ocular malformations of the chick embryo produced...

Ocular malformations of the chick embryoproduced by photocoagulation

David O. Jesberg

Heat-produced necrotizing lesions of the chick embryo pigment epithelium and retina arerepaired by regeneration in these tissues. Regeneration is usually incomplete and may bedisorderly. Quantitative effects are technically difficult, but eyes of 4 to 5 days' incubationseem more capable of recovery than those injured after 7 days of incubation. Regenerationof pigment epithelium precedes regeneration in the retina, and this suggests a trophic in-fluence. Microphthalmia results more frequently when the anterior rim of the optic cup isinsulted.

0,ur interest in the study of experi-mentally produced lesions in embryo eyeshas been intensified by the recognition ofseveral definite teratogenic systems. Wenow recognize abnormalities resulting fromfetal infection with the rubella virus andToxoplasma gonclii. These etiologicaldemonstrations help clarify such abnormali-ties as macular coloboma and atypicalcoloboma. It is noteworthy that in themost recent edition of Developmental Ab-normalities of the Eye, Ida Mann1 hastaken a strong new viewpoint by empha-sizing the infectious nature of manypreviously obscure entities.

From the Francis I Proctor Foundation for Re-search in Ophthalmology and the Departmentof Ophthalmology, University of CaliforniaSchool of Medicine, San Francisco, Calif.

This work was supported in part by ResearchGrant B-3292 from the Institute of Neuro-logical Diseases and Blindness, United StatesPublic Health Service, Bethesda, Md., and theUniversity of California Committee on Research,Herstein Fund No. 262.

There is abundant evidence from ex-perimental teratology that physical agentsmay be teratogenic, and, in particular, thework of Rugh2 in which x-ray damage ofthe central nervous system was demon-strated is most interesting. Ocular defectsranging from anophthalmia and microph-thalmia to coloboma and retinal rosetteshave been produced by differential timingof the insult.3"5

Metabolic insult is also well establishedas an active ocular teratogenic system, andthe work of Nelson0' 7 has clearly demon-strated the effects of pteroylglutamic aciddeficiency. Many experimentalists haveperformed an operation on the embryo todetect the mechanisms of teratogenesis inchick and amphibian tissues. However, theharvest in such work is often small andunrewarding.8 We have produced focaldestructive lesions in chick embryo eyeswith the photocoagulator. This paper pre-sents the initial results of our work whichhas proved to be easily accomplished andunique in the nature of the lesion which isproduced.

348

Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933096/ on 08/27/2018

Volume 1Number 3 Ocular malformations of chick embryo 349

Materials and methodsHatchery eggs from a single source were used.

We noted uniform fertility except during the latesummer months. The incubator was the forced-air humidified type.

Shell windows are most easily prepared whenthe chorioallantoic membrane is well developedafter 6 to 8 days of incubation. At this time thedevelopment of the eye is relatively advancedand photocoagulation is easily carried out. Win-dows prepared on the fourth day of incubationhave proved moderately successful and presentthe developing eye at a size which permits photo-coagulation but with more difficulty than at 6 ormore days. Shell windows were prepared in thefollowing manner. Eggs which had been in-cubating in the horizontal position were candledand the shell surface marked to localize theembryo. The shell was then sterilized withaqueous Virac* solution. A drill hole was madeoverlying the air space in the blunt end of theegg. A second drill hole was made over thecenter of the embryo. The hole was madethrough the shell, but not through the shell mem-brane. With blunt forceps the shell opening wasenlarged to 10 mm., the membrane moistenedwith sterile Locke's solution, and small openingswere made through the shell membrane over theembryo and into the air space. The embryo andits membranes were dropped by evacuating theair space with a suction bulb. The opening overthe embryo was then enlarged, and paraffinswabbed around the opening. A sterile glass coverslip was placed on the paraffin and sealed inplace. The opening in the blunt end was closedwith paraffin. After moderate practice, this pro-cedure takes about 5 minutes per egg (Fig. 1).

Coagulations were made with the iris- andsurface-coagulating equipment on the Zeiss photo-coagulator through the glass window by holdingthe egg in one hand and the coagulator handlein the other. It was usually possible to rotate theegg so that the eye was just under the glass. Inalmost every case the right eye presented. If theembryo did not move during treatment, a singleshort application produced visible coagulation(Fig. 2).

Eggs were returned to the incubator promptlyafter coagulation and sacrificed at scheduled in-tervals by decapitation. The whole head wasfixed in Bourn's fluid for 24 to 48 hours and thenwashed in 70 per cent ethanol. The heads weredehydrated with ethanol and cleared in anilinefollowed by methyl salicylate after which theywere examined grossly, photographed, and stored(Figs. 3 and 4). The heads were embedded inparaffin for sectioning and cut serially. Repre-sentative parts of the series of sections were

Fig, 1. Shell window ready for photocoagulation.

AM

SK

"By Ruson Laboratory, Inc., Portland, Ore.

VIT.

