Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

evolved. An arc transducer follows the curvature of the cornea and permits the visualization of the entire cornea and anterior segment. The analog signal is digitized and can be used to construct a 3D-representation of the anterior segment. The required use of a water bath – as a fluid coupler – may limit the applicability of the UBM.

UBM is used to examine many pathological conditions of the cornea, ocular adnexa, tumors of the anterior segment, intraocular lens, and diseases of the sclera. Because the UBM can image intraocular structures, it can be used to view intraocular cysts, narrow anterior chamber angle, and intraocular foreign bodies.

Cornea section [14], 12/6/2007, OD

University of Minnesota

# 1/1: 285 m

(e)

Cornea section [7], 12/6/2007, OD

University of Minnesota

# 1/1: 536 m

(f)

Figure 2 Confocal microscopy permits the examination of each layer of the cornea. (a) The superficial corneal epithelium appears as a cellular mosaic of nucleated cells. (b) The basal corneal epithelial cells are smaller than the more superficial layers

Cornea section [10], 2/28/2008, OS

University of Minnesota

# 1/1: 581 m

Figure 3 A confocal micrograph of the corneal endothelium of a patient with Fuchs’ dystrophy. The scant number of corneal endothelial cells is evident.

Anterior Segment Optical Coherence

Tomography

Anterior segment optical coherence tomography (OCT) is an imaging technique that can provide detailed in vivo visualization of the anterior chamber. This noninvasive, noncontact device uses OCT to produce direct crosssectional images that can be measured and used for diagnostic purposes.

Using low coherence tomography, the light is set along two different optical paths: a sample path into the eye and a reference path of the interferometer. The light source is a 1310-nm superluminescent light-emitting diode (SLD). The light returning from the sample and reference paths are then combined at the photo-detector. The strength of the return signal is a measure of the reflectance of a small volume of

of epithelium. (c) Just below the corneal epithelium lies the sub-basal nerve layer. This fine meshwork of corneal nerves is responsible for the exquisite sensitivity of the cornea.

(d) Confocal images of the anterior corneal stroma reveal the nuclei of the keratocytes. Contrasting the density of the anterior stromal keratocyte nuclei with the keratocyte density in (e), demonstrates that there is a greater density of keratocytes in the anterior stroma. (e) This confocal image of deep corneal stroma reveals a field of keratocyte nuclei and a corneal nerve (hyper-reflective linear structure). (f) The hexagonal mosaic of the corneal endothelium is seen with the confocal microscope.

S/W version: 1.2.0.1 Patient ID: Gopher, Goldie High res. corneal

180

University of Minnessota

OD OS

0

0.14 mm

tissue. Varying the optical lengths of the reference paths at each of the scanning points determines the axial depth of the tissue signal. By moving the scanning spot across the eye, multiple A-scans align to form a two-dimensional image.

There are many versatile uses of this device in assessing the anterior segment. The anterior chamber dimensions (depth, diameter, etc.) can accurately be measured. The iris and pupil – as well as the crystalline, pseudophakic, and refractive lens implants – can also be evaluated. A highly detailed evaluation of the angle structures can be assessed as well. In vivo measurements of the cornea can be evaluated in aid of surgical and refractive procedures as well as in diagnosing pathological processes. The device can measure post-LASIK corneal flap and residual stromal bed thickness, as well as produce full-thickness pachymetry maps which can assist both in refractive and glaucoma surgical planning (Figure 4). With the growing number of Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) surgeries being performed, this tool has found an additional role in post-surgical lenticle-placement evaluation.

Despite its numerous uses, using light as its medium limits its penetration into the eye. For example, ciliary body tumors cannot always be adequately visualized. In addition, image quality can be greatly reduced when imaging dense corneal opacities.

Acknowledgments

We would like to acknowledge the support of Research to Prevent Blindness and the Minnesota Lions Club.

See also: Corneal Dystrophies; Corneal Endothelium: Overview; Corneal Epithelium: Cell Biology and Basic Science; Corneal Nerves: Anatomy; Corneal Scars; The Corneal Stroma; Ocular Mucins; Overview of Electrolyte and Fluid Transport Across the Conjunctiva; Refractive Surgery and Inlays; The Surgical Treatment for Corneal Epithelial Stem Cell Deficiency, Corneal Epithelial Defect, and Peripheral Corneal Ulcer.

