Refractive and Structural Measures of Lid-Suture Myopia in Tree Shrew

Transcription

1 Investigative Ophthalmology & Visual Science, Vol. 30, No. 10, October 1989 Copyright Association for Research in Vision and Ophthalmology Refractive and Structural Measures of Lid-Suture Myopia in Tree Shrew Wendy L. Marsh-Toorle* ond Thomos T. Nortonf In order to study more thoroughly the refractive and structural changes associated with lid-suture myopia, five tree shrews were raised for approximately 16 weeks with monocular visual experience produced by lid closure. Four animals raised with normal laboratory visual experience served as a control group. Compared to the paired open eye, lid-sutured eyes were myopic ( 12.1 ± 6.3 diopters by retinoscopy), corneas were flatter (0.26 ± 0.18 mm radius increase by photokeratometry) and axial lengths were greater (0.32 ± 0.17 mm longer by A-scan ultrasonography). The axial length increase was due to elongation of the vitreous chamber (0.38 ± 0.19 mm longer by A-scan ultrasonography). The open eyes of experimental animals were not significantly different than the normal eyes of control animals. Two of these treatment effects, namely, refractive state changes and axial length increases, were demonstrated with independent techniques: streak retinoscopy was compared to coincidence optometry, and A-scan ultrasound was compared to axial measurements of photographs of frozen, sectioned eyes. The three main ocular effects of eyelid closure were stable over three measurement sessions completed within a 4 week period. Additional refractive and A-scan measurements taken 7.5 months later showed no significant changes. Optical modelling showed that the observed myopia of the lid-sutured eye is consistent with the observed elongation of the vitreous chamber coupled with the flattened cornea although other changes could not be ruled out. We conclude that an axial myopia is produced reliably in tree shrews by raising them with eyelid closure and that the measurement techniques used in this study have sufficient resolution to study the development of myopia in this species. Invest Ophthalmol Vis Sci 30: , 1989 The possibility that visual experience may be important in the development of both emmetropia and ametropia has long been a topic of interest. Recently, it has been discovered that tree shrews, 1 monkeys, 2 " 4 chickens 5 and possibly cats 6 " 10 raised with one or both eyelids closed during the early postnatal period can develop myopia in the deprived eye. Sherman et al 1 demonstrated that monocular and binocular lid closure produce myopia in the deprived eyes of the tree shrew, but did not describe in detail the refractive and structural characteristics of the open and the lid-sutured eyes. The purpose of the From the Departments of *Optometry and tphysiological Optics, School of Optometry/The Medical Center, The University of Alabama at Birmingham, Birmingham, AL Presented in part at the annual Spring meeting of the Association for Research in Vision and Ophthalmology, May 1983, Sarasota, Florida. Supported in part by U.S. Public Health Service Grant RO1 EY-05922, RO3 EY-04507, EY (CORE) and BRSG grant RR05807 awarded by the National Institutes of Health, Bethesda, Maryland. Submitted for publication: July 11, 1988; accepted January 19, Reprint requests: Dr. Wendy L. Marsh-Tootle, School of Optometry, University of Alabama at Birmingham, Birmingham, AL present study was to provide more detailed information on the refractive state, corneal curve, and axial dimensions of normal eyes and the open and lid-sutured eyes of experimental animals in order to characterize the experimentally-induced myopia in this species. Tree shrews (Tupaia belangeri, see Fig. 1) are small ( g adult weight), scansorial squirrel-like mammals thought to be closely related to the primate line." Their visual system has been the subject of many anatomical, electrophysiological and behavioral investigations. 12 In addition, the development of their visual system has been intensively investigated. 13 In comparison with other animal models of myopia, tree shrews offer a number of advantages. Like chickens, they breed readily and grow to maturity in about 4 months. Thus it is possible to use sufficient numbers of them for meaningful statistical comparisons. Like monkeys, their similarity to humans makes it more likely that mechanisms found to produce emmetropia and ametropia in tree shrew will be applicable to humans. A concern in this study was whether our measurement techniques are sensitive enough to provide reliable and valid measurements of changes in tree shrew eyes, which are normally about 8 mm in axial length. 2245

2 2246 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1989 Fig. 1. Photograph of an adult tree shrew showing the laterally placed eyes and binocular visual field of approximately 55 Vol. 30 trimmed and then gently apposed using three mattress sutures of 6-0 ophthalmic silk. A small nasal opening was left for drainage. The eyelids healed with minimal scarring in all eyes, leaving a translucent covering that allowed only diffuse illumination to reach the retina. The control (left) eyes in the experimental group opened naturally at around day 21. Thus, the period of monocular visual exposure began at about 3 weeks of age. The animals received approximately 16 weeks of monocular visual experience before refractive and structural measurements began at 19 weeks postnatal. By this age, the animals appeared to be fully mature. Head restraint: Two weeks prior to the first measurement session, the experimental and control animals underwent minor surgery. Under anesthesia (25 mg ketamine HC1 supplemented by Halothane) an acrylic "cap" containing a small machine nut was attached to the skull with four to six stainless steel screws to allow rigid positioning of