Comparative Medicine Copyright 2014 by the American Association for Laboratory Animal Science

Vol 64, No 4 August 2014 Pages 300–308

Case Study

Correction of Refractive Errors in Rhesus Macaques (Macaca mulatta) Involved in Visual Research Jude F Mitchell,1,† Chantal J Boisvert,3,† Jon D Reuter,2 John H Reynolds,1 and Mathias Leblanc2,* Macaques are the most common animal model for studies in vision research, and due to their high value as research subjects, often continue to participate in studies well into old age. As is true in humans, visual acuity in macaques is susceptible to refractive errors. Here we report a case study in which an aged macaque demonstrated clear impairment in visual acuity according to performance on a demanding behavioral task. Refraction demonstrated bilateral myopia that significantly affected behavioral and visual tasks. Using corrective lenses, we were able to restore visual acuity. After correction of myopia, the macaque’s performance on behavioral tasks was comparable to that of a healthy control. We screened 20 other male macaques to assess the incidence of refractive errors and ocular pathologies in a larger population. Hyperopia was the most frequent ametropia but was mild in all cases. A second macaque had mild myopia and astigmatism in one eye. There were no other pathologies observed on ocular examination. We developed a simple behavioral task that visual research laboratories could use to test visual acuity in macaques. The test was reliable and easily learned by the animals in 1 d. This case study stresses the importance of screening macaques involved in visual science for refractive errors and ocular pathologies to ensure the quality of research; we also provide simple methodology for screening visual acuity in these animals.

Macaques are the most commonly used nonhuman primate models in neuroscience. These models are invaluable in understanding how the brain generates complex cognitive behaviors such as memory and visual perception. These animals also enable us to study important primate-specific motor behaviors, such as hand and finger movements. Essential methodologic advantages of these models are their ability to be trained for performing specific and complex tasks for rewards, and their behavioral performance can be correlated with single-neuron recording.35 Typically, data from several hundred neurons can be recorded over a period of several months to years in a single monkey, thus allowing detailed analysis of a brain region from very few animals. The scientific validity of these visual experiments relies on an intact visual system to relay information from video monitors or other outside stimuli to the animal’s visual cortex for further processing. As such, the visual health of these experimental models should be monitored closely throughout their research careers, to ensure scientifically sound research. Whereas naturally occurring ocular diseases are uncommon in captive macaques, several conditions, which may affect research outcome, occur in both young and aged monkeys. For example, primary open-angle glaucoma and spontaneous esotropia have been described in young animals, whereas aging macaques are believed to develop similar ocular diseases as those in humans, Received: 16 Dec 2013. Revision requested: 09 Feb 2014. Accepted: 09 Mar 2014. 1 Systems Neurobiology Laboratory and 2Animal Resources Department and Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 3 Rady Children’s Hospital San Diego, in affiliation with the University of California, San Diego School of Medicine, San Diego, California. † These authors contributed equally to the authorship of this article. *Corresponding author. Email: [email protected]

including cataracts, glaucoma, focal atrophy of the retinal pigment epithelium, and age-related macular degeneration.50,51 In addition, macaques are susceptible to systemic conditions that potentially affect vision, such as atherosclerosis, hypertension, and diabetes.33 Furthermore, refractive errors can alter visual acuity and contrast sensitivity, potentially affecting macaques’ ability to perform cognitive tasks and thereby interfering with research. In fact, refractive development and optical organization in macaques are very similar to those in humans,5,18,36 and macaques have frequently been used as a model of experimentally induced myopia. Extreme cases of spontaneous myopia and presbyopia in macaques have been reported in the literature,14,39 but few studies have examined systematically the cross-sectional incidence of refractive errors in captive macaque colonies.14 In the present study, we performed ocular examinations of our research colony and report 2 cases of myopia, including one that was corrected successfully with prescription lenses. We then compared the performance of the myopic macaque with and without corrective lenses and with that of a control animal on 2 different behavioral tracking tasks. We suggest a simplified behavioral task that requires minimal training and that can be used to screen visual acuity in research macaques.

Materials and Methods

Subjects. A group of 21 male rhesus macaques (Macaca mulatta; age, 2.6 to 15.8 y [mean ± SEM, 8.7 ± 4.6 y]; weight, 7.6 to 14.1 kg [10.5 ± 2.2 kg]) were examined (Table 1). All monkeys were originally acquired from the California National Primate Research Center (University of California, Davis, CA) and were of Indian descent. Exact birth dates and complete captivity history were well documented. All monkeys were in good

300

cm13000166.indd 300

9/8/2014 3:53:23 PM

Refractive errors in macaques

Table 1. Refraction and ocular examination in the rhesus macaque colony. Cycloplegic refraction Macaque

Age (y)