Further Reading

Chiou, A. G., Beuerman, R. W., Kaufman, S. C., and Kaufman, H. E. (1999). Confocal microscopy in lattice corneal dystrophy. Graefe’s Archive for Clinical and Experimental Ophthalmology 237: 697–701.

Chiou, A. G., Kaufman, S. C., Beuerman, R. W., Maitchouk, D., and Kaufman, H. E. (1999). Confocal microscopy in posterior polymorphous corneal dystrophy. International Journal of Ophthalmology 213: 211–213.

Dhaliwal, J. S., Kaufman, S. C., and Chiou, A. G. (2007). Current applications of clinical confocal microscopy. Current Opinion in Ophthalmology 18: 300–307.

Kaufman, S. C., Musch, D. C., Belin, M. W., Cohen, E. J., Meisler, D. M., Reinhart, W. J., Udell, I. J., and Van Meter, W. S. (2004). Confocal microscopy: A report by the American Academy of Ophthalmology. Ophthalmology 111(2): 396–406. (Review.)

Ledford, J. and Sanders, V. (2006). The Slit Lamp Primer, 2nd edn. New York: Slack Inc.

Ramos, J. L., Li, Y., and Huang, D. (2009). Clinical and research applications of anterior segment optical coherence tomography – a review. Clinical and Experimental Ophthalmology 37(1): 81–89.

Wylegała, E., Teper, S., Nowin´ska, A. K., Milka, M., and Dobrowolski, D. (2009). Anterior segment imaging: Fourier-domain optical coherence tomography versus time-domain optical coherence tomography. Journal of Cataract and Refractive Surgery 35(8): 1410–1414.

Glossary

Adherens junctions – Protein complexes that occur at cell–cell junctions, involving calcium-dependent homophilic interactions of a family of transmembrane proteins called cadherins.

Connexin – Gap-junction proteins; family of structurally related transmembrane proteins that assemble to form vertebrate gap junctions. Each gap junction is composed of two hemichannels, or connexons, which are themselves each constructed out of six connexin molecules.

Corneal dystrophies – Group of disorders characterized by a noninflammatory, inherited, bilateral opacity of the cornea.

Crystallins – Water-soluble structural proteins found in the lens of the eye, accounting for the transparency of the structure.

Ectomesenchyme – It has similar properties to mesenchyme. The major difference is that ectomesenchyme arises from neural crest cells, which are a critical group of cells that form in the cranial region during early vertebrate development. Glycan – It refers to a polysaccharide or oligosaccharide. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan. Glycosaminoglycans – Long, linear carbohydrate polymers that are negatively charged under physiological conditions, due to the occurrence of sulfate and uronic acid groups.

Hurler’s syndrome – Known as mucopolysaccharidosis type I (MPS I), Hurler’s disease, or gargoylism, this genetic disorder results in the buildup of mucopolysaccharides due to a deficiency of alpha-L iduronidase, an enzyme responsible for the degradation of mucopolysaccharides in lysosomes. Without this enzyme, a buildup of dermatan sulfate occurs in the body. Symptoms appear during childhood and early death can occur due to organ damage.

Keratan sulfate – Also called keratosulfate, it is any of several sulfated glycosaminoglycans (structural carbohydrates) found especially in the cornea, cartilage, and bone. Keratan sulfates are large, highly hydrated molecules which, in joints, can act as a cushion to absorb mechanical shock. Keratocytes – The basic cell type found in the corneal stroma. The keratocytes are sparse in

distribution, occupying less than 5–10% of the stromal volume.

Lamellae – A lamella is a thin plate-like structure, often one among many lamellae very close to one another, with open space between.

Lumican – Also known as LUM, it is a human gene. This gene encodes a member of the small, leucinerich proteoglycan (SLRP) family that includes decorin, biglycan, fibromodulin, keratocan, epiphycan, and osteoglycin. In these molecules, the protein moiety binds collagen fibrils and the highly charged hydrophilic glycosaminoglycans regulate interfibrillar spacings. Not only is lumican the major keratan sulfate proteoglycan of the cornea, but it is also distributed in interstitial collagenous matrices throughout the body. Lumican may regulate collagen fibril organization and circumferential growth, corneal transparency, and epithelial cell migration and tissue repair.

