Intraocular Pressure, Tear Production, and Ocular Echobiometry in Guinea Pigs (Cavia porcellus)

Abstract

The purpose of this study was to evaluate intraocular pressure (IOP) by means of rebound tonometry, to assess tear production by using the endodontic absorbent paper point tear test (EAPTT) and phenol red thread test (PRTT), and to determine the effects of time of day on IOP and tear production in guinea pigs. The study population comprised 24 healthy adult guinea pigs (12 male, 12 female; 48 eyes) of different breeds and ranging in age from 12 to 15 mo. IOP and tear production were measured at 3 time points (0700, 1500, and 2300) during a 24-h period. Overall values (mean ± 1 SD) were: IOP, 6.81 ± 1.41 mm Hg (range, 4.83 to 8.50); PRTT, 14.33 ± 1.35 mm (range, 12.50 to 16.83); and EAPTT, 8.54 ± 1.08 mm (range, 7.17 to 10.0 mm). In addition, ultrasound biometry was performed by using a B-mode system with linear 8-MHz transducer. This study reports reference values for IOP and tear production in guinea pigs.

Guinea pigs (Cavia porcellus) are members of the Caviidae family of the order Rodentia. These animals have been used for many years as laboratory rodent models, to the extent that its very name is used as a term denoting experimental subject.^8,31 Guinea pigs are believed to have been domesticated more than 5000 y ago by the natives of the Andean region of South America.²⁹

Guinea pigs have long been a valuable animal model for studying various tissues of the eye, including the cornea, lens, and retina, as well as various eye disorders.²⁴ For example, vision researchers have taken advantage of the fact that guinea pigs, like humans, require vitamin C in their diet and thus can be made scorbutic.⁵ In addition, the guinea pig is possibly the best nonprimate model for investigating certain aspects of human cataractogenesis.²⁷ Unlike those of mice and rats, the lenses of guinea pigs display key biochemical similarities to the human lens.²⁴ In addition, guinea pigs are frequently used for retinal research. Unlike several other experimental animals (for example, rats, mice, cats, dogs, and rabbits), guinea pigs are born with their eyes open and therefore can be studied by electroretinography from birth. Because key aspects of fetal retinal development are very similar between humans and guinea pigs, this laboratory species is a useful model to study the effects of adverse intrauterine conditions on retinal development.^14,20,21

The guinea pig eye is similar to that of other rodents of similar size except for one major difference: the retinal vasculature of guinea pigs is paurangiotic but to a degree where, by fundoscopy, the retina appears devoid of blood vessels.³⁰ The cornea of guinea pigs is large and occupies 85% to 90% of the interpalpebral fissure. Corneal sensitivity is very low in guinea pigs, and the cornea is not protected nor covered by the third eyelid, because it is vestigial. ¹⁵ The tear film of guinea pigs is likely similar to that of rabbits. Guinea pigs blink very infrequently—only 2 to 5 times every 20 min—thereby suggesting pronounced tear film stability.²⁶

The tear film is vital to the normal function of the eye and for the maintenance of corneal clarity. The tear film serves as the anterior refracting surface of the eye and provides the nutrition for the corneal surface.^11,23 Lack of sufficient tear production can lead to the formation of keratoconjunctivitis sicca and corneal ulceration.³¹

The phenol red thread test (PRTT) is a quantitative tear-film test. This test is performed by placing a 75-mm cotton thread that is impregnated with pH-sensitive phenol dye in the ventral fornix of the eye for 15 s. The cotton thread is less irritating to the corneal surface than is the Schirmer tear test, and the short duration of the PRTT and the small size of the thread make it more suitable for small mammals and birds.²³ The endodontic absorbent paper point tear test (EAPTT), first reported in 2012, ¹⁰ is an alternative method for tear film measurement, in which a standardized absorbent paper point is inserted in the lower conjunctival fornix of eye for 1 min; the paper point is then removed, and the wetted portion of the paper is measured by using a digital caliper graduated in millimeters.

Intraocular pressure (IOP) is controlled and regulated by the CNS, which maintains a balance between aqueous humor production and outflow.^4,6 The assessment of IOP is crucial for a complete ophthalmic examination, because some ocular diseases, such as glaucoma and uveitis, are associated with abnormal IOP.^2,22

Infectious conjunctivitis is likely the most common primary ocular disease of guinea pigs.⁷ Chlamydophila psittaci and C. caviae are the most common bacteria associated with conjunctivitis in guinea pigs.⁹ Other bacterial invaders responsible for conjunctivitis include Listeria monocytogenes, Staphylococcus aureus, Pasteurella multocida, and Salmonella spp.⁹ Antimicrobial treatment for guinea pigs must be selected carefully to avoid antimicrobial-associated enterotoxemia. Substantial amounts of antimicrobials can be absorbed systemically after the application of topical formulations for the treatment of conjunctivitis.⁷

