corneal development. i. corneal transparency

CORNEAL DEVELOPMENT

I. CORNEAL TRANSPARENCY ' ALFRED .J. COULOMBRE AND J A N E JJ. COULOMBRE

Department of Amtomy, Yale] Unzversity School of Medicine, New Haven 11, Connecticut

SIX FIGURES

INTKOUUCTION

It is held that the vertebrate cornea is transparent from the time it first appears until it matures, (Mann, '50). A casual examination of the chick cornea during its development reveals that this is not so. The cornea, like other embryonic tissues, is, in fact, highly translucent at very early stages; but i t becomes progressively more opaque as development proceeds, until a reversal occurs and the cornea acquires the adult transparency (van den Hooff, '51). Failure to observe intermediatc stages may have led to the carlier misconceptions.

The fact that the cornea achieves adult transparency duriiig a restricted period of development invites both descriptive and experimental investigation. For , although much work has berii done with adult corneas in an attempt to uiicovcr the factors miderlying corneal transparency, ( eg, Hart and Cliardlcr, '48a, '48b; Davson, '55; Cogan and Kinsey, '42, etc.) the study of adult tissues has limitations. Thus, in the fully developed cornea a large complex of factors and conditions coexist in time and are difficult to separate experimentally; while in the developing cornea factors come into operation, and conditions into being, in a n orderly temporal sequence. A kno\vledgc

* This study was aided in part by a grant (United Stntcs Public Health Rervicxc B-870) from the National Institute of Neurological Diswser and Rlindnws, ancl in part by a grant from tlic Jnnies ITudson Brown Mcmorinl F~uld, Yale School of Medicine.

1 JOURNAL OF CET~I , l l lAR A \ T > C703rI'4RdTIVC FHVSTOLORY, VOL 5 I , U O 1

PERItUARY 1 9 58

2 COl~LOiMBRF, AND COULOMBRE

of the order in which events occur during corneal development, or detection of their coiicurraiice, would be of great value in cstablishiiig the physical basis for corneal transparency.

Studies of adult cornea have implicatcd a nuinbcr of factors affecting corncal transparency. Of these, corneal hydration, stromal mucopolysaccliarides arid the degree of packing of the stromal collagcii arc most often mentioned. The present study describes the order of appearance of these factors and conditions during the development of corneal transparency in the chick emllryo.

Both quantitative and qualitativcl rncthods were used to de- termiiic the time course of development of transparency, the chaiige in corneal water content as transparency develops, the localizatioii of stromal rrictacliromatic mucopolysacchar- ides at different ages, and the developnierital changes in stro- rrial collagen. The results, of intrinsic interest, contribute, in udditioii, to the background of information available f o r experimental studies of tlic tlcvclopmental physiology of the cor11c;;I.

MATERTAT,H A N D METIIODS

Tlic animals iisecl were chick embryos from eggs incubated a t 37.5"C in R foiwd draft incubator. All ages are given as days following the onsct of incubation.

Two i*cllatcd optical properties of tlie cornea, transparency and liglit transmission, were investigated. Corneal transparency was demonstratcd at different ages by a photographic procedure. Corneas of c1acli age wcre removed quickly f roni the embryonic eye and laid, convcx side up, over a copper grid (180 perforations per ern) in tlie bottom of a flat glass dish filled with a 0.05 ?tl aqueous solution of NaCl. Using an optical system wdiich magnified six times, the grid was photographed through the cornea under staiidardized conditions of transmitted lighting and exposure. The csposccl films mere developed arid printed under uniform conditions. This method was used solely to illustrate qualitative cliariges in transparency dui*ing drvchlopmen t.

COl<iY EAL TRANSPARENCY 3

To measure the percent of incident light transmitted by corneas of different ages, a photometric procedure was employed. The corneas were isolated and immersed as descriloed above except that the copper grid was omitted. The glass dish with its coiitaiiied cornea was placed in a photometer in such a way that a narrow, columnated beam of white light passed through the center of the cornea before striking the photocell. Since the photometer had previously been balanced at a 100% read- ing with the fluid filled dish alone in the light path, readings could he read directly as percent light transmission hp the cornea.

To deterniiiie changes in corneal water content, measurements of corneal wet weight and dry weight were nrade. Thc ease with which the cornea is isolated aiid the accuracy of corneal weight determinations were increased by tlre nature of the corneal linibus of tlre chick during the period uiider study. A thin band of collagen surrounds the chick corrrw and separates it from the row of prri-corneal cartilages. Wit11 the use of watchmakers forceps the cornea separates cleanly from the eye along this collagenous band. Corneas thus isolated were blotted on filter paper and weighed immediately to the nearest 0.01 mg in aluminum tares, on a torsion spring balance (25 mg capacity). Subsequently the corneas, still in their tares, were dried at 110°C for one hour (a procedure which reduced the corneas of all the ages studied to constant weight) and reweighed. These measurements were made 017

ten corneas on each day between the ninth aiid twenty-fourth inclusive. From the mean \vet weights and dry weights so obtained, corneal water content was calculated as mg of water per mg of dry weight.

To localize corneal metachromatic mucopolysaccharide in time and space toluidine blue staining was used. Eyes were fixed in Rossman’s fluid a t daily intervals from the tenth to the twenty-fourth clays. These eyes \\-ere embedded in par- affin, sectioned at 6 p, affixed to slides, deparaffinized in xylene and hydrated through a series of alcohol solutions. Sections so prepared were stained in a 0.033% aqueous solution of tolui-

4 COULOMBRE: AND COULOMBBE

cline-blue-0 a t 1)H 3.5-4.0 and then dehydrated and mounted in balsam.

12 E 8 UL T S

It was necessary at the outset of this study to determine ac- curately the time course of development of corneal transparency. This information permitted a more directed search for correlated events.

Gh cr ng es in co rn eal t r(i 11 s p ti re $ 1 CIJ dzi ring d ev el o 11 Inend. The chick cornea is opalescent prior to tlic fourteenth day, but

Fig. 1

Fig. 2

Fig. 3

Copper grid photographed by transmitted light without an overlying

Copper grid photogrnphrd by transmitted light through a 13 day chick

Copper grid photographed by traiisniitted light through x 20 day chick

cornea.

embryo cornea.

embryo cornea.

thereafter becomes inore transparent until, by the nineteenth day, it assumes the glasslike transparency characteristic of the adult cornea. The photographs taken to demonstrate the changes show a scarcely discernible grid image prior to the fourteenth day. Between the fourteenth and the nineteenth day the grid images heconie increasingly clear (figs. 1-3) due to an increase in the sliarpness of the image and to an increase in the contrast between the images of the grid bars and the grid interstices. The latter observation suggested that in ad- dition to an iiicrease in transparency, per se, there is also a

CORNEAL TRANSPARENCY 5

related increase in the amount of light transmitted by the cornea.

Chamges in corneal light tra.msmission dwimg developme nt. To measure the transmission of light by the developing cornea, the photometric technique described above was employed. Measurements of per cent transmission were made on 10 corneas on each day between the tenth and twenty-first days in-

r SO-

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t / 2 2 10- x 5,

301 1 . HATCHING . I

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=+ri I 0 . I2 I4 16 18 20 22 24

AGE (DAYS) Fig. 4 The percent of incident light transmitted by the chick embryo cornea

a t different ages. Each point represents the mean of measurements on 10 corneas. The vertical lines indicate standard deviation.

AGE (DAYS) Fig. 4 The percent of incident light transmitted by the chick embryo cornea

a t different ages. Each point represents the mean of measurements on 10 corneas. The vertical lines indicate standard deviation.

elusive (fig. 4). Before the fourteenth day the chick cornea transmits about 40% of incident light. After the fourteenth day the amount of light transmitted rises sharply until the nineteenth day, when the adult level (about 96% under our conditions of measurement) is achieved. Once the increase has begun it proceeds without change in rate until it levels off at 19 days. Changes in both transparency and transmission are begun and are completed rather abruptly. We are thus sup-

6 COULOMBRE AND COULOMBRE

plied with well defined times in development when changes can be sought that are correlated with the development of a transparent cornea.

Changes in corneal water content during development. The importance of the amount of water in the cornea in relation to transparency has been emphasized by many investigators dealing with the adult cornea. It seems, therefore, desirable to know something of the changes in the relative water content of the cornea during the period in development when it is becoming transparent. To obtain this information, wet and dry weights were determined for each day during the last half of embryonic development.

The dry weight of the cornea increases steadily as development proceeds (fig. 5 ) . The wet weight, by contrast, increases rapidly until about the fourteenth day, and thereafter at a markedly slower rate (fig. 5). Correlated with this change is the fact that prior to the fourteenth day, the cornea is relatively thick, being more plano-convex than concavo-convex, and that following the fourteenth day the cornea appears less swollen and its epithelial and endothelial borders become progressively more parallel throughout their extent.

These findings suggest that there is a progressive, relative dehydration of the cornea as development proceeds. This is confirmed when corneal water content is calculated at different ages (fig. 6). There is a relative loss of water until about the nineteenth day, when it becomes stabilized at the adult value. Relative water loss is most rapid between the fourteenth and nineteenth days.

Corneal nzetachrornatic mucopolysaccharid~es. The search for other events correlating in time with the development of corneal transparency led to an investigation of the appearance during development of metachromatic, stromal mucopolysac- charides. Sections of corneas from embryos less than fourteen days old which are stained with toluidine blue reveal no metachromatic material in the corneal stroma. After the fourteenth day metachromasia is detected first at the endothelial border of the stroma just inside Descemet’s membrane. As develop-


ment proceeds the metachromatic zone intensifies and spreads toward the epithelial border of the stroma. The entire ,Aroma is metachromatic by the eighteenth or nineteenth day.

Coextensive with the spread of metachromatic material tightly packed, linearly oriented bundles or plates of collagenous fibers are laid down in successive portions of the stroma. This is in contrast to the sparse and disoriented condi1,ion of collagen fibers prior to the advent of metachromasia.

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I .o "s 0.9 s 0.0 I- 0.1 ,O 0.6 I

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0 OA

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0.06

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w w . . . . . . P 10 I1 I2 I3 14 15 16 I? 18 19 20 21 22 23 24

AG€ (DAYS)

Fig. 5 Semi-log plots of corneal dry weight (upper curve), and c o r n d wet weight (lower curve) as functions of age. Each point represents the mean of measurements on 10 corneas. The vertical lines indicate standard deviation.


A qualitative observation correlates well with the above facts. Before the fourteenth day the cornea is soft and is easily torn apart. On the fourteenth day it begins to toughen and becomes more difficult t o tear with forceps as development proceeds and more tightly packed collagen is laid down.

HATCHING , 1 1 1 , 1 , , , , , ~ , , ,

9 10 I I 12 13 14 15 16 17 18 19 20 21 22 23 24 INWEATION AGE IN MYS

Fig. 6 Semi-log plot of the amount of water per unit dry weight of the cornea as a function of age. Each value is caIculated from the wet weight and dry weight data in figure 5.

DISCUSSION

The sequence of events that emerges from this descriptive study affords clues of the relative roles played by each of several factors in the development of corneal transparency. The establishment of causal sequences must await the more critical evidence of experimental studies of the developing cornea. I n the meantime, the data presented here not only suggest the experiments which must be done, but indicate the


times at which important changes occur in the developing cornea, and at which experimental interference would prove more informative.

Prior to the fourteenth day the cornea is relatively thick, opaque, contains relatively disoriented collagen, holds a large amount of water relative to dry weight and contains .no de- tectable metachromatic material. The cornea shows an increase in light transmission and in transparency beginning on the fourteenth day. At the same time a group of associated changes are initiated. Metachromatic material appears in the stroma. Densely packed stromal collagen is seen for the first time. Corneal water loss is accelerated. The cornea becomes thinner. As the cornea increases in transparency between the fourteenth and nineteenth days there is a progressive increase in the absolute and relative stromal volume occupied by metachromatic material and dense collagen, and a continued loss of water relative to dry weight. On the eighteenth or nineteenth days metachromatic material and dense collagen fill the stroma. By the nineteenth day the water content levels off at the adult value.

Simultaneously with the publication of an abstract of these findings on the chick (Coulombre '56), Smelser and Ozanics ('56) published an abstract of a similar study of the rabbit cornea. In all major respects the results reported in both ab- tracts are similar. Therefore the concurrence of a relative loss of water, a deposition of metachromatic material, the appearance of closely packed collagen and the developme n t of transparency may also be the general course of events in rnam- mals during the development of the cornea.

The general increase in transparency between the fourteenth day and hatching reported by van den Hooff ('51) has been confirmed in the present study. The transient decreas,e in transparency at nineteen days which he describes did not occur under our conditions of measurement. This discrepancy in results is not yet explained.

The cornea begins to show a relative loss of water, without a concomittant increase in transparency, before the pivotal


fourteenth day. This suggests that, while an appropriate level of corneal dehydration is a necessary condition for transparency, it is not a sufficient condition. The first appearance of metachromasia and packed collagen at the same time that corneal transparency begins to increase, suggests that these events are of more central importance in the development of corneal transparency. The facts that packed collagen and metachromatic mucopolysaccharide appear at the same time and in the same place, and spread at the same rate through the thickness of the cornea, suggest an intimate relationship between metachromatic mucopolysaccharide and the oriented packing of the collagen fibers.

Electron micrographs of chick embryonic cornea reveal stromal collagen at very young ages (personal communica- tion from Dr. Marie Jakus). I t is still an open question whet- her the changes in stromal collagen which begin on the fourteenth day represent an increase in the rate of collagen syn- thesis or simply a packing of preexisting collagen plus new collagen being laid down at an unchanged rate. I f the former alternative holds one would expect an abrupt change in the rate of increase of corneal dry weight. Since no such abrupt change occurs (fig. 5) we are inclined to accept tentatively the latter alternative.

SUMMARY

1. I n the chick, corneal transparency and light transmission begin to increase on the fourteenth day of incubation, and achieve adult levels by the nineteenth day.

2. The relative water content of the cornea decreases from a level of well over 90% of wet weight at 9 days to the adult level of 76% at nineteen days. The period of most rapid de- crease lies between the fourteenth and nineteenth days.

Metachromatic mucopolysaccharide appears in the stroma for the first time on the fourteenth day, just inside Dece- met’s membrane. It spreads throughout the stroma during the ensuing 4 or 5 days.

3.


4. Densely packed stromal collagen appears at the same time and in the same place as the metachromatic material, and spreads through the stroma at the same rate.

LITERATURE CITED

COGAN, D. G., AND V. E. KINSEY 1942 The cornea. V. Physiologic aspects.

COULOMBRE, A. J. 1956 Development of corneal transparency. Anat. Re’:., 164 :

DAVSON, €I. 1955 The hydration of the cornea. Biochem. J., 59: 24-28. ITART, W. M., AND B. F. CHANDLER 1948a The cornea. I. Swelling properties

of the fibrous tunics of the eye. Arch. Ophthal., 2nd Seric,s, 40:

19481-3 The cornea. 11. Factors affecting the transmission of visible light by the fibrous tunics of the eye. Arch. Ophthal., 2nd Series, 40:

MA”, IDA 1950 The development of the human eye. 2nd Ed., Gruiie and

SMELSER, K., AND V. OZANICS Studies on the differentiation of the cornea

VAN DEN HOOFF, A. 1951 De Doorzichtigheid van de Cornea. Ned. Tydschrift

Arch. Ophthal., Chicago, 28: 661-669.

278.

601-611.

6 12-62 3.

Stratton, Inc., N. Y. pp. 38-39. 1956

aiid sclera of the rabbit. Anat. Rec., 124: 362.

voor Geneeskunde, 95: 2491-2494.

corneal development. i. corneal transparency

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