Macular corneal dystrophy – An autosomal recessive condition, which is the least common but the most severe of the three major stromal corneal dystrophies. It is characterized by multiple, graywhite opacities that are present in the corneal stroma and that extend out into the peripheral cornea. Mesenchyme – Loosely organized connective tissue present in the embryo regardless of origin. Viscous in consistency, mesenchyme contains collagen bundles and fibroblasts.

Myofibroblast – Cell with a phenotype between a fibroblast and a smooth muscle cell in differentiation. It can contract by using smooth muscle-type actin–myosin complex, rich in a form of actin called alpha-smooth muscle actin. These cells are then capable of speeding wound repair by contracting the edges of the wound.

Neural crest – Transient component of the ectoderm, located between the neural tube and the epidermis of an embryo during neural tube formation. Neural crest cells migrate during neurulation, an embryological event marked by neural tube closure. Proteoglycan – Special class of glycoproteins that are heavily glycosylated. They consist of a core protein with one or more covalently attached glycosaminoglycan (GAG) chain(s).

Scheie’s syndrome – Mildest form of mucopolysaccharidosis type I (MPS I) (see Hurler’s syndrome).

Sutural fibers – The elasmobranch (sharks, skates, and rays) cornea resists swelling because of unique structural adaptations in the stroma called sutural fibers. These are collagen fibers running anterior-to- posterior, perpendicularly to the stromal lamellae, tying the anterior limiting lamella to Descemet’s membrane.

Transforming growth factor-beta – Transforming growth factor-beta (TGF-b) is a small protein-growth factor with a broad array of functions. It controls proliferation, cellular differentiation, and other functions in most cells. It plays a role in immunity, cancer, and heart disease.

Stromal Anatomy

In humans, the cornea has a diameter of about 11.5 mm and a thickness of 0.5–0.6 mm in the center and 0.6–0.8 mm at the periphery. Almost 90% of the human cornea is composed of stroma (Figure 1(a)). By weight, water in the extracellular matrix makes up 65% of the stroma and cellular water 11%. This hydration is roughly similar to that of cartilage, but surprisingly, it is higher than that of bone, muscle, or adipose tissue. As discussed below, the proteoglycans of the stroma are hydrophilic and, given free access to water, the tissue will imbibe up to 10-fold its normal content of water. Thus the tissue composition, particularly its hydration, is a dynamic property.

The majority of the stroma consists of thin sheets (lamellae) of tightly packed collagen fibrils. In human corneas, there are up to 200–250 such lamellae, each about 2-mm thick. Within each lamella, the collagen fibrils run parallel to the corneal surface, parallel to one another, and regularly spaced. Ends of the fibrils are not abundant and thus fibrils extend essentially the entire width of the cornea. The lamellae are largely self-contained, but occasional bundles of fibrils extend from one lamella to another. Directional orientation of the fibril layers varies between neighboring lamellae. In the posterior stroma the fibril orientation is almost perpendicular from one layer to the next. Anteriorly, it is more oblique. This arrangement of layers of parallel rigid rods in a friable matrix is not unlike that of composite structural materials such as fiberglass or reinforced concrete. Such an arrangement gives the cornea its remarkable toughness and tensile strength.

Numerous electron micrographic studies have documented the striking regularity of the collagen in the stroma. The collagen fibrils in central stroma have a diameter of about 31 nm and the distribution of diameters is narrow, giving a remarkably homogeneous distribution of collagen within each fibril bundle. Within the fibril bundles electron-dense material, identified as proteoglycans,

encase the individual fibrils and form bridges between neighboring fibrils (Figure 1(b)). In central stroma of normal human cornea, the proteoglycan bridges occur at highly regular intervals, and the bridging structures are of uniform lengths, about 1.8 nm. Such tight interaction between fibrils is considered to participate in generating the parallel alignment of the fibrils in each bundle and the highly regular spacing between neighboring fibrils. As discussed below, this lattice-like structure of the collagen fibrils in the central stroma is thought to be essential for corneal transparency.

