corneal hydration comparative physiology of fish and mammals

Corneal hydrationComparative physiology of fish and mammals

The Proctor Award Lecture

George K. Smelser

Corneas of all species studied were basically similar in structure, consisting of an orderlyarrangement of collagen fibers bounded by epithelia. All were metachromatic, indicating thecommon possession of mucopolysaccharides. The corneas of teleost fish were found to swellin various physiologic salt solutions as did those of mammals. The degree of corneal swelling(in distilled water) was greater in the scup which is adapted to life in sea water and less inthe carp which lives in a hypotonic medium. The elasmobranch cornea differs from those ofall other species so far studied. It is not hydrophilic, and was not found to swell in any ofthe media studied. No evidence of anaerobic metabolic control of corneal hydration, such asis characteristic of mammals, was demonstrable in the teleost fish (scup). Corneal swellingioas inhibited in the presence of salts, less by monovalent than by bivalent cations, and wasenhanced when bivalent unions were present. Scup corneas were found to swell readily inaqueous solutions of polyvinyl-pyrrolidone (PVP) in the absence of salts. In the presence ofsalts, scup corneas could be maintained deturgesced in solutions of these large polymers (PVP).It is postulated that salts affect the macromolecular structure of the mucopolysaccharides sothat that of the mucoid ground substance is more compact, stable, or rigid, and that this isa major factor in the maintenance of normal corneal hydration generally. It is suggested thatthe xoater balance of teleost corneas and, perhaps, of those of other animals is maintained inthe presence of salts by mucopolysaccharides in the aqueous humor which counteract theswelling pressure of the cornea. Descemet's endothelium serves in this system as a membranepermeable to salts and xoater but not to the colloids of the aqueous humor.

I£ the literature on corneal physiology ofthe past twenty years is examined, it willhe found that this tissue has excited enor-mous interest and stimulated many ingeni-ous experiments with intent to understandhow its normal degree of hydration is main-tained. This problem is intriguing primarilybecause hydration is inextricably related totransparency and, in addition, because thephysiologic processes involved in maintain-

From the Department of Ophthalmology, Collegeof Physicians and Surgeons, Columbia Univer-sity.

This investigation was supported by Research GrantB 492 from the National Institute of Neurolog-ical Diseases and Blindness, National Institutesof Health, United States Public Health Service.

ing water balance in connective tissue arefundamental ones and of great importancein diseases of the connective tissue in gen-eral. The ultimate objective in these experi-ments, as well as those reported here, is toacquire an understanding of how the cor-nea maintains its transparency. The manycontributions to this subject are well sum-marized in the symposium on The Trans-parency of the Cornea,1 and by John Harrisin The First Friedenwald Award Lecture.2

The problem was first uncovered clinicallyby the observation that swelling of thecornea was accompanied by loss of trans-parency. Therefore, the first investigationswere carried out in ophthalmic laboratorieswhere the observers were clinically ori-ented, thus leading them to study comeal

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12 SmelserInvestigative Ophthalmology

February 1962

hydration in mammals. These researches, asexemplified by those of Cogan and Kinsey,3

were invaluable, and actually introducedthis subject to physiologists. Their workled to the concept that the osmotic pressureof the fluids bathing the cornea was anessential factor in the maintenance of itsturgescence. This line of thought resultedin consideration of corneal physiology asan exclusively mammalian, or at least as anaerial-corneal, problem. It is obvious, how-ever, that aquatic vertebrates* have trans-parent corneas adapted to media of ex-tremely varied osmotic pressure and thatthe "physiologic" problem, therefore, is anextremely broad one. By studies utilizingdiverse forms adapted to varying environ-ments, one may uncover the mechanism inthese "natural experiments" which wouldescape us if attention were restricted toone complicated system.

Adaptation of the cornea to salty tears,the sea, or fresh water is not an isolatedphysiologic problem. Rather, it is a part ofthe larger one of how animals in generalare able to adapt themselves to life inmedia which vaiy enormously in osmoticpressure. This subject has been compre-hensively presented by Baldwin.4 Thebodies of fresh-water fish continually im-bibe water, as many of us have supposedthe cornea to do, and dispose of the excessthrough the kidneys. In contrast, salt-waterfish live in a desert, for most sea water ishypertonic to their tissue fluids. Most ofthem would become relatively dehydratedif they did not regularly excrete the excesssalts which continuously enter their bodieswith the water absorbed through the gills,the intestine, and perhaps the skin. In onegroup of marine fish, the elasmobranchs,the problem of living in a strong salt solu-tion has been solved by the maintenance ofa high internal osmotic pressure with ureaand trimethylamine. These animals, there-fore, are in a situation somewhat analogousto that of fresh-water fish, in that their ex-

°The experiments on marine fish were made at theMarine Biological Laboratory, Woods Hole, Mass.

ternal environment is relatively hypotonicto the internal one. For this reason andbecause of the low position they occupy inthe phylogenetic scale, the physiologic be-havior of the corneas of these species alsois obviously of considerable interest.

The manner in which aquatic corneasare adapted to their environment, the dif-ference between those exposed to hypo-tonic fresh water and to hypertonic seawater, and the mechanism for maintainingtransparency and a normal corneal watercontent in these species constitute thesubject of this report.

Materials and methods

Three types of aquatic corneas werestudied: that of a typical teleost fresh-water fish, carp (Cyprinus carpio), of amarine teleost, scup (Stenotomus), andtwo species of elasmobranchs, dogfish(Mustelus canis) and skate (Raja erinacea).For comparison, some experiments werealso carried out on guinea pig and rabbitcorneas. No guarantee can be had that thespecies selected provide corneas which re-act typically for the large groups theyrepresent, i.e., fresh-water and marineteleosts, but we may assume that this isa reasonable possibility. All fish werereceived alive and in good condition in thelaboratory where they were maintained inaquariums until used. The eyes were care-fully checked by staining with fluoresceinto insure that only undamaged corneaswere used.

Experimental

Transparency. It should be emphasizedthat the corneas of all of the species stud-ied are transparent. Measurement of trans-parency in vivo is difficult, and, when themethod requires removal of the cornea forin vitro measurement, some question mayarise concerning the effect of the procedureon the property being measured. Photo-graphs of the eyes of teleosts and elasmo-branchs can be compared (Figs. 1 and 2).Details of the structure of the iris, sharp-ness of the pupillary border, and edge of


Corneal hydration 13

Fig. 1. Photograph of scup (Stenotomus) eye. Note the detail of the iris and the edge of thelens seen through the pupil, which indicate the clarity of the cornea. (Photograph by Mr.George Lower, Westtown, Pa.)

Fig. 2. Photograph of dogfish (Mustelus) eye. The detail visible in the iris demonstrates thehigh degree of transparency in this species. The notches on the inferior aspect of the pupilare normal. Note the well-developed lower lid which is movable in this fish. (Photograph byMr. Lou Gibson, Rochester, N. Y.)


