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Keywords:

  • Mus musculus ;
  • Rattus norvegicus ;
  • Sicalis flaveola ;
  • Sporophila angolensis ;
  • standardized endodontic absorbent paper points tear test;
  • Trachemys scripta elegans

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The aqueous fraction of the tear film and the horizontal palpebral fissure length (HPFL) were measured in exotic and laboratory animals, specifically saffron finches (Sicalis flaveola), chestnut-bellied seed-finches (Sporophila angolensis), red-eared sliders (Trachemys scripta elegans), rats (Rattus norvegicus) and mice (Mus musculus). These species possess small eyes making it difficult to perform the typical Schirmer tear test. Measurement of the aqueous fraction of the tear was performed using the standardized endodontic absorbent paper point tear test (PPTT), accomplished with manual restraint by a single operator. The following results were obtained: saffron finches (n = 42)—HPFL (4.46 ± 0.09 mm) and PPTT (5.10 ± 0.26 mm); chestnut-bellied seed-finches (n = 38)—HPFL (4.77 ± 0.05 mm) and PPTT (4.11 ± 0.34 mm); red-eared sliders (n = 56)—HPFL (8.59 ± 0.08 mm) and PPTT (8.79 ± 0.38 mm); rats (n = 60)—HPFL (6.45 ± 0.09 mm) and PTT (6.18 ± 2.06 mm); and mice (n = 22)—HPFL (3.59 ± 0.27 mm) and PPTT (4.39 ± 1.45 mm).


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The aqueous fraction of the tear film is an important physiological parameter evaluated during the ophthalmic examination of animal species of veterinary interest. There are few literature sources for normal ocular parameters in wild animals due to the great number of species. Therefore, it is important to establish reference values for the tests routinely used in domestic animals.[1, 2] In many species, the small size of the globe hinders the use of conventional techniques for measuring the aqueous fraction of the tear film, necessitating alternative measurement techniques for which there are no standard reference values. This is true even for well-known laboratory animals such as mice.[3] Schirmer tear test (STT), originally developed for human medical use[3, 4] is the most widely used and accepted method to measure the aqueous fraction of the lacrimal film in veterinary medicine as well.[5-7] STT uses standard absorbent five-mm-wide paper strips. In human patients, STT was originally carried out in 5 min. To minimize ocular discomfort and save time, Nelson[8] proposed shortening the standard Schirmer I test by performing it in 1 min instead of 5 min. In animals, the test time also was reduced to 1 min to facilitate handling and reduce stress.[4] STT cannot be performed in animals with small eyes, having a palpebral fissure length smaller than the width of the strip (0.5 cm) or in species whose tear production is lower than could be measured in the STT. To avoid these difficulties, alternative techniques have been proposed by several authors, including aseptically cutting the Schirmer strips in half or in different widths or alternatively reducing the STT time.[1, 2, 9, 10] These method variations, however, may preclude comparison of the results[3] and sometimes require optical equipment for reading the results.[10] Another alternative is the cotton thread embedded with phenol red. It is a commercially available test that can be applied, usually using small forceps, in the lateral canthus for 60 s. Nevertheless, the high standard deviation recorded for this method in the literature suggests the need for further refinements. Despite the lack of available data, the phenol red cotton thread test may be used in animal models of dry eye, such as the MRL/I mice and rats, because of the reduced size (width) of the cotton threads compared to the filter paper of the Schirmer test.[3] The paper points tear test (PPTT)[11] aims to overcome these limitations and enable the comparison among species. The PPTT uses sterile standardized paper cones or tips, originally developed for endodontics. These sterile paper cones are standardized with a distal diameter of 0.30 mm,[12] allowing insertion into the conjunctival fornix even in the smallest species of interest to the veterinary ophthalmologist. It was originally proposed by Lange et al.[11] as an alternative to overcome the difficulties found while measuring tear production in black-tufted marmosets (Callithrix penicillata). The present article is a logical step forward from the original work, providing reference PPTT values for two bird species, the chestnut-bellied seed-finch—Sporophila angolensis—and the saffron finch—Sicalis flaveola, for one reptile, the red-eared slider—Trachemys scripta elegans, and for two laboratory species, the mouse—Mus musculus—and the rat—Rattus norvegicus. HPFL was measured in all these species as well.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was approved by the Ethics Committee on Animal Use from the Sector of Agricultural Sciences, Federal University of Paraná.

