Journal of the American Association for Laboratory Animal Science Copyright 2015 by the American Association for Laboratory Animal Science
Vol 54, No 6 November 2015 Pages 783–787
Evaluation of Lacrimation Characteristics in Clinically Normal New Zealand White Rabbits by Using the Schirmer Tear Test I Alexandra L Whittaker1,* and David L Williams2 Rabbits are a common animal model in eye research and in safety testing of novel chemical agents. In addition, ocular disease is a routine presentation in clinical practice. However, few studies have quantitatively examined lacrimation kinetics in this species. This study used a noninvasive method of tear measurement (the Schirmer tear test, STT) to quantify values for basal and reflex tearing and to determine the kinetic nature of tear production in 76 New Zealand white rabbits. We obtained a value of 7.58 ± 2.3 mm/min for the standard 1-min STT. Calculated values for mean residual tear volume and reflex tear flow were 1.95 µL and 0.035 µL/s, respectively. In addition, this study provides preliminary evidence for an interaction effect between eyes given that higher STT values were obtained from the second eye tested. Abbreviation: STT, Schirmer Tear Test.
Rabbits are widely used in ophthalmic research and are increasing in popularity as pets. However few reports evaluate lacrimal parameters in this species,1,5,12,10 despite the relatively high incidence of ocular disease in rabbits. Typically, ocular disease in rabbits presents as an overproduction of tears due to ocular foreign-body irritation or nasolacrimal duct blockage with resultant epiphora. Accurate diagnosis of the cause of tear overproduction is imperative for quickly and efficiently treating the underlying pathologic mechanism. The precorneal tear film is essential to the maintenance of corneal health and consists of 3 components: lipid, aqueous, and mucin. Aqueous tears account for the majority of the tear volume.23 In lagomorphs, 5 glands contribute to the precorneal tear film: the Harderian and nictitating glands associated with the medial canthus and the lacrimal, infraorbital, and exorbital glands of the outer canthus.27 The orbital lacrimal gland is credited with being the primary source of the aqueous components of tears and with the production of proteins that protect the ocular surface against bacteria.20 The Schirmer tear test (STT) is a technique widely used in ophthalmic examination to evaluate tear production as an aid to diagnosing either keratoconjunctivitis sicca or overproduction of tears. General clinical use of the test involves the placement of a standard notched filter paper into the lower conjunctival fornix of the eye and reading the amount of wetness produced after 1 min.23 Specific diagnoses are made by comparing the recorded value with the mean normal value for the species. The STT1, which is performed without the use of topical anesthesia, provides a measure of both basal and reflex tearing, whereas the STT2, which is performed after topical anesthesia with a drop of tetracaine, demonstrates basal tearing alone. Basal tears are those produced in the absence of stimulation and are lacrimal secretions that maintain the ocular surface. Reflex tears Received: 16 Dec 2014. Revision requested: 09 Feb 2015. Accepted: 02 Mar 2015. 1School of Animal and Veterinary Science, University of Adelaide, Roseworthy Campus, Australia, and 2Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom *Corresponding author. Email: [email protected]
are produced after stimulation, usually as a result of an irritant to the ocular surface, such as a foreign body or inflammatory response.25 The tear-test strip itself likely acts as a source of irritation to increase tear production in the STT1.31 It has been demonstrated that the turnover rate for reflex tearing is greater than that during basal tear-flow kinetics, and thus the protein profile of the 2 samples may differ.8 Because of the small volume of tears produced, the STT1 might be more useful in the evaluation of the increased tear production associated with ocular irritation than it is for the diagnosis of underproduction, as in the case of clinical keratoconjunctivitis sicca. 1 This criticism has led some clinicians to make greater use of the phenol red test (especially in small patients). Compared with the STT, the phenol red test requires a smaller tear volume to achieve test-thread wetting, thus minimizing variability in reading.5 Despite these advantages, the STT remains the most commonly used quantitative tear-assessment test in veterinary species.16,33 Previous studies in rabbits have evaluated standard 1-min STT1 readings1,5 and 5-min readings with topical anesthesia,12 yielding 1-min values (mean ± 1 SD) of 5.3 ±2.96 mm/min,1 4.85 ±2.9 mm/min,5 and a range of 0 to15 mm/min.1,5 In one study,5 the sample population consisted of 26 New Zealand White rabbits; another investigation1 involved a variety of breeds (which differed in size), with the New Zealand White subpopulation comprising 28 rabbits. That study1 found that, in general, neither sex nor breed significantly affected STT values, except that values of those Netherland dwarf and New Zealand Black rabbits were higher and lower, respectively, than the results for the remainder of the study population. Nevertheless, sample sizes of these breeds were small (n = 2 animals), so data should be interpreted with care. In comparison, interbreed differences in STT1 have been demonstrated in dogs.14 In rabbits, no significant difference on comparison of STT values between eyes nor any evidence of an interaction between eyes was found.1 Studies involving tear-flow rates in humans have used several different methods.7,23 For example, strips of specialized absorbent paper (Periopaper, Oraflow, Smithtown, NY) have been used to collect basal tears; the volume of tears collected is 783
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evaluated by using an electronic device (Periotron, Oraflow), which measures the charge across 2 electrodes as an indication of volume.7 In the same study, reflex tear production was evaluated by using a STT strip, reading the wetted length after 5 min, and then comparing that value with the wetted lengths obtained for known volumes of egg-white lysozyme solution.7 Fluorophotometric means for measuring the human tear-turnover rate have been based on the disappearance of fluorescent dye over time.23 Tear-flow rates in rabbits have been calculated by cannulating the excretory duct of the lacrimal gland under general anesthesia, to collect lacrimal gland fluid. 11 The tear-flow rate was determined by dividing the volume by the collection time. However, the primary aim of that study11 was to determine the effect of the stimulation of flow on the osmolarity of lacrimal fluid, rather than to investigate flow rates specifically. The object of the current exploratory study was to extend previously published work and demonstrate the kinetics of tear flow in a single, widely used strain of rabbit (New Zealand white) and to quantify values for reflex tear production by using a simple and clinically relevant method, the STT1. A second aim was to use the data to assess whether tear flow volumes or kinetics differed between the left and right eyes and whether an interaction effect existed between eyes. We hypothesized that tear production would be increased in the second eye in which the test was performed compared with the first.