Fig. 2. Normal 5 day chick showing tissue rela-tionships 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 wereexamined after 1 hour and at intervals up to 3weeks after the treatment.Results

Immediate lethal effect. In occasional in-stances at all ages the coagulation wasattended by massive hemorrhage and deathoccurred promptly. In most instances thesite of hemorrhage could be localized inthe head of the embryo. Such specimensand all others which died before sacrificewere discarded.

Cross effects. Although in some speci-mens the defects were easily seen throughthe window, the extent of the lesion was


350 JesbergInvestigative Ophthalmology

June 1962

Fig. 3. Ventral view of normal cleared 8 daychick to show both eyes.

Fig, 4. Cleared right eye oF 7 day normal chick.C.F., Plane of choroid fissure and developingpecten.

Fig. 5. Right microphthalmia in chick of totalincubation of 11 days. Photocoagulation clone onseventh day. R, Right side.

Fig. 6. Right microphthalmia with distortion ofanterior rim and choroid fissure. Treatment onsixth day with total incubation 7 days. R, Rightside.

best observed in the cleared specimen. Themost striking gross defect was microph-thalmia of the treated eye (Fig. 5). Thisresult occurred more frequently in eyeswhich were coagulated at or near the rimof the optic cup. It was seen in eyes whichwere treated at 4 to 7 days' incubation,and most frequently in the group coagu-lated on the fifth and sixth days of incuba-tion. There was distortion of the developingrim of the optic cup in some microph-thalmic eyes, and the lips of the choroidfissure showed minor areas of incompleteor disturbed closure (Fig. 6). Not all eyesbecame microphthalmic in spite of sizableand extensive coagulation lesions. Acoloboma of the iris and ciliary body wasthe usual result of an anteriorly placedcoagulation (Fig. 7). Coagulation posteriorto the equator failed to produce microph-thalmia in some eyes.

The white lesion resulting from coagula-tion was immediately visible and remainedwhite for 24 hours. There was often a zoneof hyperemia or hemorrhage at its margin(Fig. 8). The center of the lesion becamedepigmented during the first 24 hourperiod; the loss of pigmentation usuallyremained. Some distinct lesions produced



Fig. 7. Anterior segment coloboma of the righteye of chick incubated total of 11 days followingphotocoagulation on the seventh day.

on the fourth day were not detectable inthe cleared specimen. This suggested thatthe effect was reversible in eyes of 4 daysor less and irreversible after the fifth dayof incubation.

Microscopic effects. The early effects ofphotocoagulation at all incubation agesstudied were similar. Necrosis, fragmenta-tion of cells, dispersion of pigment gran-ules, interstitial hemorrhage, and separa-tion of tissue layers were regularly seen.The superficially placed scleral cartilageand skin were usually devoid of necroticchanges although occasionally a zone ofheat-damaged cartilage was apparent inthe solera (Fig. 9). Major destruction oc-curred in the pigment epithelium andretina. The choriocapillaris was absent inthe areas where pigment epithelium wasdestroyed (Fig. 10). The zone of coagu-lated retina usually became elevated in afold (Figs. 11 and 12). The immediateeffects were noted to be present for severaldays after injury and there was no ap-parent cellular reaction to the injury indi-cating localized mobilization of inflamma-tory cells. The debris became dispersedand it is presumed that some of it musthave been actively removed by phagocyticcellular action or fluid circulation, but thiswas not clearly demonstrated.

HEM

I

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.)


352 JesbergInvestigative Ophthalmology

June 1962

B

RET.

Fig. 10. Area of photocoagulation defect after4 days. Pigment epithelium is absent between Aand 73. The retina (RET.) demonstrates markednecrosis. Total incubation, 14 days. (x63. Re-duced Mi.)

HEM.

RET.

Fig. 11. Defect 24 hours after photocoagulationshowing interstitial hemorrhage (HEM.), pigmentepithelium (PE), and retinal (RET.) necrosis.<x63. Reduced %.)

Fig. 12. Photocoagulation defect placed anteriorlyand producing a retinal fold with necrosis.Coagulation on seventh day. Total incubation, 11days. (x25. Reduced Vs.)

Eyes coagulated on the fourth and fifthdays frequently failed to reveal a lesioneither grossly or microscopically afterseveral days, and it is believed that theyrepresent minimal insults where the re-generation was extremely prompt. Theregeneration of pigment epithelium wasfirst observed 4 days after coagulation onthe fourth to eighth days. The regenerativeprocess started from the undamagedepithelium (Figs. 13 and 14). The re-generating tissue extended incompletelyover the defect. The cells showed de-creased size and reduced pigment contentat the advancing edge. The advancing cellswere accompanied by an advancing chorio-capillaris.

Regeneration in the sensory retina wasfirst observed 6 days after injury and ap-peared to be initiated in situ although cellmigration from undamaged areas mighthave occurred (Fig. 15), The regeneratingretinal cells did not organize into normal-appearing retina. The celJs of the outerretinal layer formed into circular groupshaving the appearance of rosettes (Fig.16). This was apparently a result of thegreater rate of growth in these cells (Fig.17). This effect was observable 15 daysafter injury.

