Horses have large prominent corneas, the surface of which is predisposed to a variety of diseases such as superficial keratitis and epithelial keratopathy. Clinically normal corneas have a precorneal tear film that protects and nourishes the ocular surface and helps provide an optically clear surface for vision. In horses, keratopathies without obvious etiologies (eg, infectious, parasitic, or neoplastic1,2) are often presumed to be immune mediated on the basis of their variable response to topical administration of corticosteroids.3,4 The underlying cause frequently cannot be identified for many of those presumptive immune-mediated keratopathies,4 which suggests that those conditions would be more aptly described as idiopathic keratopathies. In humans, many corneal abnormalities such as Sjögren and non-Sjögren dry eye,5 anterior blepharitis,5 meibomian gland dysfunction,6 superior limbic keratoconjunctivitis,7 diabetic keratoepitheliopathy,8 and graft-versus-host disease dry eye9 cause pathological changes similar to those in horses with idiopathic keratopathies and are associated with changes in the tear film osmolality and composition. We theorized that some keratopathies in horses may also be associated with tear film abnormalities.
Deficiencies in the aqueous component of the tear film are referred to as quantitative deficiencies, are diagnosed on the basis of STT results, and have been infrequently described in horses.10–12 Abnormalities of the biochemical or biophysical properties of the tear film are referred to as qualitative deficiencies, can cause tear film instability, and are more subtle clinically than are quantitative deficiencies This makes them difficult to diagnose13 and likely under recognized.14 Simple and easily repeatable methods for assessment of the quantitative and qualitative properties of the tear film in horses would facilitate the diagnosis and monitoring of subtle tear film and corneal abnormalities and provide a basis for the development of appropriate treatment regimens. In human medicine, tear osmolarity or osmolality (which are interchangeable parameters in aqueous solutions such as the tear film) is considered to be an objective variable for the assessment of qualitative and quantitative tear film characteristics.15,16 In addition to osmolality, the ionic composition of the tear film may affect its qualitative properties. Divalent cation concentrations have been linked to increased stability in the tear film of rabbits but not of humans.17 Because of the relatively large exposed surface area of the equine cornea, horses require tear film stability for ocular health. We hypothesized that the tear film in horses would have a relatively low osmolality and high divalent cation concentration to promote tear film stability. The purpose of the study reported here was to determine the tear film osmolality and electrolyte composition in healthy horses.
Materials and Methods
Animals
Fifteen adult horses from the University of Tennessee teaching herd were evaluated for study enrollment. None of the horses were receiving medical treatment at the time of the study, and all horses were determined to be healthy on the basis of a physical examination. Horses underwent a complete ophthalmic examination that included an STT, applanation tonometry, slit-lamp biomicroscopy, and indirect ophthalmoscopy. Horses with abnormal eyelid conformation or other substantial ophthalmic abnormalities were excluded from the study. All the horses enrolled in the study were mares and had a mean (SD) age of 15.07 (4.89) years. Breeds represented included American Quarter Horse (n = 8), Tennessee Walking Horse (5), American Paint Horse (1), and mixed breed (1). All study procedures were reviewed and approved by the University of Tennessee Institutional Animal Care and Use Committee.
Tear sample collection and analysis
All horses were examined and tear samples were collected between 9:20 AM and 5:00 PM during 2 weeks in February 2014. Because the horses were accustomed to frequent handling, they were not sedated and were simply restrained in standard stocks for tear sample collection. Horses that did not comply with tear sample collection were excluded from the study. The stocks were located in an indoor facility where the temperature was maintained between 20.0° and 21.1°C and the humidity ranged between 20% and 46%.a
No topical or local anesthetic blocks were performed prior to tear collection. The ionic composition analysis required approximately 200 μL of tears, and the osmolality measurement required approximately 10 μL of tears. To ensure that an adequate volume was acquired from each horse, tears were collected from both the left and right eyes by the use of 70-μL plain microhematocrit tubes 3 times at 5-minute intervals. During tear sample collection, the eyelid was manually restrained, and 1 end of the microhematocrit tube was placed in the ventral portion of the conjunctival fornix. The tears were drawn into the microhematocrit tube by capillary action. The extent of compliance with the tear collection process varied among horses and affected the volume of tears collected. After the last tear sample was collected, fluorescein stain was applied to each eye to ensure that the cornea was not inadvertently traumatized during the tear collection process.
