Ophthalmology Yanoff Corneal Anatomy, Phisiology and Wound Healing - C. 4.1

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INTRODUCTION
A healthy cornea, together with the overlying tear film, is necessary to
provide a proper anterior refractive surface and to protect the eye
against infection and structural damage to the deeper components of
the eye. In the adult, the average horizontal diameter is 11.5–12 mm1
and about 1.0 mm larger than the vertical diameter. The anterior
refractive power is 43–43.50 diopters. The shape of the cornea is
prolate, being flatter in the periphery and steeper centrally, which cre-
ates an aspheric optical system.
Embryology, Anatomy, and
Physiology of the Cornea
Epithelium
The corneal epithelium is derived from surface ectoderm at approxi-
mately 5–6 weeks of gestation. It is composed of nonkeratinized, non-
secretory, stratified squamous epithelium (Fig. 4-1-1), which is 4–6 cell
layers thick (40–50 μm). The epithelium is covered with a tear film of
7 μm thickness, which is optically important in smoothing out micro-
irregularities of the anterior epithelial surface. The tear–air interface,
together with the underlying cornea, provides roughly two-thirds of the
total refractive power of the eye. The mucinous portion of tears, which
forms the undercoat of the tear film and is produced by the conjunctival
goblet cells, interacts closely with the corneal epithelial cell glycocalyx
to allow hydrophilic spreading of the tear film with each eyelid blink.
The tear film also helps protect the corneal surface from microbial
invasion, as well as from chemical, toxic, or foreign body damage.
Thus, the ocular surface tear film and the corneal epithelium share an
intimate mutual relationship, both anatomically and physiologically.
Ayad A. Farjo, Matthew V. Brumm, H. Kaz Soong
4.1Corneal Anatomy, Physiology,
and Wound Healing
SECTION 1 Basic Principles
PART 4 CORNEA AND OCULAR SURFACE DISEASES
Definition: The cornea represents the transparent anterior wall of the
globe.
Key features
■ With the tear film, the cornea is the major refractive surface of
the eye.
■ It provides structural integrity for the anterior part of the eye.
■ It is a key barrier against infection.
Fig. 4-1-1 Cross-sectional view of the
corneal epithelial cell layer.
superficial
cells
apical microvilli
glycocalyx layer
tear film
wing cells
basal cells
basement
membrane hemidesmosomes tight junctions
CROSS-SECTIONAL VIEW OF THE CORNEAL EPITHELIAL CELL LAYER

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CORNEAANDOCULARSURFACEDISEASES
the corneal thickness. The stroma differs from other collagenous struc-
tures in its transparency and biomechanical properties. These func-
tional properties result from the precise organization of stromal fibers
and extracellular matrix.7–9
The fibers are aligned in a parallel fashion
within each lamella, and arranged at angles relative to fibers in adjacent
lamellae.10,11
This network reduces forward light scatter and contrib-
utes to the mechanical strength of the cornea. The peripheral stroma is
thicker than the central stroma and the collagen fibrils may change
direction to run circumferentially as they approach the limbus.11,12
Bowman’s membrane is the acellular condensate of the most anterior
portion of the stroma.
The stromal collagen fibrils, which provide the major tensile
strength to the cornea, are composed mostly of type I collagen, but
require a heterodimeric complex with type V collagen to obtain their
unique and narrow diameter.13–15
They are surrounded by specialized
proteoglycans, consisting of keratan sulfate or chondroitin sulfate/
dermatan sulfate side chains, which help regulate hydration and struc-
tural properties. Keratocytes are the major cell type of the stroma and
are involved in maintaining the extracellular matrix environment.16
More keratocytes are situated in the anterior stroma than in the poste-
rior stroma. Morphological differences between the anterior and poste-
rior stromal keratocytes, such as fenestrations, have been identified.17
Corneal “crystallins,” representing 25–30% of soluble protein in kera-
tocytes, appear to be responsible for reducing backscatter of light from
the keratocytes and maintaining corneal transparency.18
Corneal shape and curvature are governed by the intrinsic biome-
chanical structure and extrinsic environment (Fig. 4-1-3). Anterior
corneal stromal rigidity in particular appears to be important in main-
taining the corneal curvature.19
Organizational differences in the col-
lagen bundles of the anterior stroma may contribute to a tighter
cohesive strength in this area and may explain why the anterior curva-
ture resists change to stromal hydration much more than the posterior
stroma, which tends to more easily develop folds. Corneal nerves and
sensation are derived from the nasociliary branch of the first (ophthal-
mic) division of the trigeminal nerve. In the superficial cornea, the
nerves enter the stroma radially in thick trunks forming plexiform
arrangements, which eventually perforate Bowman’s membrane to
provide a rich plexus beneath the basal epithelial layer.20
The fibers
appear to directly communicate with keratocytes and epithelial cells21
and may play an important role in corneal homeostasis.