the head during refractive and ultrasound measurements. Measurement Procedures In such small eyes, minor changes in curvature or separation of the refractive and imaging surfaces could produce major changes in dioptric measure. Thus, although dioptric changes could be detected in the tree shrew eyes, the corresponding structural changes might be too small to be reliably detected and characterized. Materials and Methods Subjects The experimental group consisted of five tree shrews (two male, three female) randomly selected from pups born in our breeding colony. The control group consisted of four age-matched males. The animals were hand-reared following procedures developed in Dr. Irving Diamond's laboratory at Duke University.14 Following weaning at approximately 6 weeks of age, pups were raised in the animal colony on an 18 hr light-on/6 hr light-off schedule. Care and use of the animals adhered to the ARVO Resolution on the Use of Animals in Research. Preparation Eyelid closure: The eyelids of the right eye of the experimental animals were sutured at approximately 19 days after birth, just prior to the time of natural eye opening. The animals were anesthetized with Halothane and the lids of the right eye were opened between the tarsal plates. The lid margins were At approximately 19 weeks postnatal, we began a series of measurement sessions spaced approximately 2 weeks apart. The experimental and control animals were examined on three occasions. Additional refractive and ultrasound measures were obtained in experimental animals during a fourth measurement session, occurring approximately 7.5 months after the third session. The repeated measurement sessions were intended to assess the reliability of our measures in normal animals and to learn whether there were any changes in the eyes of the experimental animals after the opening of the eyelids. The eyelids of the deprived eyes were carefully opened at the first measurement session and were not resutured. There was drooping of the lid in some animals during the subsequent weeks of measurements, but in no case did we have to reestablish the palpebral aperture. The deprived eyes will be referred to as "lid-sutured" to denote the condition that obtained during the postnatal period of development preceding measurement sessions. In each measurement session, corneal curves, refractive measures (coincidence optometry and streak retinoscopy) and A-scan ultrasonographic measures were obtained. Anesthesia (25 mg ketamine HC1 and 3.75 mg promazine hydrochloride administered subcutaneously and supplemented with Halothane) was necessary because awake animals would not remain quiescent during the measurement procedures. Corneal curves: Photokeratometry (Wesley/Jessen PEK 2000, Chicago, IL) was performed at the outset

3 No. 10 EXPERIMENTAL MYOPIA IN TREE SHREW / Morsh-Toorle and Norron 2247 of each measurement session. Throughout the session, the tear film was maintained with frequent application of artificial tears (Adapettes). Excess fluid was removed before each measurement. The eyelids were gently retracted to allow photography while minimizing distortions of the eye. In photokeratometry, a set of concentric, illuminated rings were projected onto the cornea and a photograph was taken of their image. The two best photokeratographs (selected for clarity and centering of the rings on the cornea) were analyzed by Wesley/ Jessen at and orthogonal to the meridian initially identified as the least powerful. Corneal photographs too steep for standard analysis by Wesley/Jessen (radius < mm) were hand measured. To do this, photographs were magnified X25 and the central chord was measured with a ruler at the angle specified by the original analysis. This length was converted to an estimated radius using a linear conversion obtained from measurements of ball bearings of known radii. A slope of 0.95 and a correlation coefficient of 0.97 was obtained between hand measurements and the central radii reported by Wesley/Jessen in eight randomly selected photographs that had been previously analyzed. Refraction: Refractions were determined along the horizontal meridian, first with a Hartinger coincidence optometer and second with streak retinoscopy. The Hartinger optometer was equipped with a +8 D (later a +10 D) extending lens to allow clear imaging in the small eyes. A calibration curve was generated to allow conversion of the dial readings to actual refractive error. An accessory circular fluorescent light mounted on the optometer produced a bright corneal reflection that allowed more precise positioning. The optometer was first positioned along the pupillary axis using the constricted pupil as an aid to alignment. Partial cycloplegia was produced with 1 % tropicamide preceded by proparacaine hydrochloride to enhance corneal penetration. Refraction was measured 15 to 20 min later. Following measurements with the coincidence optometer, streak retinoscopy was performed along the horizontal axis at a 50 cm working distance. A concerted effort was made to ensure that measures were taken along the same incident angle for both coincidence optometry and streak retinoscopy. Axial length measures: Measurements of axial lengths were obtained by A-scan ultrasonography using a Panametrics 5052 pulser/receiver and a 4 mm diameter 10 MHz unfocused transducer. The ultrasonic pulses from the transducer were coupled to the cornea through a saline-filled plexiglass standoff that contained a 2 mm inside-diameter rubber aperture. This procedure narrowed the ultrasound beam and increased the resolution of the axial length measurements taken from the steeply concave surfaces (posterior lens and sclera). A continuous flow of saline replaced the fluid lost to leakage as the standoff was placed gently against the cornea without applanation. Echoes from ten independent