Materials and Methods
In vitro wetting of the STT strip. The wetting of the STT strip was investigated in vitro to determine the kinetics of fluid movement along the strip. This data was used to convert wetting measured in the in vivo studies (in mm), to volume of tears (in μL). By so doing, an estimate of reflex tear-flow volumes in New Zealand white rabbits was made. Saline volumes of 5, 7.5, 10, and 12.5 µL plus an unlimited supply were added to the strip, and the wetted length of the strip was determined at 10-s intervals for 3 min. Animals. The study population comprised 76 (n = 152 eyes) New Zealand White laboratory-stock rabbits (Harlan, Leicestershire, United Kingdom). Rabbits were health-screened at the source and were deemed specific pathogen-free according to the FELASA recommendations for health monitoring of laboratory rabbits. Because the study used a relatively noninvasive procedure (the STT) that is used as a standard in clinical veterinary practice, ethics approval under the Animals (Scientific Procedures) Act 1986 (United Kingdom) was not required. However, approval for the study was obtained through the institutional ethics and welfare committee. The rabbits were housed in floor pens on dust free bedding and were clinically healthy, with no known ocular disease. Food (Harlan Teklad Rabbit Diet, Harlan) and water were provided free choice, by using wall-mounted hoppers and drinking nipples. All rabbits except 2 were female; however, previous studies have indicated no difference in 1-min STT readings between does and bucks.1 Rabbits ranged in age from 3 to 10 mo (average group weight range, 4.06 to 4.55 kg) and were housed in the same room on the basis of this age grouping. In vivo tests. Each rabbit was manually restrained in a standard commercial polycarbonate rabbit restrainer and tested by using the STT1, which does not involve topical anesthesia. Performance of the test involved insertion of the notched end of a commercial tear-test paper over the lower lid margin at the juncture of the temporal and middle third of the lid. The eyelids were held shut for the period of the test. The length of tear strip wetted was recorded (in mm), as shown by the advancement of the blue dye on the marked standardized scale (Figure 1), every
Figure 1. A standard Schirmer tear-test strip, showing dye movement and gradation scale.
10 s for a total of 3 min. Test strips were held in place throughout the period of recording. The eyes were tested sequentially, with recording for the second eye commencing immediately after completion of testing in the first. All test strips were from the same lot and manufacturer (Schering–Plough Animal Health, Kenilworth, NJ). The test is noninvasive and causes minimal distress to the rabbits. Tests were performed indoors in an environment of constant thermostatically controlled temperature and humidity. The same researcher tested all rabbits between 0900 and 1200. Previous studies have suggested that diurnal fluctuations in STT readings are not biologically significant.4 To assess interaction between eyes, the 76 rabbits were assigned randomly to groups according to the order in which both eyes were tested. The STT was performed as described previously. Results from rabbits in which the left eye was tested first were compared with those in which the right eye was tested first. Statistical analysis. Data were analyzed by one-way ANOVA (Predictive Analytics Software PASW, SPSS (2009), version 17.0, Chicago) to compare the mean basal tear volumes and reflex flow rates over the 3-min period and differences in rate between eyes, to correlate tear production and weight or age of the rabbit, and to identify any interaction between eyes. Statistical significance was defined as a P value of less than 0.05.
Results
In vitro tear strip wetting. When volumes of saline were added to the test strips, the results showed an approximately linear relationship between the length of wetting and time in both limited and unlimited supply situations Figure 2. The total wetted length was proportional to the volume of solvent applied. Each point on the graph represents a mean value for 5 separate tests, and a plot of the maximal wetted length against the volume of saline yielded a linear relationship with the equation (1)
y = 1.9844x ,
where y is the wetted length (in mm), and x is volume of saline (in µL). This relationship enables the conversion of in vivo values of length of tear strip wetting into volumes of tear production, assuming that fluid characteristics have a negligible influence on the wetting rate and that the secretion rate remains below the maximal uptake rate of the paper strip.
