Corneal hydration
Comparative 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 orderly
arrangement of collagen fibers bounded by epithelia. All were metachromatic, indicating the
common possession of mucopolysaccharides. The corneas of teleost fish were found to swell
in 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 in
the carp which lives in a hypotonic medium. The elasmobranch cornea differs from those of
all other species so far studied. It is not hydrophilic, and was not found to swell in any of
the media studied. No evidence of anaerobic metabolic control of corneal hydration, such as
is characteristic of mammals, was demonstrable in the teleost fish (scup). Corneal swelling
ioas inhibited in the presence of salts, less by monovalent than by bivalent cations, and was
enhanced when bivalent unions were present. Scup corneas were found to swell readily in
aqueous solutions of polyvinyl-pyrrolidone (PVP) in the absence of salts. In the presence of
salts, 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 so
that that of the mucoid ground substance is more compact, stable, or rigid, and that this is
a major factor in the maintenance of normal corneal hydration generally. It is suggested that
the xoater balance of teleost corneas and, perhaps, of those of other animals is maintained in
the presence of salts by mucopolysaccharides in the aqueous humor which counteract the
swelling pressure of the cornea. Descemet's endothelium serves in this system as a membrane
permeable to salts and xoater but not to the colloids of the aqueous humor.
I
ing water balance in connective tissue are
fundamental ones and of great importance
in diseases of the connective tissue in general. The ultimate objective in these experiments, as well as those reported here, is to
acquire an understanding of how the cornea maintains its transparency. The many
contributions to this subject are well summarized in the symposium on The Transparency of the Cornea,1 and by John Harris
in The First Friedenwald Award Lecture.2
The problem was first uncovered clinically
by the observation that swelling of the
cornea was accompanied by loss of transparency. Therefore, the first investigations
were carried out in ophthalmic laboratories
where the observers were clinically oriented, thus leading them to study comeal
£ the literature on corneal physiology of
the past twenty years is examined, it will
he found that this tissue has excited enormous interest and stimulated many ingenious experiments with intent to understand
how its normal degree of hydration is maintained. This problem is intriguing primarily
because hydration is inextricably related to
transparency and, in addition, because the
physiologic processes involved in maintainFrom the Department of Ophthalmology, College
of Physicians and Surgeons, Columbia University.
This investigation was supported by Research Grant
B 492 from the National Institute of Neurological Diseases and Blindness, National Institutes
of Health, United States Public Health Service.
11
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Investigative Ophthalmology
February 1962
12 Smelser
hydration in mammals. These researches, as
exemplified by those of Cogan and Kinsey,3
were invaluable, and actually introduced
this subject to physiologists. Their work
led to the concept that the osmotic pressure
of the fluids bathing the cornea was an
essential factor in the maintenance of its
turgescence. This line of thought resulted
in consideration of corneal physiology as
an exclusively mammalian, or at least as an
aerial-corneal, problem. It is obvious, however, that aquatic vertebrates* have transparent corneas adapted to media of extremely varied osmotic pressure and that
the "physiologic" problem, therefore, is an
extremely broad one. By studies utilizing
diverse forms adapted to varying environments, one may uncover the mechanism in
these "natural experiments" which would
escape us if attention were restricted to
one complicated system.
Adaptation of the cornea to salty tears,
the sea, or fresh water is not an isolated
physiologic problem. Rather, it is a part of
the larger one of how animals in general
are able to adapt themselves to life in
media which vaiy enormously in osmotic
pressure. This subject has been comprehensively presented by Baldwin.4 The
bodies of fresh-water fish continually imbibe water, as many of us have supposed
the cornea to do, and dispose of the excess
through the kidneys. In contrast, salt-water
fish live in a desert, for most sea water is
hypertonic to their tissue fluids. Most of
them would become relatively dehydrated
if they did not regularly excrete the excess
salts which continuously enter their bodies
with the water absorbed through the gills,
the intestine, and perhaps the skin. In one
group of marine fish, the elasmobranchs,
the problem of living in a strong salt solution has been solved by the maintenance of
a high internal osmotic pressure with urea
and trimethylamine. These animals, therefore, are in a situation somewhat analogous
to that of fresh-water fish, in that their ex°The experiments on marine fish were made at the
Marine Biological Laboratory, Woods Hole, Mass.
ternal environment is relatively hypotonic
to the internal one. For this reason and
because of the low position they occupy in
the phylogenetic scale, the physiologic behavior of the corneas of these species also
is obviously of considerable interest.
The manner in which aquatic corneas
are adapted to their environment, the difference between those exposed to hypotonic fresh water and to hypertonic sea
water, and the mechanism for maintaining
transparency and a normal corneal water
content in these species constitute the
subject of this report.
Materials and methods
Three types of aquatic corneas were
studied: that of a typical teleost freshwater fish, carp (Cyprinus carpio), of a
marine teleost, scup (Stenotomus), and
two species of elasmobranchs, dogfish
(Mustelus canis) and skate (Raja erinacea).
For comparison, some experiments were
also carried out on guinea pig and rabbit
corneas. No guarantee can be had that the
species selected provide corneas which react typically for the large groups they
represent, i.e., fresh-water and marine
teleosts, but we may assume that this is
a reasonable possibility. All fish were
received alive and in good condition in the
laboratory where they were maintained in
aquariums until used. The eyes were carefully checked by staining with fluorescein
to insure that only undamaged corneas
were used.
Experimental
Transparency. It should be emphasized
that the corneas of all of the species studied are transparent. Measurement of transparency in vivo is difficult, and, when the
method requires removal of the cornea for
in vitro measurement, some question may
arise concerning the effect of the procedure
on the property being measured. Photographs of the eyes of teleosts and elasmobranchs can be compared (Figs. 1 and 2).
Details of the structure of the iris, sharpness of the pupillary border, and edge of
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Corneal hydration
Fig. 1. Photograph of scup (Stenotomus) eye. Note the detail of the iris and the edge of the
lens 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 the
high degree of transparency in this species. The notches on the inferior aspect of the pupil
are normal. Note the well-developed lower lid which is movable in this fish. (Photograph by
Mr. Lou Gibson, Rochester, N. Y.)