positionings of the transducer were recorded from each eye and coded for later blind analysis. Ultrasonograms were photographed from the screen of a Tektronix 5112 oscilloscope that simultaneously displayed time marks at 1 jtsec intervals (Fig. 5). The location of echoes from the anterior cornea, anterior lens, posterior lens and anterior sclera were magnified approximately X30 and entered into a PDP-11 /34 computer using a digitizing pad. The anatomical origins of the echoes were verified in enucleated eyes from animals perfused for other studies. Echo times were converted to distance using standard conduction velocities determined in humans 15 with a transducer of similar frequency at body temperature. Because the posterior corneal surface was not perfectly resolved in all ultrasonograms, it was not analyzed as an independent measure. Instead, a weighted conduction velocity of mm/jusec for the anterior segment (front of the cornea to front of the lens) was calculated from the published values for cornea (1.610) and aqueous (1.540) assuming a cornea:aqueous ratio of 1:3. Conduction velocities used for the lens and vitreous were and 1.540, respectively. Frozen sections: After the last measurement session, eyes from four experimental animals and two normal animals were prepared for sectioning on a freezing microtome to allow direct measures of axial distances. While the animals were deeply anesthetized, the eyes were carefully enucleated, marked at the 12:00 location and frozen quickly in 68 C ethyl alcohol. They were placed in a 10% sucrose solution (with red food coloring to enhance contrast), positioned for horizontal sectioning and frozen to the stage of the microtome in the surrounding solution. Color 35 mm slide photographs were taken as 25 /*m sections were removed. Pictures taken midway through the lens were magnified XI5. Corneal and lenticular radii were determined by matching the central arcs to circular templates for later use in optical modeling. Anterior segment depth, lens thickness and vitreal chamber depth were determined from X25-X30 drawings of the slides. Values determined from frozen sections were compared with the distances calculated from the A-scan ultrasonography. Statistical tests: Nonparametric statistics were used because of our small sample size and the unknown sampling distribution of experimental eyes. Differences between independent samples: experimental

4 2248 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / October 1989 Vol. 30 (lid-sutured or open) against normal (right or left) eyes were analyzed using the Mann-Whitney U Test. Differences between matched samples (lid-sutured against open, or right against left) were analyzed with the Wilcoxon Matched-Pairs Signed-Ranks Test. The Friedman Two-Way Analysis of Variance by Ranks Test was used to test for trends over time. In this case, each animal's scores on a particular measurement were tested across three or four measurement sessions. Results One of our goals was to investigate differences between experimentally myopic eyes and normal eyes. To this end, we report the means of our various measures in each group of eyes, along with the group standard deviations across all measurement sessions. From this we assessed whether significant alterations occurred in the eyes of the experimental animals. A second goal was to examine the precision of our measurement techniques. To this end, we also present the average within-animal standard deviations of the measurements taken over the several measurement sessions (eg, the standard deviations of the measurement across sessions for each animal were averaged). For the normal animals, this provides an estimate of the errors of measurement. For the experimental animals, it provides an estimate of the stability of the lid-suture effects, assuming the measurement errors remain the same. Refraction Streak retinoscopy: In normal animals, the refractive values obtained in the right and left eyes with streak retinoscopy were very similar to each other (Fig. 2A). The mean values averaged across the three measurement sessions (Days 1, 2 and 3 in the Fig) were +3.5 diopters (D) in the right eye and +3.4 D in the left (see Table 1 for summary values). Repeated measures of normal eyes agreed within an average of 0.8 D (average within-animal standard deviation of right and left eyes), which represents the practical limits of precision of this technique for an eye of approximately 8 mm axial length. In experimental animals, each lid-sutured eye was always myopic relative to the paired open eye (Fig. 2B, Table 1). Retinoscopic values for the lid-sutured eyes averaged across animals and sessions (mean = -8.0 D) were more variable and significantly more myopic than those for the open eyes (mean = +4.1 D; P = 0.05, by 1-tailed Wilcoxon Signed-Ranks Test). Overall, the lid-sutured eyes averaged ± 6.3 D relative myopia compared to the paired open eye. The increased variability of refraction in animals oc Q_ O Q >- H X rr B Q_ O Q Q a: -10 CO I Q '-20 RETINOSCOPY CONTROL I LEFT EYE (DIOPTERS) EXPERIMENTAL rdday I A DAY 2 ODAY 3 "ODAY 4 -O O I OPEN EYE (DIOPTERS) Fig. 2. Refractive values obtained with streak retinoscopy. In this and subsequent graphs, the left eye of an animal was plotted against the right eye of the same animal. The straight line represents equal values for the paired eyes. Day 1, Day 2, etc. refer to the first, second, etc. measurement sessions. (A) Normal animals. The hyperopic values were approximately those expected based on the error of retinoscopy in small eyes. 19 (B) Values in experimental animals, showing that the lid-sutured eye was myopic relative to the open eye. Note the expanded Y-axis scale in (B) needed to plot the values for the lid-sutured eyes and the similarity of the values in the open eye to those in the normal eyes.