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Figure 2. In vitro wetting of Schirmer test strips for 5 different volumes of saline, showing mean ± SEM values for 5 tests. Figure 3. Schirmer tear test results (mean ± SEM) over period of test for both eyes analyzed separately (n = 76)
In vivo tear strip wetting. STT was studied in 76 rabbits free from ocular disease. Data from each eye were analyzed separately, and STT values (mean ± 1 SD) were similar for both eyes, with no statistically significant difference between right compared with left eyes over the whole population (P = 0.29). Lacrimation kinetics are shown in Figure 3. Because the data from both eyes were similar, they were combined for further analysis Figure 4.22 After combining the data for both eyes, we obtained a 60-s STT value of 7.6 ± 2.3 mm (range, 3 to 14 mm); the 120-s. STT value was 8.7 mm ± 3.1 mm (range, 3 to 16 mm). (Figure 4) reveals 3 linear trends representing different phases of strip wetting. Values derived from these trend data provide that when x = 10 s, y = 3.9 mm, which corresponds to a value of 1.9 µL, according to equation 1. Reflex tear production is calculated from the gradient of the initial slope in (Figure 4) (indicated by the arrow); y values for the gradient correspond to 2.8 mm, and the x value is 40 s. Therefore the gradient is 0.07 mm/s, which equates to 0.035 µL/s (according to equation 1). Interaction between eyes. This evaluation of the initial results assessed the mean difference in 60-s tear-test results between the second eye tested and the first (that is, STTsecond – STTfirst). The data were evaluated by plotting a frequency distribution of the mean differences (Figure 5). The mean of the mean difference between both eyes (that is, STTsecond – STTfirst) was 0.18 ± 0.04 (95% confidence limits) for the sample of 76 eyes.
Figure 4. Mean ± SEM. Schirmer tear test results over period of test for both eyes combined (n = 142). Trend lines are shown in red. The arrow illustrates the gradient used for calculation of reflex tear flow rates.
Discussion
Various authors have studied the kinetics of capillary flow in STT strips. From these studies, we have made the assumption that, in the case of limited supply (as in vivo) at low secretion rates, the rate of wetting length increase is linearly proportional to the secretion rate when evaporation is negligible.3,6,17,18,31 Because evaporation increases with increased wetting length, a steady state is reached at which the length of wetting is constant over time. This means comparisons that are based on the rate of wetting being equal to secretion rate can be assumed to be true. In addition, paper and fluid characteristics have a negligible effect on rate of wetting, so using saline as a control is justified.3,6,17,18. Using our results, we have shown that by plotting STT values over time, an estimate of reflex tearing can be obtained.31 The in vivo kinetic data (Figure 2) resemble saline control data, with an initial rapid uptake of tears (from the tear lake), followed by section that is associated with a decreased gradient indicative of the reflex tear production rate, and an eventual flattening of the graph. This flattening is caused by the increase in evaporation as wetting length increases, which eventually leads to a
Figure 5. Frequency distribution of the mean differences between eyes that is STT second eye- STT first eye.
steady-state situation.17 Values obtained for residual tear volume and reflex tear flow in New Zealand white rabbits were 1.9 µL and 0.035 µL/s, respectively. Plotting of STT results over time is easily achieved and provides a simple method for determining tear turnover based on the contribution of tears from the tear lake and for measuring tear production. This procedure therefore yields some useful information in addition to that obtained from a standard STT reading, which is taken at a single time point.31 To accurately quantify basal tear production, STT2 testing after the application of topical anesthesia is needed and might represent an avenue for further study to better quantify lacrimation kinetics in rabbits. The large SD likely reflects the large range in values (similarly mentioned in a previous study1) rather than error in the experimental method. Large ranges in the mean STT value are 785
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not unique to rabbits: SD values typically represent a quarter of the mean in dogs9 and cats2 and were even greater in guinea pigs (although mean STT values were extremely low).30 In our rabbits, the mean value for the standard 60-s STT was 7.58 ± 2.3 mm/min, with maximum of 14 mm/min and minimum of 3 mm/min. These results are in contrast to the values of 5.3 ± 3.0 mm/min1 and 4.8 ± 2.9 mm/min5 determined in previous studies. In light of the breed distribution in one of these studies,1 the other5 is a better comparator given that it involved New Zealand White rabbits. The difference between the values obtained in our current study and the previous one5 might reflect our increased sample number and a possible weight- or age-associated effect on tear production. This effect was not investigated specifically in the earlier study,5 but those animals weighed less than did those in the current study (less than 4 kg). In addition, we found no significant difference between the values for the right and left eyes when we compared the entire sample population as a whole, in concurrence with previous studies undertaken.1,5 This finding led us to combine the data from both eyes, for further statistical comparison. This approach is widely used in ophthalmologic research in both humans and veterinary species but may be statistically flawed and lead to false-positive results. Combining the data from both eyes might render the t test invalid, because the measurements from the 2 eyes of a single subject are usually related, not independent.22 This factor likely has a greater consequence in research investigating the effects of drugs on intraocular pressure or when assessing systemic parameters to make treatment recommendations but should be considered whenever studies of any ophthalmologic nature are designed. We hypothesized that if an interaction between eyes existed, tear production would be greater in the second eye in which the test was performed. The mean difference between these measurements was positive (0.18), albeit only slightly. Because the 95% confidence limits for this value do not span 0, there is some evidence to support the hypothesis proposed. This area has not received much attention in previous studies in any species, although other colleagues assessed this factor in rabbits to find no interaction between eyes.1 The neurology of lacrimation is complex and still under discussion. There is no reported neural system of communication between eyes that would allow a stimulus in one eye to influence reflex stimulation in the other and thus explain such a finding.28,26,32 It seems