Numerous glycoprotein and glycolipid molecules are embedded in the cell membrane of epithelial cells. These oligosaccharide-containing molecules form floating particles in the membrane that are collectively termed the glycocalyx and which confer hydrophilic properties on the anterior surface of the superficial epithelial cells. The glycocalyx interacts with the mucinous layer of the tear film and helps to maintain the trilayered structure of the latter.^[72],[73] Loss either of the glycocalyx of corneal epithelial cells or of goblet cells in the conjunctival epithelium results in tear film instability and the mucin-deficiency form of dry eye.

The superficial cells of the corneal epithelium are joined by desmosomes, adherens junctions, and tight junctions (see Figs 1.4, 1.5, and 1.6), which prevent the passage of substances through the intercellular space. Examination of fluorescein penetration into the corneal stroma with a fluorophotometer provides a measure of the barrier function of the corneal epithelium.^[74]

Wing cells

Beneath the superficial cells lie two to three layers of wing cells, so called because of their characteristic winglike shape. Wing cells are in an intermediate state of differentiation between basal and superficial cells and are rich in intracellular tonofilaments composed of keratin (see Table 1.2). The cell membranes of adjacent wing cells are interdigitate. Numerous desmosomes, adherens junctions, and gap junctions are present between wing cells (see Figs 1.4, 1.5, and 1.6).

Basal cells

The single layer of columnar basal cells of the corneal epithelium rests on the basement membrane. Basal cells, unlike superficial and wing cells, possess mitotic activity, and they differentiate consecutively into wing and superficial cells (see Table 1.2). Neighboring basal cells interdigitate laterally and are joined by desmosomes, gap junctions, and adherens junctions. The posterior surface of basal cells is flat and abuts the basement membrane.

Basal cells adhere to the basement membrane via hemidesmosomes that are linked to anchoring fibrils of type VII collagen secreted by themselves (see Fig. 1.4).^[75],[76] The anchoring fibrils penetrate the basement membrane and course into the stroma, where they form anchoring plaques together with type I collagen, a major component of the stroma. The adherens junctions are present at the lateral surface of the basal cells of the corneal epithelium and are thought to mediate cell–cell interaction.^[77]

Members of the integrin family of cell surface receptors for extracellular matrix components (ECM) proteins exist as heterodimers of α and β subunits.^[78] The integrin α5β1 heterodimer, which is the major receptor for fibronectin, is present at the surface of basal cells in the normal corneal epithelium.^[79–83] All epithelial cells undergoing active migration after debridement of the corneal epithelium express integrin α5β1.^[83],[84]

Basement membrane

As in epithelia in other parts of the body, basal cells of the corneal epithelium are anchored to a basement membrane. The presence of the basement membrane between the basal epithelium and the underlying stroma fixes the polarity of epithelial cells. Ultrastructurally, the basement membrane, which is 40–60 nm thick, is composed of a pale layer (the lamina lucida) immediately posterior to the cell membrane of the basal epithelial cells as well as an electron-dense layer (the lamina densa) (see Fig. 1.4). Type IV collagen and laminin are major components of the basement membrane (Fig. 1.7).

The basement membranes of the corneal and conjunctival epithelia contain different type IV collagen chains, although the functional relevance of this difference is unknown. Whereas collagen α5(IV) is present in the corneal basement membrane, collagen α2(IV) is present in the conjunctival basement membrane (as well as in the amniotic membrane) (Fig. 1.8).

Bowman's layer

An acellular, membrane-like zone known as Bowman's layer, or Bowman's membrane, is detectable by light microscopy at the interface between the corneal epithelium and stroma in humans and certain other mammals (but not in rodents). Given that this structure, which is 12 µm thick, is not a membrane but rather a random arrangement of collagen fibers and proteoglycans, the term Bowman's layer is preferable. The collagen fibers in Bowman's layer are primarily collagen types I and III. The diameter of these fibers is 20–30 nm, which is smaller than that of the collagen fibers present in the corneal stroma (22.5–35 nm) (see Fig. 1.4).

Bowman's layer is considered to be the anterior portion of the corneal stroma. The anterior surface of this layer, which faces the basement membrane, is smooth. Given that the collagen fibers in Bowman's layer are synthesized and secreted by stromal keratocytes, they appear continuous with those in the stroma.

Biological functions originally attributed to Bowman's layer are now thought to be mediated by the basement membrane. Bowman's layer does not regenerate after injury. Recent clinical experience with excimer laser photoablation demonstrates that a normal epithelium is formed and maintained even in the absence of Bowman's layer. Furthermore, many mammals do not have a Bowman's layer but still exhibit a well-organized epithelial structure. The physiological role of Bowman's layer therefore remains unclear.

Stroma of the cornea and sclera

Overview

The stroma constitutes the largest portion, more than 90%, of the thickness of the cornea. The peripheral portion of the cornea connects to the anterior sclera at the limbus, where the tissue loses its transparency. Many characteristics of the cornea, including its physical strength, stability of shape, and transparency, are largely attributable to the anatomic and biochemical properties of the stroma. The uniform arrangement and continuous slow turnover (production and degradation) of collagen fibers in the stroma are essential for corneal transparency.

The sclera is also composed mostly of collagen fibers and other matrix macromolecules, but nonuniformity in the arrangement of these fibers accounts for its lack of transparency.^[85] The thickness of the scleral stroma ranges from approximately 0.5 nm to 1.0 mm depending on the area, with the exception of the sites of insertion for the rectus muscle, where the sclera is thinnest. The toughness of the scleral stroma is essential for its role as a container of the intraocular tissues. Scleral fibroblasts are embedded within the collagen lamellae.

Cells

Keratocytes are the predominant cellular components of the corneal stroma and are thought to turn over about every 2 to 3 years. The spindle-shaped keratocytes are scattered among the lamellae of the stroma (Fig. 1.9). These cells extend long processes, and the processes of neighboring cells are connected at their tips by gap junctions (Fig. 1.10).^[86] The three-dimensional network structure of keratocytes can be observed by light microscopy in flat preparations of the corneal stroma by confocal biomicroscopy, and, after digestion of stromal collagen, by scanning electron microscopy (Fig. 1.10).^[87]

Keratocytes are similar to fibroblasts and possess an extensive intracellular cytoskeleton, including prominent actin filaments. Keratocytes are thus quiescent in the normal cornea but are readily activated and undergo transformation into myofibroblasts, that express α-smooth muscle actin, in response to various types of insult to the stroma.^[88] Myofibroblasts produce ECM, collagen-degrading enzymes, matrix metalloproteinases (MMPs), and cytokines for stromal tissue repair, and their ability to contract contributes to wound closure.

Although scleral fibroblasts are not as well characterized as keratocytes, they are thought to be similar to fibroblasts in other parts of the body. As in the corneal stroma, a slow turnover of collagen fibers by scleral fibroblasts is required for connective tissue homeostasis. Matrix degradation by scleral fibroblasts is promoted by prostaglandin derivatives, which accounts in part for the increase in uveoscleral outflow of aqueous humor and the reduction in intraocular pressure induced by such drugs.^[89] Activation of scleral fibroblasts by external stimuli, such as injury or surgery, also results in their transdifferentiation into myofibroblasts and consequent tissue fibrosis.

Collagen

The cellular components (mainly keratocytes) occupy only 2–3% of the total volume of the corneal stroma,^[90] with the remaining portion comprising mostly the ECM components collagen and proteoglycans. Collagen constitutes more than 70% of the dry weight of the cornea. Collagen in the corneal stroma is mostly type I, with smaller amounts of types III, V, and VI also present.^[91–100] Proteoglycans are distributed among the major collagen fibers.