5 No. 10 EXPERIMENTAL MYOPIA IN TREE SHREW / Morsh-Toorle ond Norron 2249 Table 1. Refractive values for streak retinoscopy and Hartinger coincidence optometer Number of sessions Normal: 3 : 4 Mean* (mm) ±SD* (group) ±SDf (average within animal) = -6.4 D), were more variable and significantly more myopic than those for the open eyes (mean = +4.6 D). Overall, the lid-sutured eyes averaged 11.0 D 1 A ± D relative myopia compared to the open eyes. Streak retinoscopy Normal (N = 4) Right Left (N = 5) Coincidence optometer Normal Right Left +3.5 D +3.4 D -8.0 D +4.1 D +3.7 D +4.6 D -6.4 D +4.6 D 1.2 D 1.6 D 6.0 D 1.5 D 1.7 D 1.5 D 5.1 D 2.8 D 0.6 D 1.0 D 2.3 D 1.1 D 1.7 D 1.0 D 2.7 D 2.2 D * N = Number of animals X number of sessions. t N = Number of animals: the standard deviations from all sessions for each animal were averaged across animals. A ^_^ CO or Q_ o 10 5 HARTINGER DDAY I ADAY 2 ODAY CONTROL with experimentally induced myopia also has been observed in other species. 16 " 18 Repeated measures showed more within-animal variability for lid-sutured eyes (2.3 D) than for open eyes (1.1 D). However, there was no consistent tendency for the group retinoscopic values to change over time (Friedman Two-way Analysis of Variance by Ranks Test). No significant differences in refractive values were found between open eyes of the experimental group and the normal eyes of the control group. Retinoscopy has been shown to yield increasingly hyperopic values in eyes of decreasing axial length. 19 The average retinoscopic refraction for normal eyes (+3.45 D) agrees with the apparent hyperopia predicted for an eye of approximately 8 mm axial length by Glickstein and Millodot (see Fig. 1 in ref. 19). They suggest that the hyperopia is produced by an error of retinoscopy introduced by the axial distance between the location of the retinoscopic reflex and the plane of the photoreceptors. Coincidence optometry: In normal animals, the refractive values obtained in the right and left eyes with the Hartinger coincidence optometer were similar to each other (Fig. 3A). The mean values averaged across the three measurement sessions were +3.7 D in the right eye and +4.6 D in the left (Table 1). Repeated measures of normal left and right eyes agreed within an average of 1.3 diopters. In experimental animals, refractive values obtained with the optometer for the lid-sutured eyes were always more myopic than the open eyes (Fig. 3B, Table 1). Optometer values for the lid-sutured eyes, averaged across animals and sessions (mean B CO 1 0 or UJ o 9 o UJ Q UJ LEFT EYE (DIOPTERS) EXPERIMENTAL DDAY I ADAY 2 'ODAY 3 ODAY 4 D d> OPEN EYE (DIOPTERS) Fig. 3. Refractive values obtained with the Hartinger coincidence optometer. (A) Normal animals, left eye plotted against the right eye. (B) animals, open eye plotted against lid-sutured eye. As in Figure 2B, the lid-sutured eye was always myopic relative to the open control eye.

6 2250 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1989 Vol. 30 Repeated measures showed about the same within-animal variability for lid-sutured (2.7 D) and open eyes (2.2 D). As with streak retinoscopy, there was no tendency for the group optometer measures to change over time in the experimental animals (Friedman Two-Way Analysis of Variance by Ranks Test). Thus, the myopia induced by rearing with eyelid closure appeared to be stable when tested after 19 weeks of age. Values obtained with the coincidence optometer tended to be more hyperopic than the values obtained by streak retinoscopy in both normal (mean = D) and experimental (mean = D) eyes (P < 0.07 and P < 0.13, respectively, by two-tailed paired t-test). Using the average refraction across sessions for each of the 18 eyes in the study, the correlation between the retinoscopic and optometer measures was Corneal Curves The average corneal radii calculated from analysis of the photokeratographs are shown in Figure 4. The values shown are the average of the values obtained for the two major meridians. In the normal animals, corneal radii averaged across the three measurement sessions were essentially identical for the right eye (3.41 mm) and the left eye (3.41 mm) (Fig. 4A, Table 2). Repeated measures of normal left and right eyes had a standard deviation of mm or 2.35 D, which represents the practical limits of precision with this technique on eyes of this size. In the experimental group, the corneas of the lidsutured eyes were flatter in both meridians than corneas of the open eyes in all but one case (Fig. 4B). Values for the lid-sutured eye averaged across the three measurement sessions (3.67 mm) were significantly greater than those obtained in the open eye (3.43 mm, P = 0.05, 1-tailed Wilcoxon Signed-Ranks Test). Repeated measures showed more within-animal variability for lid-sutured (0.08 mm) than for open (0.04 mm) eyes. No significant differences were found between the open eye in the experimental animals and the normal eyes of the control animals. Repeated measures did not show any consistent changes in corneal curvature between session 1 when the lid-sutured eye was opened and session 3, which occurred approximately 6 weeks later. Thus, by 19 weeks of age the corneal curves in the normal eyes appeared to have reached adult values and the flattening of the cornea induced by rearing with eyelid closure appeared to be permanent. 3.8 i; E3.6 UJ 1- o3.4 cc B 3? CORNEAL RADIUS - A - / 1 CONTROL D / / i i i LEFT EYE (mm) EXPERIMENTAL OPEN EYE (mm) / DAY 1 A DAY 2 ODAY 3 i DAY I A DAY 2 3 / / I Fig. 4. Corneal radii determined from photokeratometry. (A) Normal animals displaying similar radii in right and left eyes. (B) animals, showing longer radii (cornealflattening)in the lid-sutured compared to the open eye. Axial Length Measures Ultrasound: Sample ultrasonograms from right and left eyes of a normal control animal are shown in