feasible that a stimulus in one eye might travel within afferent trigeminal fibers and be mediated centrally in the brainstem, possibly in the receiving nucleus (sensory nucleus of the trigeminal nerve), to distribute bilaterally to the lacrimal glands of both eyes. However, this explanation is mere conjecture. Another possible explanation for an interaction might be a psychogenic cause, but again the paucity of evidence for a communication system between eyes blights this hypothesis. Given the lack of studies investigating reflex lacrimation and eye interaction effect and in view of our findings, this area of research requires further characterization by using larger sample sizes to elucidate both the presence (or absence) of an interaction and the neurologic pathway that might mediate such an effect. A limitation of our study is that age and weight of individual rabbits were not recorded to evaluate whether a relationship exists between these variables and tear production. Research has shown differences in tear production with age and weight in dogs. One study reported an increase in tear production in heavier animals, showing that basal tear production increased.4 Other authors conversely found a decrease in STT as dogs aged but no significant correlation of weight with STT.15 Another group demonstrated that older dogs were more likely to be
tear-deficient among a population of 460 dogs that presented for ophthalmic examination.19 However, other studies have looked at STT values in normal domestic canines and felines but found no significant difference with age or sex.14,21,29 It seems plausible that there might be a relationship between age and tear production by the developing lacrimal gland in younger animals, and that a reduction in functional capacity of the lacrimal and nictitans glands might occur in senescent animals, leading to a decrease in tear production. The effect of age on tear production does not appear to have been studied previously in rabbits and merits future evaluation by using animals that vary widely in age. The inclusion of weight as a covariate should be considered, to elucidate the contributing effect of each variable. Our current study has demonstrated that the STT, widely used in clinical practice, can provide much more information than might appear at first. Cases of naturally occurring keratoconjunctivitis sicca in rabbits have not been reported, but these animals are often used in research as a model for this disease.12,13 In such applications, a simple method of measuring progression of the model postoperatively is of value. In addition, rabbit ocular disease is a common presenting problem in clinical practice (particularly that of tear overproduction and epiphora), and it is useful to have techniques available to evaluate factors that might contribute to disease. Despite the large variability in the STT results that we obtained in the current study, the test generally is still valid because a clinical diagnosis of over- or underproduction of tears usually is made only after several abnormal tests have been obtained. In conclusion, this study has extended earlier work in this area by using a larger sample and consequently has provided a reliable reference value for the standard, 1-60 STTs in New Zealand white laboratory rabbits. We determined values for tear production rates, and we present evidence that supports the existence for an interaction between eyes in the results of the STT1. Finally, we have highlighted potential avenues for further study in rabbits and other species.
References
1. Abrams KL, Brooks DE, Funk RS, Theran P. 1990. Evaluation of the Schirmer tear test in clinically normal rabbits. Am J Vet Res 51:1912–1913. 2. Arnett BD, Brightman AH, Musselman EE. 1984. Effect of atropine sulfate on tear production in the cat when used with ketamine hydrochloride and acetylpromazine maleate. J Am Vet Med Assoc 185:214–215. 3. Beebe WE, Esquivel ED, Holly FJ. 1988. Comparison of lacrimation kinetics in dry eye patients and normals. Curr Eye Res 7:419–425. 4. Berger SL, King VL. 1998. The fluctuation of tear production in the dog. J Am Anim Hosp Assoc 34:79–83. 5. Biricik HS, Oguz H, Sindak N, Gurkan T, Hayat A. 2005. Evaluation of the Schirmer and phenol red thread tests for measuring tear secretion in rabbits. Vet Rec 156:485–487. 6. Clinch TE, Benedetto DA, Felberg NT, Laibson PR. 1983. Schirmer’s test: a closer look. Arch Ophthalmol 101:1383–1386. 7. Farris RL, Stuchell RN, Mandel ID. 1981. Basal and reflex human tear analysis, I and II. Ophthalmology 88:852–857. 8. Fullard RJ, Snyder C. 1990. Protein levels in nonstimulated and stimulated tears of normal human subjects. Invest Ophthalmol Vis Sci 31:1119–1126. 9. Gelatt KN, Peiffer RL Jr, Erickson JL, Gum GG. 1975. Evaluation of the tear formation in the dog, using a modification of the Schirmer tear test. J Am Vet Med Assoc 166:368–370. 10. Ghaffari MS, Moghaddassi AP, Bokaie S. 2009. Effects of intramuscular acepromazine and diazepam on tear production in rabbits. Vet Rec 164:147–148.
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11. Gilbard JP, Dartt DA. 1982. Changes in rabbit lacrimal gland fluid osmolarity with flow rate. Invest Ophthalmol Vis Sci 23:804–806. 12. Gilbard JP, Rossi SR, Gray KL, Hanninen LA. 1989. Natural history of disease in a rabbit model of keratoconjuncitivits sicca. Acta Ophthalmol 67:95–101. 13. Gilbard JP, Rossi SR, Gray KL. 1988. Tear film osmolarity and ocular surface disease in 2 rabbit models for keratoconjunctiviits sicca. Invest Ophthalmol Vis Sci 29:374–378. 14. Hamor RE, Roberts SM, Severin GA, Chavkin MJ. 2000. Evaluation of results for Schirmer tear tests conducted with and without application of a topical anesthetic in clinically normal dogs of 5 breeds. Am J Vet Res 61:1422–1425. 15. Hartley C, Williams DL, Adams VJ. 2006. Effect of age, gender, weight, and time of day on tear production in normal dogs. Vet Ophthalmol 9:53–57. 16. Hirsh SG, Kaswan RL. 1995. A comparative study of Schirmer tear test strips in dogs. Vet Comp Ophthalmol 5:215–217. 17. Holly FK, Lamberts DW, Esquivel ED. 1982. Kinetics of capillary tear flow in the Schirmer strip. Curr Eye Res 2:57–70. 18. Holly FK, LauKaitis SJ, Esquivel ED. 1984. Kinetics of lacrimal secretion in normal human subjects. Curr Eye Res 3:897–910. 19. Kaswan R, Pappas C Jr, Wall K, Hirsh SG. 1998. Survey of canine tear deficiency in veterinary practice. Adv Exp Med Biol 438:931–939. 20 Maitchouk DY, Beuermann RW, Ohta T, Stern M, Varnell RJ. 2000. Tear production after unilateral removal of the main lacrimal gland in squirrel monkeys. Arch Ophthalmol 118:246–252. 21. Margadant DL, Kirkby K, Andrew SE, Gelatt KN. 2003. Effect of topical tropicamide on tear production as measured by Schirmer’s tear test in normal cats and dogs. Vet Ophthalmol 6:315–320. 22. Newcombe RG, Duff GR. 1987. Eyes or patients? Traps for the unwary in the statistical analysis of ophthalmological studies. Br J Ophthalmol 71:645–646.