The lacrimal production measurements were performed in an unheated enclosure. The ambient temperature and relative humidity were measured using a digital thermo-hygrometer (Mod 30.5000.02, TFA Technology HK Limited, Hong Kong, China). The room was maintained with 78% humidity and 20.9 °C temperature for saffron finches, 81% humidity and 19.5 °C for chestnut-bellied seed-finches, 82% humidity and 19.8 °C for red-eared sliders, 90% humidity and 20 °C for rats, 86% humidity and 18.2 °C for mice.

The saffron finches (Sicalis flaveola), the chestnut-bellied seed-finches (Sporophila angolensis) as well as the red-eared sliders (Trachemys scripta elegans) were kindly provided by the Screening Center of Wildlife (CETAS) PUCPR/IBAMA, located in Tijucas do Sul, PR, Brazil. Wistar rats (Rattus norvegicus) and Swiss Webster mice (Mus musculus) were provided by the Curitiba Zoo, Curitiba, PR, Brazil. The general appearances of the animals studied are shown in Fig. 1. The numbers of animals investigated were as follows: saffron finches (n = 21)—10 males, seven females and four unknown gender; chestnut-bellied seed-finches (n = 19) – 10 males and nine females; red-eared slider (n = 28)—10 males and 18 females; rats (n = 30)—only females; and mice (n = 22)—19 males and three females.

image

Figure 1. Illustrative images of the species evaluated during the investigation, (a) saffron finch (Sicalis flaveola). (b) chestnut-bellied seed-finch (Sporophila angolensis) (photo courtesy of Zig Koch). (c) redeared slider (Trachemys scripta elegans). (d) rat (Rattus norvegicus). (e) mouse (Mus musculus).

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Horizontal palpebral fissure length measurements were performed on both eyes of the animals, using a stainless steel digital caliper with an LCD display (PD-150, 0-150 mm, accuracy: 0.01 mm; Vonder, Curitiba, PR, Brazil).

To measure the aqueous fraction of the tear film, the overall methodology for PPTT proposed by Lange et al.[11] was used. In brief, under physical restraint, one standardized absorbent paper point was inserted into the lower conjunctiva fornix of the right eye, and after approximately 20 s, another paper point was inserted in the fornix of the left eye. Both paper points are removed after 1 min. The wetted portion of the paper point was measured using a ruler with millimeter scale. The length of the wetted portion of the paper point was identified by bending the wetted end against a rigid surface.[11]

The choice of using the specific absorbent endodontic paper point (Roeko Color, number 30, Langenau, Germany) (Fig. 2) in our research was based on a dentistry study, which classified this particular paper point model like the better standardized material.[12]

image

Figure 2. Appearance of commercial packaging of the paper points (or cones) used (Roeko Color brand series 15–40). Size 30 was used in this investigation. PPTT being performed in both eyes simultaneously in different species: (b) red-eared slider (Trachemys s. elegans). (c) mouse (Mus musculus). (d) saffron finch (Sicalis flaveola).

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The saffron finches and the chestnut-bellied seed-finches were removed from their cages by manual capture and then were carefully physically restrained to allow the PPTT and HPFL procedure. A single operator used one hand to hold the bird immobilized, while the other hand could insert the paper strip in the conjunctival fornix of each eye.

The red-eared slider handling was performed as follows: the animals were removed from the water tank where they were housed at CETAS 2 h prior to the PPTT and HPFL measurements. After being removed from the water tank, the animals were dried with towels and placed in a dry plastic box where they remained until the time of the tear testing. Subsequently, for the PPTT and HPFL measurements, the red-eared sliders were physically restrained and the head was gently immobilized. During restraint, extreme care was taken so that there was no excessive discomfort or excessive compression on the animals' bodies.

For the PPTT procedure and the HPFL measurement on rats, the animals were carefully manually restrained by holding them dorsally by the cervical region with the index finger and the thumb, supporting the animal body with the other fingers of the same hand, thus preventing the paper tip withdraw by the animals' paws.

Mice were carefully restrained by the nondominant hand held with the index finger and the thumb over the back of the neck region, grasping the loose skin as close to the ears as possible, while the dominant hand proceeded to the placement of the paper tips in the palpebral fornix and the measurement of HPFL.

The animals were observed during 4 h after the test to evaluate signs of ocular disturbances and alterations on the behavior. General aspects of the PPTT measurements in the different species evaluated can be seen in Fig. 2.

