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such as latex spheres, polystyrene beads, yeast, and oil droplets.""11 Further work is in progress to determine whether the incoiporation of rod outer segments ...
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ing brain edema in vitro, AA acts predominantly by this mechanism. 7 Auto-oxidation, or peroxidation, involves formation of free-radical intermediates from lipids that cause molecular damage to the membranes, especially the cross-linkage of proteins. 8 Antioxidants are effective in preventing this process. It has recently been reported that docosahexaenoic acid-induced inhibition of nucleic acid synthesis in cultured lymphocytes could be counteracted by a-tocopherol and superoxide dismutase. 9 The RPE is known to have considerable superoxide dismutase activity10 as well as peroxidase enzyme associated with plasma membranes. 2 These enzymes within the RPE could provide a physiologic mechanism to neutralize the potential damaging effects of the PUFA-rich outer segment membranes that are shed for phagocytosis. If AA inhibits the phagocytic activity of RPE by release of auto-oxidative products, then the greater susceptibility of dystrophic RPE observed in our present study implies a defect in the protective enzyme system of the cell. The RCS rat is a model for retinitis pigmentosa in man. The degenerative changes in the retina of dystrophic animals are considered to be caused by a defect in the ability of the RPE to phagocytose outer segment material. Studies in vitro have shown a reduction rather than a total lack of phagocytosis for outer segment material.11"14 However, normally the phagocytic activity of the dystrophic RPE is not reduced appreciably for inert markers such as latex spheres, polystyrene beads, yeast, and oil droplets."" 11 Further work is in progress to determine whether the incoiporation of rod outer segments material in the culture media can sufficiently raise the level of AA (as well as other PUFAs) to approximate our experimental situation reported here. In this regard it is noteworthy that a raised level of AA is far more effective in disrupting cellular function than any other PUFA. 7 We thank Dr. Matthew M. LaVail of University of California, San Francisco, for kindly providing the original breeder pairs of rats and advice on the maintenance of the animal colony. In this project the interest and encouragement of Professor Frank W. Newell, M.D., are gratefully acknowledged. From Eye Research Laboratories, University of Chicago, Chicago, 111. Supported by a grant from the National Society to Prevent Blindness, Inc., and in part by a Block Fund of the University of Chicago and National Eye Institute grant EY-020406. Submitted for publication Aug. 22, 1980. Reprint requests: Brenda J. Tripathi, Ph.D., Eye Research Laboratories, University of Chicago, 939 East 57th St., Chicago, 111. 60637. Key words: arachidonic acid, polyunsaturated fatty

acids, retinal pigment epithelium, morphology, phagocytosis, tissue culture, normal rat, dystrophic rat, carmine, retinitis pigmentosa

REFERENCES 1. Berman ER: Biochemistry of the pigment epithelium. In The Retinal Pigment Epithelium, Zinn KM and Marmor MF, editors. Cambridge, Mass., 1979, Harvard University Press, pp. 83-102. 2. Stone VVL, Farnsworth CC, and Dratz EA: A reinvestigation of the fatty acid content of bovine, rat, and frog retinal outer segments. Exp Eye Res 28:387, 1979. 3. Newell FW: The challenge of the retinal pigment epithelium. Proc R Soc Lond 67:1233, 1974. 4. Dawson G and Newell FW: Arachidonic acid and retinal pigment degeneration. Lancet 1:1119, 1974. 5. LaVail MM, Sidman RL, and Gerhardt CO: Congenic strains of RCS rats with inherited retinal dystrophy. J Hered 66:242, 1975. 6. Bull HB and Breese K: Denaturation of proteins by fatty acids. Arch Biochem Biophys 120:309, 1967. 7. Chan PH and Fish man RA: Brain edema: induction in cortical slices by polyunsaturated fatty acid. Science 201:358, 1978. 8. Tappel AL: Biological antioxidant protection against lipid peroxidation damage. Am J Clin Nutr 23:1137, 1970. 9. Gery I: Inhibition of DNA and RNA synthesis in lymphocyte cultures by rod outer segments and its counteraction by vitamin E and other antioxidants. INVEST OPHTHALMOL VIS SCI 19:751, 1980.