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13
Investigate ', Ophthalmology
Februanj 1962
14 Smelser
BM
100
Fig. 3. Histologic section of dogfish (Mustelus) cornea. A well-developed Bowman's membrane (BM) is seen underlying a very thick, but easily detached epithelium. The stroma
consists of but one layer, as in mammals, of very regularly arranged lamellae. There is a
thin 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 thick
peripherally, but very thin centrally. The fibrous annular ligament is very different in tills
species from the epithelioid, glycogen-rich cells, which are found in this area in some other
species of fish (carp). The relation of Descemet's endothelium to the iris and angle is well
shown. Anterior chamber, AC. (Hematoxylin and eosin.)
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the lens (scup) seen through the cornea
are evidence of the high degree of corneal
transparency in these forms. Just as in
mammals, corneal transparency in fish depends in part upon the maintenance of a
normal state of hydration. In all experiments in which the cornea imbibed water,
it became turbid. This phenomenon, therefore, is a basic one common to all species
which have been studied and is an integral
factor in transparency of this tissue.
Anatomic studies. There is some diversity
in structure even though the general corneal plan in all vertebrates is quite similar.
All are covered by a stratified squamous
epithelium which appears to be less firmly
attached in some (dogfish) than in others.
This epithelium is nearly twice as thick in
some (carp and dogfish) as in others (scup
and guinea pig). Descemet's membrane is
not readily discernible in the fish studied,
but all appeal- to be bounded internally by
an endothelium, although this sheet of tissue is far less obvious in vertical sections m
the fish studied than in mammals.5 VrabecG
has demonstrated an endothelium in flat
preparations of the corneas of the carp and
other teleosts. Its existence in elasmobranchs, though questioned by some,7 was
readily demonstrated in dogfish as well as
in the scup by silver techniques which
were applied to flat preparations. Bowman's membrane, lacking in many mammals, is not apparent in the teleosts studied,
but is beautifully developed in the more
primitive elasmobranch (Fig. 3). The
stroma in all species is composed of lamellae of collagen fibers arranged in an extremely regular parallel fashion. This regularity is nowhere more clearly shown on a
light microscope level than in the dogfish.
The corneal stroma is lightly acidophilic
and that of all three species is metachromatic, which indicates that all contain some
mucopolysaccharides, the high concentration of which is almost characteristic of
this tissue. However, the degree of metachromasia, and therefore, probably of
mucopolysaccharide content, is varied.
Some portions of the cornea of the scup
Corneal hydration 15
were the most metachromatic and even exceeded that of the mammal. The cornea of
fresh-water fish was less metachromatic
than that of the mammal, although metachromasia was always clearly demonstrable. The corneas of elasmobranch fishes
were somewhat less metachromatic than
those of scup. The degree of metachromasia shown by any tissue is subject to
many variables, and its evaluation must be
made cautiously. In these studies, however,
the corneas were all fixed in an identical
fashion, sectioned at the same thickness,
and stained simultaneously, so that comparisons might be made between them.
The cornea of many teleosts, including
that of the scup, is divided into an outer
and an inner portion.7"9 The fibers of the
inner layer are very compact and easily
dissociated from those of the outer and
looser layer. The inner layer is continuous
with the sclera, or at least with the perichondrium of the scleral cartilage. The
peripheral cornea includes an annular ligament which forms a part of the inner corneal layer—thick at the edge and very thin
in the center. These features are shown in
Fig. 4. The outer and looser layer of the
cornea appears to be more closely related
to, or continuous with, the episcleral connective tissue, or conjunctiva, and has,
therefore, been called the conjunctival portion in contrast to the inner or scleral
portion. They are easily separable; the
outer, when grasped with forceps, is found
to be as mobile as the conjunctiva. Both
layers are extremely transparent, but become turbid when they are deformed by
tension. The central area of the cornea is
very thin. The proportion of the two layers
there is shown in Fig. 5.
Comparative hydrophilia. The tendency
of corneas to imbibe water in vitro was
found to exist in the teleost fish as in mammals. However, some difference in degree
and in reaction to the salt content of the
solutions was noted. These experiments
were conducted very simply. Whole corneas were freed from their epithelial and
endothelial covering, immersed in distilled
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stigntiuc Ophthalmology
February 1962
16 Smelser
1.1.
Fig. 5. Histologic section of the central area of the scup cornea. Note the division into an
outer (o.l.) and inner layer (i.l.). The posterior surface of the inner layer consists of an
endothelium (DE) and a few cells and fibers continuous with the annular ligament shown
in Fig. 4. (Hematoxylin and eosin.)
water or in various concentrations of sodium chloride solutions adjusted to pH 6.8
to 7.0 with sodium bicarbonate, and kept
in an ice bath. At intervals the corneas
were removed, blotted, carefully on dry
filter paper, and weighed on a torsion balance. The increase in weight of corneal
tissue caused by the imbibition of water is
shown in Figs. 6 and 7. Because the scup
corneal layers tended to become separated
during this procedure, the swelling of the
outer and inner layers was examined separately. Except where noted, the data in all
experiments apply to the inner layers
(which includes the annular ligament, Fig.
5) of the scup cornea. When tested in this
way in distilled water, the marine teleost
(scup) cornea was found to imbibe more
water than the others, and the cornea of
the fresh-water teleost (carp), the least.
These results might be expected in view of
the environment to which these animals are
adapted. Fish adapted to life in hypotonic
fresh water in which corneas swell the
most, possess the least hydrophilic corneas.
In addition, if, as is generally supposed,
corneal swelling is dependent upon metachromatically stainable mucopolysaccharides, the swelling curve of these tissues is
in accordance with the degree of staining
reaction which was observed. The corneas
of all three species were found to swell less
when immersed in a solution of 0.85 per
cent sodium chloride than in water. In this
instance, however, the degree of swelling
of the scup and guinea pig corneas was almost identical, but exceeded that of carp
(Fig. 7). However, when these corneas
were immersed in a salt solution isotonic
with fresh-water fish plasma, i.e., 0.5 per
cent sodium chloride, the swelling curves
obtained did not preserve the same ratios
to each other. The scup cornea imbibed
minimum fluid in this dilute salt solution,
and its swelling was exceeded by that of
both the carp and the guinea pig which
indicated that the uptake of water was not
directly related to the concentration of ions
in the solution.