14 SmelserInvestigate ', Ophthalmology

Februanj 1962

BM100

Fig. 3. Histologic section of dogfish (Mustelus) cornea. A well-developed Bowman's mem-brane (BM) is seen underlying a very thick, but easily detached epithelium. The stromaconsists of but one layer, as in mammals, of very regularly arranged lamellae. There is athin Descemet's endothelium. (Hematoxylin and eosin.)

scl

scl. p. 100

Fig. 4. Histologic section of peripheral scup cornea showing edge of the cartilaginous sclera(scl.), and bulbar conjunctiva. The relation between the conjunctival and outer corneal layer(o.l.) is well shown. The inner corneal layer (i.l.) rises from the scleral perichondrium(scl.p.) plus an internal third fibrous zone the annular ligament (a.l.) which is quite thickperipherally, but very thin centrally. The fibrous annular ligament is very different in tillsspecies from the epithelioid, glycogen-rich cells, which are found in this area in some otherspecies of fish (carp). The relation of Descemet's endothelium to the iris and angle is wellshown. Anterior chamber, AC. (Hematoxylin and eosin.)


Volume 1Number 1 Corneal hydration 15

the lens (scup) seen through the corneaare evidence of the high degree of cornealtransparency in these forms. Just as inmammals, corneal transparency in fish de-pends in part upon the maintenance of anormal state of hydration. In all experi-ments in which the cornea imbibed water,it became turbid. This phenomenon, there-fore, is a basic one common to all specieswhich have been studied and is an integralfactor in transparency of this tissue.

Anatomic studies. There is some diversityin structure even though the general cor-neal plan in all vertebrates is quite similar.All are covered by a stratified squamousepithelium which appears to be less firmlyattached in some (dogfish) than in others.This epithelium is nearly twice as thick insome (carp and dogfish) as in others (scupand guinea pig). Descemet's membrane isnot readily discernible in the fish studied,but all appeal- to be bounded internally byan endothelium, although this sheet of tis-sue is far less obvious in vertical sections mthe fish studied than in mammals.5 VrabecG

has demonstrated an endothelium in flatpreparations of the corneas of the carp andother teleosts. Its existence in elasmo-branchs, though questioned by some,7 wasreadily demonstrated in dogfish as well asin the scup by silver techniques whichwere applied to flat preparations. Bow-man's membrane, lacking in many mam-mals, is not apparent in the teleosts studied,but is beautifully developed in the moreprimitive elasmobranch (Fig. 3). Thestroma in all species is composed of lamel-lae of collagen fibers arranged in an ex-tremely regular parallel fashion. This regu-larity is nowhere more clearly shown on alight microscope level than in the dogfish.The corneal stroma is lightly acidophilicand that of all three species is metachro-matic, which indicates that all contain somemucopolysaccharides, the high concentra-tion of which is almost characteristic ofthis tissue. However, the degree of meta-chromasia, and therefore, probably ofmucopolysaccharide content, is varied.Some portions of the cornea of the scup

were the most metachromatic and even ex-ceeded that of the mammal. The cornea offresh-water fish was less metachromaticthan that of the mammal, although meta-chromasia was always clearly demonstra-ble. The corneas of elasmobranch fisheswere somewhat less metachromatic thanthose of scup. The degree of metachro-masia shown by any tissue is subject tomany variables, and its evaluation must bemade cautiously. In these studies, however,the corneas were all fixed in an identicalfashion, sectioned at the same thickness,and stained simultaneously, so that com-parisons might be made between them.

The cornea of many teleosts, includingthat of the scup, is divided into an outerand an inner portion.7"9 The fibers of theinner layer are very compact and easilydissociated from those of the outer andlooser layer. The inner layer is continuouswith the sclera, or at least with the peri-chondrium of the scleral cartilage. Theperipheral cornea includes an annular liga-ment which forms a part of the inner cor-neal layer—thick at the edge and very thinin the center. These features are shown inFig. 4. The outer and looser layer of thecornea appears to be more closely relatedto, or continuous with, the episcleral con-nective tissue, or conjunctiva, and has,therefore, been called the conjunctival por-tion in contrast to the inner or scleralportion. They are easily separable; theouter, when grasped with forceps, is foundto be as mobile as the conjunctiva. Bothlayers are extremely transparent, but be-come turbid when they are deformed bytension. The central area of the cornea isvery thin. The proportion of the two layersthere is shown in Fig. 5.

Comparative hydrophilia. The tendencyof corneas to imbibe water in vitro wasfound to exist in the teleost fish as in mam-mals. However, some difference in degreeand in reaction to the salt content of thesolutions was noted. These experimentswere conducted very simply. Whole cor-neas were freed from their epithelial andendothelial covering, immersed in distilled


16 Smelserstigntiuc Ophthalmology

February 1962

1.1.

Fig. 5. Histologic section of the central area of the scup cornea. Note the division into anouter (o.l.) and inner layer (i.l.). The posterior surface of the inner layer consists of anendothelium (DE) and a few cells and fibers continuous with the annular ligament shownin Fig. 4. (Hematoxylin and eosin.)

water or in various concentrations of so-dium chloride solutions adjusted to pH 6.8to 7.0 with sodium bicarbonate, and keptin an ice bath. At intervals the corneaswere removed, blotted, carefully on dryfilter paper, and weighed on a torsion bal-ance. The increase in weight of cornealtissue caused by the imbibition of water isshown in Figs. 6 and 7. Because the scupcorneal layers tended to become separatedduring this procedure, the swelling of theouter and inner layers was examined sepa-rately. Except where noted, the data in allexperiments apply to the inner layers(which includes the annular ligament, Fig.5) of the scup cornea. When tested in thisway in distilled water, the marine teleost(scup) cornea was found to imbibe morewater than the others, and the cornea ofthe fresh-water teleost (carp), the least.These results might be expected in view ofthe environment to which these animals areadapted. Fish adapted to life in hypotonicfresh water in which corneas swell themost, possess the least hydrophilic corneas.In addition, if, as is generally supposed,

corneal swelling is dependent upon meta-chromatically stainable mucopolysaccha-rides, the swelling curve of these tissues isin accordance with the degree of stainingreaction which was observed. The corneasof all three species were found to swell lesswhen immersed in a solution of 0.85 percent sodium chloride than in water. In thisinstance, however, the degree of swellingof the scup and guinea pig corneas was al-most identical, but exceeded that of carp(Fig. 7). However, when these corneaswere immersed in a salt solution isotonicwith fresh-water fish plasma, i.e., 0.5 percent sodium chloride, the swelling curvesobtained did not preserve the same ratiosto each other. The scup cornea imbibedminimum fluid in this dilute salt solution,and its swelling was exceeded by that ofboth the carp and the guinea pig whichindicated that the uptake of water was notdirectly related to the concentration of ionsin the solution.

