Ocular malformations of the chick embryo produced by photocoagulation. David O. Jesberg

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1 Ocular malformations of the chick embryo produced by photocoagulation David O. Jesberg Heat-produced necrotizing lesions of the chick embryo pigment epithelium and retina are repaired by regeneration in these tissues. Regeneration is usually incomplete and may be disorderly. Quantitative effects are technically difficult, but eyes of 4 to 5 days' incubation seem more capable of recovery than those injured after 7 days of incubation. Regeneration of pigment epithelium precedes regeneration in the retina, and this suggests a trophic influence. Microphthalmia results more frequently when the anterior rim of the optic cup is insulted. 0,ur interest in the study of experimentally produced lesions in embryo eyes has been intensified by the recognition of several definite teratogenic systems. We now recognize abnormalities resulting from fetal infection with the rubella virus and Toxoplasma gonclii. These etiological demonstrations help clarify such abnormalities as macular coloboma and atypical coloboma. It is noteworthy that in the most recent edition of Developmental Abnormalities of the Eye, Ida Mann 1 has taken a strong new viewpoint by emphasizing the infectious nature of many previously obscure entities. From the Francis I Proctor Foundation for Research in Ophthalmology and the Department of Ophthalmology, University of California School of Medicine, San Francisco, Calif. This work was supported in part by Research Grant B-3292 from the Institute of Neurological Diseases and Blindness, United States Public Health Service, Bethesda, Md., and the University of California Committee on Research, Herstein Fund No There is abundant evidence from experimental teratology that physical agents may be teratogenic, and, in particular, the work of Rugh 2 in which x-ray damage of the central nervous system was demonstrated is most interesting. Ocular defects ranging from anophthalmia and microphthalmia to coloboma and retinal rosettes have been produced by differential timing of the insult. 3 " 5 Metabolic insult is also well established as an active ocular teratogenic system, and the work of Nelson 0 ' 7 has clearly demonstrated the effects of pteroylglutamic acid deficiency. Many experimentalists have performed an operation on the embryo to detect the mechanisms of teratogenesis in chick and amphibian tissues. However, the harvest in such work is often small and unrewarding. 8 We have produced focal destructive lesions in chick embryo eyes with the photocoagulator. This paper presents the initial results of our work which has proved to be easily accomplished and unique in the nature of the lesion which is produced. 348

2 Volume 1 Number 3 Ocular malformations of chick embryo 349 Materials and methods Hatchery eggs from a single source were used. We noted uniform fertility except during the late summer months. The incubator was the forcedair humidified type. Shell windows are most easily prepared when the chorioallantoic membrane is well developed after 6 to 8 days of incubation. At this time the development of the eye is relatively advanced and photocoagulation is easily carried out. Windows prepared on the fourth day of incubation have proved moderately successful and present the developing eye at a size which permits photocoagulation but with more difficulty than at 6 or more days. Shell windows were prepared in the following manner. Eggs which had been incubating in the horizontal position were candled and the shell surface marked to localize the embryo. The shell was then sterilized with aqueous Virac* solution. A drill hole was made overlying the air space in the blunt end of the egg. A second drill hole was made over the center of the embryo. The hole was made through the shell, but not through the shell membrane. With blunt forceps the shell opening was enlarged to 10 mm., the membrane moistened with sterile Locke's solution, and small openings were made through the shell membrane over the embryo and into the air space. The embryo and its membranes were dropped by evacuating the air space with a suction bulb. The opening over the embryo was then enlarged, and paraffin swabbed around the opening. A sterile glass cover slip was placed on the paraffin and sealed in place. The opening in the blunt end was closed with paraffin. After moderate practice, this procedure takes about 5 minutes per egg (Fig. 1). Coagulations were made with the iris- and surface-coagulating equipment on the Zeiss photocoagulator through the glass window by holding the egg in one hand and the coagulator handle in the other. It was usually possible to rotate the egg so that the eye was just under the glass. In almost every case the right eye presented. If the embryo did not move during treatment, a single short application produced visible coagulation (Fig. 2). Eggs were returned to the incubator promptly after coagulation and sacrificed at scheduled intervals by decapitation. The whole head was fixed in Bourn's fluid for 24 to 48 hours and then washed in 70 per cent ethanol. The heads were dehydrated with ethanol and cleared in aniline followed by methyl salicylate after which they were examined grossly, photographed, and stored (Figs. 3 and 4). The heads were embedded in paraffin for sectioning and cut serially. Representative parts of the series of sections were "By Ruson Laboratory, Inc., Portland, Ore. Fig, 1. Shell window ready for photocoagulation. VIT. AM SK Fig. 2. Normal 5 day chick showing tissue relationships and course of light. AM, Amnion. SK, Skin surface. P.E., Pigment epithelium. RET., Retina. VIT., Vitreous. mounted and stained with hematoxylin and eosin. This procedure was accomplished on embryos of 4 to 11 days' incubation. The effects were examined after 1 hour and at intervals up to 3 weeks after the treatment. Results Immediate lethal effect. In occasional instances at all ages the coagulation was attended by massive hemorrhage and death occurred promptly. In most instances the site of hemorrhage could be localized in the head of the embryo. Such specimens and all others which died before sacrifice were discarded. Cross effects. Although in some specimens the defects were easily seen through the window, the extent of the lesion was