Swelling of outer and inner portions of
the scup cornea. The inner and outer portions of the scup cornea were found to
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Cornea! hydration 17
%
400
Uj
§
HOURS
Fig. 6. Comparison of the swelling (increase in weight) of mammalian, sea (inner layei
scup), and fresh-water teleost corneas in distilled water (pH about 7.0) at 0° C. The i
thelium 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 sec
and fourth hours.
— — —
Scup
• Guineo Pig
Carp
in NaCI solution
HOURS
Fig. 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 about
7.0 at 0° C. Five to seven corneas, freed of their epithelia, are represented by each curve
Note that the scup and guinea pig corneas swell to the same extent in this solution and that
no decrease in weight occurred at the fourth hour.
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Investigatioe Ophthalmology
February 1962
18 Smelser
1050
1000
950
900
/
Outer loyer in H 2 0
850
800
750
700
650
600
550
500
450
400
1
O
Inner layer in H 2 0
350
300
250
200
150
Inner layer in NaCI solution
100
50
HOURS
Fig. 8. Comparison of the swelling of epitheliumfree inner and outer layers of the scup cornea in
distilled water and in 1.23 per cent sodium chloride solutions. Solutions were pH about 7.0 and
0° C. The curves are averages of five experiments.
differ not only in morphology and tinctorial
characteristics but also in their ability to
imbibe water. The extreme difference in
behavior of the two layers made it necessary to investigate the two portions separately. The plane of cleavage between them
was definite, so that very little outer corneal tissue remained attached to the inner
layer. The difference in the hydrophilia is
well shown in Fig. 8. The outer corneal
layers were found to imbibe far greater
quantities of fluid than the inner. When the
corneas were immersed in salt solution, the
swelling of both layers was greatly reduced. The weight of the outer cornea increased tenfold in distilled water within
a few hours and was the most spectacular
example of swelling seen in any corneal
tissue. It maintained, in a sense, the same
swelling characteristics noted in earlier
studies, i.e., the swelling was almost exclusively in the anteroposterior axis. At the
end of an experimental period, the corneal
sample had the appearance of a slightly
flattened, oval, gelatinous mass, which,
however, maintained its shape in the solution much as does the vitreous humor or a
jelly fish.
Effect of epithelial or endothelial abrasion on corneal hydration in vivo. Obviously, the corneal stroma of mammals and
of fresh and marine teleost fish are all
hydrophilic although differences exist in
the degree of water imbibition not only
between the species, but, in the scup, between different parts of the cornea. Maintenance of normal water content in mammals is well known to be, in some manner,
dependent on the presence of an intact
epithelium and endothelium. The importance of these layers to corneal hydration
in fish was therefore investigated. The epithelium was removed from one cornea of
each of 9 scup, and, in another series of
17 fish, the endothelium of one eye was
thoroughly abraded. In the latter operation,
in one series, the anterior chamber was entered by means of a keratome incision at
the limbus, and the endothelium abraded
with an iris spatula. Control experiments
were performed in which the keratome incision was made, but the endothelium was
not deliberately injured. In another group,
the endothelium was abraded with a needle
which was inserted through the limbal
sclera and conjunctiva, because it was
feared that the keratome incision might
affect comeal hydration by allowing the
escape of aqueous humor or the entry of
sea water. The needle was also introduced
into the eye used as a control, but the endothelium was not touched. Following
these operations the fish were returned to
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Corneal hydration 19
the aquarium, and, at autopsy, the intactness of the epithelium was checked by
staining with fluorescein. Six hours after the
operations the corneas were removed by
very careful dissection and weighed. In
order to determine the normal variation in
weight between the left and right corneas
and the error due to dissection and weighing, the intact eyes of a control series of 17
scup were also dissected in the same manner and the corneas weighed. In this series
it was demonstrated that the average
weight of the right and left corneas did not
differ by more than 5.5 to 6.0 per cent
(Table I). Hydration of the corneas in the
experimental series was judged by the
change in weight of the cornea of the experimental eye relative to that of the control eye of the same fish.
Removal of the epithelium in scup resulted in definite swelling of the stromal
connective tissue (Table II), even though
it was bathed in a hypertonic salt solution
(sea water). Swelling of the outer layer
(129 per cent) was much greater than that
of the inner (30 per cent) which was to be
expected in view of the experiments with in
Table I. Difference in weights of the right
and left normal corneas
Left eye
Outer Inner Both
layer layer layers
12.2
25.5
10.0
22.0
33.2
26.2
22.0
17.6
25.8
22.0
42.7
28.2
29.8
64.7
64.3
19.8
14.8
33.8
13.1
31.6
35.2
29.6
28.8
22.0
28.2
22.2
56.4
28.4
24.7
62.8
69.6
11.4
27.0
59.3
23.1
53.6
68.4
55.8
50.8
39.6
54.0
44.2
99.1
56.6
54.5
127.5
133.9
31.2
Average
28.8 32.0 61.2
Difference
Right eye
Outer Inner Both
layer layer layers
12.2
23.3
11.8
22.2
30.2
23.6
20.6
17.5
22.6
24.0
46.1
19.4
21.6
59.4
46.7
20.1
15.1 27.3
32.8 56.1
11.8 23.6
31.0 53.2
33.6 63.8
29.0 52.6
29.6 50.2
21.5 39.0
29.6 52.2
26.6 50.6
55.6 101.7
25.3 44.7
27.2 48.8
63.0 122.4
59.2 105.9
12.8 32.9
26.3 31.5
57.8
- 9 % - 1 % -5.5%
Table II. Effect of epithelial abrasion on
corneal hydration in vivo* (weight in
milligrams)
Epithelium intact
Outer
Inner
layer
layer
22.1
21.7
21.6
53.6
30.4
21.8
23.8
12.0
18.5
27.7
28.2
22.1
22.4
68.6
32.6
25.8
34.2
12.6
Average 25.1
30.5
0
Epithelium abraded
Outer
Inner
layer
layer
51.6
43.8
41.2
51.2
108.2
63.2
57.2
62.2
38.2
57.4
Gain +129%
34.4
31.0
26.6
32.7
89.0
42.8
32.8
48.2
18.2
39.5
+30%
Duration of experiment 6 hours.
vitro swelling (Fig. 8). Abrasion of the endothelium also provoked definite swelling
(Table III) of both corneal layers, but the
hydration was less than when the epithelium was damaged, and, again, greater
swelling occurred in the outer layers. Some
effect on corneal hydration was apparent as
a result of opening the anterior chamber,
thus permitting loss of aqueous humor
and/or entrance of sea water.