Right eye

1

12.3

2

15.3

3

Spherical equivalent Left eye

Right eye

Left eye

Intraocular examination

+0.75/+0.50 × 90

+0.75/+1.00 × 75

+1.00

+1.25

Normal

−0.25/+0.75 × 90

Plano/+1.00 × 90

+0.13

+0.50

Normal

14.3

−4.00/+1.00 × 100

−5.00/+0.50 × 90

−3.50

−4.75

Normal

4

4.4

Plano/+1.00 × 80

+0.50/+0.50 × 160

+0.50

+0.75

Normal

5

4.6

+0.50/+0.50 × 90

+0.50/+0.50 × 90

+0.75

+0.75

Normal

6

4.3

−1.50/+1.50 × 95

−1.00/+2.00 × 95

−0.75

0.00

Normal

7

12.4

+0.75/+0.25 × 90

+1.00

+0.88

+1.00

Normal

8

4.3

+1.00/+0.75 × 95

+0.50/+0.75 × 90

+1.38

+0.88

Normal

9

11.3

Plano/+0.50 × 90

−0.50/+1.00 × 15

+0.25

0.00

Normal

10

5.6

+0.50/+0.50 × 90

Plano/+1.00 × 90

+0.75

+0.50

Normal

11

14.6

+0.25/+1.25 × 90

+0.50/+1.00 × 90

+0.88

+1.00

Normal

12

5.6

+1.50/+0.50 × 180

+1.00/+0.50 × 180

+1.75

+1.25

Normal

13

5.3

+0.50/+0.50 × 90

+0.50/+0.75 × 90

+0.75

+0.88

Normal

14

15.6

+0.50/+1.00 × 90

+1.00/+0.75 × 90

+1.00

+1.38

Normal

15

4.6

+1.50

+1.50

+1.50

+1.50

Normal

16

4.3

+1.00/+1.50 × 180

+1.00

+1.75

+1.00

Normal

17

9.6

+0.50/+0.75 × 90

plano/+0.50 × 90

+0.88

+0.25

Normal

18

8.6

−0.50/+1.00 × 90

−0.50/+1.50 × 90

0.00

+0.25

Normal

19

7.4

+0.25/+0.50 × 90

−0.50/+1.00 × 90

+0.50

0.00

Normal

20

2.6

+0.50

plano/+0.75 × 90

+0.50

+0.38

Normal

21

15.8

+0.50/+0.75 × 90

+0.25/+0.75 × 90

+0.88

+0.63

Normal

general health, as evidenced by comprehensive physical examination, including CBC, biochemistry, urine and fecal analysis performed semiannually. All primates were seronegative for SIV, simian retrovirus type D, and simian T-cell leukemia virus and were free of tuberculosis. All housing and enrichment met or exceeded Guide standards.19 Macaques were housed indoors under standard husbandry conditions in quad-unit stainless steel caging and had weekly access to unique, large (36 ft2 × 8 ft high) enrichment units. Animals were socially housed in pairs, unless singly housed either for behavioral incompatibility or justified scientific reasons. All fluid restrictions in NHP were performed in accordance with the Salk Institute IACUC Policies. As such, all procedures were scientifically justified and approved in the IACUC protocol. Consideration was given to using positive reinforcement instead of restriction whenever possible. When necessary, the lowest level of restriction was used to achieve the scientific objective. Even though the macaques typically learn to meet their entire daily fluid requirement during a working session, a number of precautions were taken to avoid the possibility of acute or chronic dehydration or clinical disease due to fluid restriction. To this end, the attending veterinarian performed a full physical examination (including CBC, biochemistry and urine analysis) prior to enrollment in an approved study involving fluid restriction. Clearance for continued participation was renewed at each semiannual physical examination. Sick animals or those on treatment were prohibited from being enrolled in fluid-restriction studies. While on restriction, each macaque received at least 20 mL of fluids/kg daily and was not fluid-restricted for more than 5 d each week. The laboratory and animal care staff monitored the animal’s health daily and maintained accurate records on total daily food and fluid consumption (including

treats in the laboratory). Abnormal behavior, decreased food consumption, weight loss, or urine specific gravity exceeding 1.040 was reported immediately to the attending veterinarian for evaluation. All procedures and social pairing exemptions were approved by the Salk Institute IACUC. Refraction and eye examination. The same experienced human ophthalmologist (CJB) performed the ocular examinations in anesthetized macaques. Briefly, macaques were premedicated with ketamine hydrochloride (10 mg/kg IM) and atropine (0.02 mg/kg IM) and maintained with isoflurane on a mask. Cycloplegia and pupillary dilation were achieved by administration of one drop of 1% tropicamide and 2.5% phenylephrine hydrochloride twice 5 min apart 30 min before the examination. Eyelids were maintained open by using a speculum (carefully placed to avoid pressure on the globe), and corneas were irrigated continuously with a sterile saline solution. Cycloplegic refraction with a streak retinoscope and handheld trial lenses was performed. Measurements were obtained at a 50-cm working distance, and the refractions of the most plus and most minus meridians were recorded in the plus cylinder form. The anterior segment examination was performed by using a Finoff ocular transilluminator, and a direct ophthalmoscope was used to evaluate the clarity of the red reflex by retroillumination. The fundi were evaluated by using an indirect ophthalmoscope and a Panretinal 2.2 lens (Volk Optical, Mentor, OH). Stimulus presentation and monitoring of eye movement. Stimuli were presented on a computer monitor (640 × 480 pixel resolution, 120 Hz; Trinitron Multiscan, Sony, Park Ridge, NJ) placed 57 cm from the eye. Eye position was monitored continuously by using an infrared eye tracking system (240 Hz; ETL-400, iScan, Woburn, MA). Experimental control was handled by using Cortex software (http://www.cortex.salk.edu/).

