Increase in axial length of the macaque monkey eye after corneal opacification

Transcription

1 Increase in axial length of the macaque monkey eye after corneal opacification Torsten N. Wiesel and Elio Raviola The cornea of one eye was opacified in two young macaque monkeys by multiple stromal injections of a suspension of polystyrene particles (latex). Ultrasound measurements showed that the eye with opaque cornea grew at a faster rate, so that after 1 year it was more than 1 mm longer than the normal eye. This difference in axial length was due to elongation of the posterior segment, since lens thickness, depth of anterior chamber, and corneal curvature were identical in both eyes. At histological examination, no pathological changes were observed in the anterior segment of the latex-injected eye except for a scant vascularization of the corneal opacity. The result of this experiment demonstrates that opacification of the cornea has effects on axial length similar to, although less marked than, those on lid fusion and therefore supports our previous conclusion that the myopia caused by lid fusion is triggered by the abnormal visual input and involves central visual pathways. Key words: myopia, axial length, corneal opacification, macaque monkeys Ltiology and pathogenesis of human myopia are not known, and there is a need for an animal model of this clinical condition to facilitate research. We observed that macaque monkeys develop a high degree of axial myopia when their lids are fused at birth in one or both eyes and they are subsequently exposed to a 12 hr light/12 hr dark cycle throughout their first year of life. l> 2 The experimental refractive error thus obtained would be of little help in understanding human myopia if it were caused by trivial mechanical or thermal influences exerted by the fused lids on the developing eye. However, we recently showed that the appearance of myopia could be prevented by raising From the Departments of Neurobiology and Anatomy, Harvard Medical School, Boston, Mass. This study was supported by grant EY01966 of the National Eye Institute, U.S. Public Health Service. Submitted for publication Feb. 26, Reprint requests: Dr. Torsten N. Wiesel, Department of Neurobiology, Harvard Medical School, 25 Shattuck St., Boston, Mass the lid-sutured animals in the dark. 3 This finding indicates that such myopia is triggered by the abnormal visual input and is mediated by the nervous system, but we could not rule out the possibility that abnormal neural influences produce the refractive error only when combined with the local effects of lid fusion. Clearly, crucial evidence that the fused lids play a secondary role in the genesis of myopia can only be obtained if eye elongation is induced without suturing the lids. We therefore decided to study the effects of opacification of the cornea on the axial length of the growing monkey eye. Materials and methods In the two female macaque monkeys, one Macaca mulatto, and one Macaco, arctoides, the cornea was opacified by injecting into the corneal stroma a 2.5% suspension of monodisperse polystyrene particles fim in diameter (Polysciences Inc., Warrington, Pa.). In order to prevent bacterial growth, 0.1 /xg/ml gentamicin sulfate (Schering Corp., Port Reading, N.J.) was added to the suspension. The procedure was as follows. A /79/ $00.50/ Assoc. for Res. in Vis. and Ophthal., Inc.

2 Volume 18 Number 12 Axial length increase after corneal opacification gauge needle mounted on a 1 ml syringe filled with latex was inserted into the corneal stroma, and the fluid was injected by gentle manual pressure. The latex slowly diffused through the corneal stroma and produced a milky spot 2 to 3 mm in diameter. The procedure was repeated in different corneal regions until most of the cornea appeared opaque. Although the distribution of the latex was not uniform, great care was taken that no transparent area he left at the center of the cornea in order to avoid a pinhole effect. In addition, the corneal opacity was large enough to completely cover the pupil when it was maximally dilated. In the M. mulatto, the cornea was opacified at 1 month of age and in the M. arctoides at 1.5 months of age. Subsequently, the monkeys were raised in regular animal quarters under a 12 hr light/12 hr dark cycle; the luminance in the cage was about 1 log cd/m 2. At intervals of 3 to 4 months the axial length of both eyes was measured by A-scan ultrasonography (Digital Biometric Ruler DBR-300; Sonometrics Systems Inc., New York, N.Y.). At the age of 13 months M. mulatto.) and 17 months (M. arctoides), the animals were perfused through the aorta with 10% formalin, and their eyes were enucleated. An intraocular pressure of 20 mm Hg was restored by inserting into the vitreous body a needle connected to a water reservoir, and the distance between anterior and posterior poles of the eye globes was measured. The eyes were subsequently transected with an equatorial incision. The lens was removed, and its weight, thickness, and diameter were measured. In the anterior segment, the sclerocorneal tunic was isolated by gently dissecting out the uvea, and the light transmittance of the cornea was compared in the opacified and normal eyes. These measurements were done by placing the convex surface of the fixed corneas on a 5 mm aperture mounted on the window of a photomultiplier and uniformly illuminating their concave surface. Since the distribution of the latex was not uniform, different areas of the cornea were examined. We assumed that fixation with formalin had a similar effect on the transparency of both corneas. Finally, the cornea, ciliary body, iris, and posterior segment of the eye globes were prepared for microscopy. Results At the time of corneal opacification, the rhesus monkey was +1.5 D hyperopic bilaterally, and both eyes had a 14.4 mm axial length. The procedure of opacification did not cause any obvious sign of inflammation in the anterior segment of the eye. When the Fig. 1. Cornea of a 13-month-old macaque monkey following stromal injection of latex at the age of 1 month. Although the distribution of the latex was not uniform, no transparent regions were present at the center of the cornea. Note that a few blood vessels invaded the opacity. animal was examined at the age of 4 months, a few blood vessels had grown from the limbus into the opaque portion of the cornea, and these had given rise to a scant vascular network. During the following months, the corneal vascularization remained stationary, and the appearance of the cornea at the age of 13 months is illustrated in Fig. 1. The axial length of both eyes was measured at 4, 7, and 13 months by ultrasonography, and the results are shown in Fig. 2. In both eyes, the axial length increased exponentially with age, but the eye with opaque cornea had a higher growth rate, so that at 13 months it was 1.24 mm longer than the control eye. The results of the ultrasound measurements at the end of the experiment are reported in Table I. This table demonstrates that the depth of the anterior chamber and the thickness of the lens are essentially identical in both eyes. Thus the difference in axial length is entirely accounted for by the elongation of the posterior segment of the eye with opaque cornea. The accuracy of the ultrasound measurements was confirmed by the findings at autopsy, which showed an interocular differ-