23. Occhipinti JRM, La MA, Motte J, Monjii GT. 1988. Fluorophotometric measurement of human tear turnover rate. Curr Eye Res 7:995–1000. 24. Ollivier FJ, Plummer CE, Barrie KP.2007. Ophthalmic examination and diagnostic procedures (part 1), p 438–484. In: Gelatt KN, editor. Veterinary ophthalmology. Ames (IA): Blackwell Publishing. 25. Petznick A, Evans MDM, Madigan MC, Markoulli M, Garrett Q, Sweeney DF. 2011. A comparison of basal and eye-flush tears for the analysis of cat tear proteins. Acta Ophthalmol 89:e75–e81. 26. Powell CC, Martin CL. 1989. Distribution of cholinergic and adrenergic nerve fibres in the lacrimal glands of dogs. Am J Vet Res 50:2084–2088. 27. Sakai T. 1989. Major ocular glands (harderian gland and lacrimal gland) of the musk shrew (Suncus murinus) with a review on the comparative anatomy and histology of the mammalian lacrimal glands. J Morphol 201:39–57. 28. Shamir MH, Ofri R, 2007. Comparative neuro—ophthalmology, p 1406–1469. In: Kirk N.Gelatt, editor. Veterinary ophthalmology. Ames (IA): Blackwell Publishing. 29. Smith EM, Buyukmihci NC, Farver TB. 1994. Effect of topical pilocarpine treatment on tear production in dogs. J Am Vet Med Assoc 205:1286–1289. 30. Trost K, Skalicky M, Nell B. 2007. Schirmer tear test, phenol red thread tear test, eye blink frequency and corneal sensitivity in the guinea pig. Vet Ophthalmol 10:143–146. 31. Williams DL. 2005. Analysis of tear uptake by the Schirmer tear test strip in the canine eye. Vet Ophthalmol 8:325–330. 32. Whitwell J. 1961. Role of the sympathetic in lacrimal secretion. Br J Ophthalmol 45: 349–445. 33. Wyman M, Gilger B, Mueller P, Norrys K. 1995. Clinical evaluation of a new Schirmer tear test in the dog. Vet Comp Ophthalmol 5:211–214.
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Evaluation of Lacrimation Characteristics in Clinically Normal New Zealand White Rabbits by Using the Schirmer Tear Test I Alexandra L Whittaker1,* and David L Williams2 Rabbits are a common animal model in eye research and in safety testing of novel chemical agents. In addition, ocular disease is a routine presentation in clinical practice. However, few studies have quantitatively examined lacrimation kinetics in this species. This study used a noninvasive method of tear measurement (the Schirmer tear test, STT) to quantify values for basal and reflex tearing and to determine the kinetic nature of tear production in 76 New Zealand white rabbits. We obtained a value of 7.58 ± 2.3 mm/min for the standard 1-min STT. Calculated values for mean residual tear volume and reflex tear flow were 1.95 µL and 0.035 µL/s, respectively. In addition, this study provides preliminary evidence for an interaction effect between eyes given that higher STT values were obtained from the second eye tested. Abbreviation: STT, Schirmer Tear Test.