Statistical analysis

Data obtained in this investigation were analyzed using descriptive statistics. The Shapiro–Wilk normality test was initially performed. Following this procedure, unpaired t-tests were used to compare two groups of independent subjects presenting PPTT values as continuous numeric data in mm of paper-wetting. Data were deemed significant when < 0.05. All tests were performed using sas v. 9.2 (SAS Institute, Cary, NC, USA). Additionally, paired t-test analyses of paired tear production were performed in the same individuals, comparing left to right eyes.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Shapiro–Wilk test showed that the data errors were normally distributed. Descriptive statistics of HPFL and PPTT are summarized in Table 1.

Table 1. Results obtained measuring the horizontal palpebral fissure length (HPFL) and performing paper point tear tests (PPTT) in different exotic and laboratory animal species
SpeciesPPTT (mm/min.)95% confidence interval (mm/min.)HPFL (mm)Weight (g)RH (%)RT (oC)
Sicalis flaveola 5.10 ± 0.264.59–5.614.46 ± 0.0920.00 ± 0.107820.09
Sporophila angolensis 4.11 ± 0.343.44–4.784.77 ± 0.0514.00 ± 0.108119.50
Trachemys s. elegans 8.79 ± 0.388.05–9.538.59 ± 0.08663.50 ± 201.688019.30
Rattus norvegicus 6.18 ± 2.062.14–10.226.45 ± 0.09151.20 ± 68.899022.00
Mus musculus 4.39 ± 1.451.55–7.233.59 ± 0.2730.45 ± 5.697819.50

For all species evaluated, no significant differences were observed comparing results obtained for PPTT and HPFL between males and females (> 0.05) or between right and left eyes (> 0.05).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The PPTT method has been shown to be easily performed on all species evaluated, even those with very small eyes such as mice and saffron finches, because of the characteristics of the material of the PPTT, which make the paper point firm, facilitating the insertion of the test. For all species of birds, reptiles and mammals tested the processes of restraint and insertion of the paper points in the conjunctival fornix of the lower eyelids could all be performed by a single operator. Additionally, measuring the aqueous fraction of the tears using the PPTT method requires a short period of restraint and produces apparently minimal discomfort for the most individuals of all species evaluated, with no signs of intense blepharospasm or great ocular discomfort, and there was no obvious adverse reaction attributable to stress like dyspnea or fainting. In cases where it is necessary to measure tears in stressed animals, such as in some mice or birds for example, one way to shorten the procedure time for a beginner operator maybe is to practice the PPTT first in other nonstressed animals from the same or other species. With the practice, the operator can reduce the total evaluation time, and consecutively the necessity of holding the animal more than necessary.

It was possible to apply the PPTT in both eyes simultaneously using a time gap of 15–30 s from the insertion of the paper strip into the conjunctival fornix to its placing into the contralateral one. This practice resulted in a reduction in restraint time for the bilateral assessment and hence a reduction in total restraint time, thereby reducing stress.

It was possible to apply the PPTT in both eyes simultaneously using a time gap near to 20 s from the insertion of the paper strip into the conjunctival fornix to its placing into the contralateral one. This practice resulted in a reduction in restraint time for the bilateral assessment and hence a reduction in total restraint time, thereby reducing stress. It is important to remember that the time gap required may vary depending on the person that performs the test.

Among the five animal species evaluated using PPTT, the two bird species were the easiest ones to perform in terms of handling. The third eyelid, which is very mobile, did not cause displacement of the somewhat unyielding paper points inserted into the palpebral fornix. On the other hand, Schirmer test strips are commonly displaced by birds' third eyelids. Although the red-eared sliders need proper restraint for the immobilization of the head, the implementation of the procedure was also very practical. We have shown the facility to perform the test in the most species; however, some individuals of this strain of mice were very aggressive before the restrain, and during the PPTT evaluation. Because of this, mice were certainly the most difficult animals to handle and perform the PPTT. Physical restraint was hard in this species due to the small body size and agility. In most cases, mice reacted aggressively to the test, trying to bite the operator's fingers. Moreover, the forelimbs had to be well immobilized; otherwise, the animals would quickly withdraw the paper points, using their paws with great dexterity. Based on our experience, we believe that restraint for some mice actually caused more stress than the PPTT evaluation, because these animals became more reluctant before the test during the manual restraining than during the PPTT itself. Nevertheless, mice are animals with high manual ability and they can easily remove the paper point if the hands are not restrained.

However, no difficulties restraining rats were encountered. When the paper points were inserted simultaneously into both eyes, animals often moved their heads more actively than when they were inserted unilaterally. So, although it is possible to measure both eyes at the same time, we suggest that in the future, PPTT should be performed individually on each eye, only in this species, to facilitate the process for the evaluation of an animal.

No complications were observed following PPTT measurement in any species evaluated during 4 h after the test. All animals returned to their enclosures without showing any signs of stress or ocular disturbances such as secretion or blepharospasm.