7 No. 10 EXPERIMENTAL MYOPIA IN TREE SHREW / Morsh-Toorle and Norton 2251 Table 2. Corneal radius measured by photokeratometry Number of sessions Normal: 3 Mean* ±SD* ±SZ>t : 3 (mm) (group) (individual) Normal (N = 4) Right Left (N = 5) * N = Number of animals X number of sessions. t N = Number of animals: the standard deviations from all sessions for each animal were averaged across animals. Figure 5A. Figure 5B shows that the average of 10 length measures taken during one measurement session in a representative normal animal are very similar for the left and right eye. The very small standard deviations show that the values within a measurement session were highly reliable. Whether taken by individual animal or when averaged across animals and across measurement sessions (Fig. 5C), axial length measurements showed no differences between right and left eyes. Sample ultrasonograms from the open and lid-sutured eyes of an experimental animal are shown in Figure 5D. Figure 5E shows that 10 length measures taken during one session in this experimental animal were very reliable, yielding low standard deviation, and that clear interocular differences were seen. Averaged across animals and sessions (Fig. 5F), the data show that the lid-sutured eyes were significantly longer (8.07 mm axial length) than the open eyes (7.74 mm). This elongation was due primarily to the greater depth of the vitreous chamber in the lid-sutured eye (mean, 3.35 mm) compared to the open control eye (2.95 mm). These differences were significant (P = 0.05 by 1-Tailed Wilcoxon Signed-Ranks Test). The within animal (individual) standard deviations shown in Table 3 represent the average of three measurement sessions in the normal animals and four sessions in the experimental animals. Each was obtained by averaging 10 same-session measures. On any one day, axial length measurements have an average standard deviation of less than 0.10 mm in normal animals, 0.07 mm for open and 0.09 mm for lid-sutured eyes. These appear to represent the practical limits of precision of this technique, at least as used in this study. As may be seen in Figure 5 and Table 3, the lid-sutured eyes tended to have shallower anterior segments than open and normal eyes. Also, the lenses were thinner front to back in the lid-sutured compared to the open eye, but the lenses of both the lid-sutured and the open eyes were thicker than the normal controls. None of these trends achieved statistical significance. The open eyes of the experimental animals did not differ significantly from the eyes of the normal animals on any A-scan measure. There was no tendency for the axial differences to change over time (Friedman Two-Way Analysis of Variance by Ranks Test). Thus, the axial elongation was permanent after 19 weeks of monocular experience. The stability of the measurements in the open and control eyes confirmed that the eyes had reached adult proportions by this age. Frozen sections: Superimposed tracings from photographs of the open (solid lines) and lid-sutured (dotted lines) eyes (Fig. 6A) illustrate the same vitreous elongation and resulting axial elongation that was found with the ultrasound measurements. The average measures from photographs of four of the experimental animals (Fig. 6B) also show an increased vitreous chamber depth and axial length in the lid-sutured eyes. The vitreous elongation (measured to the anterior sclera for best comparison with the ultrasound measures) did not achieve significance with a sample size of four animals (Table 4). However, the axial length of the lid-sutured eyes was greater than in the open eye in every animal. The same trends for the anterior segment and lens measured by ultrasound were illustrated by the frozen sections. Additionally, retinal thickness was measured for later use in optical modelling and tended to be thinner in lid-sutured eyes. Measures from photographs of the eyes of two normal animals showed the eyes to have very similar dimensions to each other. Ocular enlargement: The consistent flattening of the cornea in the lid-sutured eyes coupled with the axial elongation raises the possibility that lid-sutured eyes may undergo a symmetric enlargement rather than elongation only along the axial dimension. A comparison of equatorial dimensions measured from the frozen section photographs revealed no differences between groups. Eyes from the normal group (N - 3), open eye group (N = 4) and lid-sutured group (N = 4) all had a mean equatorial diameter of 8.6 mm. Thus, it appears that the elongation in tree shrew is an axial elongation rather than simply an ocular enlargement. Ray-Trace Modelling The measures on experimental eyes showed that the lid-sutured eyes were myopic despite their relative