301

cm13000166.indd 301

9/8/2014 3:53:23 PM

Vol 64, No 4 Comparative Medicine August 2014

Task and stimuli. Performance and visual acuity of the myopic animal with and without corrective lenses and of a control animal were assessed by using 2 behavioral tasks. The attention-demanding multiple-object tracking task was adapted from a paradigm used in humans.8,42,45 Macaques used in this study were already trained to perform this task using large, easily visible Gabor stimuli (1.2 cycles per degree spatial frequency) for neuronal recording studies.32 We adapted the task by varying the spatial frequency to evaluate visual acuity. The spatial frequency of stimuli varied among 8 values (0.8, 1.0, 1.33, 1.77, 2.67, 4.0, 5.33, and 8.0 cycles/ degree; Figure 1 A). Briefly, the animal began each trial by fixating a central point for 200 ms and then maintained fixation throughout the trial. Four identical Gabor stimuli (spatial frequency, 1 cycle/ degree; diameter, 3°; Gaussian envelope with σ = 0.75°; 80% Michelson contrast; mean luminance, 30 cd/m2; viewing distance, 57 cm) appeared at equally eccentric positions (Figure 1 B). At the beginning of each task trial, the 4 stimuli were placed at an eccentricity of 5 visual degrees, and each had an identical and easily visible spatial frequency (1 cycle/degree). One stimulus then was briefly elevated in luminance, identifying it as the target in tracking. After this cueing, the spatial frequency of all stimuli changed to 1 of the 8 sampled values selected randomly at each trial. All stimuli then moved along independent trajectories at approximately 10 degrees per second for 1500 ms, fully repositioning the stimuli to another set of locations, again of equal eccentricity of 5.0°. The final location of the stimulus after tracking was uncorrelated with its initial position (each of the 4 locations were equally likely to reposition to any of the final 4 locations). After a stopping motion, the stimuli changed back to the original spatial frequency (1 cycle/degree) to ensure that they were easily visible. The fixation point then disappeared, and the monkey made a saccade to the location corresponding to the single tracked target. Reward was delivered when the monkey acquired that target, without first foveating any of the nontargets. We evaluated the monkey’s ability to track stimuli (percentage of correct trials) as a function of the spatial frequency of the tracked Gabor stimuli. Because high spatial frequencies may be impossible to view with poor vision and because monkeys often resort to guessing strategies or simply give up when faced with difficult tasks, we were careful to staircase difficulty and did not include too many trial conditions with difficult high spatial-frequency stimuli. In each behavioral session, we began with a warm-up period, in which the difficulty was increased over successive rounds of testing. We always began by running 20 trials at each of the 2 easiest spatial frequencies (0.8 and 1.0 cycles/degree). If the macaque performed above the level due to chance (that is, more than 25% correct), we then interleaved the next 2 more difficult spatial frequencies (1.33 and 1.77) and ran until there were at least 20 trials for each of the added frequencies. We added the next 2 higher frequencies when the animal performed above chance level at either of the added frequencies. This procedure was repeated until the macaque failed to perform better than the level due to chance at either of the 2 added frequencies or until the full set of 8 frequencies was included. At this point, we fixed the set of frequencies for the session and ran 64 trials of each, with the selected frequency chosen randomly from trial to trial. The attention-demanding multiple-object tracking task required extensive training of the animal. In addition, the eccentricity of the stimuli varied from 3° to 8° during the trajectories, thus adding an additional uncontrolled variable. We therefore

developed a second simplified detection task in which a Gabor stimulus, of varying spatial frequency, appeared in 1 of 4 identical apertures at fixed eccentricity, and the macaque made a saccade to the aperture containing the stimulus (Figure 1 C). Briefly, the macaque began each trial by fixating a central point, at which time 4 identical apertures appeared. Each aperture was placed at an eccentricity of 5° and measured 3° in outer radius and 2.5° in inner radius. The monkey maintained fixation of the central point for 500 ms, and then a single Gabor stimulus was presented in one of the apertures with the aperture selected randomly at each trial. The Gabor stimulus and 8 spatial frequencies were identical to the previous attention-demanding multiple-object tracking task (Figure 1 B). After 500 ms, the fixation point and Gabor stimulus both disappeared, signaling the monkey to make a saccade to the location of the aperture that had contained the stimulus. Reward was delivered when the monkey acquired the correct aperture, without first fixating any of the other apertures. Initial training of the task used the 2 lowest spatial frequencies that were easily visible (0.8 and 1.0 cycles/degree) for 20 trials and then, as described earlier for the tracking task, pairs of the next 2 higher spatial frequencies were added for the following 20 trials as long as performance remained above the level due to chance. The primate exhibiting myopia (subject 3) was tested by using 6 different viewing conditions: binocular vision with and without corrective lenses, monocular vision of the left eye with and without corrective lenses, and monocular vision of the right eye with and without corrective lenses. Because each viewing condition required set-up of the viewing apparatus and calibration of the eyes with the infrared tracker, it was not possible to randomly interleave viewing conditions in testing. Instead, a single viewing condition was used for behavioral testing each day. Tests done with no corrective lenses, which typically had much poorer performance, were repeated at least twice to ensure the results were consistent from day to day.

Case Study

Abnormalities in the vision of one monkey (subject 3) were detected early in his research career, on the basis of behavioral performance and neuronal responses in visual tasks. First, he had participated in a neuronal recording study in which the visual response to stimuli inside neuron’s receptive fields was characterized as a function of stimulus contrast.47 By comparison with other animals in the study, this macaque had a contrast response curve that was shifted to the right with almost no response saturation at high contrast (that is, the stimuli appeared effectively dimmer). This study used relatively large square-wave grating stimuli (diameter, 2°; 2 cycles/degree) that were easily detected in the periphery. Later, in a second study, it was noticed that the animal performed worse in tasks using smaller Gabor stimuli, especially when they were of increased spatial frequency, and he had trouble maintaining fixation. In the home cage, this animal was docile, subordinated to conspecifics within the holding room, and was fairly indifferent to his environment. Notably, he was reported as moving particularly slowly and carefully within the cage or large enrichment units, staring for prolonged periods, and being less confident than other monkeys within the colony. His fine-motor skills, especially while eating and foraging, were normal. In light of these behavioral observations, we performed an ophthalmologic examination on this animal (subject 3). We found a significant myopia (OD −4.00/+1.00 × 100, OS

302

cm13000166.indd 302

9/8/2014 3:53:23 PM

Refractive errors in macaques

Figure 1. Behavioral tests to assess macaque visual acuity quickly. (A) Diagonally oriented Gabor stimuli of varying spatial frequency were used as stimuli. (B) An attention-demanding task was used to test visual acuity initially. In brief, 4 easily visible (1 cycle per degree) Gabor stimuli are displayed as the subject maintains fixation on a central point. One stimulus is cued with a high-contrast flash for 500 ms, and then all stimuli change to higher spatial frequencies. Stimuli move along random spatial trajectories (1500 ms) randomly shuffling the final positions. Then stimuli change back to an easily visible spatial frequency, fixation disappears, and the subject indicates which target was cued originally by making a saccade to its location for reward. (C) A simplified task tests visual acuity. Four apertures appear as the subject is fixating a central point (500 ms). A Gabor stimulus of variable spatial frequency is displayed in 1 of the 4 apertures for 500 ms as fixation is maintained. Then the stimulus and the fixation point disappear, cueing the macaque to saccade to the location of the stimulus.