Paper point tear test results obtained for the five species studied do not allow comparisons with the few reference values available in the literature due to the intrinsic differences in the methods used to obtain the data. For saffron finches, chestnut-bellied seed-finches and red-eared sliders, there are no measurement data of the aqueous fraction of the tear film in the literature.

Regarding the practicality, the measurement of the aqueous fraction of the tear film using PPTT was demonstrated to be highly feasible, particularly in birds, where the animal interference was practically nonexistent. Red-eared slider restraint requires greater skill than birds. Restraining rats for the PPTT was much easier than restraining mice. Although mice were more difficult to restrain, the procedure can also be performed with some previous training for handling and after acquiring some familiarity with the method. In our study, the previous training was important to restrain the animals to perform a correct exam.

This study demonstrates that PPTT can become a standardized method for measuring the aqueous fraction of the tear film, which is of greatest value in animals with small eyes (HPFL ≤ 5 mm). The data included in this paper serve as the second application of this method and will allow for future comparisons in different species. The literature does not have other parameters of tear measurements in these species that permits comparison between each one, reinforce the importance of the PPTT in assessing tear production in small species.

More experiments and the establishment of reference values for other species need to be performed to allow identification of individuals who deviate from normal physiological values and the comparison of the physiological production of the aqueous fraction of the tear among species.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Special thanks to the veterinarian MSc Grazielle Sorensini, competent manager of CETAS—Triage Center for Wildlife—PUCPR/IBAMA—for allowing access to the birds and reptiles used in this study. Great thanks also to the Biologist PhD Cristina Castellano Margarido from the Curitiba Zoo for allowing the use of animals in the vivarium and thanks to Gillian Shaw for her help in preparing this manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Montiani-Ferreira F. Ophthalmology. In: Biology, Medicine and Surgery of South American Wild Animals, 1st edn. (ed. Fowler ME, Cubas ZS) Iowa State University Press, Iowa, 2001; 37: 437456.
  • 2
    Montiani-Ferreira F. Oftalmologia. In: Tratado de Animais Selvagens – Medicina Veterinária, 1st edn. (ed. Cubas ZS, Silva JCR, Catão-Dias JL) Roca, São Paulo, SP, Brazil, 2007; 65: 10921104.
  • 3
    Barabino S, Chen W, Dana MR. Tear film and ocular surface tests in animal models of dry eyes; uses and limitations. Experimental Eye Research 2004; 79: 613621.
  • 4
    Strubbe DT, Gelatt KN. Ophthalmologic examination and diagnostic procedures. In: Veterinary Ophthalmology, 3rd edn. (ed. Gelatt KN) Lippincott Willians & Wilkins, Philadelphia, 1999; 11: 427466.
  • 5
    Marts BS, Bryan GM, Prieto DJ. Schirmer tear test measurements and lysozyme concentration of equine tears. Journal of Equine Medical Surgery 1977; 1: 427.
  • 6
    Kaswan RL, Salisbury MA, Wand DA. Spontaneous canine keratoconjunctivitis sicca. A useful model for human keratoconjunctivitis sicca: treatment with cyclosporine eyes drops. Archives of Ophthalmology 1989; 107: 12101216.
  • 7
    Trost K, Skalicky M, Nell B. Schirmer tear test, phenol red thread tear test, eye blink frequency and corneal sensitivity in the guinea pig. Veterinary Ophthalmology 2001; 10: 143213.
  • 8
    Nelson PS. A shorter Schirmer tear test. Optometry Weekly 1982; 73: 568569.
  • 9
    Fujihara T, Nagano T, Nakamura M et al. Lactoferrin suppresses loss of corneal epithelial integrity in rabbit short-term dry eye model. Journal of Ocular Pharmacology and Therapeutics 1988; 14: 99107.
  • 10
    Hoffman RW, Alspaugh MA, Waggie KS et al. Sjögren's syndrome in MRL/1 and MRL/n mice. Arthritis & Rheumatism 1988; 27: 157165.
  • 11
    Lange RR, Lima L, Montiani-Ferreira F. Measurement of tear production in black-tufted marmosets (Callithrix penicillata) using three different methods: modified Schirmer's I, phenol red thread and standardized endodontic absorbent paper points. Veterinary Ophthalmology 2012; 15: 17.
  • 12
    Pumarola-Suñé J, Solá-Vicens L, Sentís-Vilalta J et al. Absorbency properties of different brands of standardized endodontic paper points. Journal of Endodontics 1998; 24: 796798.