10. Feeney L and Berman ER: Oxygen toxicity: membrane damage by free radicals. INVEST OPHTHALMOL 15:789, 1976. 11. O'Donnell J: Retinal pigment epithelium phagocytosis in vitro. INVEST OPHTHALMOL Vis Sci 16(April

Suppl.):lll, 1977. 12. Hall MO: Phagocytosis by rat pigment epithelium cells in tissue culture. INVEST OPHTHALMOL VIS SCI

16(April Suppl.):lll, 1977. 13. Edwards RB and Szamier RB: Defective phagocytosis of isolated rod outer segments by RCS rat retinal pigment epithelium in culture. Science 197:1001, 1977. 14. Goldman AI and O'Brien PJ: Phagocytosis in the retinal pigment epithelium of the RCS rat. Science 201:1023, 1978.

The he eyes of young chickens grow toward emmetropia. JOSH WALLMAN, JULIAN I. ADAMS, AND JOSEPH N. TRACHTMAN. The distribution of refractive errors was followed in chicks from hatching to 8 weeks of age. A dramatic progressive decrease in the variability of refractions was observed over this period. In addition, there appeared to be a parallel decline in hyperopia, even when the artifactual

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hyperopia of retinoscopy was taken into account. These results are evidence for a postnatal developmental regulatory mechanism, most likely dependent on vision, which directs growth of the eye toward emmetropia.

The embryological development of the eye presents an interesting problem in size regulation in that, although strict regulation of the overall size is not important for proper function, the harmonious growth of the different refractive elements is crucial for adequate visual performance. This harmonious growth would seem to be a particular problem in view of the rather distinct embryological origins of the cornea, lens, and sclera. Since we find very little variability in the ocular refractions of adult chickens, we consider it likely that some regulatory process controls the growth of the eye or of one or more of its refractive elements. If such a regulator)' mechanism exists, it seems reasonable to assume that it operates after birth or hatching, when a signal would be available that reflects the refractive error of the eye. This reasoning led us to the hypothesis tested in this study: if a postnatal regulatory mechanism exists that corrects refractive errors of the eye, then neonates should show the greatest variability in refractive states, and this variability should decrease with age. Methods. The 121 White Leghorn chicks (Gallus gallus domesticus) used in this study were hatched in the laboratory, and groups of 10 to 40 animals were raised in commercial chick brooders from hatching to 4 weeks of age and thereafter in commercial developing cages. Room lights were on a 14 hr on: 10 hr off cycle. Refractions were done under cycloplegia by streak retinoscopy by one of the authors (J. T.), who is experienced in doing retinoscopy on small eyes. Retinoscopy was done in the horizontal plane; since this ignored astigmatism, we probably moderately overstate the variability at each age. Because avian ciliary muscle is striated, cycloplegia was obtained by general anesthesia (Chloropent; Fort Dodge Laboratories) together with topical application to the cornea of approximately 10 drops of a solution of 2.5 mg/ml curare, 1% atropine, and 0.025% benzalkonium chloride in 0.75% NaCl until complete mydriasis was obtained. The solution, a variant of that used by Campbell and Smith,1 was suggested by Drs. J. Delius and J. Emmerton of Ruhruniversitat, Bochum, W. Germany. It was not possible to do the refractions in a completely blind manner, since an 8-week-old chicken and a newly hatched chick could hardly be

Invest. Ophthabnol. Vis. Sci. April 1981

disguised to look alike. Consequently, to minimize experimenter bias, we did the refractions over a period of many months, with the subjects of this study mixed in with subjects of several other studies in which the experimental manipulations produced widely varying refractive errors. Although in some species retinoscopy of young animals presents difficulties because of lack of clarity of the optical media, we saw no difference in this respect between the eyes of newly hatched and 8-week-old birds, according to observations made during retinoscopy and during a few slitlamp examinations. Because it is easier to do retinoscopy on older animals, we were concerned that developmental changes would be confounded by a decrease in measurement error with age. Therefore we repeatedly refracted a total of seven eyes from animals of two widely separated ages about 15 times each, to permit estimation of the measurement error. Unfortunately, these refractions were made under different conditions from the other measurements. In particular, to prevent recognition of individual animals, the refractionist stayed in a darkened room and the individual animals were handed in, under anesthesia and cycloplegia and with a lid retractor in place. In addition, these repeated measurements were completed longer after anesthesia and cycloplegia than the measurements composing the main body of data. This was a cross-sectional study in which each animal was measured only once rather than a longitudinal study, since we suspected that weekly general anesthesia and cycloplegia might in some way influence the growth of the animal or of its eyes. Results. The results show two remarkably consistent trends (Fig. 1). From each age group to the next, there was a progressive decrease in the variability of the refractions. In addition, there was a similarly monotonic progression of decreasing hyperopia with age. To consider the decrease in hyperopia first, comparison of the distribution of refractions by the Mann-Whitney U test showed that all ages were significantly different from each other (p < 0.01) with the exception of three comparisons: 1 and 2 weeks, 3 and 4 weeks, and 3 and 6 weeks (p > 0.05). Unfortunately, we cannot be sure to what extent these changes in hyperopia reflect changes in functional hyperopia, since an artifact of retinoscopy makes small eyes appear more hyperopic. 2 This artifact probably involves the consequences of both the longitudinal chromatic aberration of the eye and the fact that the image