Swelling of outer and inner portions ofthe scup cornea. The inner and outer por-tions of the scup cornea were found to


Volume 1Number 1 Cornea! hydration 17

%400

Uj

§

HOURS

Fig. 6. Comparison of the swelling (increase in weight) of mammalian, sea (inner layeiscup), and fresh-water teleost corneas in distilled water (pH about 7.0) at 0° C. The ithelium and endothelium had been removed. Each curve represents the average of fiv€seven corneas. Note the decrease in weight of the guinea pig corneas between the secand fourth hours.

— — — Scup• Guineo Pig

Carp

in NaCI solution

HOURSFig. 7. Comparison of the swelling (increase in weight) of mammalian, sea (inner layer,scup), and fresh-water teleost corneas in 0.85 per cent sodium chloride solution, pH about7.0 at 0° C. Five to seven corneas, freed of their epithelia, are represented by each curveNote that the scup and guinea pig corneas swell to the same extent in this solution and thatno decrease in weight occurred at the fourth hour.


18 SmelserInvestigatioe Ophthalmology

February 1962

1O

1050

1000

950

900

850

800

750

700

650

600

550

500

450

400

350

300

250

200

150

100

50

/ Outer loyer in H 2 0

Inner layer in H 2 0

Inner layer in NaCI solution

HOURSFig. 8. Comparison of the swelling of epithelium-free inner and outer layers of the scup cornea indistilled water and in 1.23 per cent sodium chlo-ride solutions. Solutions were pH about 7.0 and0° C. The curves are averages of five experiments.

differ not only in morphology and tinctorialcharacteristics but also in their ability toimbibe water. The extreme difference inbehavior of the two layers made it neces-sary to investigate the two portions sepa-rately. The plane of cleavage between themwas definite, so that very little outer cor-neal tissue remained attached to the innerlayer. The difference in the hydrophilia iswell shown in Fig. 8. The outer corneallayers were found to imbibe far greaterquantities of fluid than the inner. When thecorneas were immersed in salt solution, the

swelling of both layers was greatly re-duced. The weight of the outer cornea in-creased tenfold in distilled water withina few hours and was the most spectacularexample of swelling seen in any cornealtissue. It maintained, in a sense, the sameswelling characteristics noted in earlierstudies, i.e., the swelling was almost exclu-sively in the anteroposterior axis. At theend of an experimental period, the cornealsample had the appearance of a slightlyflattened, oval, gelatinous mass, which,however, maintained its shape in the solu-tion much as does the vitreous humor or ajelly fish.

Effect of epithelial or endothelial abra-sion on corneal hydration in vivo. Obvi-ously, the corneal stroma of mammals andof fresh and marine teleost fish are allhydrophilic although differences exist inthe degree of water imbibition not onlybetween the species, but, in the scup, be-tween different parts of the cornea. Main-tenance of normal water content in mam-mals is well known to be, in some manner,dependent on the presence of an intactepithelium and endothelium. The impor-tance of these layers to corneal hydrationin fish was therefore investigated. The epi-thelium was removed from one cornea ofeach of 9 scup, and, in another series of17 fish, the endothelium of one eye wasthoroughly abraded. In the latter operation,in one series, the anterior chamber was en-tered by means of a keratome incision atthe limbus, and the endothelium abradedwith an iris spatula. Control experimentswere performed in which the keratome in-cision was made, but the endothelium wasnot deliberately injured. In another group,the endothelium was abraded with a needlewhich was inserted through the limbalsclera and conjunctiva, because it wasfeared that the keratome incision mightaffect comeal hydration by allowing theescape of aqueous humor or the entry ofsea water. The needle was also introducedinto the eye used as a control, but the en-dothelium was not touched. Followingthese operations the fish were returned to



the aquarium, and, at autopsy, the intact-ness of the epithelium was checked bystaining with fluorescein. Six hours after theoperations the corneas were removed byvery careful dissection and weighed. Inorder to determine the normal variation inweight between the left and right corneasand the error due to dissection and weigh-ing, the intact eyes of a control series of 17scup were also dissected in the same man-ner and the corneas weighed. In this seriesit was demonstrated that the averageweight of the right and left corneas did notdiffer by more than 5.5 to 6.0 per cent(Table I). Hydration of the corneas in theexperimental series was judged by thechange in weight of the cornea of the ex-perimental eye relative to that of the con-trol eye of the same fish.

Removal of the epithelium in scup re-sulted in definite swelling of the stromalconnective tissue (Table II), even thoughit was bathed in a hypertonic salt solution(sea water). Swelling of the outer layer(129 per cent) was much greater than thatof the inner (30 per cent) which was to beexpected in view of the experiments with in

Table I. Difference in weights of the rightand left normal corneas

Left eye

Outerlayer

Innerlayer

Bothlayers

Right eye

Outerlayer

Innerlayer

Bothlayers

12.225.510.022.033.226.222.017.625.822.042.728.229.864.764.319.8

14.833.813.131.635.229.628.822.028.222.256.428.424.762.869.611.4

27.059.323.153.668.455.850.839.654.044.299.156.654.5

127.5133.931.2

12.223.311.822.230.223.620.617.522.624.046.119.421.659.446.720.1

15.132.811.831.033.629.029.621.529.626.655.625.327.263.059.212.8

27.356.123.653.263.852.650.239.052.250.6

101.744.748.8

122.4105.932.9

Average28.8 32.0 61.2

Difference

26.3 31.5 57.8

-9% - 1 % -5.5%

Table II. Effect of epithelial abrasion oncorneal hydration in vivo* (weight inmilligrams)

Epithelium intact

Outerlayer

Innerlayer

Epithelium abraded

Outerlayer

Innerlayer

18.522.121.721.653.630.421.823.812.0

27.728.222.122.468.632.625.834.212.6

51.643.841.251.2

108.263.257.262.238.2

34.431.026.632.789.042.832.848.218.2

Average 25.1 30.5 57.4 39.5Gain +129% +30%

0 Duration of experiment 6 hours.

vitro swelling (Fig. 8). Abrasion of the en-dothelium also provoked definite swelling(Table III) of both corneal layers, but thehydration was less than when the epithe-lium was damaged, and, again, greaterswelling occurred in the outer layers. Someeffect on corneal hydration was apparent asa result of opening the anterior chamber,thus permitting loss of aqueous humorand/or entrance of sea water.

Similar experiments were done on guineapigs with somewhat different results. Inthis species endothelial abrasion causedgreater uptake of water than when the epi-thelium was removed. These observationsconfirm those of Maurice and Giardini.10

This reaction is the opposite of that in thefish, in which greater corneal hydration re-sulted from removal of the epithelium.

Role of metabolism in maintenance ofcorneal turgescence. In fish, the importanceof the epithelium in the prevention of cor-neal hydration may depend in part on thethickness of that tissue which may serve toimpede mechanically the movement ofwater into the cornea. However, this resultmight be achieved far more efficiently bymetabolic activity which assists in main-taining normal corneal water content.