The anterior portion of most corneal stromas is limited by an acellular layer of dense, irregularly organized collagen immediately subjacent to the epithelial basement membrane. This anterior limiting lamella (ALL) is also known as Bowman’s layer or Bowman’s membrane. In human corneas, the ALL is about 10-mm thick in the central cornea and absent in the periphery. ALL is also prominent in chickens and some other terrestrial mammals, but it is very thin or not detected in other species such as felines. Collagen fibrils in the ALL are randomly interwoven to form a dense, felt-like sheet composed primarily of collagen types I, III, and V. Collagen VII, associated with anchoring fibrils of the overlying epithelium, is also present within the ALL. The posterior of the ALL merges with the lamellar stroma via oblique fibril bundles, recently revealed using two-photon microscopy. These connecting fibers appear to serve as a stabilizing feature, anchoring the anterior lamellae to the more rigid ALL and indirectly to the epithelial basement membrane. These anterior anchoring fibers appear analogous to the wellknown sutural fibers present in corneas of some species of sharks. The sutural fibers traverse the cornea perpendicular to the orientation of the lamellae and prevent the stroma from swelling when exposed to water. The oblique anterior anchoring fibrils recent identified in human corneas may provide a similar function in that studies show the swelling of human corneas in water occurs almost exclusively in central and posterior stroma. The extremely dense collagen in the ALL has prompted speculation that it may serve as a defense against bacterial or viral infection; however, little hard evidence supports such a role.

Stromal Development

The embryonic origins of the corneal stroma were detailed in a elegant study by Hay and Revel in 1969. In developing chicks, a wave of migrating neural crest cells, destined to become corneal endothelium, moves between the overlying ectoderm and the lens during early embryogenesis. Shortly thereafter, the epithelium secretes an acellular layer of matrix, termed the primary stroma, which becomes hydrated and swells before a second wave of neural crest cells move into the stroma and begins active

secretion of the abundant extracellular matrix of the stroma. The orientation of collagen in the primary stroma is thought to direct formation of lamellae elaborated by the invading stromal cells. At day 14 (before hatching at day 21), the stroma undergoes dehydration in response to thyroxine, leading to thinning of the stroma and initiation of stromal transparency. Mammalian corneas show a somewhat different developmental pattern. No obvious primary stroma is present, and the endothelium and stromal cells are formed after a single influx of neural crest cells. After the endothelium is formed, neural crest cells in the stroma begin to elaborate new extracellular matrix. In mice and rabbits, cells in the stroma are mitotically

active until after birth. In mice, approximately at the time of eye opening (12–14 days postnatal) a decrease in the number of stromal cells occurs along with thinning and dehydration of the stroma. Simultaneously, the cornea-specific glycosaminoglycan – keratan sulfate – appears in the stroma, the stromal cells become quiescent, stromal proteins known as corneal crystallins accumulate in keratocytes, and transparency of the cornea increases significantly. Human stroma, by contrast, matures earlier in development. Keratan sulfate and corneal transparency are observed in embryos at 10–12 weeks of gestation, and infants are born with fully transparent corneas. Despite these species-related differences, the pattern of neural

crest population of the stromal space, secretion of cornealspecific matrix components following formation of the endothelial barrier, and corneal thinning due to dehydration present a common pattern of stromal development.

Stromal Cells

Cells populating the adult stroma are known as keratocytes. Although the same term has been used for fish epidermal cells, corneal keratocytes are neural crestderived mesenchymal cells (ectomesenchyme). In adult tissue, the keratocytes are located between collagenous lamellae, exhibiting a flattened cell body with numerous extended cellular processes (Figure 1(c)). Keratocytes are the population of cells responsible for deposition and maintenance of the extracellular matrix which provides strength and transparency to the cornea. This population of cells is highly interconnected via their processes with numerous junctions between neighboring cells. At sites of contact, gap junctions containing connexin 43 and adherens junctions with cadherin 11 are present. A localized stimulus, such as an epithelial scratch, activates keratocytes throughout the stroma, as indicated by uptake of the dye neutral red. Such a global response to a localized stimulus demonstrates presence of a network of active cell–cell communications maintained by interconnected keratocytes. Following maturity, keratocytes remain quiescent, almost never exhibiting mitotic nuclei or uptake of DNA precursors. Studies of keratocyte turnover in mammals find half-lives too long to estimate accurately. DNA of keratocytes in adult humans shows a high incidence of acquired chromosomal abnormalities, but that of infants and children does not. This fact suggests that keratocytes do not regularly enter the cell cycle in which DNA repair is initiated, but accumulate damage from environmental exposure to light and oxidative stresses over long periods of time. Thus, in the nonwounded cornea, keratocytes may exist for years or decades without turnover.