Endothelium
In early embryogenesis, the posterior cornea is lined with a neural
crest-derived22
monolayer of orderly arranged cuboidal cells.23
By the
78 mm stage, the cells become flattened and tightly abut one another.
At this stage, immediately anterior to the flattened layer is a discon-
tinuous, homogeneous acellular layer, which in time becomes
Descemet’s membrane.24
By the 120 mm and 165 mm stages of devel-
opment, the endothelial monolayer is uniform in thickness, spans the
entire posterior corneal surface, and fuses with the cells of the trabecu-
lar meshwork.24
Similarly, Descemet’s membrane becomes continuous
and uniform, fusing peripherally with the trabecular beams.24
The
Fig. 4-1-2 Whorl-like deposition keratopathy in corneal epithelium seen in Fabry’s
disease.
Corneal epithelial cells undergo orderly involution, apoptosis, and
desquamation. Complete turnover of corneal epithelial cells occurs in
about 7–10 days,2
with the deeper cells eventually replacing the de-
squamating superficial cells in an apically directed fashion. The most
superficial cells of the corneal epithelium form an average of two to
three layers of flat, polygonal cells. Extensive apical microvilli and
microplicae characterize the cell membranes of the superficial cells,
which in turn are covered by a fine, closely apposed, charged glycocal-
yceal layer. The apical membrane projections increase the surface area
of contact and adherence between the tear film’s mucinous undercoat
and the cell membrane. Laterally, adjacent superficial cells are joined by
barrier tight-junctional complexes, which restrict entry of tears into the
intercellular spaces. Thus, a healthy epithelial surface repels dyes such
as fluorescein and rose bengal.
Beneath the superficial cell layer are the suprabasal or wing cells, so
named for their cross-sectional alar shapes. This layer is about 2–3 cells
deep and consists of cells that are less flat than the overlying superficial
cells, but possess similar tight, lateral, intercellular junctions. Beneath
the wing cells are the basal cells, which comprise the deepest cellular
layer of the corneal epithelium. The basal cell layer is composed of a
single-cell layer of columnar epithelium approximately 20 μm tall.
Besides the stem cells and transient amplifying-cells, basal cells are the
only corneal epithelial cells capable of mitosis.3,4
They are the source of
both wing and superficial cells, and possess lateral intercellular junc-
tions characterized by gap junctions and zonulae adherens. The basal
cells are attached to the underlying basement membrane by an exten-
sive basal hemidesmosomal system. This attachment is of pivotal
importance in preventing the detachment of the multilayer epithelial
sheet from the cornea. Abnormalities in this bonding system may
result clinically in either recurrent corneal erosion syndromes or in
persistent, nonhealing epithelial defects.
The basement membrane is composed of an extracellular matrix
material secreted by the basal cells. Following destruction of the base-
ment membrane, about 6 weeks are required for it to reconstitute and
heal. The epithelial bond to the underlying, newly laid basement mem-
brane tends to be unstable and weak during this period. The epithelium
also adheres relatively poorly to bare stroma or Bowman’s layer. Under
ordinary conditions, type IV collagen and laminin are the major com-
ponents of the basement membrane; however, fibronectin production
increases to high levels during acute epithelial injury. The basement
membrane, approximately 0.05 μm in thickness, adheres to the under-
lying Bowman’s membrane through a poorly understood mechanism
that involves the anchoring fibrils and plaques.5
Epithelial stem cells – undifferentiated pluripotent cells that serve as
an important source of new corneal epithelium – have been localized to
the limbal basal epithelium. As the cells migrate to the central cornea,
they differentiate into transient amplifying cells (cells capable of multi-
ple, but limited cellular division) and basal cells. The corneal epithelial
cell layer mass appears to be the complex resultant of three phenomena.