Experiments were performed in whichthe metabolic activity of the scup corneawas reduced in vitro by low temperature or



February 1962

Table III. Effect of endothelial abrasion oncorneal hydration in vivo* (weight inmilligrams)

Corneal layer

Outer Inner

Eye intact

21.533.221.218.819.319.2

Average22.2

24.142.032.825.623.322.0

28.3

Corneal

Outer

layer

1 Inner

Endothelium scrapedwith iris

35.299.449.243.453.237.3

52.9Gain +138%

spatula30.675.052.037.235.635.6

44.3+57%

Keratome incision in both eyesEndothelium intact

62.423.545.128.035.8

Average38.9

55.421.342.014.843.5

35.4

Endothelium scrapedwith iris

81.523.481.528.062.0

55.3Gain +42%

spatula52.221.252.217.058.2

40.1+13%

Anterior chamber of both eyes entered with aneedle

Endothelium intact Endothelium abradedwith a needle

26.429.326.618.425.836.8

Average27.2

29.428.023.522.634.028.2

27.6

25.451.847.050.051.864.2

48.4Gain +78%

30.033.736.231.749.628.8

35.0+27.5%

metabolic activity was reduced by placingthese preparations in ice for IS hours. Inthe first experiment, one eye was removedas the normal control and the other chilled.The corneal weights are given in Table IV.Those which were chilled increased inweight by 9.6 per cent, a change of doubt-ful significance. In the second experiment,both eyes were chilled for 18 hours; onewas then weighed, and the other warmedto 20° C. and the sea-water medium oxy-genated for 6 hours. During the second,warm, aerobic period the corneas gained 18per cent in weight. The data, given inTable V, show no evidence that a deturges-cence accompanied the return to normalphysiologic conditions.

Experiments were also conducted inwhich one eye, the normal control, was dis-sected and weighed immediately; and theother eye was prepared as described previ-ously and kept under anaerobic conditionsby bubbling nitrogen through the mediumfor 6 hours at the aquarium temperature

Table IV. Effect of low temperature (invitro) on corneal weight

Left eye, normal weight Right eye after IS hours(mg.) at 0° C. (mg.)

65.254.853.542.5

67.661.862.544.8

"Duration of experiment 6 hours. Average 54.0Gain

59.2+ 9.6%

by the exclusion of oxygen. The entire orbitand a portion of the adjacent head struc-tures were dissected carefully so that noabrasion of the corneal epithelium or injuryto the conjunctiva occurred. This waschecked by examination with a dissectingmicroscope and fluorescein staining. Thehalf heads were placed in 50 ml. tubes ofsea water which contained 200 mg. percent glucose and were kept at the tempera-ture of the aquarium. Comparisons weremade between the right and left eyes of thesame fish. In one series of experiments,

Table V. Effect of oxygenation and warm-ing following 18 hours at 0° C. on cornealhydration (weight)

Left eye after 18 hoursat 0° C. (mg.)

30.331.136.261.4

Average 39.7

Right eyefollowing oxygena-

tion for an additioiwl6 hours at 20° C. (mg.)

32.841.841.271.4

46.8Gain +18%



Table VI. Effect of in vitro anaerobiosis oncorneal weight

Left eye normal weight(mg.)

Right eye after 6 hoursanaerobiosis (mg.)

33.857.224.449.6

36.753.324.248.4

Average 41.2Loss

40.6- 1 %

(21° C ) . The results, Table VI, show noeffect of the anaerobiosis.

In a second and larger series of experi-ments, both eyes were placed in the tubesas described. One tube was continuouslyaerated by bubbling oxygen through a sin-tered glass aerator and the other tube wasgassed with nitrogen to create an anaerobiccondition. The preparations were main-tained for 6 hours at an aquarium tempera-ture of 12° C.,* after which the corneaswere carefully dissected and weighed. Thewater content was determined by dryingthe tissue over calcium chloride in avacuum desiccator at 45° C. to a constantweight. The anaerobiosis caused a strongcontraction of the melanophores in the irisand conjunctiva so that the eyes kept undernitrogen had a silvery appearance, whereasthose exposed to the oxygenated mediumwere a normal golden color. Therefore, allcorneal layers received adequate oxygen inone series but not in the other, as judgedby the response of the melanophore. Thedifference in weight (5 per cent) betweenthe aerobic and anaerobic corneas, TableVII, was insignificant (compare with nor-mal series, Table I). The dry weight ofthese corneas was determined and no effectof anaerobiosis on water content wasfound. The difference between the twogroups was only 2 per cent. In this species(scup), therefore, a normal aerobic metab-olism of the cornea does not appear to berequisite for maintenance of corneal turges-cence as it is in the mammal.

Swelling in various salt solutions. It wasnoted in the first experiment that the swell-ing of corneas was not proportional to theconcentration of the salt solution used. Thiswas investigated further in a study of theswelling of the inner layer of the scup cor-nea. These were prepared as before,weighed, placed in the various solutions,and weighed at intervals to determine theamount of water they imbibed. The mediatested were: distilled water, 0.5 per centsodium chloride, 0.85 per cent sodiumchloride, 1.23 per cent sodium chloride(isotonic with marine teleost plasma). 3.2per cent sodium chloride (equivalent to seawater), sea water itself, marine teleostRinger's solution,11- 12 scup aqueous humor,and glucose 7.7 per cent (isotonic with 1.23per cent sodium chloride). The pH was ad-justed with sodium bicarbonate to 6.8 to7.0 in all experiments, and the flasks con-taining the corneas were immersed in anice-water bath. The swelling of the scupcornea in some of these solutions is shownin Fig. 9. It is clear that the swelling whichoccurred was not related to the osmoticpressure of the medium; for example, theswelling in distilled water and in glucosesolution was nearly identical. It would ap-

Table VII. Aerobic metabolism and cornea!hydration (weight of corneas maintained 6hours in vitro under aerobic and anaerobicconditions)

"These and one other experiment were conducted in thespring when the temperature of the sea water was low.

Aerobic (mg.)

79.674.465.458.673.257.449.852.655.353.545.438.043.843.041.4

Average 55.4

Anaerobic (mg.)

80.277.463.664.679.862.154.656.755.349.752.043.443.541.646.7

58.1Gain + 5%



February 1962

Fig. 9. The swelling of the inner layer of scup corneas in various solutions, all at pH about7.0 and 0° C. Note that swelling was least in the 0.5 per cent sodium chloride solution andmaximal in distilled water or glucose solution.

pear that the presence of ions is important,and, also, the presence of more than oneion species, because swelling was distinctlyless in Ringer's solution (shown in Fig. 14)or sea water (Fig. 9), than sodium chloridesolution of similar concentrations. There issome indication of an optimum concentra-tion of salts at which swelling is minimal(0.5 per cent). The possible significance ofthis must await further study. No tests weremade at concentrations of less than 0.5 percent sodium chloride. The swelling curvesin some media were omitted from thegraph in the interest of clarity (e.g., thecorneas in 0.85 per cent which swelled tonearly the same degree as in 1.23 per centsodium chloride solution. Those in themarine teleost Ringer's solution or in aque-ous humor swelled a little more than thosein sea water).

