CORNEAL PHYSIOLOGY AND
BIOPHYSICS
L.S. Kwok
14.1 INTRODUCfION
Our understanding of corneal physiology
and biophysics has advanced considerably
in the last few years. An accelerating factor in
the emergence of these exciting develop­
ments has been the introduction of powerful
techniques in molecular biology, cellular bio­
physics and high resolution electrophysiol­
ogy. Recent developments are emphasized in
this review, which is selective rather than
comprehensive; the treatises by Maurice
(1984), Worthington (1984) and O'Leary
(1985a, b) are recommended to complement
the present material.
14.2 CORNEAL CELLULAR PHYSIOLOGY
The mechanisms of interactions between
corneal cells, basement membranes and
extracellular matrices, are being elucidated
with new techniques in molecular biology.
These are of fundamental importance to
understanding processes such as corneal
development, cell migration and corneal
wound healing (Mattey & Garrod, 1984;
Akiyama et al., 1990b). One example is the
recent application of the polymerase chain
reaction to amplify nucleic acid sequences
such as the herpes viral genome to detect
herpes virus in the corneal epithelium
(Crouse et al., 1990).
Contact Lens Practice. Edited by Montague Ruben and Michel Guillon.
Published in 1994 by Chapman & Hall, London. ISBN 0 412 35120 X
14
New specular microscopic techniques
allow the visualization and morphometry of
superficial corneal epithelial cells in the liv­
ing eye (see Lemp et al., 1990). In older
patients in the fifth to sixth decade, Tsubota
et al. (1991) reported that the average area of
superficial corneal epithelial cells (821 ± 203)
JUll2 in diabetic aphakic patients was greater
than normal (648 ± 152 f..Ull2 average size).
Paradoxically, the corneal epithelium in the
presence of either aphakia or diabetes alone
has normal-sized cells (643 ± 125 JUll2 and
658 ± 146 ....m 2 average area, respectively).
Why corneal epithelial cells are larger in
combined diabetes and aphakia is unclear.
However, larger corneal epithelial cells (818
± 187) JUll2 are also reported in aphakic
extended soft contact lens wear (Lemp et al.,
1990). In younger phakic patients, extended
soft contact lens wear also increases the area
of superficial corneal epithelial cells (Lemp et
al., 1990). Diabetes or extended contact lens
wear probably affects the cellular physiology
of the corneal epithelium in aphakia, but the
biological significance of areally larger epi­
thelial cells remains to be established. Soft
contact lens wear in rabbits produce alter­
ations to corneal enzyme activities and epi­
thelial thinning (Cejkova et al., 1988).
The cytochemistry of the surface of the
corneal epithelium is important to under­
standing how the cornea resists invasion by
240 Corneal physiology and biophysics
tear-borne bacteria and other harmful exog­
enous entities. The sialic acid membrane
receptor has been implicated in the media­
tion of Pseudomonas aeruginosa adherence in
pre-adult mouse corneas (see Hazlett et al.,
1987), and the higher density of sialic acid
receptors in the adult cornea apparently pro­
tects against Pseudomonas invasion (Hazlett
& Mathieu, 1989). Pseudomonas appears to
bind preferentially to basal and wing cells of
cultured rabbit corneal epithelium but not to
adult superficial cells (Spurr-Michaud et al.,
1988; Klotz et al., 1989) especially at sites on
the cell periphery, whereas Staphylococcus
aureus binds randomly over the cell surface
(Panjwani et al., 1990). Apparently,
Pseudomonas has an affinity for membrane
receptors involved in cell-cell or cell­
substratum interactions; corneal adherence
is enhanced when epithelial integrity is
breached to expose deeper epithelial cells
(Klotz et al., 1989). The devastating ocular
parasite Acanthamoeba can bind to the ante­
rior corneal surface and eventually penetrate
into the corneal epithelium, even in the
absence of preceding tissue trauma (Stopak
et al., 1991). The invading Acanthamoeba
parasite consumes living corneal epithelial
cells. This profoundly disturbs the cellular
cytoskeleton, which is surrounded by tre­
mendous intracellular trafficking of water
and metabolites necessary for normal cell
function (Goodsell, 1991). Such disruption of
intracellular organization in the superficial
corneal epithelium is particularly perilous,
because destabilization of intracellular fila­
ments such as actin will affect the integrity of
the epithelial tight junctions (Rojanasakul &
Robinson, 1991) and hence compromise the
superficial epithelial cell barrier, the major
site of corneal resistance to ionic molecules
(Ehlers, 1970; Fee & Edelhauser, 1970; Klyce,
1972). Stromal penetration with poor clinical
prognosis will eventuate if the infection is
not controlled (Hirst et al., 1984; Stopak et al.,
1991). Recent evidence suggests that the
Acanthamoeba parasite might directly con­
tribute to stromal lesions by releasing colla­
genase, thus attracting neutrophil infiltrates
which release collagenolytic enzymes result­
ing in collageno lysis and stromal thinning
(He et al., 1990). Degradation and loss of
corneal stroma in inflammatory corneal dis­
ease is thought to be due mainly to the
liberation of proteolytic enzymes by invad­
ing polymorphonuclear leucocytes (PMNs)
(Trinkhaus-Randall et al., 1991).
An interesting finding is a 70 kDa collagen
binding glycoprotein in chick corneal epithe­
lial cell membranes (Sugrue, 1987). If this acts
as a collagen receptor, it could link the intra­
cellular milieu with extracellular collagen
matrices, and playa role in corneal morpho­
genesis, or wound healing (Sugrue & Hay,
1986).
The mammalian corneas system includes
the presence of the degradative enzyme ace­
tylcholinesterase. High levels of acetylcho­
line in rabbit and bovine corneal epithelium
may modulate cyclic GMP (Walkenbach &
Ye, 1991), but clear-cut physiological role has
yet to be established. In the developing chick
cornea, levels of acetylcholine peak in the
middle of corneal maturation and coincide
with events such as increased epithelial
innervation and corneal transparency
(Sturges & Conrad, 1987).
The basement membrane of the human
corneal epithelium is thinner and more uni­
form centrally. becoming thicker and inter­
digitated peripherally (McTigue & Fine,
1966). Glycoprotein microfibres are also seen
peripherally, and only appear centrally in
acute keratoconus, when the central base­
ment membrane becomes thickened (Brewitt
& Reale, 1981). The basement membrane is
characterized by numerous hemidemsos­
omes, which are areas of focal attachment of
anchoring fibrils between the corneal epithe­
lium and the underlying stroma (Gipson et
al., 1987; Tisdale et al., 1988). Extended soft
contact lens wear in cat corneas causes a
decrease in the number of hernidesmosomes
per unit length of basement membrane

(Madigan, 1989), which explains why the
contact lens wearing corneal epithelium is
less adherent than normal (Madigan et al.,
1987). Adhesion structures in the healed cor­
neal epithelium can take 12 months to
recover (Gipson et al., 1989). Contact lens
wear initiates a sequence of unknown bio­
chemical events in the cat cornea which
reduce the epithelial hemidesmosome den­
sity. Changes in extracellular matrix compo­
nents could be translated into intracellular
events in corneal epithelial cells by modu­
lated basement membrane proteins such as
integrin which are linked to the epithelial
cytoskeleton (Trinkhaus-Randall et al., 1990).
Extracellular fibronectin is involved in cell
shape and movement, as well as the forma­
tion and development of extracellular matri­
ces (Mattey & Garrod, 1984).
In the normal cornea, type I collagen fibrils
are an important contributor to stromal
structure. In the rabbit and avian cornea,
type VI collagen is located between the type I
fibrils (Linsenmayer et al., 1986; Cintron &
Hong, 1988; Hirano et al., 1989) and may play
a structural role. In connective tissues, the
collagen fibrils eventually degrade to be
replaced by new fibrils. This turnover is
achieved by the presence of type I collagen­
degrading enzymes. In cultured rabbit cor­
neal cells, Fini and Girard (1990) reported
enzymes able to degrade type IV, V and VII
collagen. The first two collagenases were
thought to be synthesized by corneal epithe­
lial cells, and the latter by stromal fibroblasts
(Fini & Girard, 1990).
The corneal stroma in keratoconus con­
tains several abnormal proteins together
with reduced levels of normal proteins (Pan­
jwani et al., 1989). A major proteoglycan of
the cornea, keratan sulphate proteoglycan
(KSPG) has an altered keratan sulphate
arrangement in keratoconus (Funderburgh et
al., 1989; Sawaguchi et al. 1991). The subnor­
mal biostructural properties of the cornea in
keratoconus are presumably linked to an
abnormal KSPG function.