According to the ‘X, Y, Z hypothesis,” X is the proliferation of basal
epithelial cells, Y is the centripetal mass movement of peripheral epithe-
lial cells, and Z is the cell loss resulting from death and desquamation.6
These three phenomena probably are not totally independent of each
other, but rather are controlled by a complex interactive feedback mecha-
nism that maintains the status quo, vis-à-vis cell density, cell distribu-
tion and polarity, and cell layer thickness. These cytodynamics are likely
to be responsible for the striking verticillate (vortex or whorl-like) bio-
chemical deposition patterns seen in Fabry’s disease (Fig. 4-1-2) and
drug deposition keratopathies (e.g., from chloroquine and amiodarone).
Langerhans cells, immunologically active dendritic macrophages derived
from bone marrow and capable of antigen processing, are present in the
peripheral corneal epithelium near the limbus. Under certain conditions
(e.g. corneal graft rejection or injury), these cells are found among the
central corneal epithelial cells. Human lymphocyte antigens are
expressed by these corneal Langerhans cells. Langerhans cells have also
been detected in the epithelial basal cell layer and in Bowman’s mem-
brane in pathological inflammatory conditions such as Thygeson’s
superficial punctate keratitis. After treatment with topical steroids,
these cells are no longer detectable by laser confocal microscopy.7
Stroma
In the seventh week of gestation, after the establishment of the primi-
tive endothelium, a second wave of neural crest cells form the early
corneal stroma. Akin to the dermis of the skin, the corneal stroma
provides important structural integrity and comprises roughly 85% of

3. CornealAnatomy,Physiology,andWoundHealing
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165
adhesion to Descemet’s membrane. Endothelial cells contain numer-
ous mitochondria and a prominent Golgi apparatus, and continuously
secrete Descemet’s membrane throughout life, beginning in utero at
the 8-week stage. The anterior portion of Descemet’s membrane
formed in utero has a distinctive banded appearance when viewed by
electron microscopy, but Descemet’s membrane produced after birth is
unbanded and has an amorphous ultrastructural texture. This mem-
brane is approximately 3 μm thick at birth, but thickens to 10 μm with
age. Endothelial cell density and topography also continue to change
throughout life. From the second to eighth decades of life, the cell
density declines from approximately 3000–4000 cells/mm2
to around
2600 cells/mm2
, and the percentage of hexagonal cells declines from
about 75% to around 60%.28
The central endothelial cell density
decreases at an average rate of 0.3% per year in normal corneas.29
As a result of endothelial activity, the stroma is maintained in a rela-
tively deturgesced state (78% water content).30
One hypothesis is that
this endothelial activity is mediated by a pump–leak process; net fluid
egress from the corneal stroma follows movement down an osmotic
gradient from a relatively hypo-osmotic stroma toward a relatively
hypertonic aqueous humor. This passive bulk fluid movement requires
no energy. The energy-requiring processes are the intracellular and
membrane-bound ion transport systems, which generate the osmotic
gradient. The two most important ion transport systems are the
membrane-bound Na+
,K+
-ATPase sites and the intracellular carbonic
anhydrase pathway.31
Activity in both these pathways produces a net
flux from stroma to aqueous humor. The barrier portion of the
endothelium is unique, in that it is permeable to some degree, permit-
ting the ion flux necessary to establish the osmotic gradient.24,26
Little, if any, in vivo mitotic potential exists within the endothelium.
Although the exact minimum number of cells/mm2
required to main-
tain corneal deturgescence is not known, corneas with cell counts
below 500 cells/mm2
may be at risk for development of corneal edema.