−5.00/+0.50 × 90) that warranted optical correction to achieve adequate visual acuity at the working distance of 57 cm (a myopia of −1.75D or more needs an optical correction at this working distance). Single-vision corrective lenses (Essilor Thin and Lite 1.6, Essilor USA, Dallas, TX) were prescribed by the ophthalmologist (OD: −4.00/+1.00; OS: −5.00/+ 0.50), cut, and mounted on a stainless steel frame in-house (Figure 2). A few parameters were essential in the design of the binocular lenses. First, the lenses were rotated to adjust for the correct axis of astigmatism. Second, the interpupillary distance was measured, and the optical center of the lenses was positioned correctly in relation to the center of the pupils. Third, the lenses were carefully cut to clear the nose bridge and prominent head crown as the vertex distance (back surface of a corrective lens to the front corneal surface) was adjusted to approximately 12 to 14 mm. Finally, the frame was fixed to an adjustable arm mounted on the working chair to allow safe manipulation and positioning of the lenses before each recording session. In addition, the lenses and working chairs were positioned in a way to avoid any diffraction or reflection of the infrared tracking system on the lenses; this effect might have interfered with eye movement monitoring or might have distracted the macaque during behavioral tasks. Furthermore, the lenses were adjusted so that the infrared camera had an unobstructed view of the eye but did not occlude the view of the monitor. Because presbyopia was not expected to occur in a 14-y-old macaque, it was

not considered. The macaque adapted well to the glasses and showed immediate behavioral improvement and performance in the tracking task. Ocular examination and refraction. After discovering this case of myopia in our colony, we decided to screen the 20 other macaques for refractive errors and ocular pathologies. The refractive errors (sphere, positive cylinder, and spherical equivalent) as well as the anterior and posterior segment examination of each eye are presented in Table 1. The intraocular examination was normal in all subjects. There were no signs of cataracts, glaucoma, or retinal abnormalities in all examined primates. The spherical equivalent (value of the spherical lens plus half the value of the cylinder added algebraically) was calculated for each eye. Across macaques, the mean refractive error was +0.56 ± 1.10D for the right eye and +0.45 ± 1.27D for the left eye. A high correlation coefficient (R2 = 0.87, P < 0.001) was found between the spherical equivalents of the 2 eyes of the same monkey. There was no correlation between the age of the monkey and the refractive errors. Hyperopia (defined as a spherical equivalent refraction of ≥+0.50D) was the most prevalent refractive error, affecting 30 (71.4%) eyes, but it never exceeded +1.50D. Myopia (as defined as a spherical equivalent refraction worse or equal to −0.50D) was found in 3 (7.1%) eyes, with 2 eyes exceeding −3.00 D in the same monkey. Astigmatism of + 0.50D or more was found in 36 (85.7%) eyes. The mean astigmatism was +0.71 ± 0.41D in the right eye and +0.75 ± 0.47D in the left eye. Astigmatism exceeding +1.00D

303

cm13000166.indd 303

9/8/2014 3:53:24 PM

Vol 64, No 4 Comparative Medicine August 2014

Figure 2. Myopic vision in a macaque corrected by using refractive lenses. (A) Forward and (B) side views of the corrective lenses. (C) Schematic of the macaque viewing the monitor and infrared eye tracker. (D) Forward view of the macaque with myopic vision wearing the corrective lenses.

was found in 3 (14.3%) subjects with a maximal value of +2.00D. A second monkey (subject 6) was diagnosed with mild myopia and astigmatism that did not necessitate visual correction for our working distance. Evaluation of visual acuity and refractive correction in primates using behavioral tasks. Using binocular vision, we tested the myopic animal (subject 3, ‘JJ’) and a control monkey (subject 2, ‘Max’) of a similar age and normal refraction (OD: −0.25/+0.75 × 90; OS: plano/+1.00 × 90) on the attention-demanding task. Both were trained and proficient in the same tracking tasks. As illustrated in Figure 3 A, the control animal was able to perform with nearly perfect performance (more than 95% correct on average), with only a modest decrease in performance at the highest spatial frequency. By comparison, the myopic animal without corrective lenses showed a dramatic drop in performance at intermediate spatial frequencies, between 2.67 and 4.00 cycles per degree. The performance improved to above 80% even at the highest spatial frequency when vision was corrected with lenses. Although the performance with visual correction remained lower on average than that of the control animal, there was no drop with increasing spatial frequency, suggesting that the lower overall performance did not reflect lower visual acuity but rather less expertise or diligence in the task.

We then compared visual performance in the myopic animal between monocular and binocular condition with and without refractive correction on the attention-demanding task (Figure 3 B). In monocular vision without refractive correction, the right and the left eyes showed a drop in visual performance at the same spatial frequency, thus suggesting that both eyes were impaired equally. This finding is consistent with the cycloplegic refraction found for the 2 eyes in this animal (Table 1). Prior experiments have found improvements in spatial frequency sensitivity with binocular viewing, relative to monocular viewing.6 Consistent with this observation, the drop-off in performance without corrective lenses in monocular vision occurred at a lower spatial frequency (between 1.33 to 1.77 cycles/degree) as compared with binocular vision (between 2.67 to 4.00 cycles/degree). In addition, failure to perform the task was much more dramatic in the monocular than binocular condition at the very highest spatial frequency. With corrective lenses, the macaque still failed at the highest spatial frequency with monocular viewing but was able to perform well under binocular viewing, further suggesting that binocular viewing provides a significant advantage when both eyes have similar acuity due to binocular summation. We then evaluated binocular visual performance and acuity by using the simplified detection task in both the control and myopic

304

cm13000166.indd 304

9/8/2014 3:53:26 PM

Refractive errors in macaques

Figure 3. Behavioral performance under different viewing conditions. (A) Performance in the attention-demanding task is shown as a function of stimulus spatial frequency under binocular viewing for a healthy control (shown in black, ‘Max’) and for the macaque with uncorrected myopic vision (shown in purple, ‘JJ’, dashed lines) and after correction with refractive lenses (purple, solid lines). (B) Performance in the attention-demanding task for the macaque with myopic vision broken out for viewing with the left eye (in red) or the right eye (in blue). Again, dashed lines indicate uncorrected vision, and solid lines corrected vision. (C) Performance in the simplified task under binocular viewing for a healthy control (in black, at ceiling) and the macaque with myopic vision without corrected vision (purple, dashed lines; squares, first session; diamond, second session; triangles, third session) and with corrected vision (purple, solid line).

animals (Figure 3 C). In these macaque, which were already proficient at fixating, this new task was learned in only a single day of training. The monkey with normal vision was able to perform this task perfectly at all 8 spatial frequencies tested. The myopic monkey similarly performed this task with very high accuracy at all spatial frequencies (>90%) when wearing corrective lenses. The test then was repeated in the myopic animal on 3 separate days without refractive correction. The visual performance showed a drop in performance at the same spatial frequencies as did the attention-demanding multiple-object tracking task, that is, between 2.67 and 4.00 cycles/degree. However, the drop in performance was steeper and fell to chance levels for the tracking task but not the simplified task. This result may reflect the added difficulty of the tracking task as well as an increased chance of losing targets as they varied in eccentricity during the tracking.