Corneal cellular physiology 241
The developing mammalian eye shows an
age-associated decrease in corneal endothe­
lial cell density (ECD), which attains an adult
value of around 250 000 cells/em/ (Bahn et al.,
1986.) The posterior surface of the cat, rabbit,
and human cornea increases in area by
210%, 130% and 17% from the infant to adult
(areas of 2.8 crrr', 2.3 ern" and 1.2 cm 2 , respec­
tively: see Kwok, 1984; Bahn et al., 1986). In
the non-human cornea, the areal increase can
account for about 80% of the adult ECD, and
since the total number of endothelial cells per
cornea is calculated to increase, mitosis is
calculated to contribute 20% of the ECD
(Bahn et al., 1986). The human cornea shows
a net loss of almost half its original endothe­
lial cells, which could account for most of the
lowered adult ECD. Bahn et al. (1986) con­
cluded that if mitosis exists in the develop­
ing human corneal endothelium it must be
have a minor effect, which is more than
compensated for by the cell loss. The analysis
of Bahn et al. (1986) can only be approximate,
because it does not allow for the possibility
that neighbouring cells may join to form
larger endothelial cells: if mitosis is present,
its contribution would be under-estimated.
The morphology of the corneal endothe­
lium is altered by contact lens wear (Schoe­
ssler & Woloschak, 1981). Intermediate
filaments which form a stable part of the
cytoskeleton In corneal endothelium {Risen
et al., 1987) must depolymerize to effect a
long-term shape change in corneal endothe­
lial cells. The migration and shape change of
rat corneal endothelial cells after a cryo­
injury is known to involve reorganization of
intracellular microtubules rather than
microfilarnents (Gordon & Staley, 1990) but
could represent a different condition. Poly­
megethous corneal endothelial changes were
reported by Carlson et al. (1988a) in patients
who had worn contact lenses for 2 to 23
years; endothelial fluorescein permeability
was unchanged, and no other significant
physiological changes were found. While a
multitude of morphological changes are
242 Corneal physiology and biophysics
reported, the biological basis and physi­
ological significance of these observations
remains poorly understood. PoIse et al. (1990)
have identified hypoxia as the primary caus­
ative factor, but the underlying aetiology is
scarcely understood. Major enzymes
involved in glucose breakdown were signifi­
cantly affected by closed-eye soft contact lens
wear (Kilp et al., 1985). Stromal oedema in
rabbit corneas during soft contact lens wear
was preceded by changes in corneal
enzymes, including impairment of corneal
endothelial Na+K+-ATPase (Cejkova et al.,
1988). Corneal pH is often mentioned as an
important facet, but stromal pH only changes
from a normal pH 7.5 (open eyes) to pH 7.4
during eyelid closure (Bonanno & PoIse,
1987). The rabbit corneal endothelium is able
to maintain normal permeability for external
pH in the range 6.5 to 8.5 (Gonnering et al.,
1979) sustaining a maximal corneal endothe­
lial potential difference (Fischbarg & Lim,
1974; Lyslo et al., 1985). When endothelial
pumping was briefly impaired by 10--{j M
ouabain, the pH of the rabbit corneal endo­
thelium remained unchanged, as did the
endothelial intracellular potential (Bowman
et al., 1984). Transient dark guttate-like spots
in the corneal endothelium of novice soft
contact lens wearers were first reported by
Zantcs and Holden (1977). The physiological
significance of this transient phenomenon is
unclear since it does not appear in adapted
lens wearers. Madigan (1989) recently
reported the same phenomenon in monkey
eyes, opening the possibility of a useful
animal model for investigation.
Sodium hyaluronate (NaHA) is injected
into the anterior chamber to protect the cor­
neal endothelium during anterior eye sur­
gery. A coating of viscous NaHA of 2 150 000
molecular mass acts as a shock absorber of
blunt trauma and also preserves the water
permeability of rabbit corneal endothelium
(Miyauchi & Iwata, 1984). Interestingly, the
mammalian corneal endothelium has surface
sites that bind hyaluronate, raising the pos­
sibility that native hyaluronate coats the pos­
terior corneal surface and that the corneal
endothelium catabolizes hyaluronate (Mad­
sen et al., 1989). Integrin receptors are
reported in human corneal endothelium
(Lauweryns et al., 1991) but their role in
endothelial adhesion and wound healing
remains to be studied further. Endocytosis in
the corneal endothelium (Kaye & Donn,
1965) was re-examined in rabbit eyes by
Raphael and McLaughlin (1990), but ques­
tions regarding possible mediation by mem­
brane receptors and the physiological
relevance to endothelial transport remain
unresolved.
Corneal oxygen is usually regarded as an
important imperative in contact lens consid­
erations but paradoxically the physiological
breakdown of molecular oxygen generates
free radical species capable of great oxidative
cell damage (Srivastava et al., 1989; Farber et
al., 1990). Oxidative free radical damage,
especially the response of the corneal endo­
thelium to H 2 0 2 has been studied intensely
because of the implications for lens cataract
formation (see Hull, 1990). Tear lactoferrin
(Boonstra & Kijlstra, 1987) may help protect
the cornea against oxidative damage by scav­
enging oxygen radicals (Kuizenga et al.,
1987). The enzyme superoxide dismutase
(SOD) is present in the non-human cornea
but its activity decreases with age (Crouch et
al., 1984). SOD scavenges the harmful super­
oxide radical, but forms intracellular hydro­
gen peroxide (H 20 2 ) , which is toxic in
greater than micromolar amounts. Fortu­
nately, H 20 2 can be vanquished rapidly by
other intracellular scavenging pathways
mediated by catalase and glutathione peroxi­
dase. Catalase is present in the mammalian
cornea (Atalla et al., 1987) and protects
against superoxide damage in the corneal
endothelium (Hull et al., 1984). Both catalase
and SOD are found in the human cornea but
their activities are very low (Mayer, 1980;
Hayden et al., 1990). Although catalase, SOD
and the enzyme glutathione peroxidase can

protect the cornea by scavenging harmful
free radicals, their interactions and specific
roles are poorly understood especially in the
human eye (see Kwok & Klyce, 1992). High
intracellular levels of oxidants such as H 20 2
can damage cell membranes through lipid
peroxidation. Aldehyde dehydrogenase in
the cornea (Holmes & VandeBerg, 1986;
Holmes et al., 1989) mitigates the effects of
membrane lipid peroxidation by detoxifying
the aldehyde by-products (Abedinia et al.,
1990).
14.3 CORNEAL WOUND HEALING
14.3.1 CORNEAL CYfOTOXICITY
Alkaline chemical injuries of the cornea dis­
rupt the basement membrane of the corneal
epithelium (Hirst et al., 1981; Gartaganis et
al., 1987). Alkali degradation of corneal col­
lagen is thought to liberate an attractant for
PMNs which are lysed, releasing hydrolytic
enzymes leading to collagen loss and pos­
sible stromal ulceration (Pfister et al., 1988). It
is possible that in the cornea injured with
sodium hydroxide, hydroxide ions can be
stored in the cornea (for later release) bonded
to corneal collagen and glycosaminoglycans
(Whikehart et al., 1991). The rabbit cornea
takes 6 months to recover normal epithelial
thickness following an alkali wound; this
appears to coincide with renonnalization of
anterior stromal hydration (Chung et al.,
1988). Chemical cautery was used by Culton
et al. (1990) in the rat cornea to re-examine
the effect of oxygen on corneal neovascular­
ization. No difference was found In the
degree of neovascularization in the rat cor­
nea under anoxia when compared with nor­
mal oxygen. The corneal cytoxicity of various
chemicals is sometimes ascertained by scan­
ning electron microscopy of the corneal epi­
thelium. Unfortunately, it may be difficult to
establish unambiguous damage thresholds
because of artefacts (Maclean & Haining,
1971). One preferred alternative is to exam-
Corneal wound healing 243
ine the effect on corneal electrical resistance
and ion transport (Burstein & Klyce, 1977).
This method offers greater sensitivity to
changes induced in the intact corneal epithe­
lium by tears-side addition of an agent.
Perturbations to the corneal epithelial mem­
branes induced by t-butyl hydroperoxide (an
organic hydrogen peroxide) are reversed
within an hour providing the dose does not
exceed 1-2 mM (Kwok & Klyce, 1992). Cor­
neal epithelial toxicity studies are often
undertaken with superficial circular wounds
used to ascertain the effect on wound closure
rate. To improve current methods in use,
Kwok (1991b) has recently proposed a more
accurate analysis of the areal closure rate
which is typically nonlinear. The healing
cornea re-epithelializes at a time-varying cell
migration rate (Kwok & Madigan, 1986) with
a shifting rate of protein synthesis (Zieske et
al., 1987). A time-varying cellular migration
rate is reported in healing corneal endothe­
lium (Miyata et al., 1990).