Rabbits are widely used in ophthalmic research and are increasing in popularity as pets. However few reports evaluate lacrimal parameters in this species,1,5,12,10 despite the relatively high incidence of ocular disease in rabbits. Typically, ocular disease in rabbits presents as an overproduction of tears due to ocular foreign-body irritation or nasolacrimal duct blockage with resultant epiphora. Accurate diagnosis of the cause of tear overproduction is imperative for quickly and efficiently treating the underlying pathologic mechanism. The precorneal tear film is essential to the maintenance of corneal health and consists of 3 components: lipid, aqueous, and mucin. Aqueous tears account for the majority of the tear volume.23 In lagomorphs, 5 glands contribute to the precorneal tear film: the Harderian and nictitating glands associated with the medial canthus and the lacrimal, infraorbital, and exorbital glands of the outer canthus.27 The orbital lacrimal gland is credited with being the primary source of the aqueous components of tears and with the production of proteins that protect the ocular surface against bacteria.20 The Schirmer tear test (STT) is a technique widely used in ophthalmic examination to evaluate tear production as an aid to diagnosing either keratoconjunctivitis sicca or overproduction of tears. General clinical use of the test involves the placement of a standard notched filter paper into the lower conjunctival fornix of the eye and reading the amount of wetness produced after 1 min.23 Specific diagnoses are made by comparing the recorded value with the mean normal value for the species. The STT1, which is performed without the use of topical anesthesia, provides a measure of both basal and reflex tearing, whereas the STT2, which is performed after topical anesthesia with a drop of tetracaine, demonstrates basal tearing alone. Basal tears are those produced in the absence of stimulation and are lacrimal secretions that maintain the ocular surface. Reflex tears Received: 16 Dec 2014. Revision requested: 09 Feb 2015. Accepted: 02 Mar 2015. 1School of Animal and Veterinary Science, University of Adelaide, Roseworthy Campus, Australia, and 2Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom *Corresponding author. Email: [email protected]
are produced after stimulation, usually as a result of an irritant to the ocular surface, such as a foreign body or inflammatory response.25 The tear-test strip itself likely acts as a source of irritation to increase tear production in the STT1.31 It has been demonstrated that the turnover rate for reflex tearing is greater than that during basal tear-flow kinetics, and thus the protein profile of the 2 samples may differ.8 Because of the small volume of tears produced, the STT1 might be more useful in the evaluation of the increased tear production associated with ocular irritation than it is for the diagnosis of underproduction, as in the case of clinical keratoconjunctivitis sicca. 1 This criticism has led some clinicians to make greater use of the phenol red test (especially in small patients). Compared with the STT, the phenol red test requires a smaller tear volume to achieve test-thread wetting, thus minimizing variability in reading.5 Despite these advantages, the STT remains the most commonly used quantitative tear-assessment test in veterinary species.16,33 Previous studies in rabbits have evaluated standard 1-min STT1 readings1,5 and 5-min readings with topical anesthesia,12 yielding 1-min values (mean ± 1 SD) of 5.3 ±2.96 mm/min,1 4.85 ±2.9 mm/min,5 and a range of 0 to15 mm/min.1,5 In one study,5 the sample population consisted of 26 New Zealand White rabbits; another investigation1 involved a variety of breeds (which differed in size), with the New Zealand White subpopulation comprising 28 rabbits. That study1 found that, in general, neither sex nor breed significantly affected STT values, except that values of those Netherland dwarf and New Zealand Black rabbits were higher and lower, respectively, than the results for the remainder of the study population. Nevertheless, sample sizes of these breeds were small (n = 2 animals), so data should be interpreted with care. In comparison, interbreed differences in STT1 have been demonstrated in dogs.14 In rabbits, no significant difference on comparison of STT values between eyes nor any evidence of an interaction between eyes was found.1 Studies involving tear-flow rates in humans have used several different methods.7,23 For example, strips of specialized absorbent paper (Periopaper, Oraflow, Smithtown, NY) have been used to collect basal tears; the volume of tears collected is 783
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evaluated by using an electronic device (Periotron, Oraflow), which measures the charge across 2 electrodes as an indication of volume.7 In the same study, reflex tear production was evaluated by using a STT strip, reading the wetted length after 5 min, and then comparing that value with the wetted lengths obtained for known volumes of egg-white lysozyme solution.7 Fluorophotometric means for measuring the human tear-turnover rate have been based on the disappearance of fluorescent dye over time.23 Tear-flow rates in rabbits have been calculated by cannulating the excretory duct of the lacrimal gland under general anesthesia, to collect lacrimal gland fluid. 11 The tear-flow rate was determined by dividing the volume by the collection time. However, the primary aim of that study11 was to determine the effect of the stimulation of flow on the osmolarity of lacrimal fluid, rather than to investigate flow rates specifically. The object of the current exploratory study was to extend previously published work and demonstrate the kinetics of tear flow in a single, widely used strain of rabbit (New Zealand white) and to quantify values for reflex tear production by using a simple and clinically relevant method, the STT1. A second aim was to use the data to assess whether tear flow volumes or kinetics differed between the left and right eyes and whether an interaction effect existed between eyes. We hypothesized that tear production would be increased in the second eye in which the test was performed compared with the first.
Materials and Methods
In vitro wetting of the STT strip. The wetting of the STT strip was investigated in vitro to determine the kinetics of fluid movement along the strip. This data was used to convert wetting measured in the in vivo studies (in mm), to volume of tears (in μL). By so doing, an estimate of reflex tear-flow volumes in New Zealand white rabbits was made. Saline volumes of 5, 7.5, 10, and 12.5 µL plus an unlimited supply were added to the strip, and the wetted length of the strip was determined at 10-s intervals for 3 min. Animals. The study population comprised 76 (n = 152 eyes) New Zealand White laboratory-stock rabbits (Harlan, Leicestershire, United Kingdom). Rabbits were health-screened at the source and were deemed specific pathogen-free according to the FELASA recommendations for health monitoring of laboratory rabbits. Because the study used a relatively noninvasive procedure (the STT) that is used as a standard in clinical veterinary practice, ethics approval under the Animals (Scientific Procedures) Act 1986 (United Kingdom) was not required. However, approval for the study was obtained through the institutional ethics and welfare committee. The rabbits were housed in floor pens on dust free bedding and were clinically healthy, with no known ocular disease. Food (Harlan Teklad Rabbit Diet, Harlan) and water were provided free choice, by using wall-mounted hoppers and drinking nipples. All rabbits except 2 were female; however, previous studies have indicated no difference in 1-min STT readings between does and bucks.1 Rabbits ranged in age from 3 to 10 mo (average group weight range, 4.06 to 4.55 kg) and were housed in the same room on the basis of this age grouping. In vivo tests. Each rabbit was manually restrained in a standard commercial polycarbonate rabbit restrainer and tested by using the STT1, which does not involve topical anesthesia. Performance of the test involved insertion of the notched end of a commercial tear-test paper over the lower lid margin at the juncture of the temporal and middle third of the lid. The eyelids were held shut for the period of the test. The length of tear strip wetted was recorded (in mm), as shown by the advancement of the blue dye on the marked standardized scale (Figure 1), every
Figure 1. A standard Schirmer tear-test strip, showing dye movement and gradation scale.
