Both the mean diameter of collagen fibers and the mean distance between such fibers in the corneal stroma are relatively homogeneous and are less than half of the wavelength of visible light (400–700 nm). This anatomic arrangement is thought to be responsible for the fact that scattering of an incident ray of light by each collagen fiber is canceled by interference from other scattered rays,^[4] allowing light to pass through the cornea. If the diameter of or the distance between collagen fibers becomes heterogeneous (as occurs in fibrosis or edema), incident rays are scattered randomly and the cornea loses its transparency.

Procollagen molecules are secreted by keratocytes into the extracellular space, after which the propeptides at both ends are cleaved to yield the mature collagen molecules. The collagen molecules self-assemble into fibrils with a diameter of 10–300 nm, and these fibrils subsequently further assemble into collagen fibers. Individual collagen fibers in the corneal stroma can be observed by transmission electron microscopy (see Fig. 1.9). As mentioned above, both the diameter of (22.5–35 nm)^[99],[101] and distance between (41.4 ± 0.5 nm)^[102] collagen fibers in the corneal stroma are highly uniform, with this regular arrangement being a major determinant of corneal transparency. At high magnification, each collagen fiber exhibits a characteristic cross-striation pattern with a periodicity of 67 nm (see Fig. 1.9). In the corneal stroma, the collagen fibers form about 300 lamellae.^[103] Each lamella courses parallel to the surface of the cornea from limbus to limbus. The turnover of collagen molecules in the cornea is slow, requiring 2 to 3 years.

The histological features of the scleral stroma are similar to those of the corneal stroma, with the scleral stroma also being composed largely of major collagen fibers and proteoglycans.^[104] The collagen types detected in the scleral stroma are also similar to those in the corneal stroma. In contrast, the matrix components present in the spaces between the major collagen fibers in the scleral stroma differ from those in the corneal stroma. This difference in the noncollagenous matrix largely accounts for the difference in ultrastructure between the cornea and sclera. Whereas the collagen fibers in the corneal stroma are highly uniform in diameter, those in the scleral stroma range in diameter from 25 to 250 nm. Furthermore, whereas collagen fibers are arranged regularly with a relatively uniform interfiber distance in the corneal stroma, the distance between collagen fibers in the scleral stroma varies. The ECM of the scleral stroma, including both collagen and noncollagenous components, is produced by the stromal fibroblasts.

Proteoglycans

Proteoglycans, the major matrix components located in the spaces among major collagen fibers in the stroma of the cornea and sclera, are composed of a core protein and glycosaminoglycan chains and are thought to modulate collagen fibrillogenesis.^[105] Glycosaminoglycans themselves also play important roles regardless of the core protein to which they are attached. The functions of proteoglycans can thus be considered from the points of view of both the core protein and glycosaminoglycans.

With the exception of hyaluronan (hyaluronic acid), the glycosaminoglycans of the corneal stroma are present in the form of proteoglycans. The most abundant glycosaminoglycan in the cornea is keratan sulfate,^[106] constituting about 65% of the total glycosaminoglycan content. The remaining glycosaminoglycans include chondroitin sulfate and dermatan sulfate. Glycosaminoglycans have the ability to absorb and retain large amounts of water. Although corneal hydration is regulated predominantly by an endothelial pump, it is also influenced by the epithelial barrier, surface evaporation, intraocular pressure, and stromal swelling pressure. The tendency of the stroma to swell results from interfibrillary imbibition of fluid and repulsion between the fixed negative charges on keratan sulfate and chondroitin sulfate. This swelling tendency has been termed the swelling pressure (SP) and is approximately 50 mmHg in the excised cornea. The negative pressure that draws fluid into the cornea is termed the imbibition pressure (IP), which, in the excised cornea, is equal to the swelling pressure. In vivo, however, the imbibition pressure is lower than the swelling pressure because of the compressive effect of intraocular pressure (IOP). The relationship between these three parameters is described by the equation:

An appreciation of the dynamics of corneal edema therefore requires an understanding of the role of the stromal ground substance (glycosaminoglycan chains of proteoglycans) in the hydration state (and hence the clarity) of the cornea. If the pump function of the corneal endothelium is lost, the corneal stroma swells, leading to a disturbance in the regular spacing between collagen fibers. The irregularity of the interfiber distance results in scattering of incident light and renders the cornea hazy.

In terms of core proteins, the corneal stroma contains lumican, keratocan, and mimecan (osteoglycin) as keratan sulfate proteoglycans as well as decorin and biglycan as chondroitin sulfate or dermatan sulfate proteoglycans (Table 1.3).^[107],[108] These core proteins are members of the family of small leucine-rich proteoglycans (SLRPs), which contain a common central domain consisting of about 10 leucine-rich repeats.^[109] They first accumulate as low-sulfate glycoproteins in the embryonic stroma and subsequently bind glycosaminoglycans to form proteoglycans typical of the adult cornea. Although the roles of specific proteoglycans in the maintenance of corneal transparency or shape under physiological conditions or in the development of corneal haziness under pathological conditions remain unclear, spontaneous mutation of a core protein gene has provided some insight. Mutation of the keratocan gene was recently shown to result in cornea plana, an anomaly characterized by abnormal corneal curvature, but it did not affect the transparency of the corneal stroma.^[110],[111]

In the normal cornea, proteoglycans are synthesized by stromal keratocytes. They are transiently synthesized by corneal epithelial cells during the early phase of wound healing.

Recent studies with transgenic or knockout mice have also provided insight into the roles of proteoglycan core proteins. Lumican-deficient mice have been shown to undergo age-dependent opacification of the corneal stroma.^[112],[113] Transmission electron microscopy revealed an irregular arrangement of collagen fibers in the posterior stroma of these animals. Humans with a mutated lumican gene have not yet been described, however. Keratocan-deficient mice show a change in the shape of the eye shell, but the transparency of the corneal stroma is not affected.^[110] Mice lacking decorin exhibit abnormal collagen fibrillogenesis in the tail tendon but not in the corneal stroma,^[114] indicating that decorin may not play an important role in maintenance of corneal stromal transparency, despite its abundance in the stroma. Such genetically modified mice not only shed light on the functions of specific molecules but also provide models of human genetic disorders of the cornea.

The main difference between the proteoglycan composition of the sclera and that of the cornea is the absence of keratocan, a specific marker of keratocyte differentiation,^[108] in the sclera. However, this difference alone does not explain the lack of uniformity in the size and arrangement of collagen fibers in the sclera. The relative amounts of proteoglycan components in the sclera are changed in an animal model of myopia.^[115] Recent studies suggest that matrix components of the cornea or sclera play specific roles in regulation of the shape or size of the eye shell. The eyeball of lumican-deficient mice is larger than that of wild-type animals, whereas that of keratocan-deficient mice is smaller.^{[110,112,113]}

Descemet's membrane

Descemet's membrane, the basement membrane of the corneal endothelium, gradually increases in thickness from birth (3 µm) to adulthood (8–10 µm) in humans. Histological analysis reveals it to be stratified into a thin (0.3 µm) nonbanded layer adjacent to the stroma, an anterior banded zone (2–4 µm), and a posterior amorphous, nonbanded zone (>4 µm), the latter of which can represent up to two-thirds of the thickness of the membrane and is laid down over time.^[116]