Fibroblastic replacement of deficienttissue was noted to be present in onespecimen 18 days after injury (Fig. IS).The iris and ciliary body were replaced bya well-organized fibrous tissue which was

it'ig. 13. Advancing edge of regenerating pigmentepithelium 18 days after coagulation. A, Pigmentepithelial cells. B, Zone devoid of pigment epithe-lium. (xlGO. Reduced %.)



PIG EP,

Fig, 14, Area of early regeneration in pigmentepithelium (PIG. EP.) mixed with residual necroticdebris 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) 6days after coagulation. (x400. Reduced VM.)

integrally related to the cornea anteriorlyand attached firmly to the lens at itsequator.

Discussion

The characteristic of developing tissueswhich distinguishes them from fully dif-ferentiated tissues is the general absenceof the cellular and fibroblastic cicatricialresponse to injury which is so characteris-tically seen in mature tissues. Embryonictissues react by dedifferentiation and re-generation and the capacity to accomplishthis decreases with the degree of differ-entiation. That dedifferentiation occursprior to regeneration is evidenced by thereduction of pigment in the regeneratinglayer of pigment epithelial cells. Recentstudies of the fine structure of pigment-

Fig. 17. Retinal rosettes (arrows) illustrating theirregular lining the regenerating cells have formed.(x400. Reduced Vs.)

ANT. CH.

Fig. 18. Fibrous tissue replacing the deficient irisof an anterior coagulation with adherence to thelens (L) and cornea (C). ANT. CH., Anteriorchamber. (xlOO. Reduced Vz.)

producing cells of the eye" demonstrateelaborate cytoplasmic structures which sug-gest that in addition to pigment these cellsmay produce secretory products whichare capable of influencing development and


354 JesbergInvestigative Oplithalmologij

June 1962

cellular activity in adjacent tissues, such asthe retina. This may be supported by thefinding here that regenerative activity isseen first in the pigment epithelium, and,following this, the choriocapillaris andretina apparently become active.

The distortion of the regenerating retinalcells into rosettes is not a unique finding;it has been observed in retinal tissue in-jured by x-ray.3 It is not unlike the rosetteswhich characterize retinal dysplasia inhuman beings and may suggest that theetiology of this condition lies in a severenecrotizing insult at an early age. Thedysplastic findings would then representregenerative activity which has failed tobecome orderly and normal in arrange-ment. The unique feature of the photo-coagulated lesion as compared with thatproduced by x-ray is the simultaneous de-struction of pigment epithelium and retina.Rugh found that with x-ray the pigmentepithelium was relatively undamaged inmouse embryos, whereas the retina demon-strated massive damage initially with sub-sequent regeneration. Recent experimentalintraocular herpes simplex virus infectionsin chick embryos in our laboratories haveshown that, when the retina was severelydamaged by virus, the pigment epitheliumwas still intact.10

The frequency of microphthalmia wasmuch greater in eyes with lesions placedat the anterior rim of the optic cup or inthe area of the choroid fissure. This find-ing emphasizes the well established factthat greater insult occurs when the mostactively developing embryonic areas areinsulted.11

The technical assistance of Miss Ruth Owenand Mr. Kevin Harrington is gratefully acknowl-edged.

REFERENCES1. Mann, Ida: Developmental abnormalities of

the eye, ed. 2, Philadelphia, 1957, J. B.Lippincott Company, pp. 162-167.

2. Rugh, R.: X-irradiation effects on the humanfetus, J. Pediat. 52: 531, 1958.

3. Rugh, R., and Wolf, J.: Resilience of thefetal eye following radiation insult, Proc. Soc.Exper. Biol. & Med. 89: 248, 1955.

4. Hicks, S. P., Brown, B. L., and D'Amato, C.J.: Regeneration and malformation in thenervous system, eye, and mesenchyme of themammalian embryo after radiation injury,Am. J. Path. 33: 459, 1957.

5. Wilson, J. C , Jordan, H. C , and Brent, R. L.:Effects of irradiation on embryonic develop-ment. II. X-rays on the ninth day of gestationin the rat, Am. J. Anat. 92: 153, 1953.

6. Nelson, M. M., Wright, H. V., Band, C. D.C, and Evans, H. M.: Effect of 36-hourperiod of pteroylglutamic acid deficiency onfetal development in the rat, Proc. Soc.Exper. Biol. & Med. 92: 554, 1956.

7. Nelson, Marjorie M.: Teratogenic effects ofpteroylglutamic acid deficiency in the rat, inWolstenholme, G. E. W., and O'Connor, C.M., editors: Ciba Foundation Symposium oncongenital malformations, Boston, 1960,Little, Brown & Company, pp. 134-157.

8. Gayer, K., and Hamburger, V.: Develop-mental potencies of eye primordia in chick,J. Exper. Zool. 93: 147, 1943.

9. Yamada, Eichi: The fine structure of pig-ment epithelium in the turtle eye, in Smelser,G. K., editor: The structure of the eye, NewYork, 1961, Academic Press, Inc.

10. Harrington, K.: Unpublished data.11. Patten, B. M.: Varying developmental mecha-

nisms in teratology, Pediatrics 19: 734, 1957.


ocular malformations of the chick embryo produced...

Documents