10 s for a total of 3 min. Test strips were held in place throughout the period of recording. The eyes were tested sequentially, with recording for the second eye commencing immediately after completion of testing in the first. All test strips were from the same lot and manufacturer (Schering–Plough Animal Health, Kenilworth, NJ). The test is noninvasive and causes minimal distress to the rabbits. Tests were performed indoors in an environment of constant thermostatically controlled temperature and humidity. The same researcher tested all rabbits between 0900 and 1200. Previous studies have suggested that diurnal fluctuations in STT readings are not biologically significant.4 To assess interaction between eyes, the 76 rabbits were assigned randomly to groups according to the order in which both eyes were tested. The STT was performed as described previously. Results from rabbits in which the left eye was tested first were compared with those in which the right eye was tested first. Statistical analysis. Data were analyzed by one-way ANOVA (Predictive Analytics Software PASW, SPSS (2009), version 17.0, Chicago) to compare the mean basal tear volumes and reflex flow rates over the 3-min period and differences in rate between eyes, to correlate tear production and weight or age of the rabbit, and to identify any interaction between eyes. Statistical significance was defined as a P value of less than 0.05.
Results
In vitro tear strip wetting. When volumes of saline were added to the test strips, the results showed an approximately linear relationship between the length of wetting and time in both limited and unlimited supply situations Figure 2. The total wetted length was proportional to the volume of solvent applied. Each point on the graph represents a mean value for 5 separate tests, and a plot of the maximal wetted length against the volume of saline yielded a linear relationship with the equation (1)
y = 1.9844x ,
where y is the wetted length (in mm), and x is volume of saline (in µL). This relationship enables the conversion of in vivo values of length of tear strip wetting into volumes of tear production, assuming that fluid characteristics have a negligible influence on the wetting rate and that the secretion rate remains below the maximal uptake rate of the paper strip.
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Figure 2. In vitro wetting of Schirmer test strips for 5 different volumes of saline, showing mean ± SEM values for 5 tests. Figure 3. Schirmer tear test results (mean ± SEM) over period of test for both eyes analyzed separately (n = 76)
In vivo tear strip wetting. STT was studied in 76 rabbits free from ocular disease. Data from each eye were analyzed separately, and STT values (mean ± 1 SD) were similar for both eyes, with no statistically significant difference between right compared with left eyes over the whole population (P = 0.29). Lacrimation kinetics are shown in Figure 3. Because the data from both eyes were similar, they were combined for further analysis Figure 4.22 After combining the data for both eyes, we obtained a 60-s STT value of 7.6 ± 2.3 mm (range, 3 to 14 mm); the 120-s. STT value was 8.7 mm ± 3.1 mm (range, 3 to 16 mm). (Figure 4) reveals 3 linear trends representing different phases of strip wetting. Values derived from these trend data provide that when x = 10 s, y = 3.9 mm, which corresponds to a value of 1.9 µL, according to equation 1. Reflex tear production is calculated from the gradient of the initial slope in (Figure 4) (indicated by the arrow); y values for the gradient correspond to 2.8 mm, and the x value is 40 s. Therefore the gradient is 0.07 mm/s, which equates to 0.035 µL/s (according to equation 1). Interaction between eyes. This evaluation of the initial results assessed the mean difference in 60-s tear-test results between the second eye tested and the first (that is, STTsecond – STTfirst). The data were evaluated by plotting a frequency distribution of the mean differences (Figure 5). The mean of the mean difference between both eyes (that is, STTsecond – STTfirst) was 0.18 ± 0.04 (95% confidence limits) for the sample of 76 eyes.
Figure 4. Mean ± SEM. Schirmer tear test results over period of test for both eyes combined (n = 142). Trend lines are shown in red. The arrow illustrates the gradient used for calculation of reflex tear flow rates.
Discussion
Various authors have studied the kinetics of capillary flow in STT strips. From these studies, we have made the assumption that, in the case of limited supply (as in vivo) at low secretion rates, the rate of wetting length increase is linearly proportional to the secretion rate when evaporation is negligible.3,6,17,18,31 Because evaporation increases with increased wetting length, a steady state is reached at which the length of wetting is constant over time. This means comparisons that are based on the rate of wetting being equal to secretion rate can be assumed to be true. In addition, paper and fluid characteristics have a negligible effect on rate of wetting, so using saline as a control is justified.3,6,17,18. Using our results, we have shown that by plotting STT values over time, an estimate of reflex tearing can be obtained.31 The in vivo kinetic data (Figure 2) resemble saline control data, with an initial rapid uptake of tears (from the tear lake), followed by section that is associated with a decreased gradient indicative of the reflex tear production rate, and an eventual flattening of the graph. This flattening is caused by the increase in evaporation as wetting length increases, which eventually leads to a
Figure 5. Frequency distribution of the mean differences between eyes that is STT second eye- STT first eye.