The effect of bivalent cations and anionson corneal swelling. The difference in swell-

ing in simple salt solutions and those con-taining many ions (sea water and Ringer'ssolution) suggested that the ionic speciespresent were of importance. To test thisconcept further, solutions of bivalent ca-tions, calcium chloride or magnesium chlo-ride, were prepared. In addition, solutionscontaining bivalent anions, sodium sulfateor sodium phosphate, were also made totest their effect on swelling. Control solu-tions of sodium chloride or potassium chlo-ride were used. All solutions were isosmoticwith 0.5 per cent sodium chloride, sincecorneal swelling had been shown to beminimal at that concentration. The pH ofall solutions was 6.8 to 7.0, and the experi-ments were conducted at 21.0° C, the tem-perature at which the scup had been living.The results of this experiment are shownin Fig. 10. The degree of corneal swellingis seen to depend in part on the charge ofthe ions which made up the solution. The



swelling was much more marked in solu-tions containing bivalent anions, phosphateand sulfate, than in solutions containingmonovalent chloride ions. In contrast, theimbibition of water was less in the pres-ence of bivalent cations, calcium and mag-nesium, in comparison with solutions ofpotassium or sodium chloride. As perhapscould be expected, an intermediate degreeof swelling resulted when both the cationand the anion were bivalent (magnesiumsulfate solution). Although variation in cor-neal swelling occurred, the difference dueto the charge on the ions was clearly sig-nificant.

Role of colloid osmotic pressure in cor-neal swelling. With the exception of theexperiments reported by Pau13 and byDohlmann and Anseth,14 most studies on

the swelling of corneal tissue have con-sidered only the role of ions on osmoticpressure. Colloids, because of their largemolecular weight, do not exert great os-motic force in comparison with that ofsmall ions, but, because they are less dif-fusible and may form part of tissue struc-ture, they are of extreme physiologicimportance. In fact, in preparations such ashave been described here, the highly dif-fusible ions may be expected to haveachieved equilibrium between the solutionand the interior of the cornea very quickly.The nondiffusible substances within thecornea then became the major factor re-sponsible for water imbibition. Therefore, aseries of experiments was conducted on theswelling of corneal stroma of the scup invarious concentrations of a colloid poly-

400

350

k.

IJJ 300

CX. 250

k. 20°

^ 150

2 io°

<O 50

-

-

i i i

s3~- - —•"" —~^ZS\i

Cl "

1 Mg+ + _

Y^-'X '(

>

= Range

HOURSFig. 10. Graph comparing the swelling of the inner layer of scup corneas in solutions con-taining bivalent cations and anions with that in solutions containing monovalent ions (KCl).The solutions were of calcium chloride, magnesium chloride, sodium phosphate (a mixtureto produce pH 7.0), and sodium sulfate, all at pH of about 7.0 and a temperature of 21° C.Each curve is the average of five corneas, and the total range of the variation is shown.



February 1962

0/

300

250

2 0 0

k j

150

100

50

HOURSFig. 11. Comparison of the swelling of the inner layer of scup corneas in 10 per cent aqueoussolutions of large and small polymers of PVP in the absence of salt. Note the greater swellingin the solution of the lower molecular weight polymer, K30. Initiation of swelling was delayedin both solutions but much more in that of the larger polymer, K90. Each curve representsfour or five corneas. The solutions were at pH about 7.0 and 21° C.

vinylpyrrolklone (PVP),* which, becauseof its inert nature, has been successfullyused as a plasma extender and is availablein various polymer sizes. They and theiraverage molecular weights are: K90,360,000; K60, 160,000; K30, 40,000; andK15, 10,000. The osmotic pressure of equalconcentrations of these polymers was, ofcourse, much greater in the case of K15than in K90. The solution of K90, however,was far more viscous than the others. Con-centrations ranging from 10 per cent to 1.25per cent were prepared in either water or1.23 per cent sodium chloride. The pH ofthese solutions was 6.8 to 6.0 (adjustedwith sodium bicarbonate) and the experi-ments were conducted at 21° C. Fig. 11shows the swelling curves which werefound when corneas were immersed in 10per cent aqueous solutions of PVP, K90and K30. The corneas swelled in both solu-

°Supplied by An turn Chemicals, New York City Divisionof General Aniline and Film Corporation.

tions but less in the more viscous, butosmotically weaker, solution of K90. Animportant difference in the shape of thecurves is seen in the first 30 minutes, inwhich shrinkage rather than swelling oc-curred. This is in sharp contrast to experi-ments with salt solutions in which swellingbegan within this time. Obviously, corneasswell in PVP solutions, but the onset of thisprocess is delayed and swelling is less inthe more viscous, osmotically weaker, solu-tion. When both salt and PVP were presentin the solution, however, the behavior ofthe swelling of the corneal stroma wasradically different (Fig. 12). Swelling wasgreatly inhibited when salt was present;in fact, in 5 per cent K90 with salt, thecorneas were deturgesced below the nor-mal value and held in a steady state withrespect to hydration for the duration of theexperiment. The transient 30 minute inhibi-tion of swelling, noted in Fig. 11, con-tinued, and, instead of swelling, a perma-nent maintenance of a near normal corneal



hydration was achieved. When solutions ofthe same concentrations of PVP were usedwithout salt, marked swelling occurred,and, although the rate was less, it wasscarcely distinguishable from that whichtook place in distilled water. Evidently thepresence of colloids has an extremely im-portant effect, but this is clearly demon-strable only in the presence of salts. Thedata in Fig. 13 demonstrate that the swell-ing power of corneal tissue may be bal-anced, or even overcome, by appropriateconcentrations of osmotically active colloidin salt solution. Swelling of the corneas oc-curred in the more dilute PVP solutions,and shrinkage in the more concentrated. Asolution of about 3.75 per cent K90 PVP, inthe presence of salt, would provide a me-dium in which the exchange of water be-tween the cornea and the outer solutionwould be in balance.

Role of aqueous humor in maintenanceof corneal turgescence. There is a verygood possibility that these experiments oncorneal swelling in solutions of PVP andsalt provide a model of the system whichis, in fact, the physiologic one in manyanimals. It is well known that the aqueoushumor of many fish and birds is viscousand contains large quantities of hyaluronicacid or similar mucopolysaccharides.15"17

Since the aqueous humor is separated fromthe corneal stroma by Descemet's endothe-lium, hyaluronic acid would not be free todiffuse into it. The existence of a mem-brane preventing the diffusion of colloidsinto the stroma is important. Inner layersof scup cornea were found to swell (70 percent in 6 hours) when immersed in freshaqueous humor of the scup. In those ex-periments, however, there was no protec-tive endothelium, and aqueous humor was

400 -

350

k.

