJ. Consideration of three types of spontaneous eyeblink activity in normal humans: during reading and video display terminal use, in primary gaze, and while in conversation. Optom Vis Sci 2001;78:712725.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Barbato G, Ficca G, Muscettola G, et al. Diurnal variation in spontaneous eye-blink rate. Psychiatry Res 2000;93:145151.

  • 18. Bacher LF, Smotherman WP. Spontaneous eye blinking in human infants: a review. Dev Psychobiol 2004;44:95102.

  • 19. Trost K, Skalicky M, Nell B. Schirmer tear test, phenol red thread tear test, eye blink frequency and corneal sensitivity in the guinea pig. Vet Ophthalmol 2007;10:143146.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Toshida H, Nguyen DH, Beuerman RW, et al. Evaluation of novel dry eye model: preganglionic parasympathetic denervation in rabbit. Invest Ophthalmol Vis Sci 2007;48:44684475.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Kirsty R, Andrew H, Meriel MC, et al. Cognitive differences in horses performing locomotor versus oral stereotypic behaviour. Appl Anim Behav Sci 2015;168:3744.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Koushan K, Skibell BC, Harvey JT, et al. Digital photography as a novel technique of measuring ocular surface dimensions. Orbit 2008;27:259265.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Doane MG. Interaction of eyelids and tears in corneal wetting and the dynamics of the normal human eyeblink. Am J Ophthalmol 1980;89:507516.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Somia NN, Rash GS, Epstein EE, et al. A computer analysis of reflex eyelid motion in normal subjects and in facial neuropathy. Clin Biomech (Bristol, Avon) 2000;15:766771.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Sforza C, Rango M, Galante D, et al. Spontaneous blinking in healthy persons: an optoelectronic study of eyelid motion. Ophthalmic Physiol Opt 2008;28:345353.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Wu Z, Begley CG, Situ P, et al. The effects of mild ocular surface stimulation and concentration on spontaneous blink parameters. Curr Eye Res 2014;39:920.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Bologna M, Marsili L, Khan N, et al. Blinking in patients with clinically probable multiple system atrophy. Mov Disord 2014;29:415420.

  • 28. Chen T, Ward DA. Tear volume, turnover rate, and flow rate in ophthalmologically normal horses. Am J Vet Res 2010;71:671676.

  • 29. Frigerio A, Cavallari P. A closed-loop stimulation system supplemented with motoneurone dynamic sensitivity replicates natural eye blinks. Otolaryngol Head Neck Surg 2012;146:230233.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Schotanus MP, Koetje LR, van Dyken RE, et al. Stratified corneal limbal epithelial cells are protected from UVB-induced apoptosis by elevated extracellular K+. Exp Eye Res 2011;93:735740.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Miller C, Utter ML, Beech J. Evaluation of the effects of age and pituitary pars intermedia dysfunction on corneal sensitivity in horses. Am J Vet Res 2013;74:10301035.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32. Evinger C, Manning KA, Sibony PA. Eyelid movements. Mechanisms and normal data. Invest Ophthalmol Vis Sci 1991;32:387400.

Supplementary Materials

  • Figure 1—

    Schematic diagram of the videography setup depicting the relationship among the horse, the primary camera that recorded only the right eye, the secondary camera that recorded both eyes, and the lights.

  • Figure 2—

    Photograph of the right eye of a horse. An ROI is outlined (red line) prior to commencement of a complete blink. The area of the ROI was calculated by use of an image-analysis system. Notice the ruler (scale in centimeters) used to calibrate the ROI data was affixed to the horse's face at a point 3 cm ventral to the right eye.

  • Figure 3—

    Plot of the SEBR versus the tear sodium concentration-to-potassium concentration ratio for 7 horses. Each circle represents results for 1 horse. Notice the strong positive correlation.

  • Figure 4—

    Mean ± SD percentage of the area of cornea exposed as a function of time for complete blinks of the superior eyelids of 10 horses. Notice that the line of best fit for the data was best described by a cubic equation. t = Time.

  • Figure 5—

    Position (A and B), velocity (C and D), and acceleration (E and F) of the superior eyelid as a function of time for eyelid closure (A, C, and E) and eyelid opening (B, D, and F). Notice that the scale on the x-axis differs between panels A and B, C and D, and E and F and that the scale on the y-axis differs between panels C and D and E and F. Eyelid closure occurred at a mean of 0.162 seconds, and eyelid opening occurred at a mean of 0.478 seconds; thus, opening required approximately twice as much time as closure. Maximum closing velocity was −16.5 cm/s and occurred at 0.0811 seconds (ie, when the slope of the curve was 0). Maximum velocity of eyelid opening was 7.40 cm/s and was attained at 0.180 seconds (approx 0.018 seconds after the initiation of eyelid opening). Acceleration during eyelid closure was greatest at the beginning and end of the closure phase (> 400 cm/s2 at both points), whereas maximum acceleration of eyelid opening was −49.7 cm/s2 and occurred at the time of complete reopening of the eyelid (0.478 seconds). For velocity and acceleration, movement of the superior eyelid in a ventral (downward) direction was defined as the negative direction, and movement of the superior eyelid in a dorsal (upward) direction was defined as the positive direction. Results for panels A and B represent mean ± SD. The line of best fit was determined for all data; all results were for analyses of complete blinks of the superior eyelids (n = 10 horses). See Figure 4 for remainder of key.

  • 1. Griepentroy GJ, Lucarelli MJ. Functions of the orbit and adnexa. In: Levin LA, Nilsson SFE, ver Hoeve J, et al, eds. Adler's physiology of the eye. 11th ed. Edinburgh: Saunders/Elsevier, 2011;333349.