All the tear samples obtained from each horse were pooled and stored at −62°C until analysis. The tear samples were allowed to thaw completely prior to analysis. Osmolality was measured with a vapor-pressure osmometer.b When the tear sample volume permitted, osmolality was measured in triplicate; however, for some horses, osmolality could be measured only once or twice because of the limited volume of the tear sample collected. For each horse, the mean was calculated when osmolality was measured in duplicate or triplicate; otherwise, the single osmolality measurement was used for analysis purposes. Immediately following osmolality measurement, the sodium, potassium, magnesium, and calcium concentrations were determined by use of an automated biochemical analyzerc for the 8 tear samples for which there was sufficient volume remaining.
Data analysis
Descriptive data were generated for each variable measured. Results were reported as the mean (SD).
Results
Most of the horses tolerated the tear collection process well. Two horses were excluded from the study, 1 because of a lower eyelid deformity and 1 because of noncompliance with the tear collection process. None of the horses had corneal fluorescein stain uptake following tear sample collection.
Tear film osmolality was measured for 13 horses, and the mean (SD) tear film osmolality was 283.51 (9.33) mmol/kg. Tear film electrolyte concentrations were determined for 8 horses. The mean (SD) sodium, potassium, magnesium, and calcium concentrations were 134.75 (10), 16.3 (5.77), 3.48 (1.97), and 1.06 (0.42) mmol/L, respectively.
Discussion
Tear film osmolality is dependent on the rate of tear production, evaporation, and composition. Human patients with evaporative dry eye have a hyperosmolar tear film, and osmolarity is considered an objective measurement to diagnose dry eye and monitor treatment response.15,16,18 Increasing osmolarity of the tear film is correlated with decreasing tear film breakup time and increasing surface tension, both indicators of an unstable tear film and subsequent corneal disease.19 In humans, tear film osmolarity varies depending on the osmometer used, but the recommended tear film osmolarity cutoff is 316 mOsm/L for the diagnosis of dry eye.18 Lacrimal fluid is isotonic when secreted, and an increase in tear film osmolality above serum osmolality is most likely the result of evaporation.20
The mean tear film osmolality for the healthy horses of the present study (283.51 mmol/kg) was similar to that for humans17 (282.5 mmol/kg) but substantially lower than the mean tear film osmolality for rabbits17 (375.83 mmol/kg), cats21 (328.5 mOsm/L), and dogsd (355.5 mOsm/L). This finding suggested that, in horses, tear film evaporation was fairly low when compared with tear production, which should promote tear film stability. Tear evaporation in horses might be less than that in other species because horses have a large mean tear volume (288 μL) and rate of tear production (33.62 μL/min).22 Tear production in the horses of the present study might have been artificially increased because of reflex tear production associated with the tear collection process; however, results of another study23 indicate that reflex tear production secondary to minor irritation is insubstantial in horses, and we believe that it was negligible in the present study. In horses, the large rate of tear production may help protect the prominent corneas from evaporative dry eye disease. Additionally, tear evaporation in horses may be low because of a fairly stable tear film and high blink rate (19 blinks/min; unpublished data obtained by our laboratory group). The STT measures only the pooled volume of tears in the inferior conjunctival cul-de-sac24 and therefore does not necessarily reflect the hydration status of the axial cornea. Determination of tear film osmolality could be used as an objective measurement of the hydration of the axial cornea and facilitate the diagnosis of evaporative dry eye in horses.
The stability of the tear film is dependent on the integrity of its lipid and mucin components and divalent cation concentration. Divalent cations decrease the surface tension of the tear film, which proportionally increases its stability.17 For example, the mean divalent cation concentrations (magnesium, 1.13 mmol/L; calcium, 0.75 mmol/L) in the tear film of rabbits are significantly higher than those (magnesium, 0.39 mmol/L; calcium, 0.36 mmol/L) in the tear film of humans,17 which may explain why the tear film of rabbits is fairly stable despite its high osmolality and the low blink rate of rabbits. For the horses of the present study, the mean magnesium (3.48 mmol/L) and calcium (1.06 mmol/L) concentrations in the tear film were substantially higher than those in the tear film of rabbits. It is likely that those divalent cation concentrations stabilize the tear film in horses in the same manner as they do in rabbits. It is interesting to note that calcium is required for the function of some matrix metalloproteinases.25,26 Horses are particularly prone to the development of matrix metalloproteinase–associated corneal melting,27 and that may be a consequence of the fairly high calcium concentration in the tear film.