Discussion

The first goal of this study was to evaluate ocular health and cycloplegic refraction in rhesus macaques enrolled in visual neu-

roscience research and to identify pathologic conditions or refractive errors that could interfere with research. Naturally occurring ocular disorders in macaques are reported only rarely in the literature and, in general, are caused by infection or trauma.43 In one report,43 only 0.8% of all macaque clinical cases were ocular, with 32% secondary to wounding. Blepharitis and conjunctivitis accounted for 29% and 21% of ocular cases, respectively, whereas corneal ulcers were diagnosed in 3% of cases. Infectious conjunctivitis in macaques can be traumatic in nature but can also be caused by viruses such as measles and Macacine herpesvirus 13,12 or can be a presenting sign of a systemic disease, such as cryptosporidiosis in SIV-infected macaques.4 Allergic conjunctivitis from latex as well as keratitis secondary to entropion has been reported in macaques.27,37 Streptococcus pneumonia is a primary cause of bacterial meningitis in nonhuman primates, including macaques, and has been shown to cause conjunctivitis and panophthalmitis.17 Macaques have been used extensively as models of human infectious diseases causing ocular infections or lesions, such as onchocerciasis, chlamydiosis, measles, and tuberculosis.7,12,16 In

305

cm13000166.indd 305

9/8/2014 3:53:26 PM

Vol 64, No 4 Comparative Medicine August 2014

addition, rhesus macaques naturally or experimentally infected with SIV may develop an AIDS-like disease, with choroidal lesions, retinal hemorrhages, retinitis, optic neuritis, and panophthalmitis.9,20 Ocular development and morphology are very similar between humans and nonhuman primates as is susceptibility to age-related diseases; consequently humans and nonhuman primates are expected to develop comparable ocular lesions.51 Several studies have documented age-related cataracts, drusen, macular degeneration, atrophy of the retinal pigment epithelium, and ocular lesions associated with diabetes in macaques.13,14,21,22,46 Glaucoma has been documented in a large, closed rhesus macaque colony with a restricted gene pool.11 Cataracts can be relatively common in captive macaques and begin developing in 20% of rhesus monkeys at 20 to 22 y old; rates increase significantly after 26 y of age.22,51 In addition, fundus lesions are relatively common and can affect up to 6.6% of captive and wild-caught cynomolgus macaques.24,48,53 Lesions include chorioretinal scarring, vasculitis, retinal hemorrhages, and ‘cotton-wool spots’ indicative of retinal inflammation and edema.53 Amyloid plaques and cerebral β-amyloid angiopathy appears in the brain of macaques older than 20 y with no obvious changes in cognitive behavior or ocular function or evidence of cerebral atrophy,51 whereas young Japanese macaques can develop an encephalomyelitis reminiscent of multiple sclerosis that can cause ocular motor paresis.1 Cynomolgus and rhesus macaques develop age-related macular degeneration similar to that in humans53 but with little influence on vision.53 Other colleagues reported 9 cases of idiopathic bilateral optic atrophy in rhesus macaques of Chinese descent and diverse ages and recommended ocular screening before involving macaques into visual research.15 In addition, macaques develop strabismus and can be useful models of this condition in humans.34 Intraocular examination in our study was within normal limits in all of our macaques. These findings might be explained by the relatively young age and general good health of our research colony. Nonetheless, spontaneous ocular diseases in captive research colonies likely are under-reported due to a lack of systematic screening by experienced ophthalmologists, the relatively young age of colonies used for biomedical research and toxicology studies, and the short experimental life of these animal models. In terms of refraction, the postnatal eye growth in macaques from birth to 5 y is very similar to that in humans, including axial eye elongation, flattening of the cornea, and reduction of the hyperopia present at birth.5 However, the emmetropization in adolescent macaques is hyperopic with an average refractive value of +2.00D, whereas that of adolescent humans is in the range of plano, that is, no refractive error (0.00D).14 This species-specific hyperopia does not decline with age but both humans and macaques display increased dispersion of refractive values with age, including rare cases of extreme myopia or hyperopia.14,39 Our mean refractive error was similar to but slightly less hyperopic as compared with previous data. This difference can be explained by the use of 1% tropicamide instead of 1% cyclopentolate hydrochloride (tropicamide is a weaker cycloplegic agent than cyclopentolate). The mean astigmatism values were also comparable to those in other studies. Although we found no cases of extreme myopia or hyperopia in our colony, at least one macaque showed significant myopia (OD −4.00/+1.00 × 100, OS −5.00/+0.50 × 90) that warranted corrective lenses. Corrective lenses immediately improved this animal’s visual acuity and performance on be-