Similar experiments were done on guinea
pigs with somewhat different results. In
this species endothelial abrasion caused
greater uptake of water than when the epithelium was removed. These observations
confirm those of Maurice and Giardini.10
This reaction is the opposite of that in the
fish, in which greater corneal hydration resulted from removal of the epithelium.
Role of metabolism in maintenance of
corneal turgescence. In fish, the importance
of the epithelium in the prevention of corneal hydration may depend in part on the
thickness of that tissue which may serve to
impede mechanically the movement of
water into the cornea. However, this result
might be achieved far more efficiently by
metabolic activity which assists in maintaining normal corneal water content.
Experiments were performed in which
the metabolic activity of the scup cornea
was reduced in vitro by low temperature or
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20
Investigative Ophthalmology
February 1962
Smelser
Table III. Effect of endothelial abrasion on
corneal hydration in vivo* (weight in
milligrams)
Corneal layer
Outer
1 Inner
Corneal layer
Inner
Outer
Eye intact
21.5
33.2
21.2
18.8
19.3
19.2
Average
22.2
24.1
42.0
32.8
25.6
23.3
22.0
Endothelium scraped
with iris spatula
30.6
35.2
75.0
99.4
52.0
49.2
37.2
43.4
35.6
53.2
37.3
35.6
44.3
52.9
Gain +138% +57%
Keratome incision in both eyes
Endothelium scraped
Endothelium intact
with iris spatula
52.2
81.5
55.4
62.4
21.2
23.4
21.3
23.5
52.2
81.5
42.0
45.1
17.0
28.0
14.8
28.0
58.2
62.0
43.5
35.8
Average
40.1
55.3
35.4
38.9
Gain +42% +13%
Anterior chamber of both eyes entered with a
needle
Endothelium intact
Endothelium abraded
with a needle
30.0
25.4
29.4
26.4
33.7
51.8
28.0
29.3
36.2
47.0
23.5
26.6
31.7
50.0
22.6
18.4
49.6
51.8
34.0
25.8
28.8
64.2
28.2
36.8
Average
35.0
48.4
27.6
27.2
Gain +78% +27.5%
28.3
"Duration of experiment 6 hours.
by the exclusion of oxygen. The entire orbit
and a portion of the adjacent head structures were dissected carefully so that no
abrasion of the corneal epithelium or injury
to the conjunctiva occurred. This was
checked by examination with a dissecting
microscope and fluorescein staining. The
half heads were placed in 50 ml. tubes of
sea water which contained 200 mg. per
cent glucose and were kept at the temperature of the aquarium. Comparisons were
made between the right and left eyes of the
same fish. In one series of experiments,
metabolic activity was reduced by placing
these preparations in ice for IS hours. In
the first experiment, one eye was removed
as the normal control and the other chilled.
The corneal weights are given in Table IV.
Those which were chilled increased in
weight by 9.6 per cent, a change of doubtful significance. In the second experiment,
both eyes were chilled for 18 hours; one
was then weighed, and the other warmed
to 20° C. and the sea-water medium oxygenated for 6 hours. During the second,
warm, aerobic period the corneas gained 18
per cent in weight. The data, given in
Table V, show no evidence that a deturgescence accompanied the return to normal
physiologic conditions.
Experiments were also conducted in
which one eye, the normal control, was dissected and weighed immediately; and the
other eye was prepared as described previously and kept under anaerobic conditions
by bubbling nitrogen through the medium
for 6 hours at the aquarium temperature
Table IV. Effect of low temperature (in
vitro) on corneal weight
Left eye, normal weight
(mg.)
65.2
54.8
53.5
42.5
Average 54.0
Right eye after IS hours
at 0° C. (mg.)
Gain
67.6
61.8
62.5
44.8
59.2
+ 9.6%
Table V. Effect of oxygenation and warming following 18 hours at 0° C. on corneal
hydration (weight)
Right eye
following oxygenaLeft eye after 18 hours tion for an additioiwl
at 0° C. (mg.)
6 hours at 20° C. (mg.)
32.8
30.3
31.1
41.8
36.2
41.2
61.4
71.4
46.8
Average 39.7
Gain
+18%
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Corneal hydration 21
Table VI. Effect of in vitro anaerobiosis on
corneal weight
Left eye normal weight
(mg.)
33.8
57.2
24.4
49.6
Average
Right eye after 6 hours
anaerobiosis (mg.)
36.7
53.3
24.2
48.4
41.2
Loss
40.6
-1%
(21° C ) . The results, Table VI, show no
effect of the anaerobiosis.
In a second and larger series of experiments, both eyes were placed in the tubes
as described. One tube was continuously
aerated by bubbling oxygen through a sintered glass aerator and the other tube was
gassed with nitrogen to create an anaerobic
condition. The preparations were maintained for 6 hours at an aquarium temperature of 12° C.,* after which the corneas
were carefully dissected and weighed. The
water content was determined by drying
the tissue over calcium chloride in a
vacuum desiccator at 45° C. to a constant
weight. The anaerobiosis caused a strong
contraction of the melanophores in the iris
and conjunctiva so that the eyes kept under
nitrogen had a silvery appearance, whereas
those exposed to the oxygenated medium
were a normal golden color. Therefore, all
corneal layers received adequate oxygen in
one series but not in the other, as judged
by the response of the melanophore. The
difference in weight (5 per cent) between
the aerobic and anaerobic corneas, Table
VII, was insignificant (compare with normal series, Table I). The dry weight of
these corneas was determined and no effect
of anaerobiosis on water content was
found. The difference between the two
groups was only 2 per cent. In this species
(scup), therefore, a normal aerobic metabolism of the cornea does not appear to be
requisite for maintenance of corneal turgescence as it is in the mammal.