8 2252 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1989 Vol. 30 CONTROL miintiiiiimim LLLLLLLL1 L-J EXPERIMENTAL ii iiiii HI 11 m Fig. 5. (A) Ultrasonograms of the left (L) and right (R) eyes of a normal animal. One millisecond time marks are also displayed above and below the ultrasound traces. Vertical lines have been drawn on the photographs to indicate the location of the (1) anterior cornea, (2) anterior lens, (3) posterior lens, and (4) anterior sclera. (B) Bar graphs representing the mean length values (± standard deviation) obtained from 10 successive positionings of the ultrasound probe within a single measurement on the left (L) and right (R) normal eyes shown in (A). Measured times were converted to length measures using published conduction velocities as described in Methods. Standard deviation bars were L-S 0 mm 0 mm 0 mm ALL ANIMALS, ALL SESSIONS ANTERIOR LENS VITREOUS L-S LUlWJOJJJJJJJJJJJJu'. 0 mm ALL ANIMALS, ALL SESSIONS B SINGLE ANIMAL, SINGLE SESSION E, SINGLE ANIMAL, SINGLE SESSION difficult to represent at this scale because of the low variability in these measures. (C) Bar graphs representing the mean ultrasound length measurements (± standard deviation) for the eyes of the four normal animals across three measurement sessions. (D) Ultrasonograms of the open (O) and lid-sutured (L-S) eyes of an experimental animal. This animal was selected for display because it exhibited the median increase in axial length of the five experimental animals. (E) Bar graphs representing the mean length values (± standard deviation) obtained from 10 successive positionings of the ultrasound probe within a single measurement session on the open (O) and lid-sutured (L-S) eyes shown in D. (F) Bar graphs representing the mean ultrasound length measurements (± standard deviation) for the eyes of thefiveexperimental animals across the three measurement sessions. corneal flattening. The only other significant difference in the sutured eyes was their vitreous elongation. We used paraxial optics to determine whether our observations were sufficient to account for the observed myopia, or whether it was likely that other undetected differences existed in the sutured eyes. For optical modelling, the thickness of the retina was measured from photographs and subtracted from the values appearing in Table 4. Because we found no differences between the open and normal eyes, we averaged the values for all open and normal eyes together to increase the numbers of observations in this (the "average eye") group. The following refractive indices were used: (cornea, 20), (aqueous, 20), 1.39 (lens cortex, 21) and (vitreous, 22). The refractive index for the lens nucleus ( ) was empirically derived to fit the results (assuming reflection from the retino-vit-

9 No. 10 EXPERIMENTAL MYOPIA IN TREE SHREW / Morsh-Toorle and Norron 2253 Table 3. Axial measures by A-scan ultrasound Number of Sessions Normal: 3 : 4 Anterior segment depth Normal (N = 4) Right Left (N = 5) Lens thickness Normal Right Left Vitreous depth Normal Right Left Axial length Normal Right Left Mean* (mm) ±SD* (group) ±SZ>f (individual) reous boundary) for the "average eye" comparison group which showed an apparent hyperopia of +3.8 D as measured by streak retinoscopy. Flattening the "average eye" by the amount observed in the lid-sutured eyes resulted in an increased hyperopia of +7 D when uncompensated by axial elongation. Then, elongating the eye with the flattened cornea by the amount observed in the lid-sutured eyes counteracted this hyperopia by 15.2 D. The final model incorporating both corneal flattening and vitreous elongation differed from the original average eye by a calculated relative myopia of 8.2 D compared to an observed difference of 11.8 D between the average (pooled open plus normal eyes) and sutured eyes. We next tried a more direct comparison of the open and sutured eyes. Instead of using average values pooled from the normal and open eyes, an index was derived for the lens cortex ( ) of just the open eyes to yield a refractive error of+4.1 D (the average observed refraction by streak retinoscopy). Using this same index with radii and distances for the lid-sutured eyes, a refractive error of 9.0 D was calculated, yielding a relative myopia of 13.1 D even when the flattened corneal values were included. The increased relative myopia in this example was produced by minor differences in the radii and distances in the lens cortex and nucleus of the comparison eye (measured in the frozen sections), which were well within our measurement error. Thus, the values used in each example are equally valid estimates of the actual values. In summary, our modelling suggests that axial elongation is a powerful, visible and probably sufficient factor to produce the myopia observed in experimental eyes. The optical modelling also showed that the lenticular shells, of which we had no direct measure, provide a major refracting component of the tree shrew eye. Because any lenticular effect would be very small compared to the total refracting power of the eye, direct measures of a large number of animals would be necessary to conclusively demonstrate any difference between groups. Thus, lens size or curve 23 also may vary between normal and lid-sutured eyes. L-S * N = Number of animals X number of sessions. t N = Number of animals: the standard deviations from all sessions for each animal were averaged across animals. B 0 L-S m i i i i t i i i i 0 mm 8 Fig. 6. Measurements from horizontal histological sections of frozen enucleated eyes. Abbreviations are as in Figure 5. (A) Superimposed tracings from photographs of the open (solid lines) and lid-sutured (dotted lines) eyes of the experimental animal whose ultrasonograms were shown in Figure 5. The tracings represent the boundaries of the cornea anteriorly and of the choroid and sclera posteriorly. (B) Bar graphs representing the mean measurements (± standard deviation) from frozen sections for the eyes of four experimental animals.