    • Search Google Scholar
    • Export Citation
  • 2. Tsubota K, Hata S, Okusawa Y, et al. Quantitative videographic analysis of blinking in normal subjects and patients with dry eye. Arch Ophthalmol 1996;114:715720.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Malbouisson JM, Messias A, Garcia DM, et al. Modeling upper eyelid kinematics during spontaneous and reflex blinks. J Neurosci Methods 2010;191:119125.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Cardona G, Garcia C, Seres C, et al. Blink rate, blink amplitude, and tear film integrity during dynamic visual display terminal tasks. Curr Eye Res 2011;36:190197.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Berke A, Mueller S. The kinetics of lid motion and its effects on the tear film. Adv Exp Med Biol 1998;438:417424.

  • 6. Garcia DM, Messias A, Costa LO, et al. Spontaneous blinking in patients with Graves’ upper eyelid retraction. Curr Eye Res 2010;35:459465.

  • 7. Cher I. Blink-related microtrauma: when the ocular surface harms itself. Clin Experiment Ophthalmol 2003;31:183190.

  • 8. Wan T, Jin X, Lin L, et al. Incomplete blinking may attribute to the development of meibomian gland dysfunction. Curr Eye Res 2016;41:179185.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Hirota M, Uozato H, Kawamorita T, et al. Effect of incomplete blinking on tear film stability. Optom Vis Sci 2013;90:650657.

  • 10. Ousler GW, Abelson MB, Johnston PR, et al. Blink patterns and lid-contact times in dry-eye and normal subjects. Clin Ophthalmol 2014;8:869874.

    • Search Google Scholar
    • Export Citation
  • 11. Best LJ, Hendrix DVH, Ward DA. Tear film osmolality and electrolyte composition in healthy horses. Am J Vet Res 2015;76:10661069.

  • 12. Korb DR, Baron DF, Herman JP, et al. Tear film lipid layer thickness as a function of blinking. Cornea 1994;13:354359.

  • 13. Fenga C, Aragona P, Cacciola A, et al. Meibomian gland dysfunction and ocular discomfort in video display terminal workers. Eye (Lond) 2008;22:9195.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Wada S, Hobo S, Niwa H. Ulcerative keratitis in Thoroughbred racehorses in Japan from 1997 to 2008. Vet Ophthalmol 2010;13:99105.

  • 15. Collins M, Seeto R, Campbell L, et al. Blinking and corneal sensitivity. Acta Ophthalmol (Copenh) 1989;67:525531.

  • 16. Doughty MJ. Consideration of three types of spontaneous eyeblink activity in normal humans: during reading and video display terminal use, in primary gaze, and while in conversation. Optom Vis Sci 2001;78:712725.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Barbato G, Ficca G, Muscettola G, et al. Diurnal variation in spontaneous eye-blink rate. Psychiatry Res 2000;93:145151.

  • 18. Bacher LF, Smotherman WP. Spontaneous eye blinking in human infants: a review. Dev Psychobiol 2004;44:95102.

  • 19. Trost K, Skalicky M, Nell B. Schirmer tear test, phenol red thread tear test, eye blink frequency and corneal sensitivity in the guinea pig. Vet Ophthalmol 2007;10:143146.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Toshida H, Nguyen DH, Beuerman RW, et al. Evaluation of novel dry eye model: preganglionic parasympathetic denervation in rabbit. Invest Ophthalmol Vis Sci 2007;48:44684475.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Kirsty R, Andrew H, Meriel MC, et al. Cognitive differences in horses performing locomotor versus oral stereotypic behaviour. Appl Anim Behav Sci 2015;168:3744.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Koushan K, Skibell BC, Harvey JT, et al. Digital photography as a novel technique of measuring ocular surface dimensions. Orbit 2008;27:259265.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Doane MG. Interaction of eyelids and tears in corneal wetting and the dynamics of the normal human eyeblink. Am J Ophthalmol 1980;89:507516.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Somia NN, Rash GS, Epstein EE, et al. A computer analysis of reflex eyelid motion in normal subjects and in facial neuropathy. Clin Biomech (Bristol, Avon) 2000;15:766771.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Sforza C, Rango M, Galante D, et al. Spontaneous blinking in healthy persons: an optoelectronic study of eyelid motion. Ophthalmic Physiol Opt 2008;28:345353.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Wu Z, Begley CG, Situ P, et al. The effects of mild ocular surface stimulation and concentration on spontaneous blink parameters. Curr Eye Res 2014;39:920.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Bologna M, Marsili L, Khan N, et al. Blinking in patients with clinically probable multiple system atrophy. Mov Disord 2014;29:415420.

  • 28. Chen T, Ward DA. Tear volume, turnover rate, and flow rate in ophthalmologically normal horses. Am J Vet Res 2010;71:671676.

  • 29. Frigerio A, Cavallari P. A closed-loop stimulation system supplemented with motoneurone dynamic sensitivity replicates natural eye blinks. Otolaryngol Head Neck Surg 2012;146:230233.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Schotanus MP, Koetje LR, van Dyken RE, et al. Stratified corneal limbal epithelial cells are protected from UVB-induced apoptosis by elevated extracellular K+. Exp Eye Res 2011;93:735740.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Miller C, Utter ML, Beech J. Evaluation of the effects of age and pituitary pars intermedia dysfunction on corneal sensitivity in horses. Am J Vet Res 2013;74:10301035.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32. Evinger C, Manning KA, Sibony PA. Eyelid movements. Mechanisms and normal data. Invest Ophthalmol Vis Sci 1991;32:387400.

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