3 1234 Wiesel and Raviola Invest. Ophthalmol. Visual Sci. December 1979 (Macaco mulatto) RE- LATEX LE-NORMAL 6 8 AGE IN MONTHS 10 Fig. 2. Effect of corneal opacification on the size of the eye globe. The axial length of the opacified (continuous line) and normal (dotted line) eye were measured by ultrasonography at different time intervals from the latex injection (arrow). In both eyes the axial length increased exponentially with age, but the eye with opaque cornea grew at a faster rate and at the end of the experiment was 1.24 mm longer than the control eye. Table I. Ultrasound measurements (mm) in M. mulatta monkey Axial length Depth of the vitreous chamber Lens thickness Depth of the anterior segment (corneal thickness + anterior chamber * Number of measurements in parentheses. Right eye ± 0.21 S.D. (51)* ± 0.03 S.D. (10) 3.58 ± 0.02 S.D. (14) 2.72 ± 0.16 S.D. (20) Left eye ± 0.09 S.D. (50) ± 0.06 S.D. (21) 3.60 ± 0.04 S.D. (23) 2.69 ±0.05 S.D. (17) Table II. Ultrasound measurements (mm) in M. arctoides Axial length Depth of the vitreous chamber Lens thickness Depth of the anterior segment (corneal thickness + anterior chamber) Right eye ± 0.16 S.D. (34)* ±0.06 S.D. (17) 3.67 ± 0.15 S.D. (21) 2.45 ± 0.06 S.D. (14) Left eye ± 0.10S.D. (16) ± 0.05 S.D. (15) 3.79 ± 0.07 S.D. (10) 2.55 ±0.03 S.D. (11) * Number of measurements in parentheses. ence in axial length of 1.14 mm. The thickness, diameter, and weight of the lens were the same in both eyes. The comparison of the light transmittance in the opaque and transparent cornea showed that the latex injection had caused a light attenuation of 0.8 to 1.2 log units. At histological examination, the corneas in both eyes did not differ in either thickness or curvature (Fig. 3). In the opaque cornea both epithelium and endothelium appeared intact and normal in structure. The latex was concentrated in the superficial half of the corneal stroma and was contained in the cytoplasm of flattened macrophages lodged in narrow clefts between the collagen lamellae. The number of blood vessels in the corneal stroma was quite small. No pathological changes were observed in the uvea or retina of the eye with opaque cornea.

4 Volume 18 Number 12 Axial length increase after corneal opacification 1235 Normal Latex Fig. 3. At histological examination, the corneas of the opacified and normal eye showed the same thickness and curvature. The latex is contained in the cytoplasm of flattened macrophages (arrows) localized in the superficial stromal layers. A small number of blood vessels (arrow-heads) invaded the cornea. (Perfusion fixation with 10% formalin; osmium tetroxide postfixation; embedment in Epon-Araldite; toluidine blue staining; 0.5 /nm sections; X145.) Also in the second monkey (M. arctoides) the eye with opaque cornea grew at a faster rate than the normal eye, so that at the age of 17 months the interocular difference in axial length was 1.05 mm. The results of the ultrasound measurement at the end of the experiment are reported in Table II. It is clear from this table the difference in axial length between the two eyes was caused again by an elongation of the posterior segment. In this animal the corneal opacity attenuated light of 0.5 to 0.7 log units. Post- mortem measurements were consistent with those obtained in vivo, and the histological examination confirmed the findings described for tlie first animal. Discussion The present study demonstrates that opacification of the cornea in young macaque monkeys caused an elongation of the posterior segment of the eye globe and therefore had effects similar to those of lid fusion.2 Previously, we reported that the eye did not

5 1236 Wiesel and Raviola Invest. Ophthalmol. Visual Sci. December 1979 elongate and myopia did not develop when animals with fused lids were raised in the dark. 3 Considered together, our previous and present experiments show that (1) the growth of the eye is normal when the lids are fused but the visual input is removed by keeping animals in the dark and (2) the axial length increases when the lids are open, but normal vision is prevented by corneal opacification. Thus the refractive error caused by lid fusion is triggered by an abnormal visual input and is mediated by the central nervous system. Since bilateral lid suture caused myopia in both eyes, we have already excluded that, in monocular closure, myopia is caused by the asymmetrical input between the two eyes. 2 It is perplexing that corneal opacification has a less marked effect than lid fusion in increasing the axial length. The two experiments, however, differ in several respects. The latex injections were done in slightly older animals and the intensity and the wavelength composition of the light which reached the retina were not the same in the two conditions. Nevertheless, we still cannot rule out the possibility that the mechanical or thermal changes which accompany lid fusion might enhance the effects of the abnormal neural influences. Neonatal lid fusion in monkeys may have a clinical correlate in the infantile hemangiomas of the eyelids which are associated with severe myopia. 4 Furthermore, the result of the present study provides additional support to our suggestion that the myopia observed in retrolental fibroplasia may be caused by the opacities in the vitreous body. 3 Therefore it would be interesting to measure the axial length in both conditions mentioned above as well as in patients who developed in their infancy alterations of the dioptic media, such as corneal scars, traumatic or congenital cataract, and vitreous hemorrhages. It should be noted that both experimental and clinical myopias represent a disturbance of the postnatal growth of the eye and concomitant development of the refractive state. In monkeys, when an abnormal visual input is presented early in life, the growth pattern of the eye is grossly distorted and myopia ensues. Thus the experimental refractive error caused by lid fusion 1 ' 2 ' 5 7 may share a common mechanism with the myopia of caged monkeys 8 and cats, 9 ' 10 or with that produced by exposing monkeys to a restricted visual space, 11 ' 12 and with those cases of human myopia in which an environmental component has been suggested. 13 There are several mechanisms by which the central nervous system can modify the postnatal growth of the eye, and these include accommodation, the tension exerted by the extraocular muscles, and regulatory influences on the intraocular pressure. We hope in future to differentiate among these possibilities. We thank the staff" of the New England Regional Primate Research Center for assistance. REFERENCES 1. Wiesel, T.N., and Raviola, E.: Myopia following neonatal lid fusion in monkeys, ARVO Abstracts, p. 96, Wiesel, T.N., and Raviola, R.: Myopia and eye enlargement after neonatal lid fusion in monkeys, Nature 266:66, Raviola, E., and Wiesel, T.N.: Effect of dark-rearing on experimental myopia in monkeys, INVEST. OP- THALMOL. VISUAL SCI. 17:485, Robb, R.M.: Refractive errors associated with hemangiomas of the eyelids and orbit in infancy, Am. J. Ophthalmol. 83:52, Sherman, S.M., Norton, T.T., and Casagrande, V.A.: Myopia in the lid-sutured tree shrew (Tupaia glis), Brain Res. 124:154, Sommers, D., Kaiser-Kupfer, M.I., and Kupfer, C: Increased axial length of the eye following neonatal lid suture as measured with A-scan ultrasonography, 17(ARVO Suppl.):295, Wallman, J., and Turkel, J.: Extreme myopia produced by modest change in early visual experience, Science 201:1249, Young, F.A.: Visual refractive errors of wild and laboratory monkeys, E.E.N.T. Digest 27:55, Rose, L., Yinon, U., and Belkin, M.: Myopia induced in cats deprived of distance vision during development, Vision Res. 14:1029, Belkin, M., Yinon, U., Rose, L., and Reisert, I.: Effect of visual environment on refractive error of cats, Doc. Ophthalmol. 42:433, Young, F.A.: The effect of restricted visual space on the primate eye, Am. J. Ophthalmol. 52:799, Young, F.A.: The effect of restricted visual space on the refractive error of the young monkey eye, IN- VEST. OPHTHALMOL. 2:571, Duke-Elder, S.: Text-book of Ophthalmology, London, 1950, Henry Kimpton, vol. 4.