Endothelial cell morphology (size and shape) appears to also correlate
with pump function. An increase in cell size (polymegathism) and an
increase in variation of cell shape (pleomorphism) correlate to reduced
ability of the endothelial cells to deturgesce the cornea.32,33
In vivo assessment of endothelial function relies on measurements
of corneal thickness or on clinical morphological studies of the
endothelial monolayer with specular microscopy. Measurement of the
corneal thickness (pachymetry) indirectly reflects endothelial function.
The average central corneal thickness is around 0.5 mm, which gradu-
ally increases toward the periphery to around 0.7 mm. Normally, as a
diurnal variation, corneas tend to be slightly thicker just after a person
awakes in the morning. This increase in thickness is the consequence
of diminished evaporation of water from underneath the closed eyelids,
and the result of reduced nocturnal metabolic activity of the endothe-
lium. Such overnight corneal swelling is more exaggerated in persons
with unhealthy endothelium, causing blurred vision in the morning
that gradually resolves later during the day.
Endothelial Responses to Stress
Mild endothelial stress may result in cell size and shape changes, while
greater stress may result in cell loss as well as irreversible alterations in
the endothelial cytoskeleton.34
Sources of stress may be metabolic
(from hypoxia or hyperglycemia), toxic (from drugs or their preserva-
tives), injury (from trauma or surgery), or alterations in pH or osmolar-
ity. For example, contact lenses cause a hypoxic stress of varying degree
to the endothelium.35
Over time, this may result in alteration of the
morphology, microanatomy, and possibly the function of the endothe-
lium.36,37
Hyperglycemia is another common metabolic stress that may
produce changes in the endothelium. When compared with age-
matched controls, the corneal endothelium in patients with type 1 and
type 2 diabetes has a lower mean cell density and greater pleomorphism
and polymegathism.38,39
Tissue manipulation, fluid flow in the anterior chamber, and intrac-
ameral pharmacological agents introduced during anterior segment
surgery may cause damage to the endothelium.40,41
Ophthalmic visco-
elastic materials (composed of hydroxypropyl methylcellulose, chon-
droitin sulfate, or sodium hyaluronate) provide significant protection
against intraoperative trauma to the endothelium.42,43
Glaucoma has been associated with endothelial cell loss. Compared
with age-matched controls, significantly lower endothelial cell counts
were noted in patients with glaucoma and ocular hypertension in one
study.44
Cell counts were inversely proportional to the mean intraocular
fusion site, known as Schwalbe’s line, is a gonioscopic landmark that
defines the end of Descemet’s membrane and the start of the trabecular
meshwork. At birth, the endothelium is approximately 10 μm thick.25
The intact human endothelium is a monolayer, which appears as a
honeycomb-like mosaic when viewed from the posterior side (Fig. 4-1-
4). The individual cells continue to flatten over time and stabilize at
about 4 μm in thickness in adulthood (Fig. 4-1-5).26
The posterior sur-
face of the endothelium is devoid of villi, except in certain pathological
conditions, in which it may develop epithelioid characteristics. Adja-
cent cells share extensive lateral interdigitations, and possess gap and
tight junctions along their lateral borders. The lateral membranes con-
tain a high density of Na+
,K+
-ATPase pump sites.27
The basal surface
of the endothelium contains numerous hemidesmosomes that promote
Fig. 4-1-3 Major corneal loading forces in the steady state. (Illustration courtesy ofWilliam
J. Dupps, MD, PhD.)
MAJOR CORNEAL LOADING FORCES IN THE STEADY-STATE
intraocular
pressure
swelling
pressure
epithelial
barrier
intralamellar
cohesive
forces
lamellar
tension
endothelial
pump
endothelial
barrier
Fig. 4-1-4 Specular photomicrograph of normal endothelium. Note the dark,
well-defined cell borders, the regular hexagonal array, and the uniform cell size.
(Bar = 50 μm.).