In spite of their quiescence, keratocytes maintain an active synthesis of the extracellular matrix. Proteoglycan secretion is maintained at a high rate throughout life. Because collagen becomes cross-linked in adult corneas, however, collagen turnover is reduced in adult keratocytes compared to that in embryonic tissue. Adult keratocytes also retain the ability to enter the cell cycle and divide. Nearly 100% of keratocytes isolated by collagenase digestion of bovine corneas enter the cell cycle when exposed to serum or mitogens. Collagenase-isolated primary keratocytes cultured in serum-free conditions maintain the keratocyte phenotype, as defined by quiescence – a dendritic morphology (Figure 1(c)) – and expression of high levels of cornea-specific products (Table 1); however, when exposed to serum or other mitogens, these cells dedifferentiate into a fibroblastic phenotype similar to

that of other mesenchymal cells cultured in serum. Following short-term culture in serum, some keratocyte properties return under quiescent conditions, but after multiple passages in culture transition to the fibroblast phenotype appears to be irreversible. When cultured corneal fibroblasts are exposed to transforming growth factor-beta (TGF-b), the cells express mRNA and protein for alpha-smooth muscle actin and assume a contractile phenotype known as the myofibroblast. TGF-b also induces a range of new gene products, many associated with corneal scarring or fibrosis in vivo. Genes and gene products which have been identified in corneal fibrotic (scar) tissue and also as products of myofibroblasts are shown in the top rows of Table 1. These include cellassociated markers such as Thy-1, matrix proteins (fibronectin, SPARC, tenascin c, etc.), collagens, proteoglycan proteins (biglycan), and glycosaminoglycans. The last four entries of Table 1 show components expressed at high levels in keratocytes which are reduced or disappear in corneal scars and are not expressed by myofibroblasts. These include corneal crystallins (such as ALDH3), the keratan sulfate proteoglycan keratocan, and the cellsurface marker CD34. As described below, the characteristic changes in collagens, crystallins, and proteoglycans occurring during keratocyte–myofibroblast transition directly impact stromal transparency. Thus, loss of transparency in extracellular matrix deposited in response to wound healing can be directly attributed to responses of the keratocytes to wounding. Smooth muscle actin-containing cells appear in healing corneas 1–2 weeks following wounding, and this appearance can be blocked with antibodies to TGF-b. This inhibition suggests that in vivo, as in vitro, TGF-b is an important inducer of fibrotic scar tissue. Several weeks following healing of corneal wounds, smooth muscle actin-containing cells are no longer seen in the wound area. It not clear, however, that the loss of smooth

Table 1 Markers of stromal fibrosis

muscle actin expression corresponds to a return to normal stromal extracellular matrix production. In fact, experimental animal studies show expression of fibrotic markers for months following wounding, and human scar tissue can persist for many decades.

In addition to keratocytes, other cell types have been observed in the stroma. Most prominent are bone marrowderived cells. Mice in which bone marrow cells express green fluorescent proteins have about 10% of stromal cells demonstrating fluorescence. Most stromal leucocytes express the CD11b marker, but not other dendrite, granulocyte, T-cell, or NK markers, placing them in the monocyte/macrophage lineage. Following minor damage to the epithelium, the number of green inflammatory cells in the stroma increases dramatically within a few hours. These transient inflammatory cells are primarily neutrophils.