havioral tasks. Another macaque (subject 6) showed mild myopia and borderline astigmatism that might also require optical correction when visual testing is performed. As with humans, macaques show a decline in visual accommodative amplitude (presbyopia) with age, but these changes typically occur in macaques older than 25 y.23 As such, presbyopia was not expected in our younger colony and was not taken into account. Overall, although the prevalence of refractive errors was low in our macaque colony, the potential for interference with behavioral and visual tasks warrants screening of visual acuity before and throughout the research career of these animal models to allow scientifically sound research. Cycloplegic retinoscopy remains the ‘gold standard’ for measuring refraction in both the clinical and research settings. 10 Autorefraction without cycloplegia by using a handheld or tablemounted autorefractor has become popular in human medicine as an easy method to screen vision in adults. Autorefraction must always be done with cycloplegia in children.40 Although noncycloplegic autorefraction provides reasonably accurate and repeatable measures in adults, this method tends to be more variable and underestimates the degree of hyperopia and overestimates astigmatism as well as myopic refraction due to accommodative efforts.10,26 Whereas cycloplegia may attenuate those differences, autorefraction—especially with handheld devices—generally is viewed as a screening tool and does not provide sufficient accuracy to allow spectacle prescription.26,40 In addition, these devices require a considerable cost investment and don’t eliminate the need for a comprehensive visual assessment by a competent veterinary or human ophthalmologist to assess visual health. Our second goal was to develop a simple behavioral task that would allow laboratories involved in visual science to easily and quickly assess visual acuity in experimental macaques. Visual acuity—the ability to see details of an object separately and in focus—depends on the optical properties of the eyes, the processing of the visual stimuli by the retina, and the transfer to and interpretation of the stimuli by the CNS.31 As such, the entire pathway or different segments can be evaluated as a whole or separately through different tests. Visual acuity traditionally has been assessed in humans and nonhuman primates by using either psychologic or behavioral methods (requiring a response from the subject) or objective methods, such as visually evoked potentials.25 The most common behavioral methods have been a combination of preferential looking and operant techniques.49 The Force Choice Preferential Looking test is based on the observation that infants and monkeys often stare fixedly at bold, high-contrast patterns and track the motion of the patterns by exhibiting compensatory eye and head movements to keep the pattern in the central vision.49 This test measures resolution acuity by using either grating targets (for example, Teller cards) or the vanishing optotype principle (Cardiff Acuity Cards). The Operant Preferential Looking test is a modification of the Force Choice Preferential Looking test in which a reward is given when the observer is able to determine the position of the target.49 Several similar operant behavioral tasks have been used to evaluate visual acuity in domestic animals including cats, dogs, horses, gerbils, and rodents.41 For example, a computer-based, 2-alternative, forced-choice visual discrimination task was developed to assess behaviorally visual function, including visual acuity, in mice and rats.41 The task relies on the ability of trained rodents to escape water by finding a submerged platform in the direction of

306

cm13000166.indd 306

9/8/2014 3:53:26 PM

Refractive errors in macaques

a monitor displaying gratings at a fixed distance. Visually evoked potentials are extracted from EEG signals initiated from brief visual stimuli and recorded from electrodes overlying the visual cortex. Visually evoked potentials are used primarily to measure the functional integrity of the visual pathways from the retina to the visual cortex.49 In most domestic mammals, visual acuity is limited more by the retina than the eyes or the processing of the stimuli by the brain.31 As such, in these species, visual acuity has been estimated by determining retinal topography and ganglion cell density on whole-retina flat mounts. Such assays have been done in several terrestrial and aquatic mammals and correlate well with visual acuity estimates obtained from behavioral and objective methods but require euthanasia of animals.28,38,52 Pattern electroretinography (PERG) is another electrophysiological test used in both humans and other mammals to assess central retinal function. In this method, the retina is stimulated with isoluminant-reversing gratings and electrodes placed on the cornea or the nearby conjunctiva record retinal cell activity. Pattern electroretinography is used as a sensitive indicator of dysfunction within the macular region and reflects the integrity of the optics, photoreceptors, bipolar cells and retinal ganglion cells.2 Clinically, pattern electroretinography is used in patients with abnormal patterns of visually evoked potentials to establish whether a central retinal disorder is present and thereby differentiate between retinal and optic nerve dysfunction as a cause for the abnormality. In addition, pattern electroretinography can be used to detect and monitor dysfunction of retinal ganglion cells due to conditions such as glaucoma, optic neuropathies, and primary ganglion cell diseases.2 The current study used 2 novel behavioral tasks that allow longitudinal assessment of visual acuity over time. The first task involved a cognitively demanding tracking task and varied the spatial frequency of tracked items to determine at what spatial frequencies the target items were lost during tracking. This task was not designed originally for testing visual acuity but was rather part of ongoing studies examining the effects of spatial attention on neuronal responses. Although effective, the task has several disadvantages as a measure of acuity, including the macaques’ difficulty in learning it and the variation in eccentricity during the tracking of stimuli. Consequently, we developed a second, simple task that required only the detection of a grating in 1 of 4 apertures. The simplified task exploited the natural salience of macaques to move their eyes to ‘pop-out targets’ and targets of higher salience.44 Similar to previous studies in human and nonhuman primates,49 this test takes advantage of the preference in primate to look toward complex stimuli against dull or uniform backgrounds. Both the normal and myopic animals readily learned the task within a single behavioral session. This simple task could be used for rapid evaluation of visual acuity in novel animals that are already implanted for head restraints with commonly used equipment for visual studies. The myopic macaque showed a drop in performance at comparable spatial frequencies using the simple task, but performance did not drop all the way to the level due to chance (25%), indicating that some residual detection was possible. Still the drop in performance was diagnostic of the visual deficit. By comparison, the macaque with normal vision and the myopic subject with corrected vision showed no signs of a drop in performance even at the highest spatial frequency tested (8 cycles/ degree) in the simplified task. The highest spatial frequency tested here (8 cycles/degree) was well below the normal acuity thresh-