14.3.2 CONTACT LENS-INDUCED CELL
DEATH
There is little doubt that contact lens wear
can lead to death of corneal cells, especially
in cells of the corneal epithelium. Soft contact
lens wear can significantly affect corneal epi­
thelial cell integrity and physiology (see
Kwok, 1983; Holden et al., 1985; Friedlander
& Zimny, 1989). In rabbits according to
Cejkova et al. (1988) soft contact lens wear
leads to corneal damage which is 'very simi­
lar to that evoked by other traumas such as
mechanical or chemical damage'. Mechanical
destruction of rabbit corneal epithelium took
longer than normal to heal in contact lens
wear (Kilp et al., 1985). Chemicals could
cause cell damage by overwhelming the cel­
lular defence mechanisms to cause cell death.
Another lethal cell mechanism is apoptosis,
where cell death is triggered by some stimu­
lus e.g. hypoxia, leading to cell/suicide' (Kerr
et al., 1972; Boobis et al., 1989). Madigan
244 Corneal physiology and biophysics
(1989) using electron microscopy found
intra-epithelial vacuoles within the wing and
superficial cells following soft contact lens
wear on the monkey cornea. Condensed or
involuted epithelial cells (a possible feature
of apoptosis) were also found in contact
lens-wearing cat corneas. Madigan's (1989)
suggestion that apoptosis may be the basis of
contact lens-induced epithelial microcysts
deserves confirmation in the human cornea.
14.3.3 SUPERFICIAL INJURIES
The cornea shows remarkable rapidity in
repairing superficial injuries. A light scratch
to the cat corneal epithelium in vitro immedi­
ately caused a fall in corneal resistance,
which was reversed within 1 h (Kwok, 1986).
The rabbit corneal endothelium shows a
similar response (Fukami et al., 1988). The
rabbit corneal epithelium is apparently able
swiftly to re-establish its barrier properties
even after superficial chemical injury
(Wolosin, 1988; Sokol et al., 1990). Presum­
ably the process includes regeneration of the
tight junction complexes between superficial
cells. The cornea in vivo is exposed to various
nutrients and agents such as epidermal
growth factor (EGF) in the tears (Ohashi et
al., 1989), which would favour rapid corneal
repair. EGF promotes epithelial wound heal­
ing and its release by the lacrimal gland is
increased during reflex tearing, but its con­
centration still falls (van Setten, 1990). A
rapid corneal repair response would greatly
assist contact lens wear to successfully sur­
mount the many possible modes of epithelial
cell injury (see Kwok, 1985b, c).
Painful stimuli which cause excitement or
annoyance were found to diminish the
mitotic rate in the rat corneal epithelium,
which is apparently an adrenergic response
since it can be simulated by adrenaline
(Friedenwald & Buschke, 1944). Signifi­
cantly, contact lens wear also affects the
mitotic rate of corneal epithelium (Hamano
& Hori, 1983; Hayashi et al., 1985). This may
impair the corneal response of contact lens­
associated epithelial cell injury, since mitosis
is the primary mechanism for re-establishing
normal corneal epithelial thickness (Thomp­
son et al., 1991). Labelling of mitosing rat
corneal epithelial cells with 3H-thymidine
indicates that about 50% of basal cells are
replaced every 3 days, with more mitosis
seen peripherally (Cenedella & Fleschner,
1990). A disturbance of corneal epithelial cell
turnover by contact lens-induced hypoxia
(Kwarecki & Krawcyzk, 1989) may present a
form of cellular stress insofar as the extended
lifetime could age the epithelium and
adversely affect epithelial cell defences.
Epithelial cells migrate to re-establish cor­
neal confluency in the healing cornea after
epithelial scraping. In the rabbit cornea. cell
regeneration and differentiation is derived
ultimately from the limbal stem cells (Chen &
Tseng, 1990). Epithelial cells at the leading
edge of movement have different mem­
brane-protein receptors than normal superfi­
cial epithelial cells (Gipson et al., 1983;
McLaughlin et al., 1986), but it is uncertain
whether these are normally deeper in the
epithelium, or represent a cell surface
change. The complex cellular events in the
healing corneal epithelium are partially
known, and involve extracellular compo­
nents such as fibronectin (Fujikawa et al.,
1981), cell surface receptors such as integrin
(Gipson & Spurr-Michaud, 1988; Trinkhaus­
Randall et al., 1990; Paallysaho & Williams,
1991) and vinculin (Soong, 1987; Zieske et al.,
1988) and intracellular enzymes such as
calcium-activated calpain (Shearer et al.,
1990). Watanabe et al. (1988) suggested that
fibronectin (FN) may be applied clinically
where adhesion of the corneal epithelium is
abnormal or retarded. In vitamin A defi­
ciency, application of FN accelerated corneal
epithelial healing in the rat (Watanabe et al.,
1991). Delayed re-epithelialization was
found in retinol deficiency (El-Ghorab et al.,
1988), and topical therapy with vitamin A is a
possibility because it is a cofactor for corneal

epithelial growth and function and is nor­
mally present as retinol in the tears (Ubels &
MacRae, 1984).
Membrane-bound receptors for low­
density lipoprotein were found in cultured
corneal endothelial cells (Elner et al., 1991). In
human corneal endothelium, cells adjacent to
a scratch injury site significantly increased
their low-density lipoprotein uptake. Most
likely the migrating or compromised cells
had increased receptor densities which
elevated the intracellular supplies of amino
acids, fatty acids and free cholesterol from
lipoprotein hydrolysis (Elner et al., 1991).
Corneal endothelial wound healing can be
pharmacologically modified by EGF (Iumb­
latt et al., 1988; Joyce et al., 1989) raising the
future possibility of clinical treatment with
growth factors like EGF. A prostaglandin
membrane receptor was found in rabbit cor­
neal endothelium (jumblatt & Paterson,
1991). The prostaglandin receptor is coupled
to cyclic AMP synthesis, and is believed to
be involved in maintaining the characteristic
polygonal endothelial cell shape (Iumblatt et
al.,1988).
14.4 PHOTOTHERAPEUTIC CORNEAL
SURGERY
The controlled application of 193 nm ultra­
violet laser energy to the living cornea is
capable of precise sculpturing of the corneal
tissue for refractive correction (Krueger et al.,
1985). The word 'precise' here is used to
denote repeatability. In regard to accuracy,
the actual correction is highly dependent on
several procedural variables (Gaster et al.,
1989). However, it is worth mentioning that
corneal incisions can be measured to within
5-10 I.I.m with femtosecond optical ranging
(Stem et al., 1989). Ocular optics consider­
ations in postoperative vision (for example,
Hemenger et al., 1990) need greater research
attention, especially to what long-term
results are actually achieved in refractive
surgery.
Photo therapeutic corneal surgery 245
Corneal scars have been recently treated
with phototherapeutic keratectomy using
193 nm excimer laser surgery (Sher et al.,
1991). The corneal epithelium was removed
prior to photoablation, and although the cor­
nea was re-epithelialized within 4 to 5 days
without induced scarring, Sher et al. (1991)
recommended in retrospect that the epithe­
lium should be kept intact for best results. In
a previous study of corneal photoablation,
Tuft et al. (1989) noted that collagen remodel­
ling in the rabbit corneal stroma was only
initiated after complete re-epithelialization.
A persistent subepithelial haze of unknown
origin was noted, which was partly amelio­
rated by postoperative steroid treatment. The
anterior proliferation of stromal keratocytes
with large vacuoules found in photoablated
rabbit cornea (Gaster et al., 1989) could
account for the subepithelial haze.
The results reported by Sher et al. (1991)
were encouraging, but several problems
appeared. The majority of patients had eye
pain, some severe, and generally at a level
greater than that experienced in radial kera­
totomy. Some patients even required nar­
cotic analgesia during the initial 24 to 48
hours after the operation. A hyperopic shift
was found in about half the patients and a
secondary hyperopic steepening procedure
was recommended after the primary myopic
ablation. Corneal scars in adult rabbit cornea
contain unusual proteoglycan structures
(Funderburgh et al., 1988). Healing takes 2
years to restore normal patterns of proteogly­
can stromal composition, and involves
movement of existing corneal proteoglycans,
de novo macromolecular synthesis, and a par­
tial reactivation of biosynthetic mechanisms
usually present in the younger, developing
cornea (Cintron et al., 1990). Thus, the age of
the scar and its varying biochemical compo­
sition at the time of surgical intervention
may affect the clinical success of treatment of
corneal scars.
The biological effects of laser energy
directed at the cornea are largely unknown,
246 Corneal physiology and biophysics
and more animal experiments are required
(see for example, Mecke et al., 1991). A thor­
ough understanding of the likely long-term
effects of phototherapeutic procedures on the
cornea is essential if tissue side effects are to
be minimized, and clinical objectives met.