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FOUR WEEKS HATCHING

10 UJ

5

EIGHT WEEKS

-5

0

+5

+10 +15 +20 -5 0 OCULAR REFRACTION (DIOPTERS)

+5

+10

Fig. 1. Distributions of refractive error of the eyes of chickens of different ages. Both eyes of each chicken were used. Arrows indicate medians. observed in retinoscopy may be reflected from the inner retinal surface rather than from the receptor cell layer.2~fi Glickstein and Millodot2 assessed the influence of eye size on the apparent hyperopia of retinoscopy by measuring adult mammalian eyes ranging widely in size (3 to 24 mm) and assuming that each species was in fact functionally emmetropic. By comparing our data with their curve, using axial lengths obtained from A-scan ultrasonography, we estimated the artifactual hyperopia of newly hatched birds (axial length of eye approximately 8 mm) to be about 4 D. Since the median refraction of these birds was 9.2 D, it is probable that some fraction of the hyperopia we observed represented functional hyperopia. Assessed in this admittedly rough manner, the functional hyperopia seemed to decline from 5 D at hatching to 3 D at 2 weeks to 1 D at 4 weeks, with emmetropia being attained at 8 weeks. To compare the variabilities of the refractions of different ages, we used Siegel-Tukey tests between each age and the next, having shifted the distributions to yield identical medians. The result

was that each age except 2 and 4 weeks was significantly more variable than the next (p < 0.05). To better describe the trend in the variability and to facilitate comparison with measurement error, we plotted the absolute values of deviations from the means for each group and for the repeatedly measured eyes. Fig. 2 clearly shows the dramatic decrease of variability with age and shows that this change was much greater than the change in measurement error. To compare the trends in error and experimental variations, lines were fitted to the experimental and error data. To fit a best line to the experimental data, a weighted least-squares regression procedure was used, which weighted each data point by the inverse of the variance of its age group. Specifically, the following equation was minimized and solved for slope (in) and y-intercept (b); F(m,b) =

- b) 2

where xi = ith age, yy = j t h absolute deviation

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VARIABILITY OF REFRACTIONS AT DIFFERENT AGES >6 5 4 3 2

0

I

2

3

4 AGE (WEEKS)

MEASUREMENT VARIABILITY

AGE (WEEKS)

Fig. 2. Top, Absolute values of the deviations from group means (from same data as in Fig. 1). Means are connected by line segments. Dashed line was fitted by weighted least-squares method. Bottom, Variability of repeated measurements at two ages. Each dot is the deviation of a measurement from the mean for that eye. Three 3-day-old eyes and four 4-week-old eyes were used.

from the mean of the ith group, and s? = variance of ith group. The resulting line for the data points had a slope of —0.25 and a y-intercept of 2.3. The equivalent line for the measurement error had a slope of —0.02 and a y-intercept of 0.65. These slopes were significantly different (t test, p < 0.05, with standard errors from unweighted least-squares lines), substantiating the impression conveyed by Fig. 2 that refractions become less variable with age. Discussion. Our hypothesis is that embryological regulatory processes can assure only approximately correct refraction of the eye and that postnatal regulatory mechanisms, dependent on the animal's visual experience, fine-tune the refractive state by selective growth of the eye or of its refractive components. Our principal finding that the variability in refractions is greatest at hatching and declines sharply in the next two months is consistent with this hypothesis. Such a decline in variablity would be expected, however,

whether the postnatal regulatory processes are dependent on vision or are simply a continuation of the prenatal embryological processes. The involvement of vision in the postnatal regulatory processes is strongly argued for by the fact that the period of life during which the variability of refraction declines is precisely the period of susceptibility to experimental myopia produced by restricting vision to the frontal visual field. '• 8 In particular, we find that 1 week of this visual restriction at hatching produces a median refraction of 20 D of myopia; in contrast, 3 weeks of such restriction at 9 weeks of age produces a change of only L I D (Wallman et al., submitted for publication). Thus both the reduction of the variability of refractions and the susceptibility to vision-related experimental myopia are highest near hatching and decline markedly over the next 9 weeks. The processes discussed here may be a general feature of vertebrate ocular development rather than being unique to chickens. There is evidence