"These and one other experiment were conducted in the
spring when the temperature of the sea water was low.
Swelling in various salt solutions. It was
noted in the first experiment that the swelling of corneas was not proportional to the
concentration of the salt solution used. This
was investigated further in a study of the
swelling of the inner layer of the scup cornea. These were prepared as before,
weighed, placed in the various solutions,
and weighed at intervals to determine the
amount of water they imbibed. The media
tested were: distilled water, 0.5 per cent
sodium chloride, 0.85 per cent sodium
chloride, 1.23 per cent sodium chloride
(isotonic with marine teleost plasma). 3.2
per cent sodium chloride (equivalent to sea
water), sea water itself, marine teleost
Ringer's solution,11- 12 scup aqueous humor,
and glucose 7.7 per cent (isotonic with 1.23
per cent sodium chloride). The pH was adjusted with sodium bicarbonate to 6.8 to
7.0 in all experiments, and the flasks containing the corneas were immersed in an
ice-water bath. The swelling of the scup
cornea in some of these solutions is shown
in Fig. 9. It is clear that the swelling which
occurred was not related to the osmotic
pressure of the medium; for example, the
swelling in distilled water and in glucose
solution was nearly identical. It would ap-
Table VII. Aerobic metabolism and cornea!
hydration (weight of corneas maintained 6
hours in vitro under aerobic and anaerobic
conditions)
Aerobic (mg.)
79.6
74.4
65.4
58.6
73.2
57.4
49.8
52.6
55.3
53.5
45.4
38.0
43.8
43.0
41.4
Average 55.4
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Anaerobic (mg.)
80.2
77.4
63.6
64.6
79.8
62.1
54.6
56.7
55.3
49.7
52.0
43.4
43.5
41.6
46.7
58.1
Gain
+ 5%
Investigative Ophthalmology
February 1962
22 Smelser
Fig. 9. The swelling of the inner layer of scup corneas in various solutions, all at pH about
7.0 and 0° C. Note that swelling was least in the 0.5 per cent sodium chloride solution and
maximal in distilled water or glucose solution.
pear that the presence of ions is important,
and, also, the presence of more than one
ion species, because swelling was distinctly
less in Ringer's solution (shown in Fig. 14)
or sea water (Fig. 9), than sodium chloride
solution of similar concentrations. There is
some indication of an optimum concentration of salts at which swelling is minimal
(0.5 per cent). The possible significance of
this must await further study. No tests were
made at concentrations of less than 0.5 per
cent sodium chloride. The swelling curves
in some media were omitted from the
graph in the interest of clarity (e.g., the
corneas in 0.85 per cent which swelled to
nearly the same degree as in 1.23 per cent
sodium chloride solution. Those in the
marine teleost Ringer's solution or in aqueous humor swelled a little more than those
in sea water).
The effect of bivalent cations and anions
on corneal swelling. The difference in swell-
ing in simple salt solutions and those containing many ions (sea water and Ringer's
solution) suggested that the ionic species
present were of importance. To test this
concept further, solutions of bivalent cations, calcium chloride or magnesium chloride, were prepared. In addition, solutions
containing bivalent anions, sodium sulfate
or sodium phosphate, were also made to
test their effect on swelling. Control solutions of sodium chloride or potassium chloride were used. All solutions were isosmotic
with 0.5 per cent sodium chloride, since
corneal swelling had been shown to be
minimal at that concentration. The pH of
all solutions was 6.8 to 7.0, and the experiments were conducted at 21.0° C, the temperature at which the scup had been living.
The results of this experiment are shown
in Fig. 10. The degree of corneal swelling
is seen to depend in part on the charge of
the ions which made up the solution. The
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Corneal hydration 23
swelling was much more marked in solutions containing bivalent anions, phosphate
and sulfate, than in solutions containing
monovalent chloride ions. In contrast, the
imbibition of water was less in the presence of bivalent cations, calcium and magnesium, in comparison with solutions of
potassium or sodium chloride. As perhaps
could be expected, an intermediate degree
of swelling resulted when both the cation
and the anion were bivalent (magnesium
sulfate solution). Although variation in corneal swelling occurred, the difference due
to the charge on the ions was clearly significant.
Role of colloid osmotic pressure in corneal swelling. With the exception of the
experiments reported by Pau13 and by
Dohlmann and Anseth,14 most studies on
the swelling of corneal tissue have considered only the role of ions on osmotic
pressure. Colloids, because of their large
molecular weight, do not exert great osmotic force in comparison with that of
small ions, but, because they are less diffusible and may form part of tissue structure, they are of extreme physiologic
importance. In fact, in preparations such as
have been described here, the highly diffusible ions may be expected to have
achieved equilibrium between the solution
and the interior of the cornea very quickly.
The nondiffusible substances within the
cornea then became the major factor responsible for water imbibition. Therefore, a
series of experiments was conducted on the
swelling of corneal stroma of the scup in
various concentrations of a colloid poly-
400
350 -
k.
IJJ
300 -
s
CX.
250
k.
20
3~-- —•"" —~^ZS\ i
Cl "
°
Mg + +
1
^
2
<O
io
>
'
Y^-'X
150
_
(
°
= Range
50
i
i
i
HOURS
Fig. 10. Graph comparing the swelling of the inner layer of scup corneas in solutions containing bivalent cations and anions with that in solutions containing monovalent ions (KCl).
The solutions were of calcium chloride, magnesium chloride, sodium phosphate (a mixture
to 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.