For the horses of the present study, the sodium concentration in the tear film was approximately equal to that in serum, but the potassium concentration in the tear film was approximately 4.75 times that in serum. Those findings were consistent with results reported for cattle,28 humans,29 and rabbits.29 A fluid rich in potassium chloride is produced in the lacrimal ductules via transporters located in apical cell membranes,20 and those transporters might be responsible for the high potassium concentration in tears. The physiologic purpose of the high potassium concentration in the tear film is unclear. However, electrolytes are essential to the health of the ocular surface, and small alterations in the electrolyte concentrations of the tear film may alter the stability of the aqueous component of the tear film.20
A limitation in the present study was the inability to consistently collect an adequate volume of tears for measurement of both osmolality and electrolyte concentrations. Results of another study22 indicate that the mean tear volume for healthy horses is 288 μL, which would have been more than sufficient for the assays we wanted to perform in the present study. Unfortunately, in some instances the capillary action was insufficient to fill the microhematocrit tubes.
In another study,d the tear film osmolarity in healthy horses as measured by a commercially available osmolarity systeme ranged from 276 to 398 mOsm/L (median, 318 mOsm/L). Although that system required a sample volume of only 50 nL, the large range of values obtained is not as clinically useful as the narrower range of osmolality values (268 to 299 mmol/kg) determined by the vapor-pressure osmometer in the present study. Additionally, measurement of osmolarity is temperature dependent because the volume of the solvent (water) changes with temperature; measurement of osmolality does not have this limitation because the weight of the solvent is not affected by temperature.30
The teaching herd of horses at the University of Tennessee is comprised primarily of mares; therefore, only mares were evaluated in the present study. The effect of sex on tear film osmolality in horses is unknown; however, sex is associated with several variables that influence tear film stability in humans.13,31,32 Consequently, we cannot be certain that the results of the present study are externally valid for the general horse population, and evaluation of tear film osmolality and electrolyte composition in stallions and geldings is necessary.
All measurements in the present study were on tear samples that were collected during the winter, albeit at fairly stable ambient temperatures and humidity. Results may vary with changing environmental conditions. Air temperature and relative humidity alter the tear film characteristics of humans who wear soft contact lenses.33 However, investigators of another study23 reported that season did not affect the quantitative tear production in horses.
Tear samples in the present study were collected from some horses in the morning and some horses in the afternoon, and we cannot rule out that diurnal variation affected our results. Although there are conflicting results regarding the effect of diurnal variation on STT measurements in horses,23,34 we are unaware of any studies that evaluated the effect of diurnal variation on tear osmolality in horses. Tear osmolarity does not vary diurnally in humans.35,36
Results of the present study indicated that the tear film osmolality in healthy horses was similar to that in humans, but the divalent cation concentrations in the tear film of horses were fairly high, compared with those in other species. Functioning in concert with a large tear film volume, those factors may have evolved to improve the stability and reduce the evaporation of the tear film in a species with a prominently exposed globe that is otherwise susceptible to injury and disease. Measurement of tear film osmolality was easy and noninvasive in the horses of this study and may be clinically useful for the diagnosis of hyperosmolar ocular surface disease in that species.
Acknowledgments
Supported by the University of Tennessee Companion Animal Fund.
Presented as a poster at the 45th Annual Conference of the American College of Veterinary Ophthalmologists, Fort Worth, Tex, October 2014.
ABBREVIATION
STT | Schirmer tear test |
Footnotes
Indoor humidity monitor 613, AcuRite, Lake Geneva, Wis.
Osmometer 5520, Wescor Inc, Logan, Utah.
Cobas c 501, Roche Diagnostics, Indianapolis, Ind.
Korth RME, Romkes G, Eule JC. Tear film osmolarity as a diagnostic tool in small animal and equine medicine? (abstr) Vet Ophthalmol 2010;13:349.
TearLab Osmolarity System, OcuSense, San Diego, Calif.
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