Previous studies have reported the results of selected ophthalmic diagnostic tests in guinea pigs,^7,26 but those from other tests are unavailable currently. The purpose of the current study was to evaluate IOP by means of rebound tonometry, to assess tear production by using the EAPTT and PRTT, and to determine the effects of time of day on IOP and tear production in guinea pigs. In addition, horizontal palpebral fissure length (HPFL) was evaluated in this species as a measure of ocular adnexal dimensions. Selected echobiometric parameters of normal guinea-pig eyes were evaluated by using B-mode ultrasonography.

Materials and Methods

Animals.

The study was approved by the Iran Society for the Prevention of Cruelty to Animals in accordance with Iranian ethical codes for studies on laboratory animals. The study population consisted of 24 (12 male and 12 female) healthy adult guinea pigs (48 eyes) of different breeds (8 American, 8 Teddy, 8 Dunkin–Hartley) bred from a captive colony and obtained from a rodent breeder. All guinea pigs ranged in age from 12 to 15 mo and weighed between 444 and 544 g (mean ± 1 SD, 497.5 ± 15.03 g).

Beginning 7 d before the first testing day, all guinea pigs were housed individually in labeled cages at constant temperature (20 to 22 °C) and humidity (45% to 50%) in an air-conditioned room and were exposed to a 12:12-h light:dark photocycle. Commercially obtained substrate in the form of paper without any dust or debris was used for bedding. The animals were fed a commercial guinea pig diet, vitamin C supplement, and green leafy vegetable and had unrestricted access to water.

Study procedures.

A full physical examination was performed on all animals in the study population, which were selected on the basis of normal physical and ophthalmic examinations, including ophthalmoscopy (Binocular Indirect Ophthalmoscope, Welch Allyn, NY), fluorescein staining (Fluorescein Glostrips, Nomax, St Louis, MO), slit-lamp biomicroscopy (Portable Slit Lamp, Depew, Reichert, NY).

Phenol red test threads (Zone-Quick, Menicon America, San Mateo, CA) and endodontic absorbent paper points (Roeko color no. 30, Coltene Whaledent, Langenau, Germany) from a single lot and manufacturer were used. The procedures were performed in the following sequence: day 1, PRTT and EAPTT; day 3, IOP; and day 5; ultrasound biometry and HPFL measurement.

To measure aqueous tear volume by using phenol red threads, the lower eyelid of both eyes of each animal was everted, and the 3-mm folded head of the PRTT cotton thread was placed into the ventral conjunctival fornix; the thread was removed after 15 s, and the portion of the thread that had changed to a red color was measured (in millimeters). After a 15-min rest period, EAPPTT was performed: a single absorbent paper point was inserted in the lower conjunctival fornix of each eye, paper points were removed after 1 min, and the wetted portion of each paper was measured by using a digital caliper graduated in millimeters (Figure 1).

Figure 1.

Photographs of ophthalmic tests in guinea pigs. (A) Phenol red thread test. (B) Endodontic absorbent paper point tear test. (C) Measurement of intraocular pressure.

IOP was measured by using a rebound tonometer (TonoVet tonometer, iCare, Tiolat, Helsinki, Finland). The tonometer with a disposable probe was held perpendicular to and 4 to 5 mm from the central corneal surface. The calibration of device was set to ‘p’. The tonometer obtained 6 consecutive measurements and displayed a reading of the mean IOP in mm Hg. The series of measurements were repeated until the tonometer indicated that an acceptable SD between the 6 measurements had been obtained. The procedure was repeated for each eye.

Ultrasound biometry was performed by using a B-mode system (Apogee 3500 Touch Veterinary Ultrasound Scanner, SIUI, Guangdong, China) with linear 8-MHz transducer; the same operator (RS) performed all evaluations. After a topical anesthetic (proparacaine hydrochloride 0.5%, Paracain, Sunways, Mumbai, India) had been applied to a guinea pig's eye, the transducer was gently applied perpendicularly to the center of the cornea by using ultrasonic transmission gel (Aquasonic, Parker Laboratories, Fairfield, NJ). The axial globe length was measured from the anterior corneal surface to the retina. The anterior chamber depth was measured as the distance between echoes from the posterior corneal surface and the anterior lens surface. The lens thickness was the distance between echoes from the anterior and posterior lens surfaces. The vitreous chamber depth was the distance between echoes from the posterior lens surface and the retina (Figure 2). To obtain the HPFL, the distance between the inner end of the caruncle and the inner end of the temporal canthus was measured by using a waterproof digital caliper with LCD display (0 to 150 mm, resolution; 0.01 mm; IP54, Guanglu, Guilin, China).