The short wavelength radiations involved
can damage cellular DNA and even jeopar­
dize long-term corneal transparency (Apple­
gate & Ley, 1991). The effect on DNA is
particularly important, as little data are avail­
able on the mutagenic effects of laser corneal
surgery.
14.5 CORNEAL BIOPHYSICS
14.5.1 CORNEAL EPITHELIAL
BIOMECHANICS
The resistance of rabbit corneal epithelium to
stretching was recently determined in iso­
lated corneas (Kwok, 1991a). The modulus of
elasticity was estimated to be of the order of
104 Pa in the tangential direction (running
limbus to limbus). This modulus is one order
of magnitude less than that reported for the
rabbit corneal stroma (jue & Maurice, 1986),
and indicates that for the same tangential
stress the epithelium will undergo greater
elongation.
Despite this difference in moduli, the pres­
ence of an intact corneal epithelium could
have an effect on the overall biomechanical
behaviour of the cornea. The corneal epithe­
lium would be expected to modify the expan­
sion of the underlying stroma by restraining
its physical excursions.
14.5.2 CORNEAL STROMAL BIOMECHANICS
The modulus of elasticity of corneal stroma is
of the order of 105 Pa, but the value deter­
mined experimentally depends on the tan­
gential stress applied to the tissue. Corneal
stress-strain relationships were examined by
Iue and Maurice (1986) in intact human and
rabbit eyes. Corneal distensions and initial
tensions of Descemet's membrane were
found to differ: the rabbit corneal stroma
began to accept additional tension only after
a calculated strain of 9% in Descemet's mem­
brane; the human corneal stroma accepted
tension immediately. The authors cautioned
against extrapolation of rabbit corneal bio­
mechanics to the human situation.
The tangential tension exerted by the
intraocular pressure on the stroma is rela­
tively uniform throughout the stromal depth
(McPhee et al., 1985) and is essentially borne
by the collagen fibrils. An important implica­
tion of this distribution is that the epithelial
basement membrane, and hence the epithe­
lium, must also be under tangential tension
in the limbus-to-limbus direction (Kwok,
1991a).
14.5.3 STROMAL BIOPHYSICS
The quasi-regular spacing of the collagen
fibrils of the corneal stroma is the basis of
stromal transparency (Maurice, 1957). McCally
and Farrell (1988) have elaborated on various
aspects of the biophysics of minimal light
scattering as the basis of stromal transparency.
The corneal stroma contains proteoglycans
(PGs) in the interfibrillary matrix, the two
major types being the keratan sulphate pro­
teoglycan (KSPG) and the chondroitin sul­
phate proteoglycan (CSPG). These are large
macromolecules approaching 50 000 units of
molecular mass, and are largely responsible
for the stromal swelling pressure (see below).
One end of the protein core of the proteogly­
can is covalently attached to the collagen
fibril (Scott & Haigh, 1985), while the other
end containing the gIycosaminoglycan
(GAG) side-chains is projected radially. At
physiological pH, carboxylic and sulphonic
acid groups in the GAGs dissociate and
adopt a net negative charge. The bulk charge
for most corneas at pH 7.4 is around
0.05 mole e-/kg stromal fluid (see Maurice,
1984). Since the PGs are attached at regular
intervals along the collagen fibril (Scott &

Haigh, 1985; Hirsch et al., 1989) each collagen
fibril roughly resembles a bottle brush in
three dimensions. If the PGs have sufficient
rigidity, it is possible that the PG structures
could generate long-distance influence on
neighbouring collagen fibrils. In particular, a
structural influence of the corneal PGs (Hart
& Farrell, 1971) could explain the curious
spatial uniformity of interfibrillary spacing
during corneal swelling. Prolonged immer­
sion of pieces of corneal stroma produces a
biphasic time course of swelling (Kwok,
1986). The biphasicity could be explained by
an uncoupling of the stromal PG complex
due to salt degradation and subsequent loss
of long-distance interaction between col­
lagen fibrils (Kwok, 1990).
In the intact cornea, the compressive effect
of the intraocular pressure on the stroma is
greatest at the endothelial side falling to zero
at the epithelium (Berkley, 1971). A gradient
in fluid pressure (or imbition pressure IP)
was found in the rabbit corneal stroma with
a central pressure of - 5.7 kPa (- 42.7 mmHg)
falling to - 2.4 kPa (- 17.8 mmHg) peripher­
ally (Wiig, 1989). Since IP = IOP-SP (see
Maurice, 1984), where lOP is the intraocular
pressure and SP is the swelling pressure, an
IP of greater magnitude in the central cornea
would imply a greater SP centrally than
peripherally if the same lOP operated at both
locations. This could not be accounted for bv
differences in local stromal hydration, which
is similar anteriorly (central versus periph­
eral) and in the wrong direction posteriorly
(Kikkawa & Hirayama, 1970). Otherwise, a
greater lOP effect must be postulated periph­
erally.
Microfibres of type VI collagen in the
interfibrillary space (Linsenmayer et al.,
1986; Cintron & Hong, 1988; Hirano et al.,
1989) may also play a structural role. An
orthogonal network of microfibrils is
reported in the chick corneal stroma (Bruns et
al., 1987) but the chick cornea is a poor
biostructural model for the human corneal
stroma, which is less organized. Clusters of
Corneal biophysics 247
proteinaceous microfibrils of unknown func­
tion are reported between stromal lamellae
and near keratocytes in the rabbit cornea
(Carlson & Waring, 1988). The abnormal col­
lagen arrangement in macular corneal dys­
trophy (Quantock et al., 1990) could be due to
the breakdown of PG structures mediating
interfibrillary collagen spacing.
Recent developments in confocal micros­
copy have enabled high resolution imaging
of the cornea in vitro (Masters & Paddock,
1990; Xiao et al., 1990). Fine filaments were
observed throughout the corneal stroma (but
were highest near Descemet's membrane), as
well as extensive interconnecting processes
between stromal keratocytes; even nerve
plexuses were resolved.
The corneal stromal fluid is comprised of
two fractions: the 'free' water is the major
portion and behaves similarly to bulk water,
and 'bound' water wherein dissolved mol­
ecules move around less freely. These frac­
tions can be discriminated with a variety of
techniques including proton NMR spectros­
copy (Masters et al., 1983), differential scan­
ning calorimetry (DSC) Castoro et al., 1988)
and isothermal thermogravimetry. Unfortu­
nately, the estimated fractions will depend
on the method of measurement: the DSC
method measures non-freezable water. The
'bound' water is associated with structural
elements such as collagen fibrils, perhaps
arranged like a cylinder concentric with the
long axis of the fibril. Each measurement
technique can be imagined to intersect dif­
ferent cylinders around the fibril leading to
different estimates of 'bound' water. The
exchange between the free fluid fraction and
external fluid will be more rapid than the
exchange between the two stromal fractions.
However, of paramount importance is the
physiological relevance and plausibility of
the fraction estimated as 'bound' water. As
far as mobile small ions are concerned,
around 8-10% of total stromal fluid can be
considered 'bound' at normal hydration
(3.4 kg H 20/kg dry mass). The free fraction
248 Corneal physiology and biophysics
of stromal fluid is the solvent volume for
osmotic effects and is important to stromal
swelling pressure (see below).
14.6 CORNEAL PERMEABILITY
14.6.1 NORMAL CORNEA
The cornea is significantly more permeable
to nonelectrolytes than the conjunctiva, by at
least one order of magnitude (Huang et al.,
1989; see Maurice (1991) for a discussion of
this article). Damage to the anterior or poste­
rior epithelial layers of the cornea leads to
tissue swelling (Maurice & Giardini, 1951).
The corneal epithelium contains the site of
greatest electrical resistance in the cornea
(Fee & Edelhauser, 1970; Klyce, 1972). The
superficial cells, interconnected by tight
junctions fonn the primary site (Klyce, 1972).
The superficial cells in the rabbit cornea are
relatively more dehydrated than the basal
and wing cells, with the dry mass content of
the superficial cells being 50% greater than
that of basal cells (Chung et al., 1988). The
hydration of superficial epithelial cells is
normally comparable with the hydration of
the anterior stroma (Chung et al., 1988).
Desquamation of superficial epithelial
cells is a normal event in the turnover of
corneal epithelium, but the mandatory des­
mosomal detachment of the departing cell
(Hazlett et al., 1980) is exquisitely timed with
the re-establishment of epithelial integrity
(Wolosin, 1988; Sokol et al., 1990). The
desquamating epithelial cell undergoes
changes to its plasma membrane, as evi­
denced by increased binding of the lectin
probe concanavalin A (Bonvicini et al., 1983).