old for 75% detection performance in normal macaques at this eccentricity (5°), which ranges from 15 to 25 cycles/degree.29 We would not have expected any drop in performance over the range tested unless normal vision was impaired. By using this simplified task, it may be feasible for other laboratories to quickly screen macaques for impaired visual acuity. Other methods for more extensive measurements of visual acuity have been used in previous studies. The acuity function in macaques has been estimated over a set of different eccentricities from fixation, with macaques trained to fixate a central point and indicate the left or right location of a Gabor stimulus.30 In the cited studies, the spatial frequency of the stimulus was increased in small steps by using a staircase procedure until a threshold of 75% performance was reached (chance level, 50%). The threshold acuity was plotted as a function of the retinal eccentricity of the stimulus with the procedure repeated at several difference eccentricities. At an eccentricity of 5 degrees, macaques can detect gratings in the range of 15 to 25 cycles/degree. One drawback of the procedure is that it required some training of the animal to ensure that the macaque performed the task with a high degree of vigilance, given that the threshold estimate will vary considerably if macaques vary in their effort (that is, if a macaque were content with liquid reward in 50% of trials, no estimate would be possible). Our simplified procedure tested a fixed set of spatial frequencies that should be easily visible at the given eccentricity and evaluated whether there was a drop in performance at higher spatial frequencies, which result is indicative of visual acuity impairment. Weak vigilance in the task would have led to an overall drop in performance at all frequencies, a scenario that could easily be differentiated from poor visual acuity at higher spatial frequencies only. Therefore, compared with other techniques, our method may require less training to be effective in macaques, even when vigilance in the task is weak initially. In conclusion, the prevalence of refractive errors appears to be low in macaque colonies. However, the potential for interference with behavioral and visual tasks warrants screening of visual acuity before and throughout the research career of these animal models to ensure scientifically sound research. Our simplified task could be used to detect potential visual deficits that may warrant further evaluation and treatment.

Acknowledgments

This research was supported by grants from the NIH R01 EY013802 and the Gatsby Charitable Foundation (to JHR). We thank Katie Williams for surgical assistance and animal care.

References

1. Axthelm MK, Bourdette DN, Marracci GH, Su W, Mullaney ET, Manoharan M, Kohama SG, Pollaro J, Witkowski E, Wang P, Rooney WD, Sherman LS, Wong SW. 2011. Japanese macaque encephalomyelitis: a spontaneous multiple sclerosis-like disease in a nonhuman primate. Ann Neurol 70:362–373. 2. Bach M, Brigell MG, Hawlina M, Holder GE, Johnson MA, McCulloch DL, Meigen T, Viswanathan S. 2013. ISCEV standards for clinical pattern electroretinography (PERG): 2012 update. Doc Ophthalmol 126:1–7. 3. Bailey CC, Miller AD. 2012. Ulcerative cheilitis in a rhesus macaque. Vet Pathol 49:412–415. 4. Baskin GB. 1996. Cryptosporidiosis of the conjunctiva in SIV-infected rhesus monkeys. J Parasitol 82:630–632.

307

cm13000166.indd 307

9/8/2014 3:53:26 PM

Vol 64, No 4 Comparative Medicine August 2014

5. Bradley DV, Fernandes A, Lynn M, Tigges M, Boothe RG. 1999. Emmetropization in the rhesus monkey (Macaca mulatta): birth to young adulthood. Invest Ophthalmol Vis Sci 40:214–229. 6. Cagenello R, Arditi A, Halpern DL. 1993. Binocular enhancement of visual acuity. J Opt Soc Am A Opt Image Sci Vis 10:1841–1848. 7. Capuano SV, Croix DA, Pawar S, Zinovik A, Myers A, Lin P, Bissel S, Fuhrman C, Klein E, Flynn J. 2003. Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestation of human M. tuberculosis infection. Infect Immun 71:5831–5844. 8. Cavanagh P, Alvarez GA. 2005. Tracking multiple targets with multifocal attention. Trends Cogn Sci 9:349–354. 9. Conway MD, Didier P, Fairburn B, Soike KF, Martin L, MurpheyCorb M, Meiners N, Insler MS. 1990. Ocular manifestation of simian immunodeficiency syndrome (SAIDS). Curr Eye Res 9:759–770. 10. Choong YF, Chen AH, Goh PP. 2006. A comparison of autorefraction and subjective refraction without and without cycloplegia in primary school children. Am J Ophthalmol 142:68–74. 11. Dawson WW, Brooks DE, Dawson JC, Sherwood MB, Kessler MJ, Garcia A. 1998. Sings of glaucoma in rhesus monkeys from a restricted gene pool. J Glaucoma 7:343–348. 12. El Mubarak HS, Yuksel S, Van Amerongen G, Mulder PGH, Mukhtar MM, Osterhaus ADME, de Swart RL. 2007. Infection of cynomolgus macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta) with different wild-type measles viruses. J Gen Virol 88:2028–2034. 13. Engel HM, Dawson WW, Ulshafer RJ, Hines MW, Kessler MJ. 1988. Degenerative changes in maculas of rhesus monkeys. Ophthalmologica 196:143–150. 14. Fernandes A, Bradley DV, Tigges M, Tigges J, Herndon JG. 2003. Ocular measurements throughout the adult lifespan of rhesus monkeys. Invest Ophthalmol Vis Sci 44:2373–2380. 15. Fortune B, Wang L, Bui BV, Burgoyne CF, Cioffi GA. 2005. Idiopathic bilateral optic atrophy in the rhesus macaque. Invest Ophthalmol Vis Sci 46:3943–3956. 16. Gardner MB, Luciw PA. 2008. Macaque models of human infectious disease. ILAR J 49:220–255. 17. Gibson SV. 1998. Bacterial and mycotic diseases, p 59–102. In: Bennett BT, Christian RA, Henrickson R, editors. Nonhuman primates in biomedical research: diseases. San Diego (CA): Academic Press. 18. Greene PR. 1990. Optical constants and dimensions for the myopic, hyperopic, and normal rhesus eye. Exp Eye Res 51:351–359. 19. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 20. Johnson JK, Warren KA, Berman NE, Narayan O, Stephens EB, Joag SV, Raghavan R, Mracario JK, Cheney PD. 2004. Manifestation of SIVinduced ocular pathology in macaque monkeys. J NeuroAIDS 2:1–13. 21. Johnson MA, Lutty GA, McLeod DS, Otsuji T, Flower RW, Sandagar G, Alexander T, Steidi SM, Hansen BC. 2005. Ocular structure and function in an aged monkey with spontaneous diabetes mellitus. Exp Eye Res 80:37–42. 22. Kaufman PL, Bito LZ. 1982. The occurrence of senile cataracts, ocular hypertension, and glaucoma in rhesus monkeys. Exp Eye Res 34:287–291. 23. Kaufman PL, Bito LZ, DeRousseau CJ. 1982. The development of presbyopia in primates. Trans Ophthalmol Soc U K 102:323–326. 24. Kuhlman SM, Rubin LF, Ridgeway RL. 1992. Prevalence of ophthalmic lesions in wild-caught cynomolgus monkeys. Prog Vet Comp Ophthalmol 2:20–28. 25. Leat SJ, Yadav NK, Irving EL. 2009. Development of visual acuity and contrast sensitivity in children. J Opt 2:19–26. 26. Liang CL, Hung KS, Park N, Chan P, Juo SH. 2003. Comparison of measurements of refractive errors between hand-held Retinomax and on-table autorefractors in cyclopleged and noncyclopleged children. Am J Ophthalmol 136:1120–1128. 27. Macy JD, Huether MJ, Beattie TA, Findlay HA, Zeiss C. 2001. Latex sensitivity in a macaque (Macaca mulatta). Comp Med 51:467–472. 28. Mass AM, Supin AY. 1995. Ganglion cell topography of the retina of the bottlenosed dolphin, Tursiops truncates. Brain Behav Evol 45:257–265.