10 2254 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1989 Vol. 30 Table 4. Axial measures from frozen section photographs Anterior chamber depth Normals* Lens thickness Normals Vitreous depth Normals Axial length Normals *N = 3. t, N = 4; open, N = 4. Mean (mm) Discussion Characterization of the Refractive and Structural Changes ±SD (group) Tree shrews were found in this study to have reliable responses to rearing with monocular eyelid closure. The variability of the effects is typical of this manipulation. 16 " 18 Lid-suture produces an eye which is myopic, has a longer vitreous chamber and total axial length, and has flatter corneal curves than its paired open eye or normal control eyes. Optical modelling demonstrated that the axial elongation was responsible for most or all of the myopia observed, but our measurements were not sensitive enough to rule out a possible role for the changes in the lens that were observed by McKanna and Casagrande. 23 ' 24 The lid-suture-induced changes seemed permanent following 16 weeks of monocular visual deprivation. In young chickens, recovery of the vitreal chamber elongation has been reported. 17 Since our animals were older, we cannot rule out the possibility that younger tree shrews might show a similar recovery. The occurrence of these changes in one eye of a genetically identical pair demonstrates the susceptibility of the developing tree shrew eye to environmental influences. The refractive and anatomical changes found in the tree shrew are consistent with those described in primates. 2 ' 4 Thus, the tree shrew may serve as a useful mammalian model of myopia. As previously demonstrated in the rhesus macaque, 25 the myopic effects of lid-suture in the shrew are not due to mechanical factors or temperature changes produced by the closed eyelid. Eleven darkreared tree shrews raised with eyelid closure showed highly variable refractions (ranging from -10 D to +8 D) but no consistent myopia in the monocularly lid-sutured eye. 26 Greater, bidirectional interocular differences were seen in both refraction and axial length of the dark-reared tree shrews in comparison with normal control animals. 26 This further suggests that the isometropia normally seen in human and animal populations can be disrupted by the absence of light as well as by the absence of clear images on the retina. The myopia and axial elongation are also dependent upon normal cholinergic activity in the tree shrew eye, since daily doses of atropine underneath the closed lid prevent the myopia and axial elongation The accommodative system was examined in the tree shrew by McKanna and Casagrande who found zonular dysplasia 23 and reduced lens weight 24 in lid-sutured tree shrews and proposed a feedback mechanism 23 in which accommodation is involved in self-regulation of ocular growth. Such a mechanism is consistent with observations that the prevalence of human myopia is related to the number of years spent in school, 29 and that even adults may show myopic shifts if they must read extensively during graduate school. 30 It is apparent that both visual and nonvisual mechanisms must underlie the enlargement of the eye to emmetropia or ametropia. In avian myopia, some regulation of ocular growth can occur in the absence of accommodation. Asymmetric axial enlargement occurs in chickens in response to restricted form vision in the corresponding visual field 17 ' 3132 even when the optic nerve is cut, 33 suggesting that local retinal factors mediate differential axial elongation. 34 Also, stroboscopic illumination reduces this myopia, further suggesting that retinal activity is important in signalling the eye to stop enlarging. 35 This idea is consistent with findings that intravitreal doses of kainic acid to developing chickens result in eye enlargement. 36 Manipulations in the developing eye which result in reduction in the amount of induced myopia (eg, ON-channel blockade in cats by APB 37 ; atropine administration in some animals 16 ' 27 ' 28 ' 38 ' 39 and humans 40 ' 41 are more difficult to interpret, because withdrawal of the ON-channel or of cholinergic mechanisms either may interfere with growth or may allow other active regulation to proceed in a normal or enhanced fashion. For instance, atropine has been shown to produce effects in developing retinal neural

11 No. 10 EXPERIMENTAL MYOPIA IN TREE SHREW / Morsh-Toorle ond Norton 2255 and vascular cells 42 which are of unknown importance in refractive error development. In summary, eyelid-closure during development seems to produce axial myopia in the shrew by nature of the visual changes it produces. Given the changes noted previously 23 ' 24 in the lenticular apparatus of lid-sutured eye, the tree shrew may serve to clarify the role that accommodation plays in refractive error development. Conversely, further work may show that the same mechanisms for emmetropization demonstrated in chickens also occur in tree shrews. Precision of Measurement Techniques The optical and structural changes in tree shrew were readily detectable with the techniques we used. Because one goal of this study was to examine methods that might be useful in future studies of developing small eyes, some discussion of technical considerations seems appropriate. Refraction: tropicamide versus atropine cycloplegia: In order to ensure that comparable refractive measures were obtained with both retinoscopy and coincidence optometry across several measurement sessions, some compromises in our cycloplegia technique were made. Tropicamide was chosen for its short duration to allow any possible recovery from the lid-suture-induced myopia to proceed without interference from a disabled accommodative mechanism. Atropine