steady-state situation.17 Values obtained for residual tear volume and reflex tear flow in New Zealand white rabbits were 1.9 µL and 0.035 µL/s, respectively. Plotting of STT results over time is easily achieved and provides a simple method for determining tear turnover based on the contribution of tears from the tear lake and for measuring tear production. This procedure therefore yields some useful information in addition to that obtained from a standard STT reading, which is taken at a single time point.31 To accurately quantify basal tear production, STT2 testing after the application of topical anesthesia is needed and might represent an avenue for further study to better quantify lacrimation kinetics in rabbits. The large SD likely reflects the large range in values (similarly mentioned in a previous study1) rather than error in the experimental method. Large ranges in the mean STT value are 785
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not unique to rabbits: SD values typically represent a quarter of the mean in dogs9 and cats2 and were even greater in guinea pigs (although mean STT values were extremely low).30 In our rabbits, the mean value for the standard 60-s STT was 7.58 ± 2.3 mm/min, with maximum of 14 mm/min and minimum of 3 mm/min. These results are in contrast to the values of 5.3 ± 3.0 mm/min1 and 4.8 ± 2.9 mm/min5 determined in previous studies. In light of the breed distribution in one of these studies,1 the other5 is a better comparator given that it involved New Zealand White rabbits. The difference between the values obtained in our current study and the previous one5 might reflect our increased sample number and a possible weight- or age-associated effect on tear production. This effect was not investigated specifically in the earlier study,5 but those animals weighed less than did those in the current study (less than 4 kg). In addition, we found no significant difference between the values for the right and left eyes when we compared the entire sample population as a whole, in concurrence with previous studies undertaken.1,5 This finding led us to combine the data from both eyes, for further statistical comparison. This approach is widely used in ophthalmologic research in both humans and veterinary species but may be statistically flawed and lead to false-positive results. Combining the data from both eyes might render the t test invalid, because the measurements from the 2 eyes of a single subject are usually related, not independent.22 This factor likely has a greater consequence in research investigating the effects of drugs on intraocular pressure or when assessing systemic parameters to make treatment recommendations but should be considered whenever studies of any ophthalmologic nature are designed. We hypothesized that if an interaction between eyes existed, tear production would be greater in the second eye in which the test was performed. The mean difference between these measurements was positive (0.18), albeit only slightly. Because the 95% confidence limits for this value do not span 0, there is some evidence to support the hypothesis proposed. This area has not received much attention in previous studies in any species, although other colleagues assessed this factor in rabbits to find no interaction between eyes.1 The neurology of lacrimation is complex and still under discussion. There is no reported neural system of communication between eyes that would allow a stimulus in one eye to influence reflex stimulation in the other and thus explain such a finding.28,26,32 It seems feasible that a stimulus in one eye might travel within afferent trigeminal fibers and be mediated centrally in the brainstem, possibly in the receiving nucleus (sensory nucleus of the trigeminal nerve), to distribute bilaterally to the lacrimal glands of both eyes. However, this explanation is mere conjecture. Another possible explanation for an interaction might be a psychogenic cause, but again the paucity of evidence for a communication system between eyes blights this hypothesis. Given the lack of studies investigating reflex lacrimation and eye interaction effect and in view of our findings, this area of research requires further characterization by using larger sample sizes to elucidate both the presence (or absence) of an interaction and the neurologic pathway that might mediate such an effect. A limitation of our study is that age and weight of individual rabbits were not recorded to evaluate whether a relationship exists between these variables and tear production. Research has shown differences in tear production with age and weight in dogs. One study reported an increase in tear production in heavier animals, showing that basal tear production increased.4 Other authors conversely found a decrease in STT as dogs aged but no significant correlation of weight with STT.15 Another group demonstrated that older dogs were more likely to be
tear-deficient among a population of 460 dogs that presented for ophthalmic examination.19 However, other studies have looked at STT values in normal domestic canines and felines but found no significant difference with age or sex.14,21,29 It seems plausible that there might be a relationship between age and tear production by the developing lacrimal gland in younger animals, and that a reduction in functional capacity of the lacrimal and nictitans glands might occur in senescent animals, leading to a decrease in tear production. The effect of age on tear production does not appear to have been studied previously in rabbits and merits future evaluation by using animals that vary widely in age. The inclusion of weight as a covariate should be considered, to elucidate the contributing effect of each variable. Our current study has demonstrated that the STT, widely used in clinical practice, can provide much more information than might appear at first. Cases of naturally occurring keratoconjunctivitis sicca in rabbits have not been reported, but these animals are often used in research as a model for this disease.12,13 In such applications, a simple method of measuring progression of the model postoperatively is of value. In addition, rabbit ocular disease is a common presenting problem in clinical practice (particularly that of tear overproduction and epiphora), and it is useful to have techniques available to evaluate factors that might contribute to disease. Despite the large variability in the STT results that we obtained in the current study, the test generally is still valid because a clinical diagnosis of over- or underproduction of tears usually is made only after several abnormal tests have been obtained. In conclusion, this study has extended earlier work in this area by using a larger sample and consequently has provided a reliable reference value for the standard, 1-60 STTs in New Zealand white laboratory rabbits. We determined values for tear production rates, and we present evidence that supports the existence for an interaction between eyes in the results of the STT1. Finally, we have highlighted potential avenues for further study in rabbits and other species.
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