29. Merigan WH, Katz LM, Maunsell JH. 1991. The effects of parvocellular lateral geniculate lesions on the acuity and contrast sensitivity of macaque monkeys. J Neurosci 11:994–1001. 30. Merigan WH, Nealey TA, Maunsell JH. 1993. Visual effects of lesions of cortical area V2 in macaques. J Neurosci 13:3180–3191. 31. Miller PE. 2008. Structure and function of the eye. In: Maggs DJ, Miller PE, editors. Slatter’s fundamentals of veterinary ophthalmology, 4th ed. New York (NY): Elsevier. 32. Mitchell JF, Sundberg KA, Reynolds JH. 2007. Differential attentiondependent response modulation across cell classes in macaque visual area V4. Neuron 55:131–141. 33. Modi KK, Chakravarti RN. 1975. Spontaneously occurring hypertension in wild rhesus monkeys. Klin Wochenschr 53:363–367. 34. Narasimhan A, Tychsen L, Poukens V, Demer JL. 2007. Horizontal rectus muscle anatomy in naturally and artificially strabismic monkeys. Invest Ophthalmol Vis Sci 48:2576–2588. 35. Newsome WT, Stein-Aviles JA. 1999. Nonhuman primate models of visually based cognition. ILAR J 39:78–91. 36. Qiao-Grider Y, Hung LF, Kee CS, Ramamirtham R, Smith EL 3rd. 2007. A comparison of refractive development between 2 subspecies of infant rhesus monkeys (Macaca mulatta). Vision Res 47:1668–1681. 37. Peiffer RL, Johnson PT, Wilkerson BJ. 1980. Peripalpebral folds and entropion in a male crab-eating macaque (Macaca fascicularis). Lab Anim Sci 30:113–115. 38. Pettigrew JD, Dreher B, Hopkins CS, McCall MJ, Brown M. 1988. Peak density and distribution of ganglion cells in the retinae of microchiropteran bats: implications for visual acuity. Brain Behav Evol 32:39–56. 39. Pleasker R, Hetzel U, Schmidt W. 2005. Two cases of spontaneous high myopia in a colony of rhesus macaques (Macaca mulatta). Verh. Ber. Erkrg Zootiere. 42:148–152. 40. Prabakaran S, Dirani M, Chia A, Gazzard G, Fan Q, Leo SW, Ling Y, Eong KG, Wong TY, Saw SM. 2009. Cycloplegic refraction in preschool children: comparisons between the handheld autorefractor, table-mounted autorefractor and retinoscopy. Ophthalmic Physiol Opt 29:422–426. 41. Prusky GT, West PWR, Douglas RM. 2000. Behavioral assessment of visual acuity in mice and rats. Vision Res 40:2201–2209. 42. Pylyshyn ZW, Storm RW. 1988. Tracking of multiple independent targets: evidence for a parallel tracking mechanism. Spat Vis 3:179–197. 43. Ribka EP, Dubielzig RR. 2008. Multiple ophthalmic abnormalities in an infant rhesus macaque (Macaca mulatta). J Med Primatol 37 Suppl 1:16–19. 44. Schiller PH, Lee K. 1991. The role of the primate extrastriate area V4 in vision. Science 251:1251–1253. 45. Sears CR, Pylyshyn ZW. 2000. Multiple object tracking and attentional processing. Can J Exp Psychol 54:1–14. 46. Stafford TJ, Anness SH, Fine BS. 1984. Spontaneous degenerative maculopathy in the monkey. Opthalmology 91:513–521. 47. Sundberg KA, Mitchell JF, Reynolds JH. 2009. Spatial attention modulates center–surround interactions in macaque visual area v4. Neuron 61:952–963. 48. Suzuki MT, Cho F. 1986. Normal and abnormal findings in ocular fundi of cynomolgus monkeys. J Toxicol Sci 11:452–457. 49. Teller DY. 1983. Measurement of visual acuity in human and monkey infants: the interface between laboratory and clinic. Behav Brain Res 10:15–23. 50. Tychsen L, Richards M, Wong A, Foeller P, Burhkalter A, Narasimhan A, Demer J. 2008. Spectrum of infantile esotropia in primates: behavior, brains, and orbits. J AAPOS 12:375–380. 51. Uno H. 1997. Age-related pathology and biosenescent markers in captive rhesus macaques. Age (Omaha) 20:1–13. 52. Wassle H, Grunert U, Rohrenbeck J, Boycott BB. 1990. Retinal ganglion cell density and cortical magnification factor in the primate. Vision Res 30:1897–1911. 53. Williams DL. 2012. The eye of other mammalian exotic pet species, p 109–118. In: Williams DL, editor. Ophthalmology of exotic pets. Oxford (UK): Blackwell Publishing. . 54. Zeiss CJ. 2010. Animals as models of age-related macular degeneration. An imperfect measure of the truth. Vet Pathol 47:396–413.

308

cm13000166.indd 308

9/8/2014 3:53:27 PM

Correction of refractive errors in rhesus macaques (Macaca mulatta) involved in visual research.

Macaques are the most common animal model for studies in vision research, and due to their high value as research subjects, often continue to particip...
4MB Sizes 1 Downloads 11 Views