Chapter 8

Cornea

MICHAEL K. SMOLEK and STEPHEN D. KLYCE

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SURFACE ANATOMY

TEAR FILM

EPITHELIUM

ANCHORING STRUCTURES

BOWMAN'S LAYER

STROMA

DESCEMET'S MEMBRANE

ENDOTHELIUM

CONCLUSION

ACKNOWLEDGMENT

REFERENCES

As a portion of the ocular tunic, the cornea

protects the delicate intraocular contents with

its tough, yet pliable, collagen structure. It is

remarkable that a tissue with this ability to

resist injury can provide the essential optics

and transparency to focus an image on the retina.

In this chapter the gross, microscopic, and

ultramicroscopic anatomic structure of the cornea

is described. Corneal physiology is not

discussed, except when structural features can be

more clearly defined by a description of their

physiologic significance. An effort has been made

to cite references primarily for normal adult

human corneas; references to other species are

made when human information is lacking or

ambiguous. Citations to embryonic development are

included when they provide better insight into

the adult anatomy of the cornea.

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SURFACE ANATOMY

The anterior and posterior surfaces of the human

cornea are often approximated in schematic eye

calculations by radii of curvature of 7.8 and 6.5

mm, respectively, compared to the external

surface of the scleral globe, which has a radius

of approximately 11.5 mm.1 The anterior corneal

surface becomes slightly flatter in the

periphery, giving the overall cornea a naturally

prolate shape. If the cornea were perfectly

spherical, it would suffer from considerable

spherical aberration in which the rays passing

through the peripheral parts of an optical system

do not come to the same focal point as the rays

near the central axis of the system. Peripheral

corneal flattening reduces, but does not entirely

eliminate, the corneal contribution of spherical

aberration to the optical system of the eye. The

crystalline lens provides additional correction

of the residual spherical aberration of the

cornea, depending on the accommodative power of

the lens. The radius of curvature of the anterior

surface translates into a vergence power of

approximately 48.8 diopters (D), which accounts

for roughly three quarters of the total

refractive power of the eye's optical system. The

posterior corneal surface adds negative power so

that the total power of the typical, normal

cornea is approximately 43 D.

The difference in curvature between the

relatively flatter anterior surface and the

relatively steeper posterior surface is

associated with the central cornea being thinner

than the periphery. Maurice reported a central

thickness value of 0.52 mm and a peripheral value

of approximately 0.65 mm in adult humans when

measured with an optical pachometer.2 Abnormal

tissue thinning may be indicative of a corneal

dystrophy, such as keratoconus, Terrien's

marginal degeneration, or pellucid marginal

degeneration.

The cornea protrudes slightly beyond the limits

of the scleral globe because of the difference in

curvature between the relatively steep cornea and

the relatively flat globe. A shallow sulcus is

formed at the intersection of the corneal and

scleral surfaces, which roughly demarcates a

region called the limbus. The limbus is typically

defined in one of two ways. The histologic limbus is

the full-thickness annular interface that

separates the optically transparent corneal

stroma from the opaque sclera. The surgical limbus

is the annular region bound by a line from the

anterior surface termination of Bowman's layer to

the posterior surface termination of Descemet's

membrane and by a line oriented perpendicular to

the external scleral surface that intersects

Schlemm's canal in the angle of the anterior

chamber.

Externally, the cornea appears elliptical with

its vertical chord shorter than its horizontal

chord (10.6 versus 11.7 mm for males and 9.6

versus 10.7 mm for females).2 This difference

arises from opaque scleral tissue extending over

the anterior corneal margin slightly more along

its superior and inferior aspects. When viewed

from within the dissected globe, the posterior

cornea appears circular with a diameter of 11.7

mm. The external corneal surface area is

approximately 120 mm2, or one-fourteenth the

total area of the ocular globe.3 Surprisingly,

the surface area is not significantly increased

by the localized distension found in keratoconus,

although keratoglobus conditions do appear to

have a greater surface area.

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TEAR FILM

The precorneal tear film is approximately 7 μm

thick with a volume of 6.2 ± 2 μL during normal

tear production.4 Tear fluid is typically

produced at a rate of 1.2 μL/minute, with a major

portion drained from the palpebral fissure

through the nasolacrimal duct and a smaller

volume lost through evaporation from the ocular

surface (approximately 3 μL/hour at 30% relative

humidity).5 Tear chemistry is complex;

ingredients include various electrolytes,

metabolites, proteins, enzymes, and lipids.6 The

functional significance of the tear film is

broad. It provides lubricating qualities and a

smooth optical interface with the air. It also

protects the epithelium from airborne

contaminants and provides natural immunity to

infectious agents through secretory

immunoglobulin molecules.7

The tear film is composed of the cellular

exudates of separate structures of the ocular

adnexa and has been difficult to measure

precisely because of its delicate fluid

nature.8 The anterior-most layer is the lipid or

oily layer derived from secretions of the

meibomian glands located in the eyelid and

caruncle and is between 0.1 and 0.5 μm thick. The

aqueous lacrimal tear layer is at least 5 μm

thick and often thicker, depending on tear

production by the lacrimal glands located in the

superiotemporal margin of the orbit. The

posterior mucous layer is approximately 1 μm

thick and is derived from secretions of

conjunctival goblet cells. The hydrophilic nature

of mucus substantially reduces surface tension

and provides a smooth, wettable surface for the

aqueous tear layer. The separation between the

middle lacrimal tear layer and the posterior

mucous layer is actually indistinct, making

definitive thickness measurements of the two

components difficult. It is highly likely that

these two components form a graded mixture, with

the posterior mucous component gradually blending

into the anterior aqueous lacrimal tear

component.

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EPITHELIUM

The corneal epithelium is the anterior-most cell

layer of the cornea (Fig. 1). It is typically

several cell layers thick, consisting of the

apical cell squamous layer, the multilayered,

polygonal-shaped wing cells beneath the apical

layer, and the posterior-most layer of basal

cells (Fig. 2). The wing cell layer is two or

three cells thick in the central cornea, but

tends to be four to five cells thick in the

periphery. In total, the epithelium is

approximately 50 μm thick in the central human

cornea.

Fig. 1. Full-thickness view of the normal

human cornea. From top: epithelium,

Bowman's layer (arrow), stroma,

Descemet's membrane (arrow), and endothelium

(hematoxylin-eosin stain, 80×). (Courtesy of Drs.

Rodrigues, Waring, Hackett, and Donohoo.)

Fig. 2. Full-thickness electron

micrograph of corneal epithelium. Note

the cell shape change with depth, the

variation of cell membrane

interdigitation, and the intracellular

differences between cell types. S, apical surface

cells; W, wing cells; and B, basal cells. Also

note the microvilli seen along the apical

membrane of the surface cells (3,620×). Inset:

Epithelium (E) overlies a thin, dense basement

membrane (arrow) with no discernible laminar

appearance (periodic acid-Schiff [PAS] stain,

330×). (Courtesy of Drs. Rodrigues, Waring,

Hackett, and Donohoo.)The epithelium is in a constant state of

turnover, with exfoliating apical cells being

replaced by underlying wing cells. During normal

exfoliation, desquamating cells are released only

after the replacement cell has established new

tight junctions with neighboring cells and the

new apical membrane is capable of maintaining

continuity of the tear film.9 Studies of induced

exfoliation of a monolayer of epithelial cells

with a biologic detergent indicate recovery of

the paracellular barriers and transepithelial

electric resistance in approximately 1 hour.10 The

epithelium completely turns over in approximately

7 days.11 Once injured, a high degree of motility

ensures coverage of a denuded area by adjacent

basal cells, followed by replacement of the

normal complement of cell layers. Basal cells are

the only epithelial cells capable of mitosis;

however, many epithelial cells originate as the

progeny of limbal stem cells and migrate

centripetally to supplement or replace cells lost

through normal desquamation or injury.12–14 Using

immunohistochemical staining for antibodies to

keratins, Wiley and associates found regional

heterogeneity indicating that the superior

corneal periphery and limbus have the greatest

numbers of stem cells producing replacement

epithelial cells.15 Limbal stem cell deficiency

may result in conjunctival epithelium invasion of

the cornea, leading to vascularization, the

appearance of goblet cells, and an irregular or

unstable epithelium that reduces visual acuity

and may produce pain or discomfort.16

The epithelium is known to chemically interact

with keratocyte cells of the stroma. These

interactions appear to be dominated by cytokines

such as interleukin-1 (IL-1) and soluble Fas

ligand that are released by injured epithelial

cells. It would appear that IL-1 is a master

regulator for corneal wound healing given its

effect on keratocyte apoptosis and the modulation

of matrix metalloproteinase and growth factors

such as keratinocyte growth factor (KGF) and

hepatocyte growth factor (HGF). The Fas ligand

system is known to influence the immune

privileged state of the cornea. In addition to

the epithelial-to-keratocyte communication,

keratocytes influence the state of the epithelium

via HGF and KGF, which affect cell turnover,

motility, and proliferation.17

APICAL CELLS

Apical surface cells appear broad and flattened:

4 to 5 μm thick and 40 to 50 μm in diameter.

Freshly emerged surface cells appear bright

during specular microscopy and have relatively

small numbers of microvilli covering their apical

membrane (Fig. 3).18

Fig. 3. Scanning electron micrograph

of corneal epithelium surface. Note

the numerous microvilli and the

cellular margins (12,900×).(Courtesy of Drs.

Rodrigues, Waring, Hackett, and Donohoo.)As the cell matures, its specular microscopic

appearance tends to darken (Fig. 4). This may be

due to changes in surface texture, because

microvilli densely cover the apical membrane at

this stage of development.4,19 Prior to cell

exfoliation, apical surface margins tend to

appear smooth, with microplicae clustered only

near the center. In its final stages the

biomicroscopic appearance of the cell surface

appears darker than in its earlier stages (Fig.

5).

Fig. 4. Confocal microscopic transverse

image of the human surface epithelium in

vivo. The squamous cell nuclei and some cell

margins are visible (500×). (Courtesy of Nidek

Technologies.)

Fig. 5. Scanning electron micrograph of

the anterior surface of the corneal

epithelium. Note that the darker cells

have fewer microvilli or microplicae near the

cell margins (1,500×). (Courtesy of Drs.

Rodrigues, Waring, Hackett, and Donohoo.)The 300-nm thick glycocalyx (buffy cell coat) of

the apical membrane can be preserved intact for

histologic evaluation.7,8,19 The glycocalyx is

composed of glycoprotein material, and numerous

separate fine filaments become visible on the

apical surface after tannic acid staining (Fig.

6). These filaments cover the tips and sides of

the microplicae and microvilli extensively,

inserting into the cell membrane. Angular bends

and filament branching are evident, as well as a

beaded substructure.19 The shortest filaments are

150 nm in length in the central cornea, while

filaments 300 nm long are found on the

conjunctival cell surfaces.19 The glycocalyx binds

loosely with the overlying mucous layer and

provides binding sites for immunoglobulin present

in the tears.7

Fig. 6. Transmission electron micrograph

showing the apical membrane of the

surface epithelium and intercellular

junctions along the lateral membranes.

Arrows indicate fine, branching filaments of the

glycocalyx emerging from the surface microvilli.

A tight junction (box) and densely staining

desmosomes are seen along the lateral membranes.

Glycogen is evident (circle), as well as numerous

vesicles (23,000×). Inset: Arrows point to

microvilli from two adjacent surface cells

(64,500×). (Courtesy of Drs. Rodrigues, Waring,

Hackett, and Donohoo.)The margins of the apical cell membrane possess

the important tight junctions surrounding the

cell circumference near the apical margin.20 This

junction complex is the correlate of the

paracellular pathway of high resistance to ion

flow. The lateral and basal membranes of the

apical cells have gap junctions, numerous

desmosomal junctions, and numerous membrane-bound

vesicles. The cytoplasm contains a flattened

nucleus (which probably disintegrates prior to

desquamation), few organelles, and a notable

increase in tonofilaments compared with the

underlying cells. There are some aggregates of

glycogen granules, small mitochondria, sparsely

distributed free ribosomes, and poorly developed

Golgi's complexes. Cytoplasmic vesicles are

fairly numerous.21

WING CELLS

Wing cells are distinguished by a variety of

polygonal shapes and by their large ovoid nuclei.

The cells are roughly 12 to 15 μm in diameter,

and their cytoplasm contains few rough

endoplasmic reticulum cisternae, mitochondria, or

Golgi's complexes. The large numbers of

cytoskeletal tonofilaments are approximately 8 nm

in length, and numerous interdigitations exist

along the cell membranes.21 Desmosomal and gap

junctions are seen between adjacent wing cells

and between basal and apical cells (Fig. 7).

Fig. 7. Transmission electron

micrograph of elaborate

interdigitations between epithelial

wing cell membranes. Desmosomes are

seen along the cell walls, as well as

intracellular tonofilaments (arrow).

Intracellular rough endoplasmic reticulum,

scattered mitochondria, and ribosomes are seen in

addition to the prominent nuclei (13,800×).

Inset: Desmosome junction (49,500×). (Courtesy of

Drs. Rodrigues, Waring, Hackett, and Donohoo.)BASAL CELLS

Basal cells appear as elongated polygonal cells

approximately 10 μm in width and 15 to 20 μm in

height, with prominent ovoid nuclei. The

polygonal nature can be readily discerned in

confocal microscopy images of the basal cell

layer (Fig. 8). Buschke and associates22 showed

that cell mitosis occurs in only 1 of 250 basal

cells of the rat epithelium. In their study,

mitosis occurred in irregular clumps of three to

six cells, and mitotic cells were much more

numerous in the periphery. The basal cell

cytoplasm appears similar to that of the wing

cells, as do the anterior and lateral cell

membranes with their complement of desmosomal

attachments. However, the basal membrane is

notable for the presence of hemidesmosomes, which

are discussed below.

Fig. 8. Confocal microscopic transverse

image of the human basal epithelium in

vivo. The cells are outlined by a fine polygonal

mesh which is the source of haloes around bright

lights at night (Sattler's veil) that occurs with

epithelial edema (500×). (Courtesy of Nidek

Technologies).

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ANCHORING STRUCTURES

HEMIDESMOSOMES

The epithelial basal cell hemidesmosome is a

multilayered junctional structure associated with

the basal membrane, with intracellular keratin

filaments (intermediate filaments) entering along

its cytoplasmic aspect. As basal epithelial cells

migrate in response to wound healing,

hemidesmosome junctions are observed to

disassemble and their integrin components

diffusely distribute across the basal cell

membrane.23 In vitamin A-deficient rat models, the

severity of epithelial sloughing was found to

correspond to the size and frequency of residual

hemidesmosomes.24

Kurpakus and colleagues25 found a monoclonal

antibody, mAb6A5, that was directed against a

200-kd polypeptide component on the cytoplasmic

exposure of the hemidesmosome. This component may

be associated with the electron-dense plate that

is seen crossing through the intermediate

filaments just proximal to the hemidesmosome

plaque.26 Short filaments pass from the dense

plate into the plaque. Polypeptides of 180 and

230 kd were isolated by Owaribe and co-

workers26 and localized to the hemidesmosome

plaque. The 230-kd component was found to be the

bullous pemphigoid antigen (BPA), while the 180-

kd component was heavily glycosylated, making it

a candidate for a transmembrane glycoprotein. In

addition, major polypeptides of 120, 200, and 480

kd were isolated from the region and may be

hemidesmosome structural components as well.26

Stepp and associates,27 using direct

immunofluorescence of frozen sections, found β1,

β4, and β5 integrin subunits and α3, α5, α6, and αv

subunits localized in the epithelium.

Specifically, α5, α6, and β4 subunits of integrin

heterodimers were found to be localized on the

basal membrane side of the hemidesmosome site,

while β1, β5, α2, α3, and αv were localized to

sites associated with cell-t-cell contact.27 A

125-kd polypeptide was isolated to the basal side

of the hemidesmosome by Kurpakus and co-

workers28 with the monoclonal antibody mAbHD. This

polypeptide may be a protein associated with the

anchoring filaments that span the lamina lucida

from the hemidesmosome plaque to the basal

lamina.

Fluorescence confocal imaging of the integrin

subunits α6 and β4 indicated that the α6 subunits

were localized to both the basal and lateral

surfaces of the basal epithelial cell, but the

β4 subunits were present only along the basal

floor of the cell. The number and distribution of

hemidesmosomes do not appear to vary with age;

however, discontinuous staining for the

α6 subunits in corneal epithelium of individuals

over the age of 70 has been noted.29

BASAL LAMINA

Basement membrane is secreted extracellularly by

the epithelial cells and forms one of several

structural components associated with cell

adhesion to Bowman's layer or stroma. With light

microscopy, a densely stained basement membrane

approximately 75 to 100 nm thick is

visible.21 Under the high magnifications of

electron microscopy, this layer appears

distinctly laminated. Anteriorly, the lamina

lucida appears as a clear, electron-lucent zone

approximately 23 nm thick, while the lamina densa

that lies apposed to Bowman's layer is an

electron-dense region approximately 48 nm

thick.21 It has not been established how basal

lamina adheres to Bowman's layer. A third

component of the basement membrane is the

reticular lamina, which lies distal to the lamina

densa and is within Bowman's layer. This region

includes the anchoring fibrils and plaques and

other electron dense materials associated with

the lamina densa (Fig. 9).

Fig. 9. Transmission electron

micrograph of epithelial basal

lamina in the human cornea,

Bowman's layer is seen below,

with basal epithelial cells

above. Note the desmosome junction seen centrally

between two basal cells (arrowhead), Magnified

view appears in upper right box. Multiple

hemidesmosomes are seen along the basal lamina

(curved arrows) with platelike features seen

within the lamina lucida. Magnified view of a

hemidesmosome is seen in the box, lower right,

Bar = 0.5 μm. (Courtesy of Roger Beuerman, Ph.D.,

New Orleans, Louisiana.)Numerous electron-dense, fine filaments are

observed clustered within the lamina lucida; they

course from the hemidesmosomes of the basal

epithelium and insert into neighboring regions on

the lamina densa. Within the lamina lucida, a

dense plate often can be seen interposed along

the filaments and lying parallel to the

hemidesmosome plaque. The precise composition of

these filaments and the dense plate is uncertain,

but the 125-kd polypeptide described previously

may be involved.28 In addition, the antihuman

monoclonal antibody, 19-DEJ-l, was found to be

reactive to the antigenic epitope of either the

anchoring filaments or the dense plate through

the filaments.30

ANCHORING FIBRILS AND PLAQUES

Within the anterior aspect of Bowman's layer,

fine filaments travel distally from the lamina

densa near the proximal insertion of the

anchoring filaments and immediately coalesce to

form striated anchoring fibrils approximately

0.15 μm in width (Fig. 10).31 These anchoring

fibrils then course posteriorly to an average

depth of 0.60 μm in the adult human and terminate

in electron-dense regions called anchoring

plaques.31,32 Maximum penetration depths of 2.05 μm

were reported for the anchoring fibrils in

humans. Some anchoring fibrils may terminate

among the type I collagen fibrils31 or return

proximally to reinsert into the lamina densa at a

neighboring site.32 There also appear to be

plaque-to-plaque anchoring fibrils. Anchoring

fibrils found in Bowman's layer appear similar to

those found in the stroma of the rabbit, a

species that lacks Bowman's layer.31

Fig. 10. Transmission electron

micrograph of the epithelial

basal lamina in rabbit cornea.

Large micrograph shows anchoring

filaments within the lamina lucida, oriented

between the hemidesmosomes of the basal

epithelial cells (HD) and the lamina densa.

Anchoring fibrils (AF) travel distally (large

arrows) from the lamina densa to insert into

electron-dense anchoring plaques (small arrows)

(73,900×). Inset right: Cross-banding on the

anchoring fibril. Inset left: An anchoring fibril

inserting into two adjacent lamina densa sites.

(From Gipson IK, Spurr-Michaud SJ, Tisdale AS:

Anchoring fibrils form a complex network in human

and rabbit cornea. Invest Ophthalmol Vis Sci

28:212, 1987.)Anchoring fibrils were found to be type VII

collagen filaments in bovine32 and human

corneas31 using immunogold labeling and

immunofluorescence staining techniques,

respectively. Type VII collagen is a dimer of

high molecular weight.33 Each monomer has a

helical domain and a globular, carboxyl domain.

The dimer is created by disulfide bonds linking

the tails of the monomers together, and striated

fibrils are formed by the nonstaggered

arrangement of the helical filaments. The

globular domains were found localized within the

anchoring plaques, which also label for type IV

collagen.30 Gipson and co-workers31 and Keene and

colleagues32 presented similar models for the

architecture of the anchoring fibril network and

indicated that the striated type I collagen

fibrils in Bowman's layer must be interwoven and

trapped by the anchoring fibril network (Fig.

11).

Fig. 11. Schematic representation of

the anchoring fibril network.

Separate collagen filaments emerge

from the lamina densa, coalesce into banded

anchoring fibrils (arrows), and travel distally

to insert into anchoring plaques (AP). Note the

anchoring filaments between the lamina densa and

the hemidesmosomes (HD) with a dense plate

feature lying across the filaments within the

lamina lucida. Large type I collagen fibrils are

interwoven with the anchoring fibril network.

(From Gipson IK, Spurr-Michaud SJ, Tisdale AS:

Anchoring fibrils form a complex network in human

and rabbit cornea. Invest Ophthalmol Vis Sci

28:212, 1987.)BASEMENT MEMBRANE CONTROVERSY

Basement membranes throughout the body are formed

primarily from type IV collagen, yet evidence for

type IV collagen within corneal lamina densa

remains sketchy and controversial. Marshall and

associates were unable to demonstrate the

presence of type IV collagen in the human basal

lamina using immunoelectronmicroscopy labeling

with colloidal gold.34 Indirect immunofluorescence

labeling for anti-type IV antigens was shown

within a subepithelial band by Konomi and

colleagues,35 but the layer did not appear

strongly labeled. Newsome and co-

workers36 indicated strong immunofluorescence

labeling, but the corneal location was not

specified. Nakayasu and associates37 showed strong

immunofluorescence labeling of a subepithelial

layer near the limbus but did not report type IV

labeling for the concomitant

immunoelectronmicroscopy portion of the study.

In a project designed to resolve this

controversy, Kolega and colleagues38 found that

basement membrane type IV collagen was

differentially stained according to location in

the adult human. There was no immunofluorescent

staining for type IV collagen in the central

cornea using three different monoclonal

antibodies, and only weak staining in the corneal

periphery. However, there was intense staining in

the conjunctiva and across the limbal region.

Cleutjens and colleagues39 and others40,41 also

found no type IV collagen in the central cornea

of the adult but did find it in the periphery.

Furthermore, type IV collagen was found to be

strongly labeled in the fetal central cornea.39

Linsenmayer and associates42 had previously

discussed findings of heterogeneous type IV

collagen distribution in a variety of basement

membranes throughout the body, including the

crystalline lens capsule.42 Thus, it would be

unusual, but not unexpected, for corneal

epithelial basement membrane to exhibit a

heterogeneous distribution. If type IV collagen

is missing, binding mechanisms in the lamina

densa would have to be reconsidered. This also

would have implications for how laminin, heparan

sulfate proteoglycan, and fibronectin binding

operate in the absence of type IV collagen.

It is possible that the type IV collagen present

in the human cornea does not react to any anti-

type IV antibodies known to date.43 A second

possibility may be that type IV collagen is

masked to specific immunolabels by proteoglycans

surrounding the collagen.40,41,44 Finally, type IV

collagen that is seen in the embryologic basement

membrane may diminish with maturation of the

tissue. Cleutjens and co-workers39 have stated

that the underlying Bowman's layer, because it is

acellular, may not provide the appropriate

signals to the epithelium to stimulate type IV

production. Any type IV that is present could be

either remnant embryonic material or produced by

progeny cells of the limbal stem cells. This

would explain the differential localization of

type IV and indicate that progeny cells of the

stem cells lose their ability to produce type IV

collagen during migration toward the central

cornea.

To further complicate the issue, Cleutjens and

colleagues45 noted an absence or thinning of

lamina densa within the central human adult

cornea, despite electron-dense plaques, anchoring

fibers, and so forth, being present. They

speculate that there may be a correlation between

epithelial migration, the thinned appearance of

the basal lamina, and the missing type IV

collagen. However, Kolega and associates38 found

that the loss of type IV labeling was not

spatially correlated with the labeling of the

monoclonal antibody AE-5, a marker of 64-kd basic

keratin differentiation in epithelium. Finally,

it has been suggested that type VII collagen

anchoring fibrils may not even need a complete

basal lamina structure as part of the anchoring

complex.45

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BOWMAN'S LAYER

When viewed with electron microscopy, Bowman's

layer appears as a felt-like composite of

randomly oriented, striated collagen fibrils

dispersed throughout an amorphous matrix (Fig.

12). In an adult, this layer is approximately 8

to 12 μm thick, being slightly thicker in the

corneal periphery.21 Bowman's layer is acellular,

except for nerve axons coursing toward the

epithelium.46 Historically, electron microscopy

has suggested a lack of keratocyte (fibroblast)

cells within Bowman's layer, which called into

question its ability to regenerate after injury

or that keratocytes could migrate into Bowman's

layer. In confocal microscopy views of the living

eye, numerous keratocytes are observed at the

level of Bowman's layer (Fig. 13).

Fig. 12. Bowman's layer of the

human cornea. Stromal collagen

fibers (S) insert into the

posterior aspect of Bowman's

layer (B). The anterior aspect of Bowman's layer

appears smooth but slightly irregular. The lamina

densa appears as a continuous, dense band

adjacent to numerous hemidesinosomes along the

basal epithelium (E). Bar = 2 μm. (Courtesy of

Roger Beuerman, Ph.D., New Orleans, Louisiana.)

Fig. 13. Confocal microscopic transverse

image of the human Bowman's layer in

vivo. Nerve axons course freely through

the structure and keratocyte nuclei appear

numerous and more refractile than deeper in the

stroma. (500×). (Courtesy of Nidek Technologies.)Bowman's layer exists in primates and a few

additional species (most notably in birds). This

is of interest, particularly in terms of the

biomechanical strength and stiffness that

Bowman's layer appears to contribute to the

cornea. Nonprimate animals are poor models

compared to primates when predicting clinical

results with keratorefractive surgical

procedures. Biomechanical computer modeling of

the cornea with anisotropic material properties

is still in its infancy, but recent efforts may

be extended for a fuller appreciation of the

contribution of Bowman's layer to corneal shape

after surgery.47

Bowman's layer is composed of collagen types I,

III, V, and VI as shown by

immunoelectronmicroscopy34,37,48 and

immunofluorescence microscopy,35,36 with type I

constituting the bulk of collagen present. Type

IV and VII collagens are present in association

with the anchoring complexes described in the

previous section. While type I collagen is absent

from the amorphous background matrix,34 it has

been localized with immunogold labeling to

striated fibrils having uniform diameters of 20

to 25 nm and 67-nm banding.46 Use of

immunofluorescence microscopy for antigens

specific to type I collagen has proven less

reliable, because there have been reports of both

its presence36 and absence37 in Bowman's layer.

The existence of type III collagen anywhere in

the eye has been controversial, but recent

immunoelectronmicroscopy findings support its

presence in Bowman's layer in the normal human

adult.34,36 Type III collagen was confirmed in the

embryonic chick cornea with immunofluorescence

labeling, but was found lacking in adult avian

tissue.49 It has been found in diseased and

wounded human corneas.36,37 The function of type

III collagen has been linked to control of fibril

diameter and uniformity when hybridized with type

I collagen.37

Type V collagen also has been implicated as a

fibril diameter-controlling collagen in

heterotypic association with type I in the

embryonic chick cornea.50,51However, type V

labeling sites on the predominantly type I fibril

could be found only after considerable mechanical

disruption of these fibrils. Conversely,

immunofluorescence labeling for type V collagen

occurred throughout Bowman's layer in the human

adult without mechanical disruption37 and was

confirmed by immunoelectronmicroscopy.37,48 The

dichotomy between species may be explained by

labeling sites being more exposed on the human

fibrils than on avian fibrils, and this also may

depend on masking by the surrounding

proteoglycans.

Using immunogold labeling, type VI collagen was

found abundantly distributed as fine filaments

throughout Bowman's layer.48 It was not associated

with type I collagen striated fibrils. As with

many other collagens, the precise function of

type VI collagen is unknown, but it is ubiquitous

in connective tissues throughout the body33 and

may contribute to the collagen matrix in which

striated collagens are embedded. It is not found

in the primary stroma of the embryonic avian eye

until after fibroblasts arrive to lay down the

secondary stroma.52

The anterior surface of Bowman's layer is sharply

defined by its interface with the lamina densa of

the overlying basal lamina; however, this

interface is not necessarily smooth.46 When viewed

with scanning electron microscopy, the surface

appears undulating, with a woven texture and

occasional pores. These pores are 0.5 to 1.5 μm

in diameter and are likely to be the channels

through which corneal nerve axons travel to enter

the epithelium.46

In the posterior aspect of Bowman's layer,

striated collagen fibrils from the underlying

stroma become contiguous with Bowman's layer.

Individual fibrils splay outward into the

amorphous matrix.46 Occasionally, relatively large

bundles of stromal fibrils course obliquely from

the midstromal regions and terminate eventually

in Bowman's layer.53 These fibril bundles

presumably provide considerable cohesion between

the underlying stroma and Bowman's layer;

Bowman's layer cannot be stripped away from

stroma as a continuous sheet as can Descemet's

membrane. As a consequence of the random

insertions by the stromal fibrils, there is only

a roughly defined interface between the two

layers when viewed under high magnification.

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STROMA

The bulk of the cornea consists almost entirely

of the corneal stroma, a fibrous tissue layer

approximately 450 μm thick in the central cornea

(Fig. 14). As determined by biochemical and

immunohistologic methods, the stroma is composed

predominantly of type I collagen with types III,

V, and VI also in evidence.34,37,48 Immunogold

labeling was intense for collagen type VI, which

is associated with the interfibrillar matrix only

and not localized to the striated fibrils.48 Type

III and V collagens are codistributed with

striated type I collagen. This codistribution is

similar to the codistribution of types III and V

in Bowman's layer. Type V and III collagens also

have been labeled at the interfacial matrix

separating stroma and Descemet's membrane.34,48

Fig. 14. Stromal collagen fibrils (c) in

uniform spatial arrangement within

orthogonal lamellae. A portion of a

keratocyte is seen within the interlamellar

space. Granular material (asterisk) is visible

adjacent to the cell body (30,000×). (Courtesy of

Drs. Rodrigues, Waring, Hackett, and Donohoo.)Type I collagen is organized into striated

fibrils 25 to 35 nm in diameter with periodic

banding according to electron microscopy.46 Type V

collagen appears colocalized with type I in the

striated fibers.54 Using atomic force microscopy,

corneal collagen was reported to have a D-

periodicity of 63.9 to 68.5 nm.55,56Scleral

periodicity banding occurs at 67-nm intervals.

Fibrils are composed of 4-nm microfibril

components arranged in a right-hand helix and

tilted at 15 degrees to the fibril axis in the

cornea and 5 degrees in the scleral. This

striated banding is the result of the offset,

stacked arrangement of individual procollagen

triple-helix molecules into units of five (the

microfibril), which are further combined to form

the basic unit of the collagen fibril. The 67-nm

axial repeat was shown to correspond to the N–

and C- telopeptids and the D-band periodic gap

zone.57

These fibrils are combined into highly ordered,

sheet-like bundles called lamellae, which lie

essentially parallel to the corneal surfaces.

Lamellae vary in width and thickness throughout

the stroma, with a tendency to have smaller

dimensions anteriorly (0.5 to 30 μm wide and 0.2

to 1.2 μm thick) and larger dimensions

posteriorly (100 to 200 μm wide and 1 to 2.5 μm

thick).46 Hundreds of individual lamellae can be

discerned in a cross-sectional slice of the full-

thickness central cornea.

LAMELLAR ORGANIZATION

Fibrils within a given stromal lamellae appear to

run without interruption along the length of the

lamellae presumably to become contiguous with

scleral fibrils at the limbus. Normally occurring

fibril terminations are observed only in

association with extracellular compartments on

the processes of fibroblasts in which collagen

molecules are organized into fibril strands

during stromal remodeling.58

Fibrils in adjacent lamellae tend to be oriented

at highly oblique angles relative to one another.

The orientation of lamellae as a function of

depth has been well studied in the developing

chick cornea in which the orientation is nearly

orthogonal.59 There have been reports of nonrandom

orientations of lamellae in the human. At

midcentral stroma, lamellae tend to orient along

the vertical and horizontal axes.60 In the far

periphery evidence exists for circumferential

annular orientation that is parallel to the

limbus.61 Because it is impractical to track

single bundles of fibrils for any great distance

in the stroma, these fibrils may actually be

organized with swirling arc-like orientations

near the limbus that collectively appear to be a

continuous belt of fibrils.62

Cross-sectional views of the central cornea give

a first impression of a highly ordered structure

with little interweaving among lamellae. Closer

examination of the histologic evidence, combined

with mechanical strength tests of lamellar

organization, indicates that a much more complex

network of interweaving and lamellar bifurcations

does exist.63 In particular, the anterior one-

third of the stroma appears more disorganized

than the posterior two-thirds when seen with

light microscopy. Similarly, the peripheral

regions of the stroma are more disorganized than

the central regions in the human, as shown by

interlamellar cohesive strength tests and

concomitant histology.63 Polarized light

micrographs of relatively large expanses of the

stroma in cross section reveal the extent of

interweaving and bifurcation more readily.53

Some collagen fibril bundles are not truly

lamellar but are oriented at oblique angles to

the surface-parallel lamellae.53,63 These bundles

also appear to bifurcate frequently, with

portions becoming contiguous with surface-

parallel lamellae at various depths. A measure of

cohesive strength that structurally ties the

anterior stroma to more posterior regions of

stroma is imparted by these depth-varying fibril

bundles. These oblique bundles have not been

observed in rabbit stroma, which may account for

the greater stiffness and shearing strength seen

with isolated human stroma compared with the

rabbit.2 However, when swollen stroma is examined

in thick sections, fine collagen bundles are

found between the lamellar bundles in the rabbit,

but not in the human. In the corneas of the

elasmobranchs, suture fibers oriented

perpendicularly to the corneal surface are seen.

These observations of depth-varying collagen in

diverse species may indicate analogous structural

features that resist shearing forces, but they

are not homologous structures.

PROTEOGLYCANS

The hydrophilic mucopolysaccharide ground

substance in which collagen fibrils are embedded

takes the form of the proteoglycan, polypeptide

protein cores to which glycosaminoglycans (GAGs)

are covalently bonded. GAGs are large

polysaccharide groups consisting of repeating

disaccharide units, with glucosamine or

galactosamine on the first monosaccharide and

galactose, glucuroninc acid, or iduronic acid on

the second monosaccharide. Proteoglycans can

assume a wide variety of forms depending on the

number and types of GAGs per molecule, as well as

the inclusion of additional side chains such as

oligosaccharides. Some of the GAGs found in the

stroma are keratan sulfate, dermatan sulfate,

chondroitin sulfate, chondroitin, and the

atypical, noncovalently bound hyaluronic acid. A

discussion about the complexity and subtle

species distinctions of corneal proteoglycans is

beyond the scope of this chapter, but the subject

has been reviewed elsewhere.64,65

The most abundant corneal stromal proteoglycans

are lumican with keratan sulfate GAG side chains

and decorin with chondroitin/dermatan sulfate GAG

side chains. In vitro analysis of fibril

formation indicates that both lumican and decorin

appear to have an inhibitory effect on collagen

fibrillogenesis because of the core proteins of

the PGs and not the GAG side chains.66

Keratan sulfate and dermatan sulfate GAGs appear

to bind along the collagen fibrils at regularly

spaced sites and tend to be oriented

perpendicularly to the fibril. The less prevalent

chondroitin sulfate and hyaluronic acid are

localized within interfibrillar spaces without

evidence of binding to the fibril collagen.67 In

the developing cornea, proteoglycan-GAG complexes

vary in distribution and orientation with

time.67 The primary role of GAGs appears to be the

maintenance of interfibrillar spacing.

GAGs are heterogeneously dispersed throughout the

cornea. Castoro and co-workers found dermatan

sulfate to be more prevalent in the anterior

portion of the bovine stroma, and keratan sulfate

more prevalent in the posterior portion.68 This

distinction was based on differential water

content within the stroma. It was theorized that

more total water was found in the posterior

stroma because keratan sulfate readily absorbs

and releases water. Conversely, the anterior

stroma contained relatively less extractable

water, presumably because dermatan sulfate GAGs

bind to water molecules more tightly than do

keratan sulfate GAGs. However, Klyce and Russell

have shown that this anterior-posterior hydration

gradient can be predicted entirely by

consideration of the transport and permeability

characteristics of the epithelium and

endothelium.69 Borcherding and associates63 found a

reduction of keratan sulfate at the corneal

limbus of the human stroma with a corresponding

rise in dermatan sulfate. Dermatan sulfate was

not found centrally but was a major GAG within

the sclera. Chondroitin was found centrally but

not peripherally, while chondroitin sulfate was

found only in the periphery and limbus.70

TRANSPARENCY

Recently, Müller and colleagues71 reported that

the distance between adjacent collagen fibrils is

20 nm on average, and given a fibril diameter of

23 nm, the interfibrillar spacing by her protocol

using fresh, unswollen tissue is only 43 nm on

average. They also found that the ring-like

structures of proteoglycans surrounding each

fibril (when viewed in cross section) have a mean

diameter of 45 nm, while the distance between

proteoglycans along the axial length of each

fibril is 42 nm. Each proteoglycan has an average

length of 54 nm based on her measurements and a

thickness of 11 nm.71

The small, regular diameter of the individual

collagen fibrils and their highly ordered spatial

arrangement with interfibrillar spacing much

smaller than that of visible light (400 to 700

nm) has been implicated as the basis for corneal

transparency.72 In contrast, the sclera has

collagen fibrils that are not uniformly arranged

and have varying diameters, which results in an

opaque tissue.

Maurice72 recognized that because of the

difference in refractive index between the

stromal collagen fibrils and ground substance, up

to 94% of incident light on the cornea would be

scattered, making the cornea virtually opaque. He

proposed that collagen fibrils are in a lattice-

like arrangement with perfect dimensional order

and that each fibril scatters an individual

wavelet of light. By the process of destructive

wavefront interference, the light scatter from

individual fibrils would be cancelled by one

another, and the cornea would remain transparent

as long as the lattice arrangement was

maintained. This theory is an example of long-

range spatial order in which even fibrils

relatively distant from one another are precisely

spaced in accordance with the spatial dimensions

of the lattice.

Histologic views of the stromal fibrils in the

normal cornea suggest that they possess a quasi-

regular spatial arrangement with short-range

interfibrillar order but not long-range

order.73 It is generally believed that short-range

order must be accounted for in any description of

corneal transparency and light scatter because of

the unique arrangement of the fibrils. Of the

short-range order models, that of Hart and

Farrell is among the most rigorous.73

In the case of physiologically induced light

scatter, Benedek74 noted the histologic appearance

of regions devoid of fibrils, which he called

"lakes." Light scatter could be generated by a

difference in the local refractive index of the

stroma, for example by a pocket of localized

edema, as compared to the surrounding tissue.

Lakes were suspected to be artifacts of

histologic processing, but Farrell and associates

have shown that these lakes theoretically could

induce light scatter equivalent to observed

empiric data.75 This supports the theory that

edematous light scatter is generated by lakes

with critical dimensions approaching one-half

wavelength of light and not merely by a random

disruption of fibrillar order. However, models

are subject to limitations, and edema may induce

a continuum of disruption, including lakes and

short-range order disruption. Also, Bowman's

layer, which has randomly distributed fibrils, is

transparent. The mechanisms responsible for this

clarity appear to be the lack of scattering

elements spatially arranged with dimensions that

approach the critical distance of one-half

wavelength of light and the fact that the overall

thickness of the layer is relatively small

compared to the stroma.

KERATOCYTES

Occupying 3% to 5% of the total stromal volume,

keratocytes are interspersed throughout the

corneal stroma and form a communicating network

through their branching stellate processes. With

confocal microscopy, keratocytes appear to be

less densely dispersed than in Bowman's layer

(Fig. 15). In the deep stroma, keratocytes appear

spindle-shaped and more numerous than in the

midstroma (Fig. 16).

Fig. 15. Confocal microscopic transverse

image of the human mid-stroma in vivo.

The keratocyte nuclei appear less dense than in

Bowman's layer and less refractile. Note the

highly refractile nucleus in the upper part of

the image. This may be an activated keratocyte

(500×). (Courtesy of Nidek Technologies.)

Fig. 16. Confocal microscopic transverse

image of the human deep-stroma in vivo.

Many of the keratocyte nuclei at this level are

spindle-shaped and appear dense in numbers

compared to the central cornea (500×). (Courtesy

of Nidek Technologies.)Tight junctions have been seen between tips of

processes, although usually a 20-nm intercellular

space is seen.21 Gap junctions are functional

based on microinjections of fluorescent dyes that

pass from cell to cell.76 Occasionally, portions

of keratocyte processes appear to lie within a

lamellar bundle; however, the majority of

keratocytes reside within the potential

interlamellar spaces. The stromal density of the

keratocytes demonstrates a linear loss as a

function of age, which tends to parallel the loss

of endothelial cells over time.77

The keratocyte is approximately 2 μm thick at the

cell body with a disproportionately large nucleus

compared to its cytoplasmic volume. Free

ribosomes, rough endoplasmic reticulum, and

Golgi's complexes are found in the cytoplasm.

Immunoelectronmicroscopic antigen labeling

specific for types III, V, and VI collagen was

found localized to keratocytes; however, type III

labeling was weak.34,48 Keratocytes have a

remarkable presence of a network of fenestrations

on their cell walls that appear to be

functionally related to the diffusion and

mechanical attachment of collagen fibers to the

cell body.78

Keratocytes are normally spatially quiescent,

albeit constantly maintaining the extracellular

matrix. They appear to have a clockwise,

corkscrew-like appearance in terms of their

distribution with depth in the stroma. It is

speculated this has some function with respect to

communication in depth, while maintaining optimum

corneal transparency.78

However, keratocytes have a considerable degree

of mobility; when activated they are able to

migrate into wound margins rapidly to synthesize

new collagen and glycoproteins for tissue repair.

In vitro results indicate that a wide variety of

growth factors increase keratocyte chemotaxis.79 A

major difference between activated and quiescent

keratocytes is the organization of the

contractile cytoskeletal proteins. In the

quiescent form, contractile proteins appear to be

associated with f-actin for the maintenance of

cell shape and interconnectivity. In the

activated form, a putative contractile apparatus

comprised of f-actin, myosin, and α-actinin is

organized into muscle-like stress fiber

bundles.80 Chondroitin sulfate and collagen types

V and VI were shown to have an inhibitory effect

on activated keratocyte migration, while

fibronectin increased migration significantly.

Integrins had a mediating effect on migration,

presumably affecting cell–matrix interactions.81

Keratocytes can vacate the anterior stroma

rapidly after de-epithelialization.82 The signal

for this response is unknown, but the observation

underscores the potential responsiveness of this

usually unremarkable cell. When keratocytes do

respond to a stromal insult, they are more

appropriately termed fibroblasts to reflect the

diminution in their stellate appearance and their

increased ability to generate procollagen for

subsequent fibril construction. There is strong

circumstantial evidence that keratocyte

activation is correlated to a higher level of

reflected light scatter from the stroma,

specifically from the cell bodies themselves, and

this may constitute much of the haze observed

after photorefractive keratectomy (PRK).83 It has

been well documented that tears are cytotoxic to

keratocyte cells, and it has been shown that

apoptosis is reduced by surgical removal of the

extraorbital lacrimal gland, although some

compensatory cytotoxicity appears to be derived

from the intraorbital lacrimal gland in the mouse

model.84

Birk and Trelstad85 investigated the mechanisms

and regulation of fibrillogenesis in embryonic

chicken cornea. They found that extracellular

fibroblast cell surface compartmentalization was

responsible for the organization of the fibrils

into lamellar bundles. Furthermore, the

orthogonal organization of lamellae in the chick

was due to the orthogonality of the surface

compartments of the fibroblast processes.

Fibroblasts apparently orient themselves during

the deposition of the secondary stroma by taking

spatial cues existing in the primary stroma.

Thus, a single fibroblast cell is able to

dispense bundles of collagen fibrils along axes

that are oriented at or near 90 degrees to one

another; these bundles then form into lamellae.

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DESCEMET'S MEMBRANE

Descemet's membrane can be thought of as the

basal lamina of the endothelium, and it varies in

thickness in the human from approximately 3 μm at

birth to 8 to 10 μm in adulthood (Fig. 17).86 The

age-related growth and renewal of the membrane

after injury indicate that it is an extracellular

secretion of the endothelium. Descemet's membrane

is stratified into distinct layers according to

histologic appearance and immunohistochemical

labeling: a thin, unbanded zone immediately

adjacent to the interfacial matrix of the stroma

(approximately 0.3 μm); a banded anterior zone (2

to 4 μm); and an unbanded posterior zone that may

be as much as two-thirds the total thickness of

Descemet's membrane in adults (> 4 μm). The

banded and unbanded zone thicknesses vary,

depending on the age of development and the

species.66 Because the unbanded posterior zone is

laid down by the endothelium over the course of a

lifetime, it is a temporal record of the

physiologic state of the endothelium. For

example, unusual striated collagen banding occurs

in the posterior zone in association with corneal

disease such as Fuchs' dystrophy.87

Fig. 17. Full-thickness view of

Descemet's membrane. Stromal collagen

(C) with a keratocyte (F) is seen at

top. Endothelium (E) is seen at bottom.

Large box indicates anterior banded zone

of 100-nm spaced collagen. Smaller boxes indicate

occasional foci in the amorphous posterior

unbanded zone. Arrows point to vesicles on the

endothelial basal membrane. (Courtesy of Drs.

Rodrigues, Waring, Hackett, and Donohoo.)As noted previously, Descemet's membrane does not

adhere strongly to the stroma, and it can be

surgically dissected as a sheet. The randomly

oriented collagen fibers in the interfacial

matrix of the stroma have a densely matted

appearance,40 and 22-nm thick fibers arising from

the matrix penetrate Descemet's membrane to a

depth of only 0.16 to 0.21 μm.88

The banded anterior zone is composed of numerous

precisely aligned sheets forming a three-

dimensional array or lattice.21 Each sheet of the

array is constructed from triangular elements

with densely staining nodes at the apices. The

separation between nodes is approximately 100 to

110 nm. The highly ordered arrangement of the

nodes causes the banded appearance seen in cross

section. In the posterior unbanded zone, the

appearance is of a homogeneous, fibrillogranular

region. The anterior unbanded zone also has a

fibrillogranular appearance but is extremely

difficult to discern visually (Fig. 18).86

Fig. 18. Descemet's membrane is a

composite of a nonbanded posterior

portion with a granular appearance (DM)

and an anterior portion with a banded

appearance (arrows). At the interface between the

stromal collagen (above) and Descemet's membrane

is a narrow, unbanded, granular-appearing region.

The endothelium (E) is seen below (37,000×).

(Courtesy of Drs. Rodrigues, Waring, Hackett, and

Donohoo.)The composition of Descemet's membrane is

primarily collagen. Marshall and

colleagues34,48 found immunogold labeling for

collagen types III and IV in the posterior

unbanded zone, type IV in the anterior banded

zone, and types V and VI in the anterior unbanded

zone and at the interfacial matrix of the stroma.

In addition, long striated collagen V labeled

filaments were seen within Descemet's membrane

immediately adjacent to the endothelial cells.

These filaments may be the same as those

occasionally seen with specular microscopy2 and

by electron microscopy86 but do not appear to be

associated with the extensive striated

distribution found with disease-related states.

Tamura and associates89 also found collagen type

VIII strongly labeled in the anterior banded zone

using immunohistochemical localization for

monoclonal antibodies.

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ENDOTHELIUM

The human corneal endothelium is a single layer

of 400,000 to 500,000 cells. Confocal microscopy

provides views of this cell layer that surpass

the details seen under specular microscopy (Figs.

19 and 20). Cells are 4 to 6 μm in height and 20

μm in width, and their posterior surfaces are

predominantly hexagonal when viewed under

specular microscopy (Fig. 21). Cross-sectional

views with electron microscopy show that cell

lateral walls are extremely tortuous and

interdigitate with extensive folds and finger-

like projections. It has been estimated that the

total paracellular path length may be 10 times

longer than the total height of the

cell.90 Numerous gap junctions along the lateral

membranes provide cell-to-cell cytoplasmic

communication as evidenced by the presence of

connexin 43 and the spreading of fluorescent dye

from an injected cell to surrounding cells (Fig.

22).91

Fig. 19. Confocal microscopic transverse

image of the human corneal

endothelium in vivo. In the young normal cornea,

the majority of the cells will have a hexagonal

outline and they will be fairly uniform in size.

The dark spots near the center in many of the

cells may represent the central endothelial

cilium (500×). (Courtesy of Nidek Technologies.)

Fig. 20. Confocal microscopic transverse

image of the human corneal endothelium

in vivo after penetrating keratoplasty. Note

cellular enlargement, polymegathism, and

polymorphism. Cell nuclei are clearly visible.

Normally non-dividing, endothelial cells enlarge

slowly with age to compensate for cell loss,

maintaining a continuous lining on Descemet's

membrane. Cataract surgery and transplants

generally exhibit cell loss. Below about 400

cells per mm2 endothelial decompensation can

occur with ensuing edema (500×). (Courtesy of

Nidek Technologies.)

Fig. 21. Scanning electron micrograph of

corneal endothelium. Note the regular

hexagonal arrangement of the cells

(1,170×). (Courtesy of Drs. Rodrigues,

Waring, Hackett, and Donohoo.)

Fig. 22. Scanning electron micrograph of

endothelial cell intracellular junctions

(8,100×). (Courtesy of Drs. Rodrigues,

Waring, Hackett, and Donohoo.)The apical portion of the lateral cell membranes

facing the anterior chamber possesses small flap-

like or leaflet-like features that overlap

adjacent cells and harbor segmented tight

junctions that form an incomplete seal around the

apical cell margin (Fig. 23). Megamolecules

(greater than 80 kd) are essentially prevented

from penetrating the paracellular pathway, but

molecules such as horseradish peroxidase and

lanthanum are able to cross the apical

junctions.92,93While these tight junctions appear

dimensionally larger than those found in the

apical cells of the epithelium, they are not as

efficient.90 Nevertheless, these junctions,

combined with the closely apposed paracellular

pathway, prevent excessive passive transport of

anterior chamber fluid into the stroma. Any

normal leakage that may occur around the

endothelial cells tends to be counterbalanced by

the active ion transport processes in the cell

membranes. With pathologic cellular damage or

substantial reduction in cell density from a

normal value between 1,400 and 2,500 cells/mm2 to

a critical value of approximately 400 to 700

cells/mm2, the endothelial transport capability

becomes overwhelmed, and chronic stromal edema

can ensue.90 Endothelial cell density normally

increases from the center to peripheral cornea by

approximately 10%, with the superior peripheral

quadrant increasing by nearly 16%.94

Fig. 23. Extensively interdigitated

lateral membranes of endothelial

cells. Near the apical margin, a

tight junction is formed (arrow). Bar = 0.5 ×m.

(Courtesy of Drs. Rodrigues, Waring, Hackett, and

Donohoo.)The normal central corneal endothelium has been

shown to have a reduction in the cell density

over a 10-year period. Repeated specular

microscopy in the same individuals over a 10-year

period with the same camera and processing method

indicates that cell density decreased from a mean

of 2,715 ± 301 to 2,539 ± 284 cells per square

millimeter (p < 0.0001). The percentage of

hexagonal cells dropped over the same time period

from 67% to 64% (p = 0.003), while the

coefficient of variation increased from 0.26 ±

0.05 to 0.29 ± 0.06 (p < 0.001).95

A single cilium often can be seen on the apical

endothelial membrane in a location geographically

associated with the intracellular centriole

pair.96 It is unclear whether this cilium is a

vestigial artifact or if it has an important

regulatory or receptive function. Small numbers

of short microvilli also are seen on the apical

surface.

It has often been reported that the endothelial

cell monolayer does not proliferate in vivo.

However, endothelium does possess the ability to

proliferate, but this capacity is normally turned

off in the natural state due to the arrest of the

G1 phase of the cell cycle. The endothelial cells

appear unable to respond to autocrine or

paracrine stimulation even though they express

mRNA and the protein for growth factors and their

receptors. Importantly, cell-to-cell contact

appears to inhibit endothelial cell proliferation

during development and later maintains the

nonproliferative monolayer state of the adult

cornea. Ethylenediaminetetraacetic acid (EDTA)

releases cell-to-cell contact inhibition and

promotes proliferation in cell cultures of older

corneas when exposed to appropriate growth

factors.97–99

It was once believed that the basal cell surface

facing Descemet's membrane was irregular in

appearance due to the complexity suggested by the

lateral walls. In recent views provided by

scanning electron micrographs of stripped bovine

and human endothelium, the cell mosaic facing

Descemet's membrane appeared hexagonal in a

manner similar to the mosaic facing the anterior

chamber, although the cell outlines appeared

broader and less distinct.100 The cell margins

adjacent to Descemet's membrane were ruffled,

with overlapping, flattened processes, some of

which appeared to insert into adjacent cell

membranes, while many were free and damaged

presumably during histologic preparation.

It has been shown that the glycosaminoglycans

keratan sulfate (KS) and chondroitin sulfate (CS)

are associated with the endothelial cell surface

in a variegated pattern (some cells have high

levels of KS and adjacent cells may have none,

while CS levels tend to show an inverse

relationship to KS levels) and likely play a role

in the ability of the cells to adhere and migrate

across the basal lamina.101

The intracellular contents of the endothelial

cell are dominated by a large nucleus and

numerous organelles, including mitochondria,

smooth and rough endoplasmic reticulum, and

Golgi's apparatus.21 The obvious metabolic

activity of these amitotic cells indicated by

their morphology is related to the energy

requirement of the endothelial ion transport

system that plays a major role in the regulation

of corneal hydration.

INNERVATION

Sensory innervation of the cornea occurs

primarily through the ophthalmic branch of the

trigeminal nerve, which inserts into the

posterior globe as the long ciliary nerve fibers

and as a portion of the short ciliary nerves

through the ciliary ganglion. The limbal region

is supplied by these nerve fibers passing

anteriorly within the suprachoroidal space with

possible branching connections between the fibers

of the long and short ciliary fibers.90 These

fiber trunks begin branching near the ora serrata

region to form a circumferential plexus near the

corneoscleral junction. Branches from this plexus

travel anteriorly to innervate the adjacent

conjunctival and limbal epithelium.102 Some minor

innervation of the peripheral cornea also occurs

through superficial branches in the episcleral

and subconjunctival regions.

Primary branching from the plexus occurs as 60 to

70 nerve fibers radially enter the midstromal

cornea with approximately half containing 15 to

30 axons and the remainder having fewer than 15

axons per fiber.103 Most fibers lose their myelin

sheaths 2 to 3 mm within the cornea; however, a

few branches may retain a Schwann cell covering

for some distance into the stroma. Loss of myelin

sheathing would aid in making the cornea

optically transparent. Considerable branching,

including recurrent branching, occurs among the

midstromal fibers. Relatively few nerve fibers

have been found to branch into the posterior

third of the stroma, and no innervation of the

endothelium or Descemet's membrane has been found

in humans.104

As the midstromal fibers travel toward the

central cornea, axons become finer, and beaded

features are seen along the filaments prior to

their termination. Collaterals from the

midstromal fibers branch anteriorly at 90 degrees

to create an extensive sub-Bowman's layer plexus.

Fibers from the plexus travel anteriorly through

Bowman's layer toward the epithelium, where they

again turn at 90 degrees and travel parallel to

the corneal surface just posterior to the

epithelium. Based on confocal microscopy, the

majority of fibers in the subbasal plexus of the

central cornea appear to be oriented along the

superior-inferior axis. There are approximately

5400 to 7200 nerve bundles in the subbasal

plexus; because each bundle may contain several

axons, the total number of axonal fibers may be

as high as 44,000.105 Each fiber immediately forms

numerous, elaborate leashes with up to several

dozen beaded axon terminals per leash and some

additional unbeaded fibers. The beaded appearance

was at one time considered an artifact of tissue

processing or trauma; however, confocal

microscopy has shown these to be genuine features

(Fig. 24). The unbeaded straight terminals are

not seen entering the epithelium; however, the

beaded terminals of the leashes diverge

vertically and obliquely among the epithelial

cells in a complex manner before terminating at

the level of the apical cells (Fig. 25).102 The

diameter of the individual nerve fibers in the

subbasal plexus are between 0.05 and 2.5 μm with

most being in the range of 0.1 to 0.5 μm.

Fig. 24. Confocal microscopic transverse

image of the human basement epithelial

membrane in vivo demonstrates

unmyelinated beaded nerve leashes. These send off

nerve terminals within the corneal epithelium

(500×; courtesy of Nidek Technologies).

Fig. 25. Nerve axons (N) are visible

in the basal cell layer of the

epithelium (49,500×). (Courtesy of

Drs. Rodrigues, Waring, Hackett, and

Donohoo.)Sympathetic innervation of the cornea occurs

through fibers with cell bodies in the superior

cervical ganglion.106,107 It has been demonstrated

in some species such as the rabbit by using

neuroanatomic mapping methods and observing the

functional changes in epithelial ion transport

induced by sympathectomy. Sympathetic innervation

is considered rare in all primates, including

humans. Parasympathetic innervation has not been

demonstrated in humans.

The corneal epithelium is one of the most highly

innervated structures in the body. Sensitivities

are 300 to 600 times that of skin, and a corneal

surface area of 0.01 mm2 may contain as many as

100 terminal endings.102 For the human cornea with

a surface area of approximately 120 mm2, there

may be as many as 1.2 million terminal endings.

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CONCLUSION

The understanding of corneal anatomy is being

refined continuously. Studies of epithelial wound

healing have led to a clearer picture of the

hemidesmosome-anchoring fibril complex.

Similarly, refractive surgery has generated the

need for more information on stromal collagen

structure and fibroblast activity. The use of

ophthalmic lasers and the continued efforts to

improve corneal storage media have stimulated

more research in endothelial cell morphology.

Clinical research has been, and will continue to

be, a significant motivator in the study of

corneal anatomy. It is likely that the

relationship between physiologic function and

anatomic diversity will become increasingly

important as attempts are made to alter the

cornea in very subtle ways to ultimately improve

clinical results.

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ACKNOWLEDGMENT

This study was supported in part by National

Institutes of Health grants EY-014162 (M.K.S.),

EY-13311 (S.D.K.), and EY-02377 (L.S.U.) from the

National Eye Institute, Bethesda, Maryland.

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