S 300

250

IGH

T

%

?/VE

AL

8

200

150

100

50

K90- l .25% in NoCI solution _

• - - - • • K90-5% in NoCI solution

HOURSFig. 12. Graph showing the efFect of salt on swelling of the inner layer of scup corneas insolutions of PVP. Note that the corneas immersed in a 5 per cent solution of K90 with saltdid not swell, but remained deturgesced below normal, whereas those in the same concentra-tion of K90 without salt underwent maximal swelling. The swelling of corneas in distilledwater is shown for comparison. Each curve represents four corneas.


26 SmelserInrcstigativa Ophthalmology

February 1962

Fig. 13. The swelling of the inner layer of scup corneas in various concentrations of PVPK90 in 1.23 per cent sodium chloride solution is shown. The swelling which occurs in 1.23per cent salt solution and in distilled water without PVP is shown for comparison. All solu-tions were 21° C. and pH 7.0.

free to diffuse into the corneal connectivetissue. In addition, in the in vivo experi-ments when the endothelium was damaged,the corneal connective tissue exposed to theaqueous humor did swell (13 per cent to27 per cent).

The inner layers of the corneas of 12scup were weighed and inserted into nar-row, empty, but moist (with salt solution)Visking dialysis tubes immersed in freshscup aqueous humor and reweighed 6hours later. The corneas were placed in thecenter of a lengtli of dialysis tubing, whichwas bent into a U shape, the open ends ofwhich extended well above the aqueoushumor. The corneas were not subject topressure other than that induced by thestiffness of the wet membrane. They werekept at the temperature of the sea water(12° C. during the springtime experiment)in which the fish had been living. Control

experiments were made in the same man-ner, but the membranes and corneas wereimmersed in distilled water or 1.23 per centsodium chloride solution instead of aqueoushumor. The results are shown in TableVIII. The corneas dialyzed against saltsolution or water swelled by imbibing fluidthrough the semipermeable dialysis mem-brane. Those exposed to aqueous humorthrough the membrane did not swell. Pre-sumably, this resulted because the corneaswere in a normal ionic environment andthe colloid osmotic pressure of the aqueoushumor balanced that of the connective tis-sue, and the dialysis membrane adequatelyreplaced the function of the endothelium.

Swelling properties of elasmobranch cor-neas. The above experiments have beenconfined to teleost corneas, mainly, thoseof a marine form, plus some references tomammalian corneas for comparison. Similar



Table VIII. Swelling of corneas (innerlayer) during 6 hour dialysis at 12° C.

Originalweight(mg.)

Finalweight(mg.)

Dialyzed against 1.23%solution of sodiumchloride

Average

23.333.534.219.936.3

29^4

29.038.837.424.639.0

33.8Gain +15%

Dialyzed against distilledwater

Average

18.633.732.025.255.3

33.0

27.439.442.632.661.7

40.7Gain +23.3%

Dialyzed against aqueoushumor

Average

27.436.822.120.731.745.849.228.526.418.422.432.3

30.1

28.238.621.619.234.647.042.230.027.020.624.533.8

30.6Gain +1.6%

experiments on corneal swelling in waterand salt solutions were also performed ontwo species of elasmobranchs, the dogfish(Mustelus) and the skate (Raja). The be-havior of the corneas of these species wasof interest because: (1) they maintain theirinternal osmotic equilibrium in a peculiarmanner, (2) the corneal structure isvery similar to that of mammals, and(3) these species are more primitive thanare the teleosts, and for this reason onemight expect to uncover more basic prop-erties of corneal tissue.

The swelling experiments were con-ducted as before, with the same solutions,and with the addition of an elasmobranchRinger.11-18 Fig. 14 shows the result withmost of these solutions. Those omitted forthe sake of clarity in the graph did not dif-

fer from those shown. The swelling curvesof the scup cornea in distilled water andteleost Ringer are included for comparison.The comeal stroma of the elasmobranch ofboth species, did not swell in any of themedia tested. In fact, some loss in weightwas observed. The corneas of these fishdiffer from those of all other vertebrates, sofar studied, in their failure to imbibe water;in fact, it would appear that these eyeshave the reverse problem from those of allother species in that there is a need to pre-vent the loss of water from the stromarather than to prevent its enhance.

Discussion

Corneas of all vertebrates are structur-ally very similar, since they are constructedof regularly arranged collagen lamellaebounded by epithelia. The important mu-coid constituents, as shown by metachro-matic staining reactions, are present in all,but vary in concentration and possibly inkind and in degree of polymerization.

Corneas of all species which have beenstudied, except the elasmobranchs reportedhere, imbibe water from the surroundingmilieu after damage to the limiting cellularmembranes or in vitro. It has been tacitlyaccepted in recent years that maintenanceof normal turgescence involves metabolicwork, a "pump" of some sort, located in theepithelium or endothelium. It has beenshown in mammals that reduction of cor-neal metabolism by anaerobiosis or loweredtemperature adversely affects its opticalproperties,10 reduces transparency,20 andthe stroma becomes hydrated. Repeated at-tempts to demonstrate this phenomenon inscup failed. Corneas transferred to warmaerobic conditions following 18 hours incold increased in weight. In contrast, anal-ogous experiments with rabbits,2'21 showedthat corneas decreased in weight due tothe expulsion of water when they werereturned to normal temperature after re-frigeration. Scup corneas, maintained underanaerobic conditions for 6 hours, gainedonly 5 per cent in weight, whereas in anal-ogous experiments rabbit corneas22'23 in-



February 1962

Scup m Teleost Ringer solution

Elasmobranch in Ringer solution

0 . 5 % NaCI—• — — — —• H O

HOURSFig. 14. Graph demonstrating the absence of swelling of elasmobranch, Mustelus (dogfish)corneas when immersed in various media. The epithelium and endothelium had been removed.Swelling of the inner layer of teleost corneas (scup) in water and a salt solution is shownfor comparison. All solutions were pH about 7.0 and 21° C.

creased their water content by 50 per cent.Apparently the epithelial barriers in fishdo not house the same mechanisms as theydo in mammals. No metabolic "pump" inthe mammalian sense could be shown.However, both epithelia are of great im-portance in maintaining cornea! turges-cence in fish, because, if they are removed,the corneas imbibe water. Possibly, theyserve in a more passive or protective roleby separating the stroma from aqueous hu-mor and sea water by a relatively imper-meable coat. It is not suggested that theyare impermeable to water, but that theepithelium may serve to "retard" the en-trance of sea water, and the endotheliummay be impermeable to high molecularweight constituents of the aqueous humor,as will be discussed below.

The studies of Francois and co-workers24

and of Maurice25 showed that the collagen

fibers of swollen corneas were not in them-selves swollen, but the interfibrillar spaceswere enlarged. Heringa and others20 foundthat corneas from which the mucopoly-saccharides had been extracted did notswell, and concluded that it was the con-nective tissue ground substance which wasmainly responsible for the hydrophilia.The mucopolysaccharides are long, nega-tively charged polymers of chondroitin, itssulfates, and keratosulfates, which arefirmly bound to protein. These are fixed tocollagen fibrils, are part of the structureof the cornea, and are not free to diffusefrom it. Because of this, they contributematerially to the Donnan effect. They at-tract and hold water which diffuses intothe cornea.