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that the same general trends reported here occur in human infants. Banks,9 in an extensive review of many studies, found a general tendency for neonatal refractions to be more variable than those of older children. In addition, by employing a correction for the artifact of retinoscopy similar to that used here, he found that neonates probably have some functional hyperopia that declines as they grow, a result similar to ours. Mohindra and Held10 have reported that the standard deviation of noncycloplegic refractions declines from 3.25 D at birth to 0.99 D at 6 months of age, and I. Mohindra (personal communication) states that cycloplegic refractions confirm this trend. On the other hand, A. Fulton and V. Dobson (personal communication) did not find a decline in standard deviations in a retrospective study of cycloplegic refractions made in a hospital clinic. We have argued here that the refractive state of the eye is fine-tuned postnatally by a growth regulatory mechanism that uses some signal from the visual system as an error signal. We would like to speculate that the most appropriate candidate for this error signal is one related to the mean level of accommodation, since in most circumstances this would be a reliable indicator of refractive state of the eye. If high levels of accommodation caused growth in the myopic direction or if low levels caused growth in the hyperopic direction, emmetropia would be achieved. We greatly appreciate the assistance of Ms. Debbie Rosenthal, Ms. Charlotte Ledoux, and Mr. Andre Washington in carrying out this study. From the Biology Department, City College of the City University of New York, New York, N. Y. This study was supported by National Institutes of Health grant EY-02727. Submitted for publication Aug. 22, 1980. Reprint requests: Josh Wallman, Biology Department, City College of the City University of New York, New York, N. Y. 10031. Key words: refractive errors, emmetropia, accommodation, development, birds, chickens, hyperopia, myopia

REFERENCES 1. Campbell HS and Smith JL: Pharmacology of the pigeon pupil. Arch Ophthalmol 67:501, 1962. 2. Clickstein M and Millodot M: Retinoscopy and eye size. Science 168:605, 1970. 3. Charman WN and Jennings JAM: Objective measurements of the longitudinal chromatic aberration of the human eye. Vision Res 16:99, 1976. 4. Millodot M and Sivak J: Hypermetropia of small animals and chromatic aberration. Vision Res 18:125, 1978.

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5. Nuboer JFW, Bos N, van Genderen-Takken H, van den Hoeven H, and van Steenbergen JC: Retinoscopy and chromatic aberration. Experientia 35: 1066, 1979. 6. Hughes A: The artifact of retinoscopy in the rat and rabbit eye has its origin at the retina/vitreous interface rather than in longitudinal chromatic aberration. Vision Res 19:1292, 1979. 7. Wallman J, Turkel J, and Trachtman J: Extreme myopia produced by modest change in early visual experience. Science 201:1249, 1978. 8. Wallman J: Developmental characteristics of experimental myopia produced by altered visual experience.

INVEST OPHTHALMOL VIS SCI 18(April

Suppl.):251, 1979. 9. Banks MS: Infant refraction and accommodation. In Electrophysiology and Psychophysics: Their Use in Ophthalmic Diagnosis, Sokol S, editor. Int Ophthalmol Clin 20:205, 1980. 10. Mohindra I and Held R: Development of human refraction. In Abstracts of Third International Conference on Myopia, Copenhagen. 1980.

Emmetropization: a vision-dependent phenomenon. JEFF RABIN, RICHARD C. VAN SLUYTERS, AND RAFI MALACH. Anomalous visual experience during development has been shown to induce myopia in several species of animals. A retrospective analysis of refractive error among htimans subjected to various ocular anomalies that disrupt pattern vision revealed a significant degree of myopia. This result suggests that emmetropization is a vision-dependent phenomenon.

Neonatal eyelid fusion induces myopia in a variety of animals including monkeys,1 tree shrews,2 cats, 3 and chicks.4 This phenomenon apparently is unrelated to eyelid fusion per se, since it also occurs in monkeys subjected to corneal opacification at an early age 1 and in chicks reared wearing masks containing translucent occluders that attenuate patterned vision in all or part of the visual field.4 It fails, however, to occur in dark-reared, lid-sutured monkeys. 6 Thus, in animals, anomalous patterned visual experience during development is thought to be a relevant factor for producing myopia experimentally. However, the validity of using these results from animal studies to help explain the genesis of myopia in man was questioned in a recent study of refractive error in patients afflicted with ptosis early in life.7 Despite a significant increase in the incidence of myopia in ptotic eyes, corrected visual acuity was not consistently reduced. The absence of amblyopia in these ptotic eyes led the

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