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Investigative Ophthalmology
February 1962
24 Smelser
0/
300
250
200
150
kj
100
50
HOURS
Fig. 11. Comparison of the swelling of the inner layer of scup corneas in 10 per cent aqueous
solutions of large and small polymers of PVP in the absence of salt. Note the greater swelling
in the solution of the lower molecular weight polymer, K30. Initiation of swelling was delayed
in both solutions but much more in that of the larger polymer, K90. Each curve represents
four or five corneas. The solutions were at pH about 7.0 and 21° C.
vinylpyrrolklone (PVP),* which, because
of its inert nature, has been successfully
used as a plasma extender and is available
in various polymer sizes. They and their
average molecular weights are: K90,
360,000; K60, 160,000; K30, 40,000; and
K15, 10,000. The osmotic pressure of equal
concentrations of these polymers was, of
course, much greater in the case of K15
than in K90. The solution of K90, however,
was far more viscous than the others. Concentrations ranging from 10 per cent to 1.25
per cent were prepared in either water or
1.23 per cent sodium chloride. The pH of
these solutions was 6.8 to 6.0 (adjusted
with sodium bicarbonate) and the experiments were conducted at 21° C. Fig. 11
shows the swelling curves which were
found when corneas were immersed in 10
per cent aqueous solutions of PVP, K90
and K30. The corneas swelled in both solu°Supplied by An turn Chemicals, New York City Division
of General Aniline and Film Corporation.
tions but less in the more viscous, but
osmotically weaker, solution of K90. An
important difference in the shape of the
curves is seen in the first 30 minutes, in
which shrinkage rather than swelling occurred. This is in sharp contrast to experiments with salt solutions in which swelling
began within this time. Obviously, corneas
swell in PVP solutions, but the onset of this
process is delayed and swelling is less in
the more viscous, osmotically weaker, solution. When both salt and PVP were present
in the solution, however, the behavior of
the swelling of the corneal stroma was
radically different (Fig. 12). Swelling was
greatly inhibited when salt was present;
in fact, in 5 per cent K90 with salt, the
corneas were deturgesced below the normal value and held in a steady state with
respect to hydration for the duration of the
experiment. The transient 30 minute inhibition of swelling, noted in Fig. 11, continued, and, instead of swelling, a permanent maintenance of a near normal corneal
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Volume 1
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Corneal hydration 25
hydration was achieved. When solutions of
the same concentrations of PVP were used
without salt, marked swelling occurred,
and, although the rate was less, it was
scarcely distinguishable from that which
took place in distilled water. Evidently the
presence of colloids has an extremely important effect, but this is clearly demonstrable only in the presence of salts. The
data in Fig. 13 demonstrate that the swelling power of corneal tissue may be balanced, or even overcome, by appropriate
concentrations of osmotically active colloid
in salt solution. Swelling of the corneas occurred in the more dilute PVP solutions,
and shrinkage in the more concentrated. A
solution of about 3.75 per cent K90 PVP, in
the presence of salt, would provide a medium in which the exchange of water between the cornea and the outer solution
would be in balance.
Role of aqueous humor in maintenance
of corneal turgescence. There is a very
good possibility that these experiments on
corneal swelling in solutions of PVP and
salt provide a model of the system which
is, in fact, the physiologic one in many
animals. It is well known that the aqueous
humor of many fish and birds is viscous
and contains large quantities of hyaluronic
acid or similar mucopolysaccharides.15"17
Since the aqueous humor is separated from
the corneal stroma by Descemet's endothelium, hyaluronic acid would not be free to
diffuse into it. The existence of a membrane preventing the diffusion of colloids
into the stroma is important. Inner layers
of scup cornea were found to swell (70 per
cent in 6 hours) when immersed in fresh
aqueous humor of the scup. In those experiments, however, there was no protective endothelium, and aqueous humor was
400 -
350
k.
S
300
IGHT
250
?/VEAL
%
8
200
K 9 0 - l . 2 5 % in NoCI solution
_
150
100
•---••
K90-5% in NoCI solution
50
HOURS
Fig. 12. Graph showing the efFect of salt on swelling of the inner layer of scup corneas in
solutions of PVP. Note that the corneas immersed in a 5 per cent solution of K90 with salt
did not swell, but remained deturgesced below normal, whereas those in the same concentration of K90 without salt underwent maximal swelling. The swelling of corneas in distilled
water is shown for comparison. Each curve represents four corneas.
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Inrcstigativa Ophthalmology
February 1962
26 Smelser
Fig. 13. The swelling of the inner layer of scup corneas in various concentrations of PVP
K90 in 1.23 per cent sodium chloride solution is shown. The swelling which occurs in 1.23
per cent salt solution and in distilled water without PVP is shown for comparison. All solutions were 21° C. and pH 7.0.
free to diffuse into the corneal connective
tissue. In addition, in the in vivo experiments when the endothelium was damaged,
the corneal connective tissue exposed to the
aqueous humor did swell (13 per cent to
27 per cent).
The inner layers of the corneas of 12
scup were weighed and inserted into narrow, empty, but moist (with salt solution)
Visking dialysis tubes immersed in fresh
scup aqueous humor and reweighed 6
hours later. The corneas were placed in the
center of a lengtli of dialysis tubing, which
was bent into a U shape, the open ends of
which extended well above the aqueous
humor. The corneas were not subject to
pressure other than that induced by the
stiffness of the wet membrane. They were
kept 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 manner, but the membranes and corneas were
immersed in distilled water or 1.23 per cent
sodium chloride solution instead of aqueous
humor. The results are shown in Table
VIII. The corneas dialyzed against salt
solution or water swelled by imbibing fluid
through the semipermeable dialysis membrane. Those exposed to aqueous humor
through the membrane did not swell. Presumably, this resulted because the corneas
were in a normal ionic environment and
the colloid osmotic pressure of the aqueous
humor balanced that of the connective tissue, and the dialysis membrane adequately
replaced the function of the endothelium.
Swelling properties of elasmobranch corneas. The above experiments have been
confined to teleost corneas, mainly, those
of a marine form, plus some references to
mammalian corneas for comparison. Similar
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Corneal hydration 27
Table VIII. Swelling of corneas (inner
layer) during 6 hour dialysis at 12° C.
Dialyzed against 1.23%
solution of sodium
chloride
Average
Dialyzed against distilled
water
Average
Dialyzed against aqueous
humor
Average
Original
weight
(mg.)
23.3
33.5
34.2
19.9
36.3
29^4
Final
weight
(mg.)
29.0
38.8
37.4
24.6
39.0
33.8
Gain +15%
18.6
33.7
32.0
25.2
55.3
27.4
39.4
42.6
32.6
61.7
33.0
40.7
Gain +23.3%
27.4
36.8
22.1
20.7
31.7
45.8
49.2
28.5
26.4
18.4
22.4
32.3
30.1
28.2
38.6
21.6
19.2
34.6
47.0
42.2
30.0
27.0
20.6
24.5
33.8
30.6
Gain +1.6%
experiments on corneal swelling in water
and salt solutions were also performed on
two species of elasmobranchs, the dogfish
(Mustelus) and the skate (Raja). The behavior of the corneas of these species was
of interest because: (1) they maintain their
internal osmotic equilibrium in a peculiar
manner, (2) the corneal structure is
very similar to that of mammals, and
(3) these species are more primitive than
are the teleosts, and for this reason one
might expect to uncover more basic properties of corneal tissue.
The swelling experiments were conducted as before, with the same solutions,
and with the addition of an elasmobranch
Ringer.11-18 Fig. 14 shows the result with
most of these solutions. Those omitted for
the sake of clarity in the graph did not dif-
fer from those shown. The swelling curves
of the scup cornea in distilled water and
teleost Ringer are included for comparison.
The comeal stroma of the elasmobranch of
both species, did not swell in any of the
media tested. In fact, some loss in weight
was observed. The corneas of these fish
differ from those of all other vertebrates, so
far studied, in their failure to imbibe water;
in fact, it would appear that these eyes
have the reverse problem from those of all
other species in that there is a need to prevent the loss of water from the stroma
rather than to prevent its enhance.
Discussion
Corneas of all vertebrates are structurally very similar, since they are constructed
of regularly arranged collagen lamellae
bounded by epithelia. The important mucoid constituents, as shown by metachromatic staining reactions, are present in all,
but vary in concentration and possibly in
kind and in degree of polymerization.
Corneas of all species which have been
studied, except the elasmobranchs reported
here, imbibe water from the surrounding
milieu after damage to the limiting cellular
membranes or in vitro. It has been tacitly
accepted in recent years that maintenance
of normal turgescence involves metabolic
work, a "pump" of some sort, located in the
epithelium or endothelium. It has been
shown in mammals that reduction of corneal metabolism by anaerobiosis or lowered
temperature adversely affects its optical
properties,10 reduces transparency,20 and
the stroma becomes hydrated. Repeated attempts to demonstrate this phenomenon in
scup failed. Corneas transferred to warm
aerobic conditions following 18 hours in
cold increased in weight. In contrast, analogous experiments with rabbits,2'21 showed
that corneas decreased in weight due to
the expulsion of water when they were
returned to normal temperature after refrigeration. Scup corneas, maintained under
anaerobic conditions for 6 hours, gained
only 5 per cent in weight, whereas in analogous experiments rabbit corneas22'23 in-
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Investigative Ophthalmology
February 1962
28 Smelser
Scup m Teleost Ringer solution
Elasmobranch in Ringer solution
0 . 5 % NaCI
—• — — — —•
HO
HOURS
Fig. 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 shown
for comparison. All solutions were pH about 7.0 and 21° C.
creased their water content by 50 per cent.
Apparently the epithelial barriers in fish
do not house the same mechanisms as they
do in mammals. No metabolic "pump" in
the mammalian sense could be shown.
However, both epithelia are of great importance in maintaining cornea! turgescence in fish, because, if they are removed,
the corneas imbibe water. Possibly, they
serve in a more passive or protective role
by separating the stroma from aqueous humor and sea water by a relatively impermeable coat. It is not suggested that they
are impermeable to water, but that the
epithelium may serve to "retard" the entrance of sea water, and the endothelium
may be impermeable to high molecular
weight 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 themselves swollen, but the interfibrillar spaces
were enlarged. Heringa and others20 found
that corneas from which the mucopolysaccharides had been extracted did not
swell, and concluded that it was the connective tissue ground substance which was
mainly responsible for the hydrophilia.
The mucopolysaccharides are long, negatively charged polymers of chondroitin, its
sulfates, and keratosulfates, which are
firmly bound to protein. These are fixed to
collagen fibrils, are part of the structure
of the cornea, and are not free to diffuse
from it. Because of this, they contribute
materially to the Donnan effect. They attract and hold water which diffuses into
the cornea.
When a tissue such as the cornea is
placed in water, water molecules tend to
diffuse from the medium into the tissue
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Number 1
spaces between the structural components.
If this structure is rigid, or elastic, so that
its constituents are not easily displaced by
the inward diffusing water, no swelling will
will occur; there will be simply an exchange of water between the outer medium
and that of the interstitial spaces.27 If, however, the bonds which hold the structure
together are weak, water which is diffused
into that tissue will be bound to hydrophilic sites, the structures will be displaced,
and swelling will result. In short, organization of structure combats swelling. Swelling results when the forces which tend to
hold the structure in a compact arrangement are overcome by those which attract
and bind water. For example, the present experiments show that the looser outer
layer of the cornea swells three times as
much as the more compact inner layer, although staining reactions indicate a nearly
equal mucoid content.
It seems possible to explain the data obtained in these experiments in terms of
the relationship of macromolecular anatomy to corneal structure and swelling. All
of the corneas, save those of elasmobranchs,
swelled readily in salt solutions but much
more in the absence of ions (in water, solutions of glucose, or PVP). The presence of
salts did not reduce swelling because of an
osmotic pressure effect but, it is believed,
because of their effect on corneal structure. It was clear that the ability of salt
solutions to inhibit swelling was not dependent on their concentration and, therefore, osmotic pressure for several reasons:
(1) Their effect was not proportional (in
the range studied) to concentration. (2)
Bivalent cations in isosmotic concentrations
were more effective in preventing swelling
than were monovalent ions, and the presence of bivalent anions enhanced the
swelling. (3) No osmotic pressure differential could be created by salt solutions, because they could diffuse readily into and
out of the tissue spaces. It has been shown
that they move freely in these spaces.28
Swelling in salt solutions is due largely to
the hydrophilic properties of the large non-
Corneal hydration 29
diffusible molecules. The corneas swelled
in solutions of glucose and of the small
PVP polymers K15 or K30 because these
uncharged solutes had no effect on the
hydrophilic property of the mucoid, which
continued to bind water. As long as the
concentration of the PVP in the external
medium was high, water left the cornea because of the osmotic imbalance. When the
PVP had diffused into the tissue, no osmotic difference existed, and swelling occurred again as it would in distilled water.
When the large polymers of PVP were
used, their viscosity prevented their rapid
entrance into the cornea and swelling was
delayed. In the presence of salts, it is postulated that the corneal structure became
more compact and the large polymers of
PVP could not diffuse into it and swelling
was prevented. In the experiments of Dohlmann and Anseth,14 this phenomenon was
not observed because the colloid was kept
from entering the cornea by means of a
semipermeable membrane. In the experiments of both Dohlmann and of Pau, the
colloid was studied in the presence of ions
and buffers, so that the effect of salts was
not seen.
It seems reasonable to postulate that connective tissue of the cornea swells less in
ionic solutions because of the effect of the
ions on the compactness of the charged
polymers, such as corneal mucopolysaccharides. Closely related compounds, e.g.,
hyaluronic acid, exist in free solution as
random coils and occupy a space in water
which is large in relation to their mass,29
and which is reduced when ions are present.30"33 This may come about in part because portions of the polymer chains may
repel one another because of their negative
charges. When these charges are satisfied
by the presence of cations in the solutions
the chains become more compact. In addition, the water in the interstices of the
random coil is probably, to some degree,
"organized" or held in a quasi-crystalline
form as it is in collagen.34 Cations diffusing
into such a system might be expected to
neutralize the charges on the polymer,
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30
Investigative Ophthalmology
February 1962
Smelser
allowing the random coils to become more
compact. The water held in the crystallike
configuration could also be reduced since
the forces holding water to protein or mucoids are weaker than those binding it to
simple ions. An analogy may be drawn between the cornea and nucleic acid films
which, when floated on distilled water,
have been shown to expand (swell) to a
much greater degree than when floated on
salt solutions where the expansion of the
film was inhibited.35
By analogy with the systems which have
been studied, it seems probable that in the
experiments reported here salts acted to
render the macromolecular structure of the
mucoid ground substance more compact,
stable, or rigid, so that water diffusing into
the tissue could not disperse these molecular structures so readily. This effect would
not inhibit the hydrophilic character of the
protein and mucopolysaccharide, so that
water would still enter the connective tissue interstices and some swelling would
occur, but it would be limited in degree by
the molecular anatomy, and less in amount
than expected by the Donnan formula.
This idea is well expressed by Gustavson*
in relation to dermal connective tissue:
"Both the Donnan and the Procter-Wilson
equations were worked out for diffusion of
ions into a volume sufficiently large for
surface forces to be neglected, and not for
a diffusion into a set of capillary tubes,
which skin nearly represents. It is therefore obvious that, as soon as structure factors become prominent in the system, the
simple calculations based on the Donnan
effect can no longer be applied in their
original form. The higher the restrain imposed on the structure of the solid phase,
the less free water will be present, which
accordingly implies introduction of new
and unknown factors into the simple equations."
When corneas were immersed in PVP
solution without salt the corneal structures
were more open or loose and the PVP dif°Ref. 27, p. 167.
fused in, thus negating any effect it might
have had if it had been kept outside. In the
presence of salt, the corneal tissue remained
more compact, the large viscous PVP polymer K90 could not diffuse into it, but remained outside to counteract the Donnan
effect of the structural mucoids and proteins which could not diffuse out. It is suggested that this experiment provides a
model of the system which maintains the
corneas of fish, and possibly some other
animals, in their normal state of hydration.
The endothelium may serve as a semipermeable membrane, that is, permeable to
water and to salts, but impermeable to
large molecules of the aqueous humor
which, in our experiments, was shown to
balance the colloid osmotic and swelling
pressure of the cornea so that it remained
deturgesced. It is a reasonable assumption
that the aqueous humor is constantly renewed and serves to maintain the cornea
in its normal state. Since the formation of
aqueous humor would require work, metabolic activity would still be involved in the
water regulation of the cornea.
The concept of a metabolic pump in the
corneas has led to a search for ion pumps
which would "pump out" the salt and water
which had diffused into the tissue. A sodium pump was found,36 but the sodium
was moved inward, not outward, thus adding to the puzzle. There is a possibility
that this phenomenon has no real bearing
on the problem of corneal hydration, that
it merely represents a vestigial function of
surface epithelium, developed in amphibia
where the classical sodium pump (in frog
skin) behaves as in the cornea of the rabbit. A possible function of the sodium
pump of the cornea, however, is suggested
by these experiments. It may serve in some
species (although probably not in salt
water fish) to insure a proper ionic environment of the connective tissue of the cornea, so that its structure may remain compact and stable.
In the absence of experimental data, it
is unwise to attempt to extend the concept
formulated here to mammals. Some fea-
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Volume 1
Number 1
tures of it, however, may apply, namely,
the role of salts in maintaining structural
compactness on a macromolecular scale.
An additional system may have evolved for
those species lacking a colloid osmotic
force in the aqueous humor. The biologic
reason, if any, for the change in type of
aqueous humor and mechanism for the
maintenance of corneal deturgescence
should be interesting. Perhaps even more
intriguing is the problem of how the elasmobranch cornea is made so that it possesses a high degree of transparency, presumably a reasonable content of mucopolysaccharide ground substance, but lacks the
property of hydrophilia. Having established
a seemingly ideal cornea presumably early
in the evolutionary series, it is puzzling
why more complicated systems evolved.
Corneal hydration 31
11.
12.
13.
14.
15.
16.
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