When a tissue such as the cornea isplaced in water, water molecules tend todiffuse from the medium into the tissue



spaces between the structural components.If this structure is rigid, or elastic, so thatits constituents are not easily displaced bythe inward diffusing water, no swelling willwill occur; there will be simply an ex-change of water between the outer mediumand that of the interstitial spaces.27 If, how-ever, the bonds which hold the structuretogether are weak, water which is diffusedinto that tissue will be bound to hydro-philic sites, the structures will be displaced,and swelling will result. In short, organiza-tion of structure combats swelling. Swell-ing results when the forces which tend tohold the structure in a compact arrange-ment are overcome by those which attractand bind water. For example, the pres-ent experiments show that the looser outerlayer of the cornea swells three times asmuch as the more compact inner layer, al-though staining reactions indicate a nearlyequal mucoid content.

It seems possible to explain the data ob-tained in these experiments in terms ofthe relationship of macromolecular anat-omy to corneal structure and swelling. Allof the corneas, save those of elasmobranchs,swelled readily in salt solutions but muchmore in the absence of ions (in water, solu-tions of glucose, or PVP). The presence ofsalts did not reduce swelling because of anosmotic pressure effect but, it is believed,because of their effect on corneal struc-ture. It was clear that the ability of saltsolutions to inhibit swelling was not de-pendent on their concentration and, there-fore, osmotic pressure for several reasons:(1) Their effect was not proportional (inthe range studied) to concentration. (2)Bivalent cations in isosmotic concentrationswere more effective in preventing swellingthan were monovalent ions, and the pres-ence of bivalent anions enhanced theswelling. (3) No osmotic pressure differen-tial could be created by salt solutions, be-cause they could diffuse readily into andout of the tissue spaces. It has been shownthat they move freely in these spaces.28

Swelling in salt solutions is due largely tothe hydrophilic properties of the large non-

diffusible molecules. The corneas swelledin solutions of glucose and of the smallPVP polymers K15 or K30 because theseuncharged solutes had no effect on thehydrophilic property of the mucoid, whichcontinued to bind water. As long as theconcentration of the PVP in the externalmedium was high, water left the cornea be-cause of the osmotic imbalance. When thePVP had diffused into the tissue, no os-motic difference existed, and swelling oc-curred again as it would in distilled water.When the large polymers of PVP wereused, their viscosity prevented their rapidentrance into the cornea and swelling wasdelayed. In the presence of salts, it is pos-tulated that the corneal structure becamemore compact and the large polymers ofPVP could not diffuse into it and swellingwas prevented. In the experiments of Dohl-mann and Anseth,14 this phenomenon wasnot observed because the colloid was keptfrom entering the cornea by means of asemipermeable membrane. In the experi-ments of both Dohlmann and of Pau, thecolloid was studied in the presence of ionsand buffers, so that the effect of salts wasnot seen.

It seems reasonable to postulate that con-nective tissue of the cornea swells less inionic solutions because of the effect of theions on the compactness of the chargedpolymers, such as corneal mucopolysac-charides. Closely related compounds, e.g.,hyaluronic acid, exist in free solution asrandom coils and occupy a space in waterwhich is large in relation to their mass,29

and which is reduced when ions are pres-ent.30"33 This may come about in part be-cause portions of the polymer chains mayrepel one another because of their negativecharges. When these charges are satisfiedby the presence of cations in the solutionsthe chains become more compact. In addi-tion, the water in the interstices of therandom coil is probably, to some degree,"organized" or held in a quasi-crystallineform as it is in collagen.34 Cations diffusinginto such a system might be expected toneutralize the charges on the polymer,



February 1962

allowing the random coils to become morecompact. The water held in the crystallikeconfiguration could also be reduced sincethe forces holding water to protein or mu-coids are weaker than those binding it tosimple ions. An analogy may be drawn be-tween the cornea and nucleic acid filmswhich, when floated on distilled water,have been shown to expand (swell) to amuch greater degree than when floated onsalt solutions where the expansion of thefilm was inhibited.35

By analogy with the systems which havebeen studied, it seems probable that in theexperiments reported here salts acted torender the macromolecular structure of themucoid ground substance more compact,stable, or rigid, so that water diffusing intothe tissue could not disperse these molecu-lar structures so readily. This effect wouldnot inhibit the hydrophilic character of theprotein and mucopolysaccharide, so thatwater would still enter the connective tis-sue interstices and some swelling wouldoccur, but it would be limited in degree bythe molecular anatomy, and less in amountthan expected by the Donnan formula.This idea is well expressed by Gustavson*in relation to dermal connective tissue:"Both the Donnan and the Procter-Wilsonequations were worked out for diffusion ofions into a volume sufficiently large forsurface forces to be neglected, and not fora diffusion into a set of capillary tubes,which skin nearly represents. It is there-fore obvious that, as soon as structure fac-tors become prominent in the system, thesimple calculations based on the Donnaneffect can no longer be applied in theiroriginal form. The higher the restrain im-posed on the structure of the solid phase,the less free water will be present, whichaccordingly implies introduction of newand unknown factors into the simple equa-tions."

When corneas were immersed in PVPsolution without salt the corneal structureswere more open or loose and the PVP dif-

°Ref. 27, p. 167.

fused in, thus negating any effect it mighthave had if it had been kept outside. In thepresence of salt, the corneal tissue remainedmore compact, the large viscous PVP poly-mer K90 could not diffuse into it, but re-mained outside to counteract the Donnaneffect of the structural mucoids and pro-teins which could not diffuse out. It is sug-gested that this experiment provides amodel of the system which maintains thecorneas of fish, and possibly some otheranimals, in their normal state of hydration.The endothelium may serve as a semiper-meable membrane, that is, permeable towater and to salts, but impermeable tolarge molecules of the aqueous humorwhich, in our experiments, was shown tobalance the colloid osmotic and swellingpressure of the cornea so that it remaineddeturgesced. It is a reasonable assumptionthat the aqueous humor is constantly re-newed and serves to maintain the corneain its normal state. Since the formation ofaqueous humor would require work, meta-bolic activity would still be involved in thewater regulation of the cornea.

The concept of a metabolic pump in thecorneas has led to a search for ion pumpswhich would "pump out" the salt and waterwhich had diffused into the tissue. A so-dium pump was found,36 but the sodiumwas moved inward, not outward, thus add-ing to the puzzle. There is a possibilitythat this phenomenon has no real bearingon the problem of corneal hydration, thatit merely represents a vestigial function ofsurface epithelium, developed in amphibiawhere the classical sodium pump (in frogskin) behaves as in the cornea of the rab-bit. A possible function of the sodiumpump of the cornea, however, is suggestedby these experiments. It may serve in somespecies (although probably not in saltwater fish) to insure a proper ionic environ-ment of the connective tissue of the cor-nea, so that its structure may remain com-pact and stable.

In the absence of experimental data, itis unwise to attempt to extend the conceptformulated here to mammals. Some fea-



tures of it, however, may apply, namely,the role of salts in maintaining structuralcompactness on a macromolecular scale.An additional system may have evolved forthose species lacking a colloid osmoticforce in the aqueous humor. The biologicreason, if any, for the change in type ofaqueous humor and mechanism for themaintenance of corneal deturgescenceshould be interesting. Perhaps even moreintriguing is the problem of how the elas-mobranch cornea is made so that it pos-sesses a high degree of transparency, pre-sumably a reasonable content of mucopoly-saccharide ground substance, but lacks theproperty of hydrophilia. Having establisheda seemingly ideal cornea presumably earlyin the evolutionary series, it is puzzlingwhy more complicated systems evolved.

R E F E R E N C E S

1. The transparency of the cornea: a sympo-sium, Oxford, 1960, Blackwell Scientific Pub-lications.

2. Harris, J. E.: The physiologic control ofcorneal hydration. The First Jonas S. Frieden-wald Memorial Lecture, Am. J. Ophth. 44:262, 1957.

3. Cogan, D. G., and Kinsey, V. E.: The cornea.V. Physiologic aspects, Arch. Ophth. 28:661,1942.

4. Baldwin, E.: An introduction to comparativebiochemistry, London, 1949, Cambridge Uni-versity Press.

5. Smelser, G. K., and Chen, D. K.: A com-parative study of the structure and hydrationproperties of corneas adapted to air andaquatic environments, Acta XVII Cone, ophth.1:490, 1954.

6. Vrabec, F.: Studies on the corneal and tra-becular endothelium. III. Corneal endothe-lium in Teleostei, Vestnik CeskoslovenskeZoologick6 Spolecnosti. Acta Soc. zool. Bo-hemosloven. XXIII: 161, 1959.

7. Walls, G. L.: The vertebrate eye and itsadaptive radiation, Bloomfield Hills, Mich.,1942, Cranbrook Institute of Science.

8. Rochon-Duvigneaud, A.: Les yeux et la visiondes vert6br6s, Paris, 1943, Masson & Cie.

9. Duke-Elder, Sir Stewart: System of ophthal-mology, vol. 1: The eye in evolution, St.Louis, 1958, The C. V. Mosby Company.

10. Maurice, D. M., and Giardini, A. A.: Swellingof the cornea in vivo after the destruction

of its limiting layers, Brit. J. Ophth. 35:791,1951.

11. Young, J. Z.: The preparation of isotonicsolutions for use in experiments with fish,Pubbl. stazione zool. Napoli 12:425, 1933.

12. Young, J. Z.: The innervation and reactionsof drugs on the viscera of teleostean fish,Proc. Roy. Soc, London, ser. B 120:303,1936.

13. Pau, Hans: Beitrag zur Physiologie undPathologie der Hornhaut, van Graefes Arch.Ophth. 154:579, 1954.

14. Dohlmann, C. H., and Anseth, A.: The swell-ing pressure of the ox corneal stroma, Actaophth. 35:73, 1957.

15. Balazs, E. A.: Physiology of the vitreousbody, in Schepens, C. L., editor: Importanceof the vitreous body in retina surgery withspecial emphasis on reoperations, Proc. IIConf. of Retina Foundation, St. Louis, 1960,The C. V. Mosby Company.

16. Balazs, E. A., Laurent, T. C , Laurent,U. B. G., DeRoche, M. H., and Bunney,D. M.: Studies on the structure of the vitre-ous body. VIII. Comparative biochemistry,Arch. Biochem. 81:464, 1959.

17. Barany, E., Berggren, L., and Vrabec, F.:The mucinous layer covering the corneal en-dothelium in the owl, Strix aluco, Brit. J.Ophth. 41:25, 1957.

18. Cavanaugh, G. M., editor: Formulae andmethods. IV. Woods Hole, Mass., 1956,Marine Biological Laboratory.

19. Smelser, G. K.: Relation of factors involvedin maintenance of optical properties of corneato contact-lens wear, A.M.A. Arch. Ophth.47:328, 1952.

20. Smelser, G. K.: Discussion of McGraw, J. L.,and Enoch, J. M.: Contact lenses—an eval-uating study, Tr. Am. Acad. Ophth. 58:573,1954.

21. Davson, H.: The hydration of the cornea,Biochem. J. 59:24, 1955.

22. Smelser, G. K.: Morphological and functionaldevelopment of the cornea. The transparencyof the cornea: a symposium, Oxford, 1960,Blackwell Scientific Publications.

23. Langham, M. E., and Taylor, I. S.: Factorsaffecting the hydration of the cornea in theexcised eye and the living animal, Brit. J.Ophth. 40:321,'1956.

24. Francois, J., Rabaey, M., and Vander-meerssche, G.: L'ultrastructure des tissusoculaires au microscope electronique. £tudesde la cornee et de la sclerotique, Ophthal-mologica 127:74, 1954.

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


32 SmelserInoestigative Ophtlwlmology

February 1962

26. Heringa, G. C , Leyns, W. F., and Weidinger,A.: On the water adsorption of cornea, Actaneerl. morph. 3:196, 1940.

27. Gustavson, K. H.: The chemistry and reac-tivity of collagen, New York, 1956, AcademicPress, Inc.

28. Maurice, D. M.: The use of permeabilitystudies in the investigation of submicroscopicstructure, in Smelser, G. K., editor: The struc-ture of the eye, New York, 1961, AcademicPress, Inc.

29. Ogston, A. G., and Starrier, J. E.: The dimen-sions of the particle of hyaluronic complexin synovial fluid, Biochem. J. 49:585, 1951.

30. Mathews, M. B.: Chondroitin sulfuric acid—alinear polyelectrolyte, Arch. Biochem. 43:181,1953.

31. Laurent, T. C.: Studies on hyaluronic acidin the vitreous body, J. Biol. Chem. 216:263,1955.

32. Rowen, J. W., Brunish, R., and Bishop,F. W.: Form and dimensions of isolatedhyaluronic acid, Biochim. et biophys. acta19:480, 1956.

33. Balazs, E. A.: Physical chemistry of hyalu-ronic acid, Fed. Proc. 17:1086, 1958.

34. Berendsen, H. J. C., and McCulloch, W. S.:Structural properties of cells, I960, Quart.Prog. Rep., Res. Lab. of Electronics, Massa-chusetts Institute of Technology.

35. Ambrose, E. J., and Butler, J. A. V.: Swellingand orientation phenomena with nucleo-pro-tein films, Discussions of Faraday Soc. 13:261, 1953.

36. Donn, A., Maurice, D. M., and Mills, N. L.:Studies on the living cornea in vitro. I.Method and physiologic measurements; II.The active transport of sodium across theepithelium, A.M.A. Arch. Ophth. 62:741;757, 1959.


corneal hydration comparative physiology of fish and mammals

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