Fig. 4-1-5 Light micrograph of normal endothelium (×100). Note the single-cell
endothelial layer with a Descemet’s membrane of uniform thickness (epithelial
surface at top of figure). (Courtesy of Dr David Barsky.)
endothelium
Descement's
membrane
Bowman's
membrane
epithelium

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CORNEAANDOCULARSURFACEDISEASES
and/or dystrophies), ocular surface inflammation or atopic disease,
medicamentosa, dry eyes, neurotrophic and exposure keratopathies,
conjunctival disease (e.g., pemphigoid, radiation keratoconjunctivitis,
and Stevens–Johnson syndrome), extensive damage to the limbal stem
cells, and eyelid abnormalities.
The epithelial healing problems of postinfectious (metaherpetic)
ulceration, seen after acute herpetic keratitis, are believed to be caused
by damage to the basement membrane from antiviral drug toxicity or
from overzealous iatrogenic scraping of the corneal surface using either
mechanical or chemical means.49
In neurotrophic corneas it is possible
that interruption of corneal innervation results in depletion of sub-
stance P, a neurogenic chemical known to regulate corneal physiological
functions. Diabetic corneas may manifest abnormally thickened and
easily delaminated basement membranes (Fig. 4-1-7), perhaps akin to
basement membrane abnormalities elsewhere, as in the renal glomer-
uli.45
A combination of pharmacological interruption of corneal nerve
function and damage to the epithelial cells and substrate may cause
persistent epithelial defects associated with topical anesthetic abuse.50,51
Limbal stem cell deficiency is an increasingly recognized cause of
nonhealing epithelial defects. Evidence also implicates matrix
metalloprotease-9 (gelatinase B) as a factor that may impact epithelial
healing and/or desquamation.52,53
Stromal Injury
Similar to skin, stromal wound healing consists of repair, regeneration,
and remodeling,13
involving a complex interplay of cytokines, growth
factors, and chemokines.54
Importantly, stromal repair differs from
dermatological healing in that it occurs avascularly and ideally main-
tains corneal clarity. The reparative cascade begins with activation of
stromal keratocytes (Fig. 4-1-8), which enlarge in size within the first 6
hours after injury and migrate into the injured area within 24 hours,
becoming more fibroblast-like in appearance and behavior.55
Activation
of the keratocytes may be triggered by epithelial factors.55
The repara-
tive cascade that follows typically results in corneal opacity in the
affected area. The keratocytes within the area of injury undergo apop-
tosis, peaking 4 hours after the initial insult.56
Apoptosis appears to
modulate the wound healing response by influencing the activation of
adjacent keratocytes.
Within 1–2 weeks, myofibroblasts with contractile properties enter
the area under the epithelium and become involved in the remodeling
of the stroma, with increased expression of matrix metalloproteas-
es.13,57
These cells may be responsible for the “haze” seen after corneal
injury or surgery. The remodeling process can sometimes continue over
a prolonged period of several years, and may eventually restore corneal
clarity in the affected area. Certain corneal surgeries, such as laser in
situ keratomileusis, cause a progressive loss of keratocytes over time
through an as yet unclear mechanism.58
pressure in the glaucoma and ocular hypertension groups. Mechanisms
of cell loss may include direct damage from intraocular pressure, con-
genital alterations of endothelium in glaucoma, and drug toxicity.45
CORNEAL WOUND HEALING
Epithelial Injury
Within minutes after a small corneal epithelial wound, cells at the edge
of the abrasion begin to cover the defect as rapidly as possible by a
combination of cell migration and cell spreading. A longer delay of up
to 4–5 hours is seen in larger defects. This lag phase is necessary for
the preparatory cellular changes of an anatomical, physiological, and
biochemical nature to occur before rapid cell movement. Various cell
membrane extensions, such as lamellipodia, filopodia, and ruffles,
develop at the leading edge of the wound. Anchoring hemidesmosomes
disappear from the basal cells. This early nonmitotic wound coverage
phase is remarkable for its speed; the cells have been measured to
migrate at a rate of 60–80 μm/h (Fig. 4-1-6).46
The migrating sheet of
epithelial cells is attached most firmly to the underlying substrate at
the leading margin. The relatively firmer adhesion at the leading mar-
gin suggests that the epithelial sheet movement may have “front-wheel
drive,” with the less well-anchored cells behind the leading margin
being pulled forward, possibly by intracellular contractile mechanisms
that involve actin.47
Fibronectin, a ubiquitous extracellular matrix pro-
tein present in plasma and in fresh wounds, is thought to be one of the
key elements in the mediation of cell-to-substrate adhesion and cell
migration. Present on the extracellular side of adhesion plaques, it is
thought to mediate the linkage between the vinculin–talin–integrin
complex and the substrate during epithelial migration after a wound
has occurred. Laminin, a less ubiquitous extracellular matrix protein,
is thought to serve a similar function.