A population of stem or progenitor cells has also been identified in the corneal stroma. Transplantation of avian corneal keratocytes into the neural crest migratory pathways of early embryos showed that some of the transplanted cells changed phenotype, migrating and differentiating into a variety of neural crest-derived tissues, thus demonstrating that progenitor cell potential is maintained following stromal differentiation. In mammals, similarly, stromal cells with stem cell properties can be isolated using a variety of techniques including spheroid culture, cloning, and fluorescence-activated cell sorting. These cells exhibit multipotent differentiation, clonal growth, and expression of a number of stem-cell-associated genes. These stromal stem cells do not express characteristic keratocyte markers, but can do so under selected culture conditions or when injected into corneal stroma in vivo. These stromal stem cells can restore transparency to lumican null mice with stromal haze, suggesting they may be appropriate for cell-based therapy of corneal diseases. The stromal stem cells appear to reside in the anterior region of the stroma near the limbus, but it is currently unknown what specific roles they play in corneal maintenance or repair.

Transparency

The cornea is one of the few complex biological tissues with high transparency to light, and the biophysical properties which allow such transparency have been the subject of debate for decades. In most transparent tissues such as the lens, all structures have a similar index of refraction so that light is not scattered as is passes through the tissue. Birefringence studies of the cornea, however, demonstrate that the corneal collagen has an index of refraction different from the extrafibrillar matrix surrounding it, and thus should scatter light, producing an opalescent or white appearance. David Maurice proposed, in 1957, that the highly regular structure of the collagen fibrils in the stroma

functioned like a crystal, producing destructive interference of scattered light, allowing nonscattered light to pass. Mathematical models by McCally, Farrell, and others supported this concept, suggesting that, as long as the collagen fibrils were small, tightly packed, and aligned, transparency was possible. The crystal-like regularity of the stromal collagen allows for analysis using classical techniques such as X-ray diffraction. In a large number of such studies, Keith Meek and collaborators have confirmed the high regularity of spacing of nearest neighbor collagen fibrils and loss of that regularity in virtually every instance in which the cornea loses transparency. These studies have confirmed stromal hydration as one of the most critical parameters in maintenance of transparency. Excess water is adsorbed by the proteoglycans between collagen fibrils, disrupting the critical spacing and eliminating the order essential for transparency. Stromal transparency is consequently a highly dynamic property, dependent both on the removal of water from the tissue by the pumping action of the endothelial layer and by the water-binding properties of the stromal proteoglycans.

Recently, a new aspect of corneal transparency has come to notice with the discovery of abundant soluble proteins in keratocytes and other cellular tissues of the cornea. These proteins, termed corneal crystallins, are typically enzymes with housekeeping functions present in corneal cells at concentrations vastly exceeding that in other tissues. This group of proteins includes isoforms of aldehyde dehydrogenase (ALDH), transketolase, and up to a dozen or more unrelated enzymes. The variety of proteins involved and their unusual abundance has led to the suggestion that – like lens crystallins – these proteins serve to alter the refractive index of cells, thus reducing light scatter by corneal cells. Studies by Jester and coworkers have shown light scatter by keratocytes correlates with abundance of corneal crystallins in these cells, supporting a role for crystallins in stromal transparency.

Stromal Extracellular Matrix

Collagens are the most abundant proteins in the cornea, and most of the collagen in the stroma is involved in the collagen fibrils of the lamellae. Studies by Birk and coworkers have shown that fibrils in the stroma are heterotypic, containing both types I and V collagens in the same fibril. The triple-helical domain of the type V collagen molecules is buried within the fibril with its NH2-terminal domains exposed at the fibril surface. These exposed domains alter lateral association of collagen molecules during fibrillogenesis and, therefore, limit fibril diameter. The abundance of collagen type V is a likely factor in the small diameter of the stromal fibrils compared to fibrils in dermis and sclera, which contain mostly collagen types I and III. In corneal scarring and

haze, collagen type III is elevated in the stroma suggesting a direct correlation between the abundance of the small type I and V fibrils and corneal transparency.

Type XII collagen is found associated at regular intervals with stromal fibrillar collagen. The long form of this fibril-associated (FACIT) collagen can be modified with chondroitin sulfate, making it also a fibril-associated proteoglycan. Mice expressing a truncated form of type XII show altered collagen fibril spacing in the stroma. In addition to fibrillar collagen, stroma is very rich in type VI – a nonfibrillar collagen. This protein interacts with cells and numerous components of the stroma forming a beaded network throughout the stroma. Type VI microfibrils can be visualized running perpendicularly to the fibrils in lamellae and may help stabilize the lamellar structure. Numerous other collagen types have also been identified in the stroma in small amounts. Type IV collagens, often associated with basement membrane, are seen in the stroma to be associated with keratocytes. One of these, alpha3(IV) is downregulated as keratocytes become activated by wound healing or mitogens, and thus serves as a marker of keratocyte phenotype.

