The relationship between morphology andtransparency in the nonswelling cornea!

stroma of the shark

Jerome N. Goldman* and George B. Benedek

The ultrastructure of the corneal stroma of the spiny dogfish, Squalus acanthias, was studiedin order to determine whether there was a morphologic basis for the alleged inability of theelasmobranch cornea to swell, and for the possibly related maintenance of corneal trans-parency when it is immersed in distilled water. The most apparent morphologic explanationfor the nonswelling properties was the presence of "sutural fibers" which seemingly boundthe anterior and posterior limits of the corneal stroma. The filamentous strands connectingcollagen fibers within the lamellae were more pronounced than those seen in the cornealstroma of other species. Bowman's layer ivas found to comprise 15 per cent of the totalstromal thickness, yet the structural organization of its collagen fibers was incompatible withthe "lattice theory" for corneal transparency proposed by Maurice. Densitometric studies ofa slit-lamp photograph of dogfish stroma showed that Bowman's layer scatters less light thanthe underlying tissue. Measurements of collagen fiber size and density were made at differentdepths within the corneal stroma in order to provide a basis for future physiologic studies,and to determine whether there might be an explanation for transparency consistent with thecollagenous idtrastructure of both Bowman's layer and the more organized corneal stroma.

From the Cornea Research Unit, Institute of Bio-logical and Medical Sciences, Retina Founda-tion, Boston, Mass.

This work was supported in part by United StatesPublic Health Service Research Grant NB-02220, by United States Public Health ServiceTraining Grant NB-05518, and by special fel-lowship NB-1275 from the National Instituteof Neurological Diseases and Blindness, UnitedStates Public Health Service; in part by a Fightfor Sight Grant-in-Aid of the National Councilto Combat Blindness, Inc., New York, N. Y.;and in part by the Massachusetts Lions Eye Re-search Fund, Inc.

The Phillips 200 Electron Microscope was ob-tained with the support of United States PublicHealth Service Ceneral Research Support GrantFR-05527 awarded to the Retina Foundation.

Portions of this work were reported at the EasternSectional Meeting of the Association for Re-search in Ophthalmology, Washington, D. C,March 10, 1967, and the French Ophthalmo-logic Society, Paris, France, May 8, 1967.

* Special Fellow in Experimental Pathology, sup-ported by Grant NB-1275, National Institute ofNeurological Diseases and Blindness.

R,Janvier1 first observed that the corneaof the skate does not swell in water. Hefurther noted that this cornea, unlike othersso tested, remains transparent upon immer-sion. He attributed the nonswelling prop-erty to the existence of "sutural fibers"which ran transversely from the "anteriorbasal membrane" (Bowman's layer) to theposterior surface of the cornea. His re-markable studies remained an obscure curi-osity until Smelser,2 nearly 80 years later,regenerated interest in the nonswellingproperties of both the skate and the smoothdogfish shark. Payrau3' J and associatesconfirmed the existence of the sutural fibersin several species of elasmobranchs, andelaborated on Ranvier's early descriptionswith studies including electron microscopicanalyses of these sutural components.

Phylogenetically, the elasmobranchs im-mediately precede terrestrial forms5 (Fig.1). They have the most primitive verte-

574

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Ultrastructure of corneal stroma 575

POSITION OF ELASMOBRANCHS IN VERTEBRATE PHYLOGENY

BIRDS

REPTILES

MAMMALS

ELASMOBRAf^S

Fig. 1. The family Elasmobranchii appear justbefore the major division of the vertebrate phylo-genetic tree into aquatic and terrestrial forms.They first appeared about 350 million years agoin the Silurian Era.

M LATHICKNESS

} 0FSECTION

ELECTRON BEAM

Fig. 2. Since the thickness of each section (400to 700 A) is large compared to the diameter ofthe collagen fibers, the electron beam "sees" theentire cross-sectional diameter of most of thelongitudinal fibers embedded in any one section.A fiber, a, which has been sectioned' so as to in-clude much less than half its diameter, will ob-viously be blurred on the electron micrograph,and will be selectively excluded by the observer.One would not, therefore, expect longitudinal sec-tions to yield a measurement differing from thatof cross sections.

brate cornea containing a lamellated colla-gen stroma. The construction of this stromais so simple that several authorities at-tached no significance to the presence ofthe very apparent fibers traversing thelamellae.0"8 This primitive arrangement isassociated with two properties relevant to

clinical ophthalmology: an inability toswell, and the inability to lose transpar-ency under conditions which render thecorneas of more advanced species opaque.This study introduces a series of investi-gations aimed at determining the factorsinvolved in maintaining the transparent,nonswollen state of the corneal stroma ofthe common spiny dogfish shark, Squalusacanthias, and will provide a detailed mor-phologic description which may be usefulin future studies.

Method and materialsPreparation for microscopy. The excised cornea

was fixed for 20 minutes in 3 per cent glutaralde-hyde in pH 7.4 cacodylate buffer. One millimeterstrips of cornea were cut and postfixed for 30 to60 minutes in a 2 per cent osmium tetroxide solu-tion in cacodylate buffer (pH 7.4). Dehydrationin increasing concentrations of ethyl alcohol wasfollowed by four 5 minute rinses in propyleneoxide and by overnight immersion in a mixture ofequal parts of propylene oxide and Epon 812.Epon 812 (Luft's 1:1 mixture) was used for em-bedding. Sections were cut perpendicular, oblique,and tangential to the corneal surface with glassand diamond knives on an LKB Ultramicrotome,Model 8802A, and mounted on various types ofcopper grids with and without parlodion films.The former were provided with a thin carbonlayer. Electron photomicrographs were made witha Phillips 200 Electron Microscope operating at60 kv. Kodak Contrast Projector Slide plates wereused for most of the study. Toward the end,Kodak Electron Image plates were substituted.