3 350 Investigative Ophthalmology June 1962 Jesberg Fig. 3. Ventral view of normal cleared 8 day chick to show both eyes. Fig. 6. Right microphthalmia with distortion of anterior rim and choroid fissure. Treatment on sixth day with total incubation 7 days. R, Right side. Fig, 4. Cleared right eye of 7 day normal chick. C.F., Plane of choroid fissure and developing pecten. Fig. 5. Right microphthalmia in chick of total incubation of 11 days. Photocoagulation clone on seventh day. R, Right side. best observed in the cleared specimen. The most striking gross defect was microphthalmia of the treated eye (Fig. 5). This result occurred more frequently in eyes which were coagulated at or near the rim of the optic cup. It was seen in eyes which were treated at 4 to 7 days' incubation, and most frequently in the group coagulated on the fifth and sixth days of incubation. There was distortion of the developing rim of the optic cup in some microphthalmic eyes, and the lips of the choroid fissure showed minor areas of incomplete or disturbed closure (Fig. 6). Not all eyes became microphthalmic in spite of sizable and extensive coagulation lesions. A coloboma of the iris and ciliary body was the usual result of an anteriorly placed coagulation (Fig. 7). Coagulation posterior to the equator failed to produce microphthalmia in some eyes. The white lesion resulting from coagulation was immediately visible and remained white for 24 hours. There was often a zone of hyperemia or hemorrhage at its margin (Fig. 8). The center of the lesion became depigmented during the first 24 hour period; the loss of pigmentation usually remained. Some distinct lesions produced

4 Volume 1 Number 3 Ocular malformations of chick embryo 351 HEM I Fig. 7. Anterior segment coloboma of the right eye of chick incubated total of 11 days following photocoagulation on the seventh day. on the fourth day were not detectable in the cleared specimen. This suggested that the effect was reversible in eyes of 4 days or less and irreversible after the fifth day of incubation. Microscopic effects. The early effects of photocoagulation at all incubation ages studied were similar. Necrosis, fragmentation of cells, dispersion of pigment granules, interstitial hemorrhage, and separation of tissue layers were regularly seen. The superficially placed scleral cartilage and skin were usually devoid of necrotic changes although occasionally a zone of heat-damaged cartilage was apparent in the solera (Fig. 9). Major destruction occurred in the pigment epithelium and retina. The choriocapillaris was absent in the areas where pigment epithelium was destroyed (Fig. 10). The zone of coagulated retina usually became elevated in a fold (Figs. 11 and 12). The immediate effects were noted to be present for several days after injury and there was no apparent cellular reaction to the injury indicating localized mobilization of inflammatory cells. The debris became dispersed and it is presumed that some of it must have been actively removed by phagocytic cellular action or fluid circulation, but this was not clearly demonstrated. Fig. 8. Photocoagulation effect after 24 hours. There is an area of hemorrhage at the margin (HEM). Total incubation, 7 days. Fig, 9. Zone of devitalized scleral cartilage (arrow) from coagulation on the eleventh day. Total incubation, 21 days. (xloo.)

5 352 Jesberg Investigative Ophthalmology June 1962 RET. Fig. 10. Area of photocoagulation defect after 4 days. Pigment epithelium is absent between A and 73. The retina (RET.) demonstrates marked necrosis. Total incubation, 14 days. (x63. Reduced Mi.) HEM. RET. Fig. 11. Defect 24 hours after photocoagulation showing interstitial hemorrhage (HEM.), pigment epithelium (PE), and retinal (RET.) necrosis. <x63. Reduced %.) B Eyes coagulated on the fourth and fifth days frequently failed to reveal a lesion either grossly or microscopically after several days, and it is believed that they represent minimal insults where the regeneration was extremely prompt. The regeneration of pigment epithelium was first observed 4 days after coagulation on the fourth to eighth days. The regenerative process started from the undamaged epithelium (Figs. 13 and 14). The regenerating tissue extended incompletely over the defect. The cells showed decreased size and reduced pigment content at the advancing edge. The advancing cells were accompanied by an advancing choriocapillaris. Regeneration in the sensory retina was first observed 6 days after injury and appeared to be initiated in situ although cell migration from undamaged areas might have occurred (Fig. 15), The regenerating retinal cells did not organize into normalappearing retina. The celjs of the outer retinal layer formed into circular groups having the appearance of rosettes (Fig. 16). This was apparently a result of the greater rate of growth in these cells (Fig. 17). This effect was observable 15 days after injury. Fibroblastic replacement of deficient tissue was noted to be present in one specimen 18 days after injury (Fig. IS). The iris and ciliary body were replaced by a well-organized fibrous tissue which was Fig. 12. Photocoagulation defect placed anteriorly and producing a retinal fold with necrosis. Coagulation on seventh day. Total incubation, 11 days. (x25. Reduced Vs.) it'ig. 13. Advancing edge of regenerating pigment epithelium 18 days after coagulation. A, Pigment epithelial cells. B, Zone devoid of pigment epithelium. (xlgo. Reduced %.)