sulfate was avoided because of its long-lasting effects. In tree shrews, pupillary dilation persists for 48 h or more after atropine sulfate administration. We did not expect a complete cycloplegic effect from the tropicamide, but felt that reliable interocular comparisons would be possible when it was used in anesthetized animals. To ensure that the refractive measures were valid, a comparison was made between refractions obtained using tropicamide and atropine. Refractive measures using cycloplegia induced by 1% atropine were obtained during the second measurement session for normal animals. Comparable measurements were obtained in four of five experimental animals at the fourth (terminal) measurement session approximately 7.5 months after the other measurement sessions. No significant differences were found between the refractions measured with retinoscopy or with the optometer using tropicamide or atropine in either the normal or the experimental eyes (P < 0.65, paired t-test). We conclude that our regimen of anesthesia plus tropicamide produced sufficient cycloplegia for valid refractions. Comparison of retinoscopy and coincidence optometry: Refractive values obtained with the Hartinger coincidence optometer were significantly more hyperopic than were those obtained with retinoscopy (average difference for all eyes combined = 0.82 ± 1.27 D, P < 0.02 two-tailed t-test) but were highly correlated, as reported in Results. It is not surprising that the values obtained with these two measures were somewhat different, because the projection of the coincidence optometer is very discrete compared to the broad beam used in retinoscopy. The lower values obtained with the retinoscope suggest that, in the fully dilated eye, retinoscopy may pick up more negative vergence from a source which is peripheral to the much smaller area sampled by the optometer. Comparison of ultrasound and frozen section measures: The primary goal in using A-scan ultrasound was to provide reliable, noninvasive measures of sufficient accuracy to reveal differences between paired eyes in the size and location of the ocular components. As was shown in Figure 5B and 5E, 10 consecutive ultrasonograms in each eye were virtually identical. Although the measures were reliable, the question remained as to whether they accurately measured the axial dimensions. Our main concern using A-scan ultrasonography was error due to poor lateral resolution. We stopped the ultrasound beam with a 2 mm rubber aperture and chose to use a flat transducer because it was more sensitive to misalignments than our focused transducers. The A-scan provided indirect measures of length because we did not directly measure conduction velocities. The frozen sections confirmed that the differences we reported with ultrasound arose from true differences in distances through the various tissues and not from differences in their conduction velocities. A correlation of 0.84 was found between measures of total axial length obtained with ultrasound versus those measured from photographs of frozen sections. Correlations between the A-scan and frozen sections for the lens and vitreous were poor ( 0.18 and +0.58, respectively). When these two measures were combined, the correlation of their sum rose to 0.91, suggesting that localization of the posterior lens was poor but that localization of the anterior edge of the sclera was good. In comparison with frozen-sectioned eyes, ultrasound provided consistently smaller values for the axial length (mean difference = ± 0.11 mm) and the combined lens and vitreous values (mean difference = ±0.11 mm). We attribute most of this error to poor lateral resolution rather than incorrect conduction velocities. The average difference in axial length between lidsutured and open eyes detected with ultrasound (mean = ±0.19 mm) was not significantly different from the difference measured from photo-

12 2256 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / October 1989 Vol. 30 graphs (mean = ± 0.20 mm). Thus, reasonable agreement was found in the axial elongation of the lid-sutured eyes. Because we cannot know the effect of freezing on the dimensions of these tissues, we do not know which method better reflects the in vivo dimensions. However, both methods lead us to the same conclusions, namely that there is an axial elongation in lid-sutured eyes. Based on the foregoing examination of the measurement techniques used in this study, it is clear that they are sufficient to detect the myopic refractive changes and the major structural changes in the location of refractive surfaces. Further, it appears that these techniques have sufficient precision to detect these changes as they develop during the period of monocular exposure. As demonstrated by the optical modeling, an improved estimate of lens curvature and lens focal length will be needed to provide a more complete model of the effects of rearing with eyelid closure on the optical and structural development of the eye in tree shrew. Key words: refractive error, visual optics, animal myopia, lid-suture, tree shrew, ocular development Acknowledgments We thank Mr. John T. Siegwart, Jr. for superb technical support in hand-rearing the pups, constructing mechanical apparatus and assisting during the measurement sessions. We also thank Dr. Josh Wallman for instruction in setting up the Hartinger coincidence optometer and Dr. Neville McBrien for critically reading the manuscript. The tree shrew photograph (Fig. 1) was taken by Mr. Bill Boyarski and Dr. V. A. Casagrande. References 1. Sherman SM, Norton TT, and Casagrande VA: Myopia in the lid-sutured tree shrew (Tupaia glis). 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