Determination of lamellar thickness, collagenfiber diameter, and collagen fiber density. Asingle thin section was made through the entirethickness of the central cornea and put on acopper grid on which a parlodion film had beenplaced, enabling one to see all the lamellae ofthe same section. Thus, it was possible to deter-mine at what depth of the corneal stroma (e.g.,lamella 4, 10, or 20) electron micrographs werebeing made. Care was taken to orient the tissueso that sectioning was perpendicular to the cornealsurface. The entire thickness of the stroma, in-cluding Bowman's layer, was then photographedat different magnifications.

In order to determine collagen fiber size, 8 by10 inch prints were made of the entire thicknessof each lamella and the prints were coded. Aminimum of twenty clearly demarcated collagenfibers, in either a cross or longitudinal section,were selected from each photograph. An investi-gator, knowing code numbers only, measured the

576 Goldman and Benedek Investigative OphthalmologyDecember 1967

diameters of the selected collagen fibers with anocular micrometer and an operating microscope.Where there were differences in the measurementsof two axes of the cross-sectioned collagen, onlythe narrower of the two was considered the fiberdiameter. Where both types of sections were pres-ent in one lamella, the dimensions were deter-mined for both in the same lamella (Fig. 2).

Statistical analysis of the diametric measure-ments of cross and longitudinally sectioned fiberswithin the same lamella showed that there wasno significant difference (s = <0.05) betweenthem. Values for both were, therefore, combined.Means, and standard deviations were determinedfor each of the 25 lamellae before the code wasbroken (Table I ) . Some lamellae (Nos. 6 and17) did not have fibers which could be accuratelymeasured because of the extreme obliquity oftheir course in the section.

Table I. Lamellar thickness and collagenfiber diameter in one adult elasmobranchcornea

LamellaNo.

Thick-ness(mil)

Cross-sectiondiameter, A

(mean ± S.D.)(No.)

Longitudinal-section

diameter, A(mean ± S.D.)

(No.)

Bow-man'slayer

123456789

101112131415

16171819202122

232425

18,000

1,4561,6602,0703,5353,7403,1154,3654,3654,5704,1404,9904,5705,2055,4105,410

5,8205,8205,6155,6155,2054,1403,955

3,5353,535

332

298 ±25 (20)268 ±24 (20)

327 ±21 (20)

308 + 17 (20)

318 ±22 (20)

293 +15 ( 9)

314 + 21 (20)295+ 19 (20)

280 ± 17 (20)

279+15(20)

269 + 13 (20)282 ±11 (20)

282 + 36 ( 9)

282 + 26 (20)264 + 26 (20)

296 + 31 (20)301 + 22 (20)

300 ±28 (20)

321 ±22 (20)296 + 25 (20)

288 ±25 (20)305 ±32 (20)306+11 (20)318 + 25(20)301 + IS (20)

293 + 18 (20)

261 + 24 (20)

269 + 26 (20)

290 ±24 (20)288+15 (10)287 ±13 ( 5)

For the determination of collagen fiber density(the number per given area), the same coded 8by 10 inch prints were arbitrarily divided into agrid on 1 inch ruled squares. Wherever the photo-graphic enlargement of any one lamella provideda minimum of ten 1-inch squares containing col-lagen fibers with circular cross-sectional diameters,the number of fibers in each square was countedand recorded. If a horizontally or obliquely sec-tioned fiber appeared within the outlines of thegrid, or if a marked irregularity in the patternof the fibers was found, the square was ex-cluded. Means and standard deviations and thenumber of 1 inch squares counted on eachprint were determined before the code was re-vealed.

In vivo determination of light scattered by thecorneal stroma. After a portion of the epitheliumhad been removed, slit-lamp photographs of thecornea of a living dogfish shark were taken (Fig.3) (Zeiss photoslit-lamp with Kodak Tri-X filmin a special 35 mm. camera body). Positive printenlargements magnified twelve times were studiedwith a Photovolt densitometer employing a 1.0mm. pupillary aperture. The density curve wasdrawn with an xy plotter. The translation of thephotograph was transmitted to the x motion ofthe plotter through a micropotentiometer in whichten turns corresponded to 10 mm. on the photo-graphic enlargement. This was expanded to 300mm. on the plotter.

Fig. 3. Slit-lamp photograph of the anterior seg-ment of the dogfish eye; a = the slit image ofthe lens capsule, h = the corneal slit image wherethe epithelium is in place, c = the portion ofepithelium-free cornea at which densitometry wasperformed for Fig. 26, and d = the iris.

Volume 6Number &

Ultrastnicture of corneal stroma 577

Results

When thick sections of epon-embeddedmaterial were examined with the lightmicroscope, the total thickness of the cen-tral cornea was found to be 0.20 to 0.25mm. This correlated well with the dimen-sions calculated from electron photomicro-graphs, as well as with in vivo optical de-terminations made with the Maurice-Giar-dini pachometer.0 Forty to 55 per cent ofthe total corneal thickness consisted of epi-thelium. Fifteen per cent of the remainingcorneal thickness resembled Bowman's

Fig. 4. Low power view of five lamellae in themiddle portion of the dogfish stroma. Wrinklesappear in alternate layers in which the collagenfibers have been cut in cross section. The "suturalfibers" (S) cross the lamellae in a wavy path inthis photograph. (Original magnification x6,000.)

layer in man. The main portion of thestroma contained 24 to 25 lamellae whencounted under the higher magnifications ofthe electron microscope. The thickness ofeach layer varied according to its depth inthe cornea. Alternate lamellae appearedwrinkled together, like crepe paper. Thesewrinkled lamellae consisted chiefly of crosssections of collagen. (Fig. 4).

By dissecting out one sutural fiber, Ran-vier demonstrated that the sutural fiberswere continuous through the thickness ofthe corneal stroma in the skate. With theuse of polarized light, he also showed thatthe portions of sutural fibers seen in al-ternate lamellae were continuous from onelamella to the next. Our studies confirmthose of Payrau and associates: The suturalfibers traverse as many as ten adjacentlamellae, and probably run the entirethickness of the cornea of the dogfish. Theattachments of these fibers, which werenot found by Payrau and co-workers, willbe described in detail. When the cornea

Fig. 5. Flat preparation of dogfish endothcliumfixed in 5 per cent gkitaraldehyde in dogfishRinger's solution diluted to 991 jnOsm. (20 min-utes) and postfixed with 2 per cent osmiumtetroxide (5 minutes). (Original magnificationxlOO.)

578 Goldman and Benedek Investigative OphthalmologyDecember 1967

Fig. 6. The basal lamina (b) of the epithelium (E) demonstrating the membrane (arrows)of the basal cell and the two zones described in the text. The anterior conical attachmentof the fine fibrillar bundle blends in with the posterior dense zone. The cross striations in thevertical fibers in Bowman's layer (indicated by an asterisk) are artifacts caused by super-imposed horizontal fibers in the thickness of the section. (Original magnification x35,000.)

was fixed conventionally for paraffin- orepon-embedding, no endothelial cells re-mained attached to Descemet's membrane.Since a rather typical endothelial mosaicwas discernible by specular reflection onthe slit lamp, various methods were usedin an effort to demonstrate its existence.The most successful of these required fixa-tion of the entire fish in glutaraldehyde.With this technique, we observed a cellu-lar arrangement similar to the mosaic

found in flat endothelial preparations ofhigher species (Fig. 5).

Basal lamina and Bowman's layer. Thebasal lamina compares structurally withthat found in the human cornea, but isproportionally thicker in the dogfish. Itmeasures 2,800 to 4,200 Angstrom units(A). Anteriorly, it is represented by thecell membrane of the basal epithelial cell.Posterior to this is a narrow (600 A) lightzone and a narrower (200 A) dense zone

Volume 6Number 6

Ultrastructure of corneal stroma 579

Fig. 7. The collagen of Bowman's layer. There is no apparent orientation of the fibers.{Original magnification x64,000.)

which blends into the thick, moderatelydense region comprising the major portionof the structure. The lamina ends in a con-densation of dark material which abutsthe underlying Bowman's layer. These re-lationships are demonstrated in Fig. 6. Inthis electron photomicrograph, the finefibrillar component of the sutural complexcan be seen blending into a conical evag-ination of the most posterior dense layerof the basal lamina. This cone represents

the anterior terminus of the fine fibrillarcomponent. No other specific morphologicstructure appears at this point of attach-ment.

Bowman's layer is 30 to 45 times thickerthan the basal lamina, measuring between0.01 and 0.02 mm. centrally in different in-dividuals of the species. As in the humancornea,10'1X the collagen fiber feltwork com-prising Bowman's layer has no particularorientation (Fig. 7), except in its posterior

580 Goldman and Benedek Investigative OphthalmologyDecember 1967

portion where there is a preponderance offibers aligned parallel to the corneal sur-face. Most of these fibers blend into themost anterior lamellae of the stroma. Afew of them join the fine fibrillar com-ponent of the sutural fibers within the an-terior two lamellae and pass with themthrough the thickness of at least one lam-ella (Fig. 8). The relationship of thesethick fibers to the collagen fibers compris-ing the corneal lamellae is not clear. Thecollagen fibers of Bowman's layer are thin-ner than those in the adjacent anteriorlamellae. Their diameter is about 270 A.In Bowman's layer as in other lamellaethere is no significant statistical differencebetween the diameters measured in crosssection and those measured in longitudinalsection.*

We have also estimated the volume frac-tion (d(.) of fibers in the Bowman's zoneusing the electron micrograph in Fig. 7.This was done by counting the number offibers per unit area (NA) whose axes areoriented within some pre-selected solidangle (Afi). If p is the radius of a fiber,then the volume fraction of fibers (dc) isgiven in terms of NA and AO by dc = NA

(TT/J2) (4TT/A£2). We can identify thosefibers lying in a solid angle AQ by count-ing for each unit area only those fiberswhose photographic image ranges betweena perfect circle (vertical alignment of thefiber) to an ellipse of a pre-selected length-to-width ratio. In our measurement wetook this ratio to be 3. The fibers havingthis projected length to width ratio wereinclined an angle A0IU = tan-1 (4/o/Lf)where LE is the thickness of the epon sec-tion photographed. We counted all fiberswhose orientation was between verticaland this maximum angle. The solid angle

"The terms fiber and fibril have been used interchangeablyby various authors. In discussing the elasmobranch corneait is particularly important to define these terms. Here, wewill vise fiber to refer to the mature, thick collagenousstructures, measuring about 300 A in diameter, whichcomprise Bowman's layer, the major portion of the stroma,and the thick component of the sutural complex. The finecomponent of the sutural complex, measuring about 75 Ain diameter, will be referred to as fibrils. Neither ofthese terms will be used in describing other filamentouslinear structures.

AQ which was included in our measure-ment is related to A0m by AQ = 2 TT (1 -cos A0m). In this way we found the vol-ume fractions of fibers to be about (40 ±10) per cent. The lower limit of 30 per centcorresponds to an epon section of 500 A,while the upper limit of 50 per cent cor-responds to a section of thickness 700 A.The volume fraction of fibers in the sharkstroma, dc = 35 per cent, falls within theselimits.

Stroma. The three adult dogfish corneasin which the layers of the stroma wereactually counted under the electron micro-scope each had 25 lamellae. In order todistinguish the anterior two from the pos-terior two, magnification of x20,000 wasrequired. The thickness of consecutivelamellae measured in several corneas re-vealed variation from one cornea to an-other, but showed similar distribution tothat tabulated for one cornea (Fig. 9,Table I). Every precaution was taken toinsure that sections were made perpendic-ular to the corneal surface. Any deviationfrom the perpendicular in the sections usedfor measurement or counting is probablysmall. As previously stated, the entirestromal thickness could be seen within oneparlodion-supported grid opening, so thatthe numerical position of each lamellacould be ascertained.

Within the lamellae, the collagen fibersize varied according to the location of thelamella. With the exception of Bowman'slayer, the anterior lamellae containedlarger fibers than the posterior ones. Thebest fitting parabola, D = aL2 + bL + c(where D = diameter and L = lamellanumber), was computed and fitted be-tween the measured points (Fig. 10). Theparabola was considered as three slices(slice No. 1 = lamellae 1 to 8, slice No.2 = lamellae 9 to 16, and slice No. 3 =lamellae 17 to 25) and analyzed. The col-lagen fiber diameters within each slice dif-fered statistically from those in the othertwo slices.

In analyzing the density of collagenfibers, two slices were considered (slice

Volume 6Number 6

Ultrastructure of corneal stroma 5S1

f

B

Fig. 8. The thick fibers (F) of the sutural complex originate in the posterior portion of Bow-man's layer (B). They join the fine fibrils (f) as they pass through the anterior layers (num-bered 1, 2, and 3). In this micrograph, the process (p) of a poorly fixed keratocyte accom-panies the principal bundle through the second and a portion of the third lamella. (Originalmagnification x29,000.)

No. 1 = lamellae 1 to 16, and sliceNo. 2 = lamellae 17 to 25). The two slices,which appeared to differ from one anotherwhen represented graphically (Fig. 11),were significantly different upon statisticalanalysis. The fibers were more concentratedin the posterior lamellae.

As a rule, the collagen fibers in eachlamellar ribbon of the shark stroma aremore parallel to one another than in hu-

man or rabbit corneas, but changes of di-rection (Fig. 12) and individual fiberstraveling perpendicular to the general di-rection are frequently seen. These fibersmay be part of the thick component ofthe sutural complex, which will be de-scribed in the next section. Unlike thelamellae of human and rabbit corneas, theribbons of collagen fibers do not appearto interweave. Rarely, a group of fibers is

THICKNESS OF EACH LAMELLA IN THE STROMA

OF ONE DOGFISH CORNEA

I I I I I 1 1 MI 2 3 4 B 6 7 6 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25

ANTERIOR POSTERIOR

LAMELLA NUMBERFig. 9. Graphic representation of the distribution of lamella thickness in one dogfish cornea.This pattern exists for the stroma of other adult members of this species.

DISTRIBUTION OF COLLAGEN FIBER DIAMETER

AT DIFFERENT DEPTHS OF DOGFISH STROMA

3 5 0

DIA

I\

325

300

275

_ _ _ _ X • X

x • * • • v * *"" T" —

§Q250

225 —

NORMALx - CROSS SECT.. _ L 0 N G - S E C T .

BOWMAN'S LAYER

19 . 21 23 25

LAMELLA NUMBERFig. 10. The mean diameter of collagen fibers decreases progressively in the posterior lamellae.Bowman's fibers compare in diameter with the smaller fibers found in lamellae 18 through 25.

DENSITY OF COLLAGEN FIBERS AT DIFFERENT

DEPTHS OF DOGFISH STROMA

600

550

500

450

400

— s

1

4I

(10) /

( > INDICATES NUMBER OF SQUARES COUNTEDPER ELECTRON PHOTOMICROGRAPH

1 1 1 1 1 1I 3 5 7 9 II 13 15 17 19 21 23 25

LAMELLA NUMBER

Fig. 11. The significant change in collagen fiber density in the posterior lamellae.

Volume 6Number 6

Ultrastructure of corneal stroma 5S3

v

N

P

Fig. 12. Part of a keratocyte lying between the twenty-first and twenty-second lamellae.Collagen fibers appear in perpendicular (*) and horizontal (* °) section in adjacent portionsof the same lamella. One vertically extending process has an accumulation of free ribosomesat its base; N = nucleus, p = process, g = golgi apparatus, and v = surface vesicle.(Original magnification x29,000.)

observed to pass obliquely across a lamella(Fig. 13). Occasionally, even in the cen-tral cornea, rows of collagen only one, two,or three fibers thick, which represent thelateral excursions of the thick component

of the sutural complex, are found. Thewidth of the transversely running ribbonsof collagen fibers making up one lamellaexceeded the width of the sectioned tissue,so that their dimensions could not be de-

Fig. 13. A bundle of collagen fibers of one lamella in the anterior portion of the stromacrossing obliquely to a third lamella. The individual thick fibers (single arrows) and a thinrow of thick fibers (double arrows) are also seen. (Original magnification x24,000.)

K

Fig. 14. Interstices between collagen fibers within one lamella are filled with a reticulum offine strands. The suggestion of a collagen fiber substructure appears in the micrograph.(Original magnification xl20,000.)

Volume 6Number 6

Vltrastructure of corneal stroma 585

termined. The thickness of the entire cor-nea doubled near the periphery with ap-parent proportionate increase in the thick-ness of each of the individual lamellae, butthe number of lamellae remained the same.

Fine strands connect adjacent collagenfibers within any one lamella. These arebest seen in oblique or cross sections (Fig.14). The strands resemble those seen be-tween the collagen fibers in human andrabbit corneas.12 A great many more ofthem are found in the shark than in thecorneas of the other two species. Withinthe central shark cornea, the concentrationof filamentous strands between collagenfibers is greater in the posterior lamellae.These collagen fibers occasionally displayan electron dense peripheral ring sugges-tive of a substructure (Fig. 14).

The sutural fiber complex. The suturalfiber as seen by Ranvier with the lightmicroscope was found to contain the twotypes of fibers described by Payrau, whichwill be referred to as the fine and thickcomponents of the sutural complex. Onhorizontal section, the buttonhole-shapedsutural complexes are seen to be irregu-larly spaced, their long axes parallel to oneanother within one lamella. There are

about fifteen sutural complexes per squaremillimeter (Fig. 15). The montage of suchsections (Fig. 16) demonstrates the varietyof relationships within the sutural com-plex. Thick fibers are seen only on the twopointed ends of the buttonhole, and neverwithin or around the buttonhole on theflattened sides except at the intersection oflamellae, where the sutural complex as-sumes a triangular shape. The cylindricalcore of the fine fibrils is referred to byPayrau and associates as the principalbundle. Its narrowest diameter varies from3,500 A to 4,500 A. The longitudinal diam-eter may be four times as great. One bun-dle contains 60 to 100 fibrils, each of whichmeasures about 75 A in diameter. A bun-dle attaches anteriorly to the anteriordense layer of the basal lamina and ex-tends posteriorly into Bowman's layer,where it is joined by the thick fibers. Bothcomponents terminate in an interweavingnetwork beneath the most posterior lamellaand Descemet's membrane (Fig. 17).Rarely, smaller bundles of fine fibrils areseen in cross section in the middle of aposterior lamella, isolated from the thickcomponent of the sutural complex. Thesefine fibrils do not show the collagen peri-

Fig. 15. The irregular distribution and parallel orientation of the sutural complexes arevisualized amidst the undulating collagen fibers of a portion of a lamellar ribbon seen inhorizontal section. (Original magnification xl2,000.)

586 Goldman and Benedek Investigative OphthalmologyDecember 1967

Fig. 16. The varied relationships of the fine (f) and thick (F) fibrillar components of thesutural complex. In a, b, and c interfibrillar bridges can be distinguished among the thickfibers in the apical triangle of the complex. A cross section of a vertical process (p) of akeratocyte appears in c. The manner in which the thick fibrillar components leave the com-plex near the interlamellar interface can be appreciated in d. The rounding of the principalbundle at this level suggests that its flattened appearance elsewhere is the result of lateralcompression. (Original magnification a and h, x64,000; c, x34,000; and d, x46>000.) -;

Volume 6Ntmbcr 6

Ultrastructure of corneal stroma 587

tFig. 17. The fibers of the sutural complex plunge past the process of a keratocyte to theirtermination in the network overlying Descemet's membrane (D). Croups of fine fibrils (f)can be seen amidst the thick fibers in this network. The endothelial monolayer was not pres-ent in this preparation. (Original magnification x31,000.)

odicity seen in the fibers of the thick com-ponent.

The thick fibers take several pathways,some of which are described by Payrau.As previously stated, the thick componentoriginates anteriorly in the posterior por-tion of Bowman's layer. Some thick fibersleave the sutural complex at or near everyinterlamellar interface. A few leave withinthe center of the lamella. Whether or not

any of them traverse the entire thicknessof the cornea cannot be ascertained fromobservations made here. No individualthick fiber has been traced for more thantwo lamellae. When a thick fiber has leftthe sutural complex, it blends in with themass of parallel collagen fibers of the la-mella proper. The anteroposterior path ofsome thick fibers can be quite straight forthe thickness of one or two lamellae. In

588 Goldman and Benedek Jnoestigative OphthalmologyDecember 1967

' f2

Fig. 18. The twisting path of the fibers of the thick component. Three taut bundles of finefibrils (fl, j2, and f3) can be followed as the sutural complex traverses a lamella in mid-stroma. (Original magnification xl7,000.)

the same cornea, other thick fibers showas many as four twists within one lamella(Fig. 18). This observation, when corre-lated with the direction of the thick fibersin the horizontal sections of the suturalcomplexes of Fig. 16, suggests that a largenumber, if not the bulk of the thick fibers,twist about as thev descend within the tri-

angle on one side of the core of a suturalcomplex. There is no evidence that a thickfiber on one side of the principal bundlepasses to the other side, except at the inter-lamellar interfaces.

Paraffin-embedded dogfish corneas fixedin CPC-formalin13 were histochemically ex-amined in order to determine the composi-

Volume 6Number 6

infrastructure of corneal stroma 589

Fig. 19. Sderal collagen demonstrates typical periodicity, loose organization, twisting, andvariable cross-sectional diameter of fibers. (Original magnification x60,000.)

tion of the sutural complex. There ap-peared to be slight reactivity to Periodicacid-Schiff reagent (PAS-positive), butvirtually none to Verhoeff's Elastic Tissuestain1*1 or to the Orcein Van Gieson stain.15

When Wilder's reticulin stain14 was used,

however, the sutural complex could beseen in sharp contrast. The dark, linearstaining extended through Bowman's mem-brane to the base of the epithelium in somesections. This extension suggests that thethin component was being stained. The

590 Goldvian and Benedek Investigative OphthalmologyDecember 1967

Fig. 20. Varying diameters of scleral collagen fibers seen in cross section. Fibrillar accumula-tions (arrows) adjacent to the scleral fibroblasts (Fib.) may be collections of mucopolysac-charide, protocollagen, or elastin. (Original magnification x25,000.)

acid mucopolysaccharide content of thesutural complex did not differ histochem-ically from the adjacent stroma when ex-posed to toluidine blue at pH 2.5, 4.0, and7.0, hyaluronidase digestion, and selectiveblocking of alcian blue by KC1.13>1G

Scleral collagen, in contrast to coraealfibers, usually demonstrate the typicalperiodicity and greater variation in cross-sectional diameter, They are also moreloosely organized and twist about one an-other (Figs. 19 and 20).

Volume 6Number 6

Ultrastructure of corneal stroma 591

Fig. 21. Processes of two or possibly three keratocytes (I , 2, and 3) are closely applied atthe interface of two lamellae. The thin (f) and thick (F) components of a sntural complexpass between keratocyte 2 and the process of a fourth keratocyte (4). Two double rows ofthick fibers (single arrows) are in evidence. (Original magnification x30,000.)

The keratocytes. The cellular elementsof the dogfish stroma are quite similarto the keratocytes found in normal hu-man and rabbit corneas. Usually thecell is situated between lamellae. Thereappears to be a continuous line of cellsin which keratocytes maintain actual orvirtual contact with the processes of ad-jacent keratocytes in the same plane(Fig. 21). The surrounding collagen liesclose to the cell membrane. The cellorganelles are similar to those of otherfibroblasts.

The relationship of the keratocytes tothe sutural complex suggests that the cellsaccommodate themselves to the fixed posi-tion of the components of the complex.In horizontal sections, portions of the kera-tocyte cell membrane are seen surroundingall or a portion of the sutural complex.The principal bundle of fine fibrils is oftencompletely surrounded by keratocyte cyto-plasm without any trace of the thick fibers.In the vicinity of the sutural complex, thereis a dark condensation subjacent to the

cell membrane suggestive of compressionof the cell at that site (Figs. 22 and 23).

In perpendicular section of keratocytes,the anteroposterior dimension is greaterthan that of those in human or rabbit cor-neas, so that the cell sometimes has aplump appearance. This plump appearanceis especially pronounced in the keratocyteof the embryonic shark. The sutural com-plex intersects the keratocyte. The finefibrils of the principal bundle form a bar-rier to which the external membranes ofthe cell seem to accommodate themselves.These sutural complex-keratocyte relation-ships suggest that the cell membranes maybe in motion. One unique property of dog-fish keratocytes is the manner in which theprocesses extend in an anteroposterior di-rection. These vertical processes are oftenin close association with components ofthe sutural complex (Figs. S, 16}c, and25). Sometimes an accumulation of freeribosomes is seen at the base of the verti-cal cell process within the cytoplasm of thecell (Figs. 24 and 25).

592 Goldman and. Benedek Inoestigatioe OpiUhahnologijDecember 1967

Fig. 22. Horizontal section of a keratocyte. The cell membrane (c) is apparent only whenfolded over within the cell. The thin component of the principal bundles (f) appear unaltered.The adjacent thick fibrillar component (F) is often attenuated when seen within the confinesof the keratocyte. The structure marked with an asterisk may be a mitochondrion. Islands ofkeratocyte cytoplasm (K) are seen in the midst of the collagen; N = nucleus. (Original mag-nification x23J000.)

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Ultrastructure of co-meal stroma 593

Descemet's membrane and the endothe-lium. No space is discernible between thenetwork underlying the most posterior la-mella and Descemet's membrane in sectionscut perpendicular to the corneal surface.In extremely oblique sections, a fiber-free

region is distinguishable. The loose adher-ence of the very thin Descemet's mem-brane (3,000 A to 44,000 A) to the stromamight have caused this separation to occurduring tissue processing. In the dogfish,Descemet's membrane consists of a homog-

N

ERm

\

ER

Fig. 23. Two principal bundles being engulfed by the margins of a keratocyte. There is aregion of increased electron density along the cell membrane adjacent to this region (singlearrows). N = nucleus, ER = granular endoplasmic reticulum, m = mitochondrion. (Originalmagnification x43,000.)

Goldman and Benedek Investigative OphthalmologyDecember 1967

enous layer of fine fibrils more loosely in-terwoven than those seen in mammals.Posterior to Descemet's membrane, a mono-layer of cells was seen in only one embed-ding. A morphologic description of thesecells will be waived until better fixation isachieved. A definite endothelium can beidentified on slit-lamp examination and onflat endothelial preparation.

Discussion

This study was inspired by the observa-tion that the stroma of the elasmobrancbretains its transparency even under manyof the conditions in which other corneasare known to swell and become opaque.This is not to say that the transparency ofthe dogfish stroma cannot be impaired. Aneedle passed obliquely through it leaves

\

c N

v

Fig. 24. Portion of a keratocyte with vertical processes extending anteriorly and posteriorly.At the base of each vertical process, an accumulation of free ribosomes is seen. Fine fibrillarmaterial (arrow), perhaps representing mucopolysaccharide or protocollagen, can be seenoutside the cell near the base of one of the processes. N = nucleus, g — golgi apparatus,c = centriole, V = surface vesicle. (Original magnification x23,000.)

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Ultra structure of corneal stroma 595

Fig. 25. Higher magnification of tlie region surrounding the inferior process in Fig. 24. Thecytoplasmic membrane (cm) of the process can be distinguished in tlie midst of the fibriUarcomponents of the sutural complex; g = golgi apparatus, r = ribosomes. (Original magnifica-tion x45,000.)

596 Goldman and Benedek Investigative OphthalmologyDecember 1967

an opaque line in its path. Retention oftransparency appears to be related to thenatural nonswelling properties. Measure-ments and descriptions of the cornea of areadily obtainable and easily managedmember of this unique family supplementthe earlier reports of Payrau, previouslycited, and are intended to provide a foun-dation for future experimental work oncorneal transparency, swelling mechanisms,and the flow of water and electrolytes incorneal stroma.

The knowledge the measurements andrelationship of the collagenous componentsof the cornea of Squalus acanthias permitsus to evaluate the theoretical basis forcorneal transparency. In a pioneering treat-ise, Maurice showed that if individual col-lagen fibers scattered light independentlyof one another then the corneal stromawould be opaque. The fact that the corneais transparent indicates that phase rela-tions must exist between the electric fieldsemanating from each fiber which a ray oflight strikes. These phase relations resultin a mutual destructive interference of thewavelets scattering from each fiber, andhence in a diminution of the intensity ofthe scattered light. Maurice further sug-gested that the physical basis for the phaserelationship between the scattered wave-lets was a regular or lattice arrangementof the collagen fibers.

The thickness of Bowman's layer in theshark is between 0.01 and 0.02 mm., de-pending on the individual specimen. Thisis about 15 per cent of the stromal thick-ness in the central cornea. In Bowman's,the axes of the fibers are randomly oriented(Fig. 7). Although their diameters are verynearly' equal, they are neither parallel nordisposed in a lattice. Nevertheless, thesefibers cannot be regarded as independentpoint scatterers.

If they were independent, then Mau-rice's formula (Equation 10)17 for thefraction AS of light scattered would apply:

( n ° 2 " n i 2 ) " A t -

We can predict the results of applyingthis formula in the case of Bowman's zonein the dogfish. The parameters needed aredc, the volume fraction of fibers; p, thefiber radius; A, the wavelength of light;At, the thickness of the zone; and nc andni} the indices of refraction of the collagenfibers and the ground substance. The val-ues of dc, p, and At, respectively, are: 40 ±10 per cent, 139 ± 12 A, and 0.015 ±0.005 mm. We also take X = 5000 A. Wehave not measured nc or n{ for the shark'sBowman's zone. However, if we take thevalues nc = 1.55 and n; = 1.345, the val-ues given in Maurice's paper, we find thatthe fraction AS of light scattered from anincident beam is 35 per cent, with a pos-sible range of values produced by the un-certainty in dc and At of 18 per cent at theminimum and 59 per cent at the maxi-mum. Even in the case of the minimumvalue of about 18 per cent, this degree ofscattering is inconsistent with transpar-ency.

In fact, when we examine slit-lamp pho-tographs of the Squalus stroma, we findthat the zone corresponding to Bowman'slayer is not only transparent but that itactually scatters less light than the rest ofthe corneal stroma (Figs. 3 and 26).

The calculation we have made suffersfrom the use of the values nc and ni whichwere obtained for the corneas of otherspecies. The value of nc, the collagen fiberindex, is probably the same for the sharkand the species used by Maurice. Ofcourse, nt could be different and a mea-surement of it should be made for theshark. Nevertheless, we have assumed thatnt is the same for both species. If this isthe case, then the observed transparencyof the shark's Bowman's zone forces us toconclude that a lattice arrangement of col-lagen fibrils is not a necessary conditionfor corneal transparency.

This conclusion is entirely consistentwith the principles of optics according towhich light is scattered only when theilluminated medium contains spatial fluc-tuations in the index of refraction over

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infrastructure of corneal stromu 597

Fig. 26. Densitometric tracing of optic section taken from slit-lamp photograph of dogfishcornea seen in Fig. 3. In the inset, (a) represents the lens capsule and (b) is the cornea fromwhich the epithelium lias been scraped.

dimensions comparable with the wave-length of light18-1!) The fluctuations in theindex of refraction as one passes from thecollagen fiber to the ground substance ma-trix occur over dimensions of about 500Angstrom units. This distance is smallcompared to the wavelength of light.

Under the conditions present in both thenormal Bowman's layer and the normalstroma of the dogfish shark, the trans-mission and scattering of light must becalculated by summing the dipole fieldsradiating from each illuminated point.Only those fluctuations in the index ofrefraction ' having a spatial extent com-parable with or larger than one half thedimension of the light wavelength ('—'2500A) are important in considering the rela-tionship of structure to transparency. Infact, the theory of the scattering of lightby a medium having fluctuations in itsindex of refraction shows that fluctuationshaving a wavelength of A/2 scatter lightof wavelength A toward the backwarddirection, while scattering in a direction 6away from the forward direction is pro-

duced by fluctuations of wavelength

, i.e., by fluctuations having(2 sin y2 6)a wavelength longer than A/2. One canexpect to find inhomogeneities in the re-fractive index equal to or greater than—'2500 A in corneas with reduced trans-parency. Scleral opacity results not fromthe absence of a lattice arrangement ofcollagen fibers, but rather from such largevariations in collagen fiber diameter (250A to 4,800 A in our specimens) that neigh-boring regions have different refractive in-dices. (Figs. 19 and 20.) The separationof adjacent bundles of scleral fibers fromone another by noncollagenous spaceswhich are large compared to the dimen-sions of a wavelength of light also con-tributes to scleral opacity. The operativemechanisms to be considered, in this case,are refraction and reflection.

Although various chemical and electricalfactors might contribute to nonswellingproperties, the morphologic characteristicsdescribed in this paper appear to account

598 Goldman and, Benedek Investigative OphthalmologyDecember 1967

for the restriction of swelling in the dogfishcornea. There is great species variation inthe degree of interweaving of the lamel-lae-0 and in the regularity of the arrange-ment of collagen fibers within a singlelamella viewed with the electron micro-scope. The adult dogfish demonstrates thehighest degree of regularity I have ob-served. The fine strands between the indi-vidual collagen fibers in each lamella areconsiderably more electron dense thanthose found in rabbit and human corneasprepared and examined under similar con-ditions. They are also more numerous inthe elasmobranch. If these interfibrillarstrands are actual structures, the increasedelectron density may very well representgreater strength and a greater tendency foradjacent collagen fibers to maintain theirclose relationship.

The sutural complex could account fornonswelling completely. The thin com-ponent is never kinked. It does, indeed,appear to be stretched between the basallamina of the epithelium and Descemet'smembrane. If the "sutural fibers" cannotstretch, the maximal thickness of the cor-neal stroma might be limited by the lengthof the thin bundles.

The nature of the thin fibrils is not defi-nitely known. They do not have the peri-odicity typical of corneal collagen, but thismay be related to their small diameter. Itis more difficult to demonstrate periodicityin the corneal collagen fibers than in thoseof sclera. The positive reaction of the su-tural complex to Wilder's stain, coupledwith its greater PAS reactivity, is con-sistent with the histochemistry of reticulin.The fine fibrillar component is of the sameorder (75 A) as has been described forthis chemical analogue of mature colla-gen.̂ 1

The fibers of the thick component appearto bridge only a few lamellae at most. Al-though some of them descend in a straightanteroposterior path for one or two lamel-lae alongside the thin fibrils, many of themfollow the twisting path previously de-scribed. The twisting, along with the prob-

ability that they join together only a fewadjacent lamellae, makes it unlikely thatthey fix the anterior and posterior limits ofthe stroma. They may serve to restrict theseparation of normal collagen fibers withinthe lamellae, as well as the separation oflamellae from one another. I have observedboth phenomena in human stromal swell-in!

The simple arrangement of the elasmo-branch stroma into 25 layers lends itself tothe layer-by-layer determination of lamel-lar thickness, collagen fiber size, and col-lagen fiber density. The progressive de-crease in fiber size as one proceeds poste-riorly is not linear. Statistically, the fibersin each third of the stroma are closer indiameter to one another than to any others,with the exception of the fiber size of thecollagen in Bowman's layer.

The observation that the collagen fibersin Bowman's layer are smaller than thoseof the stroma is consistent with the find-ings of others.31' 22; 23 Jakus21 found that thecollagen fibers in Bowman's layer were con-sistently smaller than those found in theunderlying stroma within the same species,but that these diameters exhibited speciesdifferences.

The different values ascribed to the sizeof corneal collagen fibers by various au-thors may be the result of variation in thepreparation of the tissue for examination.No statistical difference was found in mea-surements made of the diameters of 20longitudinal and 20 perpendicular fibers inthe same lamella. This is in disagreementwith several previous reports in which thelongitudinal width is said to exceed thecross-sectional diameter. Jakus'25 observa-tion of an increase in the thickness of thecollagen fibers in the more posterior por-tions of the human corneal stroma describesa condition which is just the reverse of theprogressive decrease in fiber diameter de-termined in this study. This species dif-ference cannot be accounted for.

The frequent occurrence of the isolatedprincipal bundle of the sutural complexsurrounded by keratocyte cytoplasm, and

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Ultrastructure of corneal stroma 599

the rare occurrence of the isolated thickfibers in such surroundings, suggest thatthe thick fibers are either displaced or di-gested by the migrating keratocytes of thenormal cornea. Since there is little evidencefor the former situation in the electronmicrographs, the latter possibility must beseriously considered. The vertical exten-sion of keratocyte processes, only whenthey are associated with the sutural com-plex, may be related to the disappearanceor renewal of the thick fibers. Whether theribosomal accumulations at the base ofthese processes are indicative of activity isunknown.

The fact that the elasmobranch stromawill not swell even in the absence of theepithelium and the endothelium makes anendothelial pump unnecessary for the regu-lation of strom al hydration. The difficultiesencountered in maintaining endothelial in-tegrity in this study may reflect the experi-ence of those in the past who have ques-tioned or denied the existence of this mono-layer. However, the presence of anendothelial mosaic observed on slit-lampmicroscopy and on endothelial flat prepa-ration has encouraged us to make furtherattempts at delineating endothelial mor-phology.

The authors are deeply indebted to Miss Bar-bara Kravitt for her suggestions and technical as-sistance in all aspects of the electron microscopy,and in the preparation of the manuscript, and toMr. Stephen Klyce and Dr. Jean Brasseur for theirhelp with the determination of collagen fiber sizeand statistical analysis. They also acknowledge thestimulating conversations regarding the problemof transparency with Mr. Oleg Pomerantzeff, Dr.Jean Brasseur, Dr. David Maurice, and Dr. DavidBerkley and, regarding morphology, with Dr.Marie A. Jakus and Professor Toichiro Kuwabara.The densitometric determinations were made byMr. Pomerantzeff and Mr. Henri de Guillebon.

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2. Smelser, G. K.: Corneal hydration. Compara-

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3. Payrau, P., Offret, G., Faure, J.-P., Pouliquen,Y., and Cug, G.: Sur la structure lamellairedes cornees de certains poissons, Arch. f.Ophth. 24: 685, 1964.

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5. Smith, H. W.: From fish to philosopher, Gar-den City, 1961, Doubleday and Co., Inc.

6. Duke-Elder, S.: System of ophthalmology,in The eye in evolution, St. Louis, 1958,The C. V. Mosby Company, vol. 1.

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14. Manual of histologic and special stainingtechniques, Armed Forces Institute of Pathol-ogy, ed. 2, New York, 1960, The BlakistonDivision, McGraw-Hill Book Company,Inc.

15. McManus, J. F. A., and Mowry, R. W.:Staining methods, New York, 1965, HoeberMedical Division, Harper and Row Pub-lishers.

16. Kitano, S., and Goldman, J. N.: Cytologicand histochemical changes in corneal woundrepair, Arch. Ophth. 76: 345, 1966.

17. Maurice, D. M.: The structure and trans-parency of the cornea, J. Physiol. 136: 263,1957.

18. Tanford, C : The physical chemistry of mac-romolecules, New York, 1965, John Wileyand Sons, Inc.

19. Mclntyre, D., and Gormick, F., editors:Light scattering from dilute polymer solu-

600 Goldman and Benedek Investigative OphthalmologyDecember 1967

tions, New York, 1964, Gordon and Breach,Publishers, Inc.

20. Ehlers, N.: On the vegetative physiology ofthe cornea, Danish M. Bull. 13: 133, 1966.

21. Robb-Smith, A. H. T.: What is reticulin?in Tunbridge, R. E., editor: Connective tis-sue symposium, Springfield, 111., 1957, CharlesC Thomas, Publisher.

22. Jakus, M. A.: The fine structure of the cor-nea, XVII, Concilium ophth. acta 2: 461,1955.

23. Teng, C. C : Fine structure of the humancornea: Epithelium and stroma, Am. J.Ophth. 54: 969, 1962.

24. Jakus, M. A.: Personal communication.25. Jakus, M. A.: The fine structure of the hu-

man cornea, in Smelser, G, K., editor: Thestructure of the eye, New York, 1961, Aca-demic Press, Inc.