6 Volume 1 Number 3 Ocular malformations of chick embryo 353 PIG EP, Fig, 14, Area of early regeneration in pigment epithelium (PIG. EP.) mixed with residual necrotic debris 4 days after coagulation, {xloo. Reduced VIT. Fig. 16. Several areas of retinal rosettes (arrows) 18 days after coagulation, (xl60, Reduced %.) Fig. 15. Area of retinal regeneration (RET. A) adjacent to the undamaged retina (RET, B) 6 days after coagulation. (x400. Reduced VM.) Fig. 17. Retinal rosettes (arrows) illustrating the irregular lining the regenerating cells have formed. (x400. Reduced Vs.) integrally related to the cornea anteriorly and attached firmly to the lens at its equator. Discussion The characteristic of developing tissues which distinguishes them from fully differentiated tissues is the general absence of the cellular and fibroblastic cicatricial response to injury which is so characteristically seen in mature tissues. Embryonic tissues react by dedifferentiation and regeneration and the capacity to accomplish this decreases with the degree of differentiation. That dedifferentiation occurs prior to regeneration is evidenced by the reduction of pigment in the regenerating layer of pigment epithelial cells. Recent studies of the fine structure of pigment- ANT. CH. Fig. 18. Fibrous tissue replacing the deficient iris of an anterior coagulation with adherence to the lens (L) and cornea (C). ANT. CH., Anterior chamber. (xloo. Reduced Vz.) producing cells of the eye" demonstrate elaborate cytoplasmic structures which suggest that in addition to pigment these cells may produce secretory products which are capable of influencing development and

7 354 Jesberg Investigative Oplithalmologij June 1962 cellular activity in adjacent tissues, such as the retina. This may be supported by the finding here that regenerative activity is seen first in the pigment epithelium, and, following this, the choriocapillaris and retina apparently become active. The distortion of the regenerating retinal cells into rosettes is not a unique finding; it has been observed in retinal tissue injured by x-ray. 3 It is not unlike the rosettes which characterize retinal dysplasia in human beings and may suggest that the etiology of this condition lies in a severe necrotizing insult at an early age. The dysplastic findings would then represent regenerative activity which has failed to become orderly and normal in arrangement. The unique feature of the photocoagulated lesion as compared with that produced by x-ray is the simultaneous destruction of pigment epithelium and retina. Rugh found that with x-ray the pigment epithelium was relatively undamaged in mouse embryos, whereas the retina demonstrated massive damage initially with subsequent regeneration. Recent experimental intraocular herpes simplex virus infections in chick embryos in our laboratories have shown that, when the retina was severely damaged by virus, the pigment epithelium was still intact. 10 The frequency of microphthalmia was much greater in eyes with lesions placed at the anterior rim of the optic cup or in the area of the choroid fissure. This finding emphasizes the well established fact that greater insult occurs when the most actively developing embryonic areas are insulted. 11 The technical assistance of Miss Ruth Owen and Mr. Kevin Harrington is gratefully acknowledged. REFERENCES 1. Mann, Ida: Developmental abnormalities of the eye, ed. 2, Philadelphia, 1957, J. B. Lippincott Company, pp Rugh, R.: X-irradiation effects on the human fetus, J. Pediat. 52: 531, Rugh, R., and Wolf, J.: Resilience of the fetal eye following radiation insult, Proc. Soc. Exper. Biol. & Med. 89: 248, Hicks, S. P., Brown, B. L., and D'Amato, C. J.: Regeneration and malformation in the nervous system, eye, and mesenchyme of the mammalian embryo after radiation injury, Am. J. Path. 33: 459, Wilson, J. C, Jordan, H. C, and Brent, R. L.: Effects of irradiation on embryonic development. II. X-rays on the ninth day of gestation in the rat, Am. J. Anat. 92: 153, Nelson, M. M., Wright, H. V., Band, C. D. C, and Evans, H. M.: Effect of 36-hour period of pteroylglutamic acid deficiency on fetal development in the rat, Proc. Soc. Exper. Biol. & Med. 92: 554, Nelson, Marjorie M.: Teratogenic effects of pteroylglutamic acid deficiency in the rat, in Wolstenholme, G. E. W., and O'Connor, C. M., editors: Ciba Foundation Symposium on congenital malformations, Boston, 1960, Little, Brown & Company, pp Gayer, K., and Hamburger, V.: Developmental potencies of eye primordia in chick, J. Exper. Zool. 93: 147, Yamada, Eichi: The fine structure of pigment epithelium in the turtle eye, in Smelser, G. K., editor: The structure of the eye, New York, 1961, Academic Press, Inc. 10. Harrington, K.: Unpublished data. 11. Patten, B. M.: Varying developmental mechanisms in teratology, Pediatrics 19: 734, 1957.