Calcium in the tears is associated with nor­
mal surface cell exfoliation in the corneal
epithelium (O'Leary et al., 1985). Perfusion of
the excised rabbit cornea with a calcium-free
165.8 mM saline solution (0.97% solution by
weight) for 150 min was found to induce
abnormal sloughing of surface epithelial cells
(Bergmanson & Wilson, 1989), but the physi­
ological implications for the living human
cornea are not known.
The corneal endothelium is the principal
entry site for corneal glucose (see Maurice,
1984; DiMattio, 1984) and is reported to have
a transmembrane-protein glucose trans­
porter which allows the passage of water
molecules (Fischbarg et al., 1987). Although
water permeation was only demonstrated for
osmotically-induced flows, the glucose trans­
porter may mediate most of the water flow
through the corneal endothelium. The cor­
neal endothelium forms a functional syncy­
tium presumably via intercellular gap
junctions (Rae et al., 1989b) but the physi­
ological ramifications of such good cell-to­
cell coupling are yet to be fully explored.
Sodium fluorescein is a widely used oph­
thalmic probe of membrane permeability.
For doses expected in clinical and research
practice, fluorescein is confirmed to have no
deleterious ocular effects as evidenced from
corneal endothelial electrical potential mea­
surements (Akiyama et al., 1990a).
14.6.2 PATHOLOGICAL CHANGES
The corneal epithelial barrier is altered in
diseases such as diabetes (Gobbels et al.,
1989) and Graves' ophthalmopathy (Khalil et
al., 1990) as evidenced by a subnormal resis­
tance to fluorescein. However, Stolwijk et al.
(1990) found a normal fluorescein permeabil­
ity in the corneal epithelium of diabetic
patients, but epithelial permeability to one
drop of 0.4% oxybuprocaine HCI was twice
the normal value. The corneal epithelium in
diabetes shows abnormal fragility (O'Leary
& Millodot, 1981), and has abnormal wound
repair (Fukushi et al., 1980). Pathophysi­
ological changes such as increased glucose
storage (Friend et al., 1981), and subnormal
oxygen uptake (Graham et al., 1981) indicate
that abnormal cell functions are associated
with the permeability changes.
The permeability to non-electrolytes of the
rabbit corneal endothelium is claimed to

increase during intraocular inflammation
(Macdonald et al., 1987). Exposure of rabbit
eyes to 300 nm UV-B radiation in doses of 0.1
to 0.5 J/cm 2 induced temporary corneal swell­
ing, which was attributed to increased
endothelial permeability due to cell mem­
brane damage (Riley et al., 1987).
14.6.3 CHANGES IN WOUND HEALING
The permeability of healed rabbit corneal
epithelium to non-electrolytes after an epi­
thelial scraping is initially elevated after
re-epithelialization (Huang et al., 1990) but
recovers to normal within a week (Thoft &
Friend, 1975; Huang et al., 1990). The regen­
erating rabbit corneal epithelium after scrap­
ing loses its adrenaline sensitivity for at least
1-2 months (Kikkawa & Morimoto, 1980).
This implies that chloride transport is
directly affected, since it is exogenous
adrenaline which normally decreases epithe­
lial chloride resistance (Klyce & Wong, 1977).
Although the regenerating corneal epithe­
lium after wounding is initially thin, electro­
physiological functions such as the
transepithelial potential remain operational
but only subnormally (Okuhara & Kikkawa,
1968). Epithelial ion transport after myopic
keratomileusis surgery in rabbit corneas
began to recover epithelial adrenaline sensi­
tivity at 25 days (Marcus et al., 1983).
The rabbit corneal epithelium has a gra­
dient of hydration with the lowest hydration
found anteriorly in the superficial cells
(Chung et al., 1988). After re-epithelialization
of an alkali wound, the rabbit corneal epithe­
lial cells become more hydrated taking up to
6 months to recover (Chung et al., 1988).
Presumably, the epithelial barrier is also
affected after regeneration.
The healed cat cornea is able to tolerate a
postoperative decrease in endothelial cell
density of 60%, following mechanical scrap­
ing (Landshman et al., 1988). A greater
decrease, where endothelial cell densities fell
below 105000 cells/emf, resulted in in-
Corneal permeability 249
creased postoperative corneal thickness. The
decompensation was attributed to greater
numbers of giant endothelial cells, which
apparently compromised the endothelial
barrier.
14.6.4 CHEMICALLY-INDUCED CHANGES
Proper regulation of epithelial cell volume is
essential in maintaining the epithelial bar­
rier function. Epithelial oedema will result if
the normal homeostatic mechanisms (see
below) are overwhelmed, as in the case of a
severe alkali wound (Chung et al., 1988). The
application of tears-side t-butyl hydroperox­
ide (tBHP) induced a permeability increase
in the rabbit corneal epithelium, as well as
abnormal oscillations of corneal thickness
(Kwok & Klyce, 1992). The rabbit corneal
epithelium responded to tBHP with immedi­
ate and reversible falls in electrical potential
difference (PD) and short circuit current,
providing that the dose did not exceed 1 mM
tBHP (Kwok and Klyce, 1992). Epithelial
Na+K+ -ATPase generates the epithelial PO
and irreversible alterations occurred at doses
exceeding 1 mM tBHP indicating H 202 modi­
fication of Na+K+-ATPase (Garner et al.,
1986). An increased epithelial conductance
was due to unnamed effects in the anterior
corneal epithelial membranes. An extremely
high dose of 8.8 mM H:z0 2 was reported to
penetrate the cornea and impede the func­
tion of the corneal endothelium (Yuan &
Pitts, 1991). Such a clinical occurrence would
be exceptional and unlikely since this dose
equals 300 ppm, and was applied for 10
min/day for 5 days (Yuan & Pitts, 1991).
Acute reactions by patients to H 202 associ­
ated with contact lens wear are occasionally
reported in the clinical literature (Knopf,
1984) but the long-term consequences of cor­
neal exposure to small amounts of H 202 are
unknown.
Addition of 50 f.LM H 202 to the aqueous
humour halves the resistance of the rabbit
corneal endothelium to the penetration of
250 Corneal physiology and biophysics
nonelectrolytes like inulin and mannitol
(Riley & Giblin, 1983). The endothelial
Na+K+ -ATPase is apparently unaffected by
small amounts of H 202 , so the altered perme­
ability is due to membrane changes (Welsh et
aI., 1985), or perhaps modulation of the bicar­
bonate pump. Glucose in the aqueous
humour can protect the rabbit corneal endot­
helium from the deleterious effects of H 202 •
14.7 CORNEAL ION TRANSPORT
14.7.1 EPITI-IELIAL ION TRANSPORT
In the rabbit corneal epithelium, chloride is
transported into the tears (van der Heyden et
al., 1975) at levels modulated by exogenous
adrenaline (Klyce & Wong, 1977). The sus­
pected location of a chloride ion channel in
the tears-side membrane of corneal epithe­
lium (Festen & Slegers, 1979) was confirmed
by Marshall and Hanrahan (1991) using
voltage-clamped gigaohm sealed patches of
cultured corneal epithelium. Possible sources
of exogenous adrenaline are the tears (Trope
& Rumley, 1984), and adrenergic nerves in
the human corneal stroma (Toivanen et al.,
1987). Adrenaline can enhance intracellular
cyclic AMP by stimulating ~-adrenergic
receptors in the corneal epithelium (Fogle &
Neufeld, 1979), which are predominantly of
the ~2 subtype (Walkenbach et al., 1984).
Increased intracellular cyclic AMP will
increase the tear-side chloride permeability
of the corneal epithelium, favouring greater
efflux of chloride into the tears (for reviews,
see Reinach, 1985; Klyce & Bonanno, 1988).
The activation of the ~-adrenoreceptor sys­
tem has the ability to thin a swollen rabbit
cornea (Klyce, 1977), but would playa sec­
ondary role since its rate is equivalent to less
than 5 % of the rate known for the endothelial
HC03- transport mechanism. Additionally,
the human corneal epithelium appears to
transport chloride into the stroma and
sodium into the tears (Fischer et aI., 1978), so
the applicability of the rabbit corneal find­
ings to human corneal function is unclear.
The basal cells of the rabbit and bullfrog
corneal epithelium contain a potassium con­
ducting channel (Klyce, 1972; Festen &
Slegers, 1979; Carrasquer et aI., 1987). The
potassium channel was thought to face the
stroma and Rae et aI. (1990a) recently con­
firmed this location using voltage-clamped
patches of basal cell membrane. Such a con­
ductance would be expected to recycle potas­
sium ions from the corneal stroma for
subsequent extrusion by the stromally­
facing Na+K+-dependent ATPase electro­
genic pump located in the basal cells. The
Na+K+-ATPase pump generates the bulk of
the transcorneal potential difference (TCP) of
2Q-40 mV in rabbits (aqueous side positive).
The cornea seems different in carnivorous
mammals: the human cornea has a far lower
TCP of around 0-1 mV (Fischer et al., 1974,
1978) and similar TCPs were reported in cat
corneas (Ehlers, 1970; Kwok, 1986). The TCP
is sensitive to oxygen (Ehlers & Ehlers, 1966)
and is reduced in contact lens wear (see
Kwok, 1983).
The pH of rabbit corneal epithelium in cul­
tured cells was found to be 6.87 ± 0.02 using a
dual wavelength photometer technique (Korb­
macher et al., 1988). This agrees with a pH of
6.5 reported by Krejci (1972), but is somewhat
lower than a pH of 7.34 ± 0.03 reported by
Bonanno and Machen (1989), who used an
identical photometric procedure. The pH of
rabbit corneal epithelium appears to be nor­
mally more acidic than the tears, which have a
pH in the range of 7-7.5 (Krejci, 1972). The
normal human tears have a pH of around 7.5
(Carney et al., 1989). Intracellular pH in rabbit
corneal epithelium is regulated by a basal cell
Na+/H+ exchanger, which extrudes intracellu­
lar protons (H+ ions) while bringing in extra­
cellular sodium ions (Korbmacher et al., 1988;
Bonanno & Machen, 1989). Intracellular pH
may affect the epithelial transport of ions like
chloride, and hence modify functions such as
hydration control (Klyce, 1977). Intracellular
pH in the rabbit corneal epithelial basal cell is

regulated by co-operative Na +-K+ and K+-H+
exchangers; inhibition of these mechanisms
will acidify the basal cell interior (Bonanno &
Machen, 1989; Bonanno, 1991). Enhancement
of anaerobic glycolysis by anoxia, or applica­
tion of cyanide elevates epithelial production
of lactate which is transported into the stroma
by a lactate-H" cotransporter (Bonanno, 1990;
Chen & Olen, 1990).
14.7.2 STROMAL ION TRANSPORT
The osmotic effect of stromal accumulation of
epithelially-generated lactate during anterior
hypoxia is widely believed to be the basis of
contact lens-induced corneal oedema (Refojo,
1975; Klyce, 1981). In the rabbit cornea,
around 95% of total lactate is normally found
in the corneal stroma (Chen & Chen, 1990).
Of the total rabbit corneal lactate formation,
47% was formed in the corneal epithelium,
32 % in the stroma and 21 % in the endothe­
lium. Epithelial lactate was shown to be
transported into the stroma by a proton­
lactate pump. That 79% of corneal lactate
production is normally associated with the
corneal epithelium and stroma is not surpris­
ing since the total stromal cell volume (Kaye,
1966) is comparable to total epithelial cell
volume (Huff, 1990b). Lactate itself was con­
firmed to ha ve no directly detrimental effects
on corneal function (Huff, 1990a). Of the total
exogenous glucose provided to the isolated
cornea, the epithelium consumed about half.
The presence of a fixed negative charge
enables the stromal movement of ions and
water to be calculated by modelling the cor­
neal stroma as an ion-exchange matrix
(Maroudas, 1968, 1975). In this model, the
fixed negative charge in the corneal stroma
attracts counterions such as Na+ to preserve
bulk electroneutrality in the stromal fluid.
Additionally, cr would be attracted to any
fixed positive charges in the corneal stroma.
In the cornea in vivo, the aqueous humour
will host many of the mobile ions. At steady
state, the mobile ions will be distributed
Corneal ion transport 251
according to the Gibbs-Donnan distribution,
the simplest form of which is:
[Na+]; X rcrj, = [Na+]o X [Cn o (eqn 14.1)
where the concentrations are in moles/litre,
subscript i refers to the stroma, and a refers
to the external solution. Experimental deter­
minations indicate an excess of Na+ in the
corneal stroma exceeding the concentration
calculated from eqn 14.1. This extra fraction
is required to neutralize the fixed stromal
charge, and the (mainly sodium) ions immo­
bilized in the stromal matrix represent an
hyperosmotic force which generates an
osmotic pressure. The fluid entering from the
external solution creates a swelling pressure.
The expansive force found in isolated corneal
stroma from the osmotic pressure is around
8 kPa (60 mmHg) but figures can vary
according to measurement methods.
Equation 14.1 can be used to derive an
expression to calculate the fixed stromal
charge and the osmotic pressure generated
(Hodson, 1971). The result indicated good
agreement with experimental swelling pres­
sures, and calculated a charge of 0.048
mole e-/l (Hodson, 1971). A recentdetermina­
tion in bovine corneal stroma reported a
charge of 0.0395 mole e-/l (Hodson et al.,
1991), somewhat lower than expected but
which could be due to the osmometric
method used. On theoretical grounds, it
appears that the stromal charge may have an
anomalous dependency on temperature
(Kwok & Klyce, 1990). A corneal stromal
charge that increased with falling tempera­
ture can account for apparently conflicting
results in the past. The molecular biophysics
of this temperature effect remains to be
found, but it probably originates in confor­
mational changes in stromal proteoglycans.
The Gibbs-Donnan result (eqn 14.1) can be
used to accurately model corneal stromal
swelling, but a previous attempt (Elliott et al.,
1980) neglected factors such as the 'bound'
fluid fraction in the stroma and calculated
low stromal charges. The situation for the
252 Corneal physiology and biophysics
cornea in vivo is different because gradients & Lim, 1974; Hodson, 1974). The corneal
of water pressure and hydration are present, endothelium shows a net transport of bicar­
and has yet to be rigorously described. bonate into the aqueous humour (Hodson &
Miller, 1976) and inhibition of bicarbonate
translocation retards water efflux (Harris et
14.7.3 ENDOTHELIAL ION TRANSPORT
al., 1956; Fischbarg & Lim, 1974) and insti­
The physiological functions of the so-called gates stromal swelling (Hull et al., 1977). The
corneal endothelium suggest that 'posterior net amount of bicarbonate solute trans­
corneal epithelium' (Stocker, 1954) is a better located into the aqueous humour can be
description. As outlined below, the barrier and calculated to satisfactorily account for experi­
ion transport properties of the corneal endot­ mentally determined 'pump' rates (Liebo­
helium are primarily directed towards control­ vitch & Weinbaum, 1981).
ling stromal hydration and preserving stromal A bicarbonate-dependent K+ pump which
transports K+ into cultured bovine corneal
transparency. In that sense, it differs little in
principle to the purpose and mission of the endothelial cells was reported by Savion et al.
corneal epithelium. The term corneal endothe­ (1989). This K+ pump is ouabain-insensitive
lium is now so well entrenched that it has but must be electroneutral because ouabain
become routine jargon, and will be used here. can completely abolish the corneal endothe­
In 1873, Theodore Leber demonstrated that lial potential difference (PO) (Fischbarg &
the corneal endothelium contains the pri­ Lim, 1974).
mary mechanism for corneal hydration con­
trol (see Stocker & Reichle, 1974). Leber's
Transendothelial potential difference
crude experiments were later refined by sev­
eral workers who confirmed the endothelial A small electrical PO of around -0.5 mV is
site of the fluid pump (for example Itoi et al., measured across the corneal endothelium of
1964; Mishima & Kudo, 1967; Hoshino, 1968; the rabbit, bovine, human and cat eye (Fisch­
Maurice, 1972). Many of the basic prin­ barg, 1972; Hodson, 1974; Wigham & Hod­
ciples governing the corneal endothelial son, 1981a,b; Kwok, 1986). The endothelial
transport of ions and fluid evolved from the PO is negative on the aqueous side - of
rabbit corneal model (see Fischbarg et al., opposite direction to the transepithelial PO,
1985). The rabbit corneal endothelium whose magnitude is ~O times larger in the
actively mediates an efflux of fluid from the rabbit eye but of comparable magnitude in
corneal stroma equal to about 7 ~lIcm2/hl the human and cat eye (see Fischer et al.,
(Baum et al., 1984; Kuang et al., 1990). 1978; Kwok, 1986). The endothelial PO is
Mathematically this is equivalent to a temperature sensitive and falls with
thickness change of 70 um/h. Opposing . decreased temperature (Hodson, 1975). The
this outwards pump is an inwards fluid PO was decreased in cultured bovine corneal
'leak' from the aqueous humor into the endothelial cells exposed to hypotonic
corneal stroma. At steady-state stromal media, and bicarbonate sensitivity was
thickness, the fluid loss due to the pump is diminished (Coroneo et al., 1989), an effect
offset by the fluid gain of the 'leak'. possibly mediated by a membrane ion chan­
nel.
The removal of stromal fluid through the
Bicarbonate dependency
corneal endothelium is coupled to bicarbon­
A bicarbonate transporter is the primary ate translocation and is also associated with
corneal endothelial mechanism responsible the small endothelial PO (Fischbarg & Lim,
for moving fluid out of the stroma (Fischbarg 1974; Hodson, 1974). However, the PO is not

generated by the bicarbonate transporter
which is electroneutral. This explains why
inhibition of endothelial bicarbonate supply
by the carbonic anhydrase inhibitor 10.. . 7 M
ethoxyzolamide represses the endothelial
pump (Barfort & Maurice, 1974), but has little
effect on the transendothelial potential dif­
ference (Fischbarg & Lim, 1974). The corneal
endothelial PO principally arises from activ­
ity of endothelial Na+K+-ATPase (see Fisch­
barg et al., 1985). Immunohistochemical
probes confirm the presence of carbonic
anhydrase, a bicarbonate-chloride exchanger
and Na+K+ -ATPase in human corneal endo­
thelium, which are located primarily on the
aqueous-side of the endothelium (Holthofer
et ai., 1991).
Transendothelial electrical resistance
The corneal endothelial electrical resistance
or conductance are good indicators of corneal
endothelial permeability, owing to the corre­
spondence between endothelial permeability
and ionic fluxes (Hodson & Wigham, 1983).
The corneal endothelium resistance is
reported in the range 20 to 70 ohm cm-2 in
the presence of adenosine (see Fischbarg &
Lim, 1984). Comparable but slightly higher
resistances were reported in the cat corneal
endothelium (Kwok, 1986). Lower resis­
tances are found when adenosine is omitted
from the Ringer solution bathing the isolated
corneal endothelium (Fischbarg & Lim,
1984). The endothelial resistance is tempera­
ture sensitive and rises with decreased tem­
perature (Hodson, 1975).
Using the patch clamp technique,· Liebo­
vitch et al., (1987) modelled the molecular
kinetics of ion channels as they gated
between open and shut states. An unconven­
..The patch clamp technique, developed in Gennany,
captures a small piece (a 'patch') of cell membrane in a
micropipette to measure an electrical current of a few
picoamps as one or more ion channels open. The mem­
brane is held at an electrically constant voltage, or voltage
'clamp' (see Rae et al., 1988; Liebovitch & T6th, 1990).
Corneal ion transport 253
tional fractal scaling model was introduced.
Ion channel kinetics of a potassium channel
and non-selective cation channel have been
characterized in the endothelial membrane
facing the aqueous in rabbit cornea (Rae et
ai., 1989a, 1990b).
Corneal endothelial pH
The rabbit corneal endothelium has an intra­
cellular pH of around 7.1 for an ambient pH
of 7.5 (Bowman et al., 1984). Endothelial PO is
greatest when the external pH is in the range
7.3 to 8 (Fischbarg & Lim, 1974; Lyslo et al.,
1985). The corneal endothelial pH is respon­
sive to external levels of H+, falling to pH 6.6
when the external pH is 7.0 (Bowman et al.,
1984). To account for this effect, a Na+/H+
exchanger is hypothesized (Fischbarg et al.,
1985), which transports H+ out of the endo­
thelium. The endothelial PO also falls with
pH (Fischbarg & Lim, 1974; Lyslo et al., 1985)
suggesting that the electrogenic endothelial
Na+K+-ATPase is indirectly coupled to the
Na+ /H+ exchanger.
The short-circuited corneal endothelium
Details of the exact mechanism of transen­
dothelial ion flow remain unresolved.
Wigham and Hodson (1985) used the classic
Ussing technique of electrically short­
circuiting the corneal endothelium to calcu­
late the net ionic flow. In an electrically
'tight' cell layer such as the corneal epithe­
lium, the short-circuit current (SCC) equals
the net flow of ions through the layer. How­
ever, the endothelium is of very low resis­
tance and the meaning of the SCC, and its
relationship to the physiological (open cir­
cuit) condition is disputed (Fischbarg &
Lim, 1984). In the young rabbit cornea,
Wigham and Hodson (1985) measured an
SCC that was equivalent to a net ionic
current of 5 X 10- 14 mcl/rn''. The net HC0 3 ­
flux was measured at 3.4 X 10- 14 mol/rn",
which could not entirely account for the
254 Corneal physiology and biophysics
endothelial SCc. The short-fall could be
made up by a net flow of another anion
such as 0- in the same direction as HC03 - ,
or by a net cation flow of Na+ in the
opposite direction. No net Cl" flux was
detected, and the net Na+ flux was only
0.08 X 10- 14 mol/rn/ into the stroma, the
correct direction, but too small to account
for the difference. It should be emphasized
that such simple balance sheet calculations
ignore several subtle mechanisms which
are operating under conditions where a
voltage is imposed across the endothelium.
One possible factor for example is the dif­
ferent mobilities of the ions, which may
affect the overall calculation. The short­
circuit technique yields different net fluxes
than reported under open-circuit condi­
tions (see Fischbarg & Lim, 1984). Other
criticisms can be advanced:
In Hodson's technique the electrical resis­
tance of the corneal endothelium R; is in
series with the corneal stroma ~tr and resis­
tance of the solutions ~I' but the electrical
current required to short the endothelium is
given by SCC = VJ R; where V e is the
open-circuit potential difference. But in each
cornea, R; is only known at the end of the
experiment, so it must be estimated at the
start; this is done by assuming that ~tr +
R so 1 is constant across all corneas so R; = R to t
- (R str + R s o1) = Rt':lt - constant, where R to t is
the total resistance for a particular cornea.
Unfortunately, stromal resistance Rs tr can
vary by up to 12% (Maurice, 1961; Green,
1967), and in the absence of solution stirring,
the unstirred fluid layer resistance R s o1 could
also vary. If high stromal resistance corneas
had high Ve values and hence high SCC
levels, then if stromal resistance is assumed
constant, the SCC estimated from VJ(R to t ­
constant) will be lower than the actual value
Ve/(~ot - R s tr - Rso1) ' Thus, in the studies of
Hodson and Wigham (1985) and Wigham
and Hodson (1985), if high resistance corneas
had high resistance stromas, it is possible
that the SCC in these specimens was under­
estimated. Overall, this would bring the SCC
and net bicarbonate fluxes closer to agree­
ment.
The endothelial 'pump-leak' controversy
The 'pump-leak' concept has been disputed
lately. Doughty and Maurice (1988) claimed
that the fluid pump in the rabbit corneal
endothelium was not bicarbonate depen­
dent; Doughty (1989) alleged that in rabbit
corneas previously stored at 4 0 C some were
able to deturgesce in the absence of measur­
able endothelial pump activity. The criticism
of both studies is the possibility of con­
founding artefacts in the apparatus used to
quantify endothelial pump rate. In that sys­
tem, corneal endothelial pump rate is
inferred from the movement of a meniscus of
a liquid column continuous with the aque­
ous side of the perfused cornea (see Fig. 1 of
Baum et al., 1984). The problem is that the
epithelium is typically removed, so the
corneal stroma will swell; the stroma is con­
strained anteriorly, so will expand post­
eriorly registering as a 'pump rate'.
Continuous stromal swelling would explain
why Doughty and Maurice (1988) measured a
gradually increasing 'pump rate' at zero
bicarbonate levels. Another possible prob­
lem with the technique is the uncertainty of
the stromal fluid compartment adjacent to
Descernet's membrane, which must be at
steady state to justify the analysis adopted.
The corneal stroma has a gradient of hydra­
tion across its thickness (Castoro et al., 1988)
and does not swell uniformly throughout its
thickness, but shows a spatial variation
(Kikkawa & Hirayama, 1970; Wilso!1 et al.,
1984). It is assumed in the Doughty and
Maurice (1988) system that stromal gradients
in hydration and swelling are dissipated,
and stromal fluid flow stabilized. However,
the absence of an epithelium would affect the
biomechanics of the corneal tissue (Kwok,
1991a) and influence stromal fluid flows. The
methods and conclusions of Doughty (1989)

can be similarly criticized. The corneal
endothelial pump rate was recently reported
to be abolished when carbonic anhydrase
inhibitors (which decrease bicarbonate lev­
els) were applied to rabbit corneas (Kuang et
al., 1990). This finding confirms the
bicarbonate-dependency of the corneal
endothelial pump, and casts a further doubt
on the contrary Doughty and Maurice (1988)
claim. To date, the 'pump-leak' hypothesis
remains the best framework for exploring
details of stromal deturgescence by the cor­
neal endothelium.
