continuing education

Anterior Segment Anatomy and Physiology

Paul White, OD
November 2000

The foundation for understanding the prevention, detection and remediation of contact lens tissue complications.

As contact lens practitioners we frequently examine the anterior segment, but we only occasionally consider in detail the underlying processes. To enhance our clinical understanding, it is helpful to review periodically the essentials of anterior segment anatomy and physiology.

The cornea is divided into five layers: epithelium, Bowman's layer, stroma, Descemet's membrane and endothelium, and it has special innervational and vascular characteristics. Overall structure combines with specific metabolic and oxygen consumption processes to produce a uniquely transparent optical medium. The pre-ocular tear film (POTF) and eyelid distribution of the POTF are intimately associated with proper corneal function.

Epithelium

This outermost corneal layer consists of non-keratinized, stratified epithelium, which is mounted on a fine basement membrane and anchored by filaments extending into the underlying collagen. Columnar basal cells are next to the basement membrane, and two or three rows of small interlocked wing cells are mounted on the basal cells. Two or three layers of surface squamous cells cap the wing cells. Thus, the epithelium is divided morphologically into three layers: superficial or squamous cell layer, middle or wing cell layer, and deep or basal cell layer. The epithelium is about 50 micrometers thick and occupies 10 percent of total corneal thickness. Scanning electron microscopy shows the epithelial surface has an uneven texture.

Functions. Major epithelial functions are physical protection, optical refraction, POTF stabilization and barrier to fluids and microorganisms. As the most external corneal structure, it has to protect the more internal structures from foreign elements and pressure from blinking, eye rubbing and contact lenses. The epithelium is both very resilient to foreign elements and repairs quickly after trauma. To fulfill optical refractive needs, the epithelium has to maintain uniformity and transparency. Optical uniformity is also dependent upon a stabilized and uniform POTF. Reduction of the epithelium's fluid barrier characteristic upsets corneal hydration and transparency, and epithelial injury can reduce the microorganism barrier and lead to infection.

Figure 1: A large epithelial wound with stain

Mitosis and Wound Healing. It is generally believed that the only epithelial cells that undergo mitosis to generate new cells are the basal cells; they are the epithelium's germinative layer. The new cells move anteriorally. As they migrate, they first change their shape to that of wing cells, and then they become the squamoused superficial cells, which disintegrate into the POTF and are sloughed away by eyelid action (the process of desquamation). The epithelium normally regenerates totally in about seven days, and this rate is accelerated in response to trauma.

Very small corneal epithelial wounds are covered in about three hours by adjacent basal cells, which send out pseudopodia to blanket the area. With larger corneal epithelial wounds, cells from all layers of the surrounding epithelium advance and flatten to cover the wound. Initially, the newly regenerated epithelium is very susceptible to damage, but a tight adhesion is established in only a few days if the basement membrane is largely intact (Figure 1). At first the epithelium is only a couple of layers thick and will regain normal thickness within several weeks.

Stabilization of the POTF. The polygonal superficial cells are 40 to 60 micrometers in diameter, and they have unique anatomical characteristics to simultaneously maintain the POTF while separating it from the cornea's extracellular spaces. The distinctive aspect of these cells' surface membranes are microscopic projections or irregularities (microvilli, reticulations and microplicae) and a thickened outer leaflet with an extensive fibrillar glycocalyx, or the buffy coat which increases surface area by its folds and increases the adherence of the POTF's mucin layer to the glycocalyx.

Fluid Barrier. The superficial layer has a tight junction complex among laterally adjacent cells, and this produces a barrier that resists fluid flow through the epithelial surface. For example, a damaged endothelium or high intraocular pressure (IOP) may allow aqueous humor to enter the stroma. The epithelium's tight junction complex traps the fluid within the epithelium, producing edema and bullae. You can use fluorescein dye to evaluate the intactness of this barrier, and weakened epithelial intactness demonstrates increased staining.

Bowman's Layer

	Figure 2. Posterior folds.

Beneath the epithelium's fine basement membrane is the very thin (about 12 micrometers) Bowman's layer, which is randomly arranged collagen fibrils. This dome-like structure is attached at the limbus and is relatively tough, but damage causes fibrous scar tissue to form. The frequency of epithelial damage without involvement of Bowman's testifies to its toughness. It is very difficult to change the shape of Bowman's layer without cutting through it into the stroma, such as in corneal refractive surgery.