At 24–30 hours after medium-sized epithelial injuries, mitosis or
cell proliferation begins and restores the rarefied epithelial cell popula-
tion. After large epithelial injuries, significant increases in cellular divi-
sion occur as late as 96 hours.48
Only the basal cells, transient
amplifying cells, and the limbal stem cells partake in this reconstitutive
mitosis.3,4
In laboratory and clinical trials, agents known to influence
epithelial migration, mitosis, apoptosis, adhesion, and differentiation
have been studied as possible therapeutic agents to enhance corneal
epithelial healing, including growth factors, fibronectin, and retinoids.
Primarily mitogenic agents and growth factors also stimulate produc-
tion of extracellular matrix components to enhance cell-to-substrate
adhesion.
Various pathological conditions may delay or prevent the normal
corneal epithelial healing process. These include the following: damage
to the cellular substrate (caused by herpetic or other infectious disease,
diabetes mellitus, chemical burns, or basement membrane injuries
Fig. 4-1-6 Light micrograph that shows the leading edge of migrating rat corneal
epithelium as it tapers to a layer of one-cell thickness. As the epithelial defect is
rapidly covered by migrating cells, it is initially coated with a thin, rarefied cell
population prior to onset of mitotic activity (hematoxylin & eosin).
Fig. 4-1-7 Recurrent erosion in a diabetic cornea. Note the abnormally thick
basement membrane (asterisk) and the intralamellar split within (arrow)
(hematoxylin & eosin).
*

5. CornealAnatomy,Physiology,andWoundHealing
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167
enlargement of the surrounding cells and their centripetal migration
into the injured region. Clinically, by slit-lamp biomicroscopy, these
endothelial lesions disappear 1–3 days after injury. With more severe
trauma, the underlying Descemet’s membrane may even be torn or
ruptured, as in forceps delivery injuries and in corneal hydrops in kera-
toconus. When injured, Descemet’s membrane curls in toward the
stroma and surrounding endothelial cells slide in to cover the defect
and produce new Descemet’s membrane. Although normal endotheli-
um does not appear to replicate in vivo, recent evidence suggests that
endothelial cells retain a degree of latent proliferative potential even
into adulthood.59
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Endothelial Injury
Nonperforating puncture injuries of the anterior cornea that do not
directly involve the endothelium (e.g., those that occur when the cor-
nea is struck by small, high-velocity particles) may cause concentric
lesions of the endothelium arising from rapid focal distortion of the cell
layer. Buckling of the endothelial layer can also result from excessive
corneal bending in large-incision surgeries and/or from lens fragments
striking the endothelium during cataract surgery, and may occasionally
produce snail-track lesions, clinically seen as serpentine, grayish lines
on the endothelium. The damaged cells are rapidly replaced by
Fig. 4-1-8 Transmission electron micrograph of the corneal stroma. Activated
keratocytes with dilated endoplasmic reticulum (inset: black arrow) and prominent
nucleoli (white arrow) 1 week after keratoplasty. Bowman’s layer is at the upper
border of the micrograph. A normal keratocyte (asterisk), noticeably smaller than the
activated cells, is visible above the scale bar. (Bar = 5.0 μm.) (Reproduced with permission
from Ohno K, Mitooka K, Nelson LR, et al. Keratocyte activation and apoptosis in transplanted human corneas in a
xenograft model. Invest OphthalmolVis Sci 2002;43:1025–31.)
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