Endothelial hypoxia
The corneal endothelium runs the risk of a
dysfunctional fluid pump under hypoxic
conditions (Mishima & Kudo, 1967; Dikstein
& Maurice, 1972) associated with contact lens
wear. Contact lenses applied to rabbit cor­
neas caused a 30% drop in endothelial oxy­
gen partial pressure (Stefansson et al., 1987),
and the corneal endothelium could theoreti­
cally approach anoxia during anterior
hypoxia (Kwok, 1985a). Under normoxic con­
ditions the corneal endothelial production of
lactate is depressed (the Pasteur effect), but
the generation of cellular energy in the form
of ATP through the tricarboxylic acid cycle
(TCA) is so efficient (36-38 mole of ATP per
mole of glucose) that oxidative phosphoryla­
tion usually accounts for less than half of
corneal glucose breakdown (Riley, 1982). The
diminution of corneal respiration will favour
greater anaerobic glycolysis and more lactate
production, but generation of ATP is less
efficient (two moles of ATP per mole of
glucose). If the corneal glucose supply can
accommodate an increased rate of consump­
tion to maintain ATP generation, the corneal
endothelial pump should be minimally
affected. The rabbit aqueous humour appar­
ently contains sufficient glucose to minimize
the effect of short-term hypoxia on corneal
ATP. The activity of rabbit corneal endothe­
lial Na+K+-ATPase as reflected in the intrac-
Corneal ion transport 255
ellular potential was apparently little affected
by the absence of external oxygen for an hour
(Bowman et al., 1984). When corneal respira­
tion was suspended by a 1 h exposure to
sodium cyanide, the ratio of ATP/ADP only
fell by 20% (Masters et al., 1989). (Cyanide
disrupts the electron transport chain impair­
ing ATP generation via oxidative phospho­
rylation; see Stryer, 1988.) Similarly, the
de-epithelialized isolated rabbit cornea
shows minimal swelling when exposed to
potassium cyanide (Riley & Winkler, 1990).
However, in that study the aqueous-side
perfusate was continuously replaced at a rate
equivalent to five times normal anterior
chamber volume per hOUT, essentially ensur­
ing a constant glucose supply. Taking the
result of Masters et al. (1989), a fall in
corneal ATP of around 20% in the living
eye during contact lens wear may impede
endothelial pumping and affect corneal
hydration. Green et al. (1990) halved the
level of oxygen exposed to the rabbit cor­
nea and found that the net bicarbonate
efflux increased by about two-thirds, while
net sodium efflux fell by about one-fifth. It
is uncertain what effects were involved,
but an increased bicarbonate flux would be
expected to create an efflux of fluid from
the corneal stroma. For obscure reasons,
the human corneal endothelial fluid pump­
ing shows long-term endurance in anterior
hypoxia with contact lens wear while
endothelial morphology shows a consider­
able transient sensitivity (Zantos &
Holden, 1977) and then an acquired poly­
megethism (Schoessler & Woloschak, 1981).
The diabetic corneal endothelium shows
long-term changes in morphology in the
human (Schultz et al., 1984), and has a
subnormal deswelling capacity in the dia­
betic rabbit (Herse, 1990). This was attrib­
uted to altered Na+K+ -dependent ATPase.
However, endothelial pumping could also
be impaired by lowered cellular ATP due to
diminished glucose uptake by the diabetic
corneal endothelium.
256 Corneal physiology and biophysics
Corneal endothelium in disease
Ouabain binds to membrane Na+K+­
ATPase and is capable of inhibiting activity,
but has little effect on the permeability of the
corneal endothelium (Mishima & Trenberth,
1968). The radio-active marker 3H-ouabain
has been used to label the location and
spread of Na+K+-ATPase. The areal density
of labelling is referred to as the 'pump site
density', a potentially confusing term since
the endothelial bicarbonate transport is the
endothelial pump directly associated with
water movement. The endothelial pump site
density was claimed to be subnormal in
dysfunctional corneas such as those with
Fuch's endothelial dystrophy, and bullous
keratopathy (McCartney et al., 1987) where
corneal detumescence is demonstrably sub­
normal (Mandell et al., 1989; PoIse et al.,
198Q). Reduced pump site density is also
reported in the corneal endothelium in
intraocular inflammation in the rabbit eye
(Macdonald et al., 1987). However, these
analyses assume that ouabain binds 1:1 to
Na +K+-ATPase, but there could be a fraction
of endothelial Na+K+ -dependent ATPase
that is ouabain insensitive. Even if the 1:1
binding assumption is correct, there is no
guarantee that the same ratio applies in the
diseased cornea. Exogenous factors such as
diet can affect corneal endothelial cell density
(Nadakavukaren et al., 1987). The Na+K+­
dependent ATPas€ activity in the cornea of
diabetic rabbits is less than normal (Herse,
1990). This was claimed to explain the
increased stromal hydration, but increased
endothelial permeability can not be ruled out
since the diabetic corneal endothelium has
increased polymegethism and pleomor­
phism (Meyer et al., 1988). The rate of the
HC03- transport, which determines transen­
dothelial fluid transport, was also not consid­
ered.
Chondroitin sulphate (CS), a GAG found
in the corneal stroma and sometimes
included into commercial corneal preserva­
tion media (Stein et al., 1988), was reported to
stimulate (in an unknown way) the rabbit
corneal endothelial potential (Koniarek et al.,
1988). If the oedematous cornea loses stromal
GAGs into the aqueous humour (Kangas et
al., 1990), an intriguing possibility arises. CS
leaching out of an oedematous corneal
stroma will encounter the corneal endothe­
lium and stimulate the endothelial potential
difference. This will increase endothelial
fluid pumping (which is correlated posi­
tively with potential difference; Barfort &
Maurice, 1974; Fischbarg & Lim, 1974) and
thus act to oppose further corneal swelling.
Could such a negative feedback loop exist in
the intact cornea as an emergency resort to
keep stromal swelling in check?
Muscarinic cholinergic receptors which
regulate intracellular cyclic GMP have been
reported in bovine corneal endothelium
(Walkenbach & Ye, 1991).
14.7.4 MACROSCOPIC ION TRANSPORT
Over 35 years ago, Kronfeld (1956) proposed
that corneal thickness be used as an indicator
of corneal physiology during contact lens
wear. The measurement of corneal thickness,
or pachometry, has become a useful research
and clinical tool. Unfortunately, pachometry­
based clinical assessment requires careful
interpretation to avoid false negatives. In
corneas with abnormally low endothelial
permeability (measured with fluorescein)
following penetrating keratoplasty, no corre­
lation is found with corneal thickness
(Bourne & Brubaker, 1983). In patients aged 5
to 79 years, the corneal endothelial perme­
ability to fluorescein was found to increase
by 23% with age, but corneal thickness was
not found to change with age (Carlson et al.,
1988b). Thus, a cornea may have an appar­
ently normal thickness when the endothelial
permeability is abnormal.
The older human cornea is reported to
show a slower recovery from induced
oedema than the deswelling rate found in

younger corneas (Polse et al., 1989). Detu­
mescence of swelled corneas with Fuch's
dystrophy is also subnormal (Mandell et al.,
1989).
It is unclear whether hyperosmotic drops
applied to the corneal surface can sustain a
useful change in corneal thickness. Chan and
Mandell (1975) instilled a total of 1000 drops
of a 2% solution of saline onto the corneas of
three subjects over 20 mins, and induced a
1-3% change in corneal thickness. However,
after an hour the thickness reversed to base­
line levels. In rabbit eyes cryo-injured to
induce corneal swelling, Insler et al. (1987)
suggested that 2% and 5% saline drops were
equally effective in deswelling oedematous
corneas. Unfortunately, they were unable to
statistically substantiate an improvement
over balanced saline control drops due to the
variability of results.
14.8 CONCLUSIONS
14.8.1 CLOSING THE CREDIBILITY GAP: THE
FUTURE
The goal of laboratory-based corneal research
is to help further our understanding of ocular
biological processes. The hope is that this
will lead to effective clinical strategies in
treatment of corneal disease. Unfortunately,
the clinician is often alienated by the esoteric
nature of much of laboratory research in
corneal physiology and biophysics. The
uncertain extrapolation of necessary animal
studies to the clinical situation is exacerba­
tory but improved research techniques will
hopefully propitiate the clinician.
The reader will correctly gather from this
selective overview that present research
findings are far from definitive. Our current
understanding of corneal physiology and
biophysics and the effects of contact lenses
remains incomplete and answers demand
analyses at the molecular level. Fortunately,
we now have the research technology avail­
able.
References 257
ACKNOWLEDGMENTS
I thank various colleagues for kindly provid­
ing reprints and preprints of their research.
This work was supported in part by a Uni­
versity of Houston Research Initiation Grant,
and my thanks to Dr Stephen D. Klyce for
postdoctoral sponsorship at the LSU Eye
Center where experiments and ideas for this
work began with support in part from
USPHS grants EY03311 and EY02377 from the
National Eye Institute.
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