Proteoglycans are the second most abundant component of the corneal stroma. The corneal proteoglycans all belong to the small leucine-rich proteoglycan (SLRP) family, consisting of proteins of about 40 kDa, decorated with several N-linked oligosaccharides and one to two glycosaminoglycan chains. Normal adult stroma has one proteoglycan protein, decorin, which is modified with dermatan sulfate and three more SLRP proteins– lumican, keratocan, and osteoglycin (mimecan) – which have keratan sulfate chains. During healing, a fifth SLRP protein, biglycan, is detected in the stroma. Biglycan joins decorin as a second dermatan sulfate-containing proteoglycan in scar tissue. These SLRP proteoglycans contain multiple leucine repeat regions (LRR) – motifs involved in protein–protein binding. Each of the stromal proteoglycans binds collagen in a repeating pattern along the length of the fibrils with the keratan sulfate-containing SLRP’s binding sites different from that of decorin. X-ray studies and molecular modeling reveal that SLRP proteins fold into compact horseshoe-shapes with the glycosylation on the convex portion of the curve and the collagen-binding region on the inner face. Thus, proteoglycan association with collagen produces glycosaminoglycan chains protruding from the fibril with a ‘bottle brush’ like appearance, as documented by deep freeze etch electron microscopy (Figure 1(b)). The interactions between proteoglycan and collagen has an effect on the rate and size of the fibrils formed during fibrillogenesis, effecting the diameter and length of the collagen fibrils, and thus the physical properties of the tissue. Decorin-knockout mice thus have weakened skin, whereas lumican-knockout mice exhibit large and heterogeneous collagen fibrils in the posterior corneal stroma leading to corneal haze. These in vivo models confirm the importance

of the SLRP proteins in maintenance of the stromal ultrastructure required for vision. In addition to structural roles, the SLRP proteins interact directly with cells and growth factors. Decorin activates the EGF receptor on cell surfaces and also binds to and inactivates TGF-b, leading to reduced fibrosis in experimental models. Lumican stimulates attachment of macrophages and also has been shown to stimulate the healing of corneal epithelial wounds. Lumican-knockout mice show reduced responsivness to lipopolysaccharide-induced septic shock, and poor induction of proinflammatory cytokines. Lumican core protein also binds the CXC-Chemokine KC (CXCL1) and thus regulates neutrophilic infiltration. The SLRP proteins, therefore, clearly play important roles in mediating the response of the stroma to injury and inflammation.

The glycosaminoglycan chains (glycans) – decorating SLRP proteins – constitute about half of the molecular weight of the proteoglycans. Because of their high level of sulfation, these glycans are hydrophilic, providing the impetus for influx of water into the stroma against which the endothelium provides an active pump. Dermatan sulfate is a widespread and abundant glycan which, in the cornea, is less highly sulfated than in skin or sclera. Keratan sulfate, while detectable in many tissues, is present in abundance only in cartilage and cornea. In addition, only in the cornea are lumican, keratocan, and mimecan glycanated with keratan sulfate, making the stromal keratan sulfate proteoglycans an abundant, structurally unique, tissue-specific class of matrix molecules. Keratan sulfate binds water differently than dermatan sulfate, and it is clear that the ratio of these glycosaminoglycans in the stroma is important for stromal transparency. In genetic diseases such as Scheie’s and Hurler’s syndromes, dermatan sulfate cannot be degraded and accumulates in the cornea. In such cases, intense corneal opacity occurs early in life. In macular corneal dystrophy, keratan sulfate is not properly sulfated. In this disease, the cornea loses transparency in the second decade of life. It is notable that, in scar tissue, keratan sulfate is absent or greatly reduced, whereas dermatan sulfate is more abundant and highly sulfated. It is thought that such long-term differences in the glycosaminoglycan content of scar tissue contributes to light scattering as a result of differential water binding by the two types of glycosaminoglycan.