Stroma

The stroma, or substantia propria, comprises about 90 percent of corneal thickness and provides most of corneal strength. A highly specialized regular structure and an absence of blood vessels in the stroma are two important bases for corneal transparency.

Lamellae. The stroma consists of several hundred stacked, layered lamellae 1.3 to 2.5 micrometers thick which extend from limbus to limbus and run parallel to the surface and to each other. The stromal lamellae are tape-like bands rich in collagen and glycosaminoglycans (GAGs). Keratocytes are the predominant stromal cell type. They occupy less than 10 percent of stromal volume, generate stromal ground substance and constantly replace GAGs lost by hydrolysis and diffusion. Almost all stromal nutrition arrives by diffusion through the endothelium from the aqueous humor.

Ground Substance and Hydration. The fibers of the lamellae are embedded in a springy ground substance. Stromal ground substance is about 78 percent water, 15 percent collagen and seven percent other elements. Thus, the stroma can be considered as a hydrogel with a Dk of about 30. Although collagen fibrils in the stromal lamellae have a highly ordered arrangement that allows good light transmission, collagen spacing and light scatter vary with the degree of corneal hydration. Six percent or greater stromal edema measurably reduces corneal transparency. In contrast to stromal swelling induced by glaucoma or endothelial dysfunction, contact lens-induced swelling begins in the posterior stromal lamellae. At about six percent edema, posterior striae manifest, and at about 10 percent or greater swelling posterior folds are produced (Figure 2). The stroma is quite tolerant to variations in oxygen tension, osmotic pressure and metabolic wastes unless its bounding membranes are compromised. Contact lens wear can cause such compromise and result in stromal edema, light scattering and neovascularization.

Corneal Thickness. Central corneal thickness of a normal eye is approximately 0.52 mm, and peripheral corneal thickness is about 0.65 mm. Fluid imbibition increases corneal thickness, and increased corneal thickness for an individual is a manifestation of edema, which can be measured with a pachometer. Sophisticated pachometers are necessary to have the required accuracy, validity and repeatability of measurements. Because central corneal thickness is a little over 0.50 mm, a 0.01 mm change equals a 2 percent change in thickness. (Figure3).

Descemet's Membrane

The endothelium produces Descemet's membrane, a basal membrane that has two layers: the anterior banded layer, which is produced during fetal development, and the posterior non-banded layer, which is produced throughout life by the endothelium at a rate about 1 to 2 micrometers per decade. Short fibrils extend from the stroma into Descemet's membrane and attach it loosely.

Figure 3. Schematic of increased corneal thickness. Dotted line indicates uniform thickness and radius changes.

Guttae are produced when endothelial cells are activated to develop increased amounts of basal lamina material. The common peripheral guttae are known as Hassall-Henle warts, while central guttae indicate endothelial cell dysfunction. Many pathologies that damage endothelial cells result in excess production of Descemet's membrane material.

Endothelium

The endothelium consists of a single thin layer of about 400,000 mostly hexagonal shaped cells, 4 to 6 micrometers thick and 20 micrometers wide. Near the posterior border of the layer, intercellular spaces are very narrow, which creates tight junctions and limits movement of substances into and out of the cornea. Endothelial cells also have tight interdigitations.

The corneal endothelium functions as a leaky membrane that has an active pump component. Fluid moves across the endothelium between the cells and into the stroma as a result of stromal swelling pressure. Swelling is countered by the endothelium's active pumping of bicarbonate ions into the aqueous, and bulk water follows. There is much still not understood about endothelial cell function and maintenance of corneal clarity, but it is of major importance to the maintenance of corneal detergescence.

Figure 4. Polymegathism, pleomorphism, and guttae are shown.

Mosaic. The normal endothelial mosaic is not perfectly regular, and there is some variation in size (polymegathism) or in number of facets and shape (pleomorphism). Endothelial mosaic variation increases with age, after some ocular traumas and for some contact lens wearers. Contact lens-induced polymegathism, usually not accompanied by reduction of cell density, seems often to be related to hypoxia and may involve a decreased stromal pH which interferes with normal endothelial metabolism. Cell density is usually expressed in number of cells per millimeter squared, and can show cell loss. Polymegathism is considered in terms of the distribution of cell areas and the ratio of the maximum to minimum cell areas. Pleomorphism may be defined by the number of cells that deviate from a hexagonal shape (Figure4).

Specular Microscopy. Clinical specular microscopy is an in vivo photography of approximately 200 to 800 micrometers of endothelium over the central 4 to 6 mm of the central cornea. More complex scanning systems have been used to photograph larger endothelial areas. Although often limited by inability to view the peripheral cornea and small sample size in each photograph, specular microscopy provides better research information than slit lamp biomicroscopy of the endothelium.

Innervation

Nerves penetrate the posterior two-thirds of the cornea, move forward, divide, penetrate Bowman's membrane and enter the epithelium. There are nerve networks within the stroma, under Bowman's membrane and in the epithelium where nerve endings are most numerous. Because corneal sensitivity conforms to corneal nerve ending density, sensitivity increases from the limbus to the central corneal zone. Nerve networks overlap, which makes localization of corneal stimuli difficult. Contact lens wear can decrease corneal sensitivity (increase corneal touch threshold [CTT]), as a result of hypoxia and slowing down of aerobic metabolism.

The nerve fibers to the corneal epithelium primarily originate from the trigeminal nerve. About 75 nerve trunks radially enter the cornea, and they lose their myelin sheaths within 1 to 2 mm after entry. The densest network of nerve terminals is located in the basal cell layer. Epithelial nerve fibers have protective and nutritional functions. As sensory nerve fibers, they respond to physical stimuli and warn the body of danger. While it is accepted that pain and touch elicit responses, heat and cold are questionable.

Vasculature

Central and paracentral areas of a healthy cornea are avascular and non-edematous, but the peripheral transition area between cornea and sclera, the limbus, contains anatomical architecture of both and is vascularized with physiologic edema. The limbus is usually less than 1 mm wide, often most prominent and widest superiorally, and has many variations from normal (Figure 5).

	Figure 5. Both the limbal architecture and vascular injection are seen.

The sclera immediately adjacent to the limbus has a vascular plexus at all levels. Of particular importance are the anterior ciliary arteries, which form the perilimbal plexus called the superficial marginal arcade. This arcade has minute branches at right angles to the limbus. Some branches of the episcleral vessels abruptly curve down to take part in the deeper plexus of vessels.

Association of fibrillar, papillae and white cell invasion together with new vessels constitute a pannus. Superficial neovascularization is sometimes referred to as a pannus of a given number of millimeters as measured from the limbus. Clinical tolerance in such measurements is large unless photographed, but anything greater than two millimeters is considered abnormal.

Most contact lens-induced pannus or neovascularization remains static within 2 mm to 3 mm of the limbus. Rectifying the cause of the problem often results in emptying blood from the vessels, but with residual ghost vessels that easily refill with blood from subsequent stress. Most contact lens-induced neovascularization involves the superficial vessels, but neovascularization of the deep vessels may occur. Hypoxia, mechanical pressure, POTF interference, accumulation of toxic by-products and infection may all contribute to contact lens-induced neovascularization. It is important to differentiate between vessel dilation and injection as opposed to true neovascularization.

Metabolism

The cornea is avascular and it derives its nutrition indirectly. With open eyes and no contact lenses, atmospheric oxygen mixes with the POTF and becomes available to the cornea for metabolism. Atmospheric pressure at sea level is about 760 mm Hg, and the oxygen partial pressure or tension component of this is about 155 mm Hg. With closed eyes, oxygen is obtained primarily by diffusion from the palpebral conjunctival blood vessels; and this is about 55 mm Hg, or about one-third of that available with open eyes. Limbal capillaries and aqueous humor provide only a very small amount of oxygen. Corneal edema from hypoxia is detectable with a conventional slit lamp when oxygen pressure in the POTF between a contact lens and the cornea is 20 mm to 40 mm Hg. The epithelial oxygen need or consumption rate ranges widely among individuals. Oxygen flux is the rate that oxygen molecules move into the cornea or a contact lens, and it depends upon the oxygen partial pressure.

Glucose. There is about 15 times more glucose in the aqueous humor than in the POTF, and aqueous humor is the cornea's and corneal epithelium's vital glucose source. Glucose metabolism provides the energy for corneal cellular activity and growth, as well as contributing to corneal detergescence and transparency. Reduction of aqueous glucose depresses stromal glucose and epithelial glycogen. The epithelium converts glucose into glycogen and stores it in granules within cells. Glycogen is a reserve energy source used during stress. Hypoxia leads to glycogen depletion, and the epithelium is less able to respond to trauma and repair itself.

Glycolysis. In aerobic glycolysis, glucose produces maximum energy via the Kreb's cycle. Full aerobic metabolism has a glycolysis phase in which glucose is reduced to pyruvic or lactic acid, and a respiration phase in which lactic acid is oxidized into carbon dioxide and water. Hypercapnia is the buildup of carbon dioxide. With hypoxia, more acid and less energy are produced. To increase energy, the glycogen stored in the epithelium's basal cells is used. The resultant increase of residual lactic acid cannot easily penetrate the epithelium, so it diffuses through the stroma, Descemet's membrane and the endothelium into the aqueous humor. Lactic acid build-up alters corneal pH and produces an osmotic imbalance, increasing corneal hydration. Depressed metabolic activity also depresses corneal sensitivity.

Critical Oxygen Values

All contact lens materials restrict corneal oxygen availability to various degrees. It is important to know when this reduction is sufficient to compromise corneal function and health. These critical oxygen requirement values vary among researchers, research techniques, individual corneas, the individual corneal layers of epithelium, stroma and endothelium, and central versus peripheral cornea.

Even after critical oxygen requirement values are established, they would need to be correlated with the oxygen transmissibility (Dk/L) and the average oxygen transmissibility (Dk/L) of contact lenses. The most commonly accepted critical Dk/L values to avoid corneal edema during open eye lens wear are 24 to 30. To limit overnight corneal edema with contact lens wear to the expected 4 percent without contact lens wear, the most accepted critical Dk/L values are 75 to 90. Thus, there is about a three to one ratio of open eye versus closed eye Dk/L values, which closely corresponds to the three to one ratio of oxygen availability with open versus closed eyes.

There are many contact lens complications related to hypoxia. The epithelium may have a reduced number of cells, a decrease in cellular metabolic activity, decreased mitotic rate, thinning, hypoesthesia, microcysts, compromised junction integrity and infectious keratitis. The stroma may manifest edema, striae, folds, acidosis, keratocyte impairment, thinning, thickening and vascularization. Blebs, polymegathism and impaired hydration control may manifest in the endothelium.

Transparency

	Figure 6. Primary movement direction of upper and lower eyelids with blinking.

The unique structure and function of the cornea must produce very good transparency. Several theories proposed over the years contribute to an understanding of corneal transparency, although there are still unknowns.

Maurice's lattice theory indicates that there is a two-dimensional lattice of collagen fibrils in the stroma, which are regular with uniform orientation and with a higher index than surrounding interstitial substance. Fibrils are spaced less than a wavelength of light apart. Wavelengths of light scattered from individual fibers cancel each other out by interference, which produces transparency in the direction of incidence. Alteration of these factors can decrease transparency. Hydration (edema) swells the spaces between the fibrils, alters interference patterns and decreases transparency. Although this theory has flaws, it helps our overall comprehension.

Cogan's and Kinsey's osmotic theory is no longer accepted as the sole factor controlling corneal transparency. However, osmosis can affect corneal turgescence and transparency. Nature wants to balance salt concentration (osmolarity) in fluids on both sides of a permeable membrane. To establish an osmotic equilibrium of 0.9 isotonic saline, fluid flows from the hypotonic to the hypertonic. With open eyes, evaporation of the POTF makes it slightly hypertonic, helps draw fluid from the cornea, and assists in detergescence. With sleep and closed eyes, the POTF is not hypertonic relative to corneal fluid and this contributes to corneal edema during sleep. This osmotic factor and hypoxia during sleep cause an average 4 percent corneal thickness increase even for non-contact lens wearers.

The primary control of corneal hydration and transparency is attributed to the endothelial pump as has been described.

Pre-Ocular Tear Film

The normal secretion and distribution of tears is essential for clear and comfortable vision. The tear film forms a smooth surface over the optically uneven corneal epithelium, provides lubrication for eyelid movement, acts as a nutrient vehicle for ocular surface metabolites, dilutes irritants, flushes away debris, provides antibacterial activity and transports white blood cells to help heal corneal injuries. These complex and interrelated functions depend upon the continuous production of tear components, their segregation into a multi-layered film over the ocular surface and frequent blinking to remove and resurface that tear film.

The stabilized POTF consists of three functional layers. The inner mucin layer is mainly composed of glycoproteins secreted by goblet cells in the surface of the conjunctival epithelium. This layer is at least 1 micrometer thick and functions as the interface between the hydrophobic surface epithelial cells and the aqueous tear layer.

The middle aqueous layer is composed mostly of water with electrolytes, IgA and proteins, some of which have antibacterial enzymatic activity. Secreted by the main and accessory lacrimal glands, which are both under neural control, it is the main component of basal and reflex tearing. This layer is about 7 micrometers thick, though recent investigations show that the inner and middle layers may be different manifestations of a single layer.

The lipid phase is a single molecular layer of cholesterol esters, low polarity lipids, free fatty acids and waxes. This layer is about 0.1 micrometer thick and floats on the aqueous layer. Produced mainly by the meibomian glands, the lipid layer thickens, stabilizes and retards evaporation of the aqueous layer.

Basic secretion, a constant slow flow of tears, is produced by all of the orbital secretory glands. Reflex secretion, an increased rate of tearing caused by neural stimulation, occurs in the main lacrimal gland. However, there is some evidence that all of the glands are regulated and can therefore respond to challenges and changes in the environment, aiding in the maintenance of a stable and constant tear film.

Corneal hydration, thickness and transparency are primarily determined by the balance of fluid entering the stroma via its epithelium and endothelium barriers and the metabolically driven egress of fluid through the endothelium and into the aqueous humor. To maintain a proper balance, the corneal epithelium must be healthy. This requires constant and adequate POTF corneal wetting. Even brief absence of this leads to epithelial cell desiccation, and more prolonged absence produces epithelial damage and stromal dehydration.

Distribution By Eyelids

The blink has a profound influence on the structure, stability and function of the tear film. In addition to its obvious protective function, blinking smoothes the anterior surface of the mucin layer, spreads and thins the lipid layer within the palpebral aperture and possibly stimulates the discharge of meibomian and goblet cell secretions. The sharply edged posterior border of the lid acts as a squeegee in removing depleted tears during the closing phase of the blink and in applying a fresh layer during the opening phase.

Tears are eliminated from the eye by a pump system which is activated by blinking. Each punctum opens into a short vertical canaliculus which turns nasally into the horizontal canaliculus. The superior and inferior horizontal canaliculi merge into the common canaliculus which opens into a reservoir, the lacrimal sac. Each blink compresses the lacrimal sac, which forces its contents down through the naso-lacrimal duct into the inferior meatus of the nose. Opening the lids re-expands the sac, creating negative pressure within the canaliculi and drawing tears in through the puncta from the lacrimal lake.

If the eyelid tear system functions properly, the periodic motion of the eyelids during blinking distributes the precorneal fluid and wets the cornea and contact lens. The upper eyelid acts as a windshield wiper, moving downward and then upward with blinking. The movement is much quicker in the downward phase than in the upward phase. Blink characteristics for a given person are somewhat consistent, but they vary significantly among people. During a blink, the lower eyelid makes a horizontal, transverse, nasal movement. Lower eyelid movement
facilitates removal of debris from the eye by carrying the marginal men-iscus toward the medial canthus for drainage into the punctum. Nearly half the precorneal fluid volume is contained within the marginal meniscus, which acts as a variable reservoir. With a full blink, the upper eyelid draws fluid from this reservoir and moves it over the eye as the upper eyelid is retracted. There is no transfer of fluid during the interblink period (Figure 6).

The problems created by dry eyes are aggravated by poor lid congruity to the front of the eye or by poor blinking. A normal blink is classically described as one in which the upper and lower eyelids come in contact during the closure phase. However, most people do not have a full blink; rather, the upper eyelid often moves about halfway over the cornea.

Blink frequency is about 10 to 15 times per minute, but it decreases when people concentrate on tasks such as reading. Exercises or training procedures to improve blink amount or frequency seldom produce long-term alterations. Contact lens wear, especially improperly fitted rigid contact lenses, can alter blink rate or amount. Blink inhibition can reduce the cleaning and wetting of the anterior surface of contact lenses, reduce contact lens movement and the interchange of precorneal fluid between the contact lens and the cornea and can lead to desiccation and staining of the cornea, especially in areas not covered by the contact lens.

Tear flow is essential to maintain the epithelial surface and to remove cellular debris. Many types of cells are in the precorneal fluid, including desquamated corneal epithelial cells, conjunctival cells, leukocytes, keratinized squamous cells and bacteria. Cell shedding is a normal part of epithelial turnover, and contact lens wear might decrease the turnover time, especially with extended wear. This may relate to corneal hypoxia and a reduction in mitosis in the basal layer.