Figure 2.

B-mode ultrasonogram of the eye of an adult female guinea pig. ACD, anterior chamber depth; AGL, axial globe length; LT, lens thickness; VCD, vitreous chamber depth.

A single examiner (SMR) performed all the ocular tests, examinations, and measurements. Tear production and IOP measurements were performed 3 times (0700, 1500, 2300) during a 24-h period. Measurements were performed under dim red-light illumination (16-W bulb) in the dark. Animals were calm and needed only minimal manual restraint them. At 7 d after completion of all examinations, all animals were examined and were free of signs of conjunctivitis, keratitis, blepharitis, corneal ulceration, and intraocular disease.

Statistical analysis.

Statistical analysis was performed by using the statistical software SPSS for Microsoft Windows (version 20.0, SPSS, Chicago, IL). Normality was tested by using a one-sample Kolmogorov–Smirnov test. Paired-samples t tests were used to compare the IOP, PRTT, EAPPTT, HPFL, and ultrasound parameters obtained from right and left eyes. The mean and SD were calculated for all eyes combined and for right and left eyes separately. Independent-samples t tests were used to compare the mean IOP, PRTT, EAPPTT, HPFL, and ultrasound parameters values according to sex and body weight in each group. Repeated-measures ANOVA was used to analyze data from 24-h measurements. Pearson correlation was used to evaluate relationship between mean IOP, PRTT, EAPPTT, HPFL, ultrasound parameters, and body weight. A P value of less than 0.05 was considered statistically significant.

Results

None of the guinea pigs demonstrated any signs of ocular discomfort during the 24 h after the measurements had been taken. All continuous numeric data obtained in the population used in this investigation were normally distributed according to the one-sample Kolmogorov–Smirnov test (P > 0.5).

The overall (that is, all eyes) mean (± 1 SD) value for IOP was 6.81 ± 1.41 mm Hg (range, 4.83 to 8.5 mm Hg), PRTT was 14.33 ± 1.35 mm/15s (range, 12.5 to 16.83 mm/15s), and EAPTT was 8.54 ± 1.08 mm/min (range, 7.17 to 10.0 mm/min), respectively. The only significant difference found was that tear production according to the EAPTT in guinea pigs differed (P = 0.003) between 0700 and 2300. For all tests, results did not differ significantly between right and left eyes or between male and female guinea pigs, and there was no correlation between measured parameters and body weight. Descriptive statistical results of IOP, EAPTT, and PRTT in guinea pigs are summarized in Table 1; biometry data are provided in Table 2.

Table 1.

Mean and range (95% confidence interval [CI]) of intraocular pressure and tear production measured at 3 time points during a 24-h period in 24 guinea pigs (48 eyes)

					Tear production
	Intraocular pressure (mm Hg)				Phenol red thread test (mm)				Endodontic absorbent paper point tear test (mm)
Time	Mean	1 SD	Range	95% CI	Mean	1 SD	Range	95% CI	Mean	1 SD	Range	95% CI
0700	5.43	0.39	4–7.5	4.50–6.37	13.50	0.84	10–17	11.50–15.49	7.93a	0.37	6.5–9.5	7.06–8.81
1500	6.87	0.72	3–9.5	5.16–8.58	14.50	0.95	9.5–19	12.24–16.75	8.31	0.54	6.5–11	7.03–9.59
2300	8.12	0.88	4 –10.5	6.03–10.21	15.00	1.26	11.5–21.5	12.01–17.98	9.37a	0.43	8–11	8.35–10.39

^aValues are significantly (P = 0.003) different.

Table 2.

Ocular echobiometry findings (mean ± 1 SD) from 24 guinea pigs

	Left eyes	Right eyes	Male eyes	Female eyes	All eyes	Range
Axial globe length (mm)	9.05 ± 0.29	8.98 ± 0.29	8.92 ± 0.22	9.11 ± 0.27	9.01 ± 0.25	8.67–9.49
Anterior chamber depth (mm)	0.85 ± 0.06	0.82 ± 0.06	0.83 ± 0.05	0.85 ± 0.03	0.84 ± 0.04	0.76–0.88
Lens thickness (mm)	4.40 ± 0.21	4.27 ± 0.25	4.31 ± 0.12	4.36 ± 0.18	4.33 ± 0.14	4.12–4.52
Vitreous chamber depth (mm)	3.26 ± 0.23	3.27 ± 0.19	3.25 ± 0.09	3.28 ± 0.30	3.26 ± 0.20	2.94–3.56
Horizontal palpebral fissure length (mm)	9.90 ± 0.66	9.99 ± 1.04	9.53 ± 0.83	10.35 ± 0.54	9.94 ± 0.78	8.59–11.12

Discussion

Obtained by using rebound tonometry, the mean IOP values of the guinea pigs in our study were lower than those obtained by using applanation tonometry, which have been reported as 18.27 ± 4.55 mm Hg.⁷ In rabbits, IOP values obtained by using applanation tonometry were higher than values from rebound tonometry.¹⁹ The mean IOP of guinea pigs measured by using rebound tonometry is lower than the values reported for rabbits.¹⁹ In chinchillas, IOP recorded by applanation tonometry¹³ are higher than those from rebound tonometry.¹⁷ Further studies designed to compare these 2 types of tonometry with manometric measurement in guinea pigs are needed.

In our study, the highest IOP in guinea pigs was obtained at 2300, and IOP increased from morning to night. Although this increase was not statistically significant in the current study, rabbits demonstrate significant time-dependent differences in IOP, with the highest value during the morning.¹⁹

Tear production according to the 15-s PRTT in guinea pigs was reported as 16 ± 4.7 mm in 2007⁷ and 21.26 ± 4.19 mm in 2008.²⁶ The PRTT value we obtained was slightly lower than previous reports. The result for this difference is unknown, but the range of PRTT values in the current study (10 to 21.5 mm) was within previously published reference intervals.^7,26

The overall mean PRTT and EAPTT values of our guinea pigs were lower than those for New Zealand White rabbits (20.88 ± 3.71 mm/15s and 13.8 ± 1.5 mm/min, respectively).^3,12 Both EAPTT and PRTT values appeared to increase slightly from 0700 to 2300, such that the EAPTT value at 0700 differed significantly from that at 2300. Although the values were statistically significantly different, a difference of less than 1.5 mm is unlikely to be clinically relevant. The reported PRTT value of chinchillas (14.6 ± 3.5 mm)¹⁶ is similar to that we obtained for guinea pigs. Whereas one previous study found statistically (but not clinically) significant differences in PRTT between male and female guinea pigs,⁷ another previous study²⁶ did not examine the effect of sex on this parameter. In our study, tear production did not differ significantly between male and female guinea pigs.

No abnormal opacity was observed in the lenses of study population by using slit-lamp biomicroscopy and ultrasonography. The anterior chamber length and axial globe length were shorter and lens thickness was less in guinea pigs than chinchillas.¹³ Vitreous chamber depth was similar between guinea pigs (3.26 ± 0.20 mm) and chinchillas (3.69 ± 0.52 mm)^1.3 The ratio of lens thickness to axial globe length in our guinea pigs (1:2) is the same as that of chinchilla⁶ and male Hartley albino guinea pig¹ but lower than that of capybara (1:3)¹⁵ and rabbits (1:2.5).²⁵ The anatomic features shared by chinchillas and guinea pigs (that is, a small eye and a proportionally thick lens) might exclude them from being useful biologic models for experimental intraocular manipulations, such as subretinal injection and posterior segment surgery.¹³

The HPFL of guinea pigs (9.94 mm) was slightly smaller than that of chinchillas (14.4 mm)¹³ and capybaras (26.35 mm).¹⁶ The mean HPFL of guinea pigs in our study was less than 2 mm shorter than that reported previously,²⁸ and this difference may reflect differences in the breeds, ages, and sizes of the animals used in the 2 studies. The normal palpebral fissure length is an important biometric feature that might facilitate correct diagnosis of various conditions (for example, ankyloblepharon, eyelid agenesis, blepharophimosis, euryblepharon, entropion, or ectropion) and assist surgeons to restore palpebral form and function by correctly positioning the upper and lower eyelids.¹³

All guinea pigs well tolerated tonometry, tear production measurements, and ultrasonography biometry, without any restlessness during gentle manual restraint in sternal recumbency. The impact of the probe induced a blink reflex in some, but not all, animals in our study. Although the discrepancy in may have been attributable to the central corneal sensitivity of guinea pigs (1.6 g/mm²),²⁶ interspecies differences in corneal innervation cannot be excluded.¹¹

The guinea pig has become a popular model for the study of human myopia.¹⁸ Risks of retinal detachment, macular degeneration, and primary open-angle glaucoma are closely associated with the degree of myopia in human.¹⁸ A previous study into the high prevalence of ocular disease in guinea pigs reported that lenticular opacities were the most common abnormality.³¹ Slit-lamp biomicroscopy and ultrasonography are necessary for assessing the condition of the lens, and our current results may be helpful for both researchers and clinicians in assessing the relationships among myopia, glaucoma, and ocular health status in guinea pigs.