Biosynthesis of glycosaminoglycans is controlled differently from that of the proteins to which they are attached. For example, TGF-b treatment of keratocytes in vitro causes little change in lumican or decorin secretion; however, keratan sulfate modifying the lumican becomes dramatically shorter and virtually unsulfated during this treatment and dermatan sulfate modifying the decorin becomes longer and much more highly sulfated. These changes in glycanation can have dramatic effects on the properties of the proteoglycan. Lumican with short, unsulfated glycan chains serves as an

attachment substratum for macrophages and stimulates cell migration. Lumican modified with highly sulfated keratan sulfate, on the other hand, is antiadhesive and serves as a barrier for migrating cells. Thus the state of the glycan chains of the SLRP proteins serves not only to regulate water binding, but also other biological properties of the molecules. Modification of synthesis of the glycosaminoglycan is complex and not yet fully understood. Initiation, elongation, and sulfation of dermatan sulfate requires the participation of up to 16 different glycosyltransferase and sulfotransferase enzymes. For keratan sulfate, not all of participating enzymes are known as yet. One important gene has been clearly identified, however. CHST6 codes for a cornea-specific keratan sulfotransferase. This enzyme is mutated or nonfunctional in macular corneal dystrophy, leading to undersulfation of keratan sulfate.

In healing wounds and fibrotic corneas, not only are keratan sulfate and dermatan sulfate altered but another glycosaminoglycan, hyaluronan (HA), is present. This glycan, not present in normal corneal tissue, appears rapidly in the stroma in most pathological conditions and remains for many months following healing. The hyaluronan biosynthetic enzyme HAS2 is upregulated in keratocytes in response to mitogens and TGF-b. Mice lacking HAS2 in the stroma do not express HA in response to induced inflammation, demonstrating that upregulation of HAS2 mRNA in the keratocytes is the source of HA during stromal pathology. Hyaluronan is a simple, unsulfated, acidic polysaccharide but is known to exhibit a large number of biological activities. It is associated with cell motility, inflammation, and with metastatic potential of cancer cells. In culture, knockdown of HAS2 mRNA reduces keratocyte ability to respond to TGF-b with fibrotic matrix components. These results suggest that HA might represent an extracellular signal that mediates scarring in the stroma.

A final class of matrix components important to stromal function are the noncollagenous matrix proteins. Some of these are listed in Table 1. Another member of this group is protein BIGH3 (TGF-b-inducible gene H3). This secreted protein interacts with collagen and contains an RGD amino-acid sequence. It promotes cell attachment and in a number of systems the BIGH3 protein inhibits cell growth and motility. In the cornea, it has been shown that mutations in the gene coding for this protein (TGFBI) lead to a variety of corneal dystrophies presenting as opaque deposits in the stroma, usually in adults. Some of the specific syndromes caused by BIGH3 are granular dystrophy, Groenouw type I, Reis–Bucklers, lattice dystrophy type I, and Avellino dystrophy. The discovery that a minor component of the stromal extracellular matrix leads to marked disruption of vision attests to the complex biophysical equation involved in maintenance of stromal transparency. There are likely to be more important components of this system yet to be discovered.

Conclusion

The corneal stroma is a physically tough tissue with the remarkable property of transparency to light. Stromal transparency is essential for vision, and corneal scarring obscures vision for millions of individuals worldwide. Although we have gained an understanding of how stromal fibrosis disrupts vision, we do not yet understand how this process might be reversed biologically. An important challenge for the future is to employ our understanding of stromal biology to design pharmaceutical or cell-based treatments to reverse the scarring process and restore the complex balance of cells, molecules, and water in order to provide vision for those affected by corneal blindness.

Acknowledgments

The authors wish to thank Kira Lathrop and Martha Funderburgh for the excellent illustrations. This work was supported by NIH Grant EY016415 and Research to Prevent Blindness Inc.

See also: Artificial Cornea; Corneal Dystrophies; Corneal Imaging: Clinical; Corneal Scars.

Further Reading

Birk, D. E. (2001). Type V collagen: Heterotypic type I/V collagen interactions in the regulation of fibril assembly. Micron 32(3): 223–237.

Farrell, R. A., McCally, R. L., and Tatham, P. E. (1973). Wave-length dependencies of light scattