Descemet's membrane is composed primarily of collagen types IV and VIII and laminin^[117] but also contains fibronectin.^[118],[119] Type VIII collagen, which is produced by the corneal endothelium, forms a hexagonal lattice that is substantially different from the structure of type IV collagen in the basement membrane. Collagen fibrils in the stroma are continuous with those in Bowman's layer but not with those in Descemet's membrane. Descemet's membrane adheres tightly to the posterior surface of the corneal stroma and reflects any change in the shape of the stroma. Rupture of Descemet's membrane by physical stress, such as compression birth injury, results in the penetration of aqueous humor into the corneal stroma and consequent stromal edema. Descemet's membrane does not regenerate after endothelial cells re-cover the ruptured area. Diseases such as Fuchs’ dystrophy are associated with an atypical striated pattern of collagen deposition in Descemet's membrane.^[120] A patient with early-onset Fuchs’ dystrophy was found to harbor a mutation in COL8A2,^[121] which encodes the α2 chain of type VIII collagen.

Endothelium

A single layer of corneal endothelial cells covers the posterior surface of Descemet's membrane in a well-arranged mosaic pattern (Fig. 1.11). These cells are uniformly 5 µm in thickness and 20 µm in width and are polygonal (mostly hexagonal) in shape. The uniformity of endothelial cell size has been evaluated by statistical analysis based on photographs taken by a wide-field specular microscope. In young adults, the cell density is about 3500 cells/mm². The coefficient of variation of mean cell area (standard deviation of mean cell area/mean cell area) is a clinically valuable marker and is about 0.25 in the normal cornea. An increase in the variability of cell area is termed polymegathism. Another morphometric parameter of the state of the endothelium is hexagonality. In the normal healthy cornea, about 70–80% of endothelial cells are hexagonal. However, endothelial damage can result in a decrease in the hexagonality value and an increase in the variability of cell area (Fig. 1.11). Deviation from hexagonality is referred to as pleomorphism.

Endothelial cells contain a large nucleus and abundant cytoplasmic organelles, including mitochondria, endoplasmic reticulum, free ribosomes, and Golgi apparatus (Fig. 1.12), suggesting that they are metabolically active. The endothelial cells interdigitate and contain various junctional complexes, including zonula occludens, macula occludens, and macula adherens. In addition, gap junctions allow the transfer of small molecules and electrolytes between the endothelial cells. The interconnected endothelial cell layer provides a leaky barrier to aqueous humor.

Loss of or damage to corneal endothelial cells results in increased imbibition of water by the corneal stroma. The endothelial cells contain ion transport systems that counteract the imbibition of water into the stroma. An osmotic gradient of Na⁺ is present between the aqueous humor (143 mEq/L) and the stroma (134 mEq/L). This gradient results in the flow of Na⁺ from the aqueous humor to the stroma and in a flux of K⁺ in the opposite direction. The Na⁺- and K⁺-dependent ATPase and the Na⁺/H⁺ exchanger are expressed in the basolateral membrane of corneal endothelial cells. Carbon dioxide also diffuses into the cytoplasm of these cells and, together with water, generates bicarbonate ions (HCO₃⁻) in a reaction catalyzed by carbonic anhydrase. The HCO₃⁻ then diffuses or is transported into the aqueous humor. Coupled with this movement of HCO₃⁻ is a flux of water across endothelial cells into the aqueous humor. Given that this ion transport system is partially dependent on cellular energy, cooling of the cornea results in its thickening and in it becoming opaque. The return of the cornea to normal body temperature, however, results in restoration of its normal thickness and clarity in a phenomenon known as temperature reversal.

Corneal endothelial cells essentially do not proliferate in humans, monkeys, and cats, but they do divide in rabbits. Endothelial cell density in the normal healthy cornea decreases with age.^[122] It is important that corneal endothelial cells are protected during surgery. The loss of endothelial cells for any reason results in enlargement of the remaining neighboring cells and their spreading to cover the defective area, without an increase in cell number. The indices based on specular microscopy fluctuate as endothelial damage is resurfaced by the migration and enlargement of the remaining endothelial cells. The coefficient of variation of mean cell area is the most sensitive index of corneal endothelial dysfunction, whereas hexagonality is a good index of the progress of endothelial wound healing.

Maintenance of Normal Corneal Integrity

Overview

Maintenance of corneal structure is crucial for the physiological roles of this tissue in refraction and biodefense. A smooth epithelium, a transparent stroma, and a functioning endothelium are all essential for clear vision. The cornea is vulnerable to various chemical or biological agents as well as physical events in the outside world. It is, therefore, equipped with an active maintenance system responsible for renewal of the corneal epithelium and wound healing.

The widespread application of corneal surgery, including keratoplasty and refractive surgery, has necessitated a more detailed understanding of recent advances in cellular and molecular biology of corneal wound healing. In most parts of the body, wound healing is initiated by the extravasation of blood constituents that accompanies disruption of blood vessels. The cornea, however, is an avascular tissue. The mechanism of wound healing in the cornea thus differs from that elsewhere in the body.

Epithelial maintenance

Role of limbal stem cells

Corneal epithelial cells renew continuously to maintain the normal layered structure of the epithelium. The centripetal movement of corneal epithelial cells has been well demonstrated, as has the fact that only the basal epithelial cells are capable of proliferation. Thoft and Friend proposed that an equilibrium exists between the centripetal movement of epithelial cells, the differentiation of basal cells into superficial cells, and the desquamation of epithelial cells from the corneal surface (X, Y, Z hypothesis).^[123] The existence of corneal epithelial stem cells at the limbus has also been postulated.^[124] Indeed, the limbal epithelium exhibits a higher proliferative activity and a lower differentiation capability than those of the corneal epithelium, and basal limbal epithelial cells are thought to be a type of undifferentiated stem cell because they do not express corneal epithelium-specific keratin (keratin 3/12).^[125]

Basal epithelial cells of the human limbus express various possible stem cell markers.^[126] The expression of p63, a-enolase, keratin 5/14, and the hepatocyte growth factor (HGF) receptor has also been shown to be higher in the limbal epithelium than in the corneal epithelium. Although no direct evidence for the existence of limbal stem cells has been obtained to date, ABCG2 appears to be the most promising surface marker for the identification of such cells. Deficiency of limbal stem cells has been suggested to result in impairment of corneal epithelial homeostasis in individuals with aniridia, inflammatory disorders of the ocular surface such as Stevens–Johnson syndrome, or severe alkali burn of the ocular surface.^[127]

Epithelial movement

Injury to the corneal surface is not uncommon and results in an epithelial defect, the rapid resurfacing of which is required for restoration of the continuity of the corneal epithelium. Repair of epithelial defects occurs in three distinct phases characterized by epithelial cell migration, proliferation, and differentiation, resulting in restoration of the stratified structure of the epithelium.

Epithelial migration is thus the initial step in the resurfacing of epithelial defects.^[128] Trauma to the corneal epithelium induces the sliding and migration of the remaining epithelial cells adjacent to the injury site toward the defective area.^[129–132] Dynamic changes in cell–cell and cell–matrix (fibronectin–integrin system) interactions, up-regulation of hyaluronan, and modulation of the ECM by newly expressed proteolytic enzymes play important roles in these two types of epithelial cell movement in response to injury. Such changes are under the overall control of growth factors and cytokines.

Fibronectin–integrin system

Fibronectin provides a provisional matrix during the first phase of epithelial wound healing in many tissues.^[133] Fibronectin appears at the newly exposed corneal surface soon after epithelial or stromal injury,^[118],[119] and epithelial cells then attach to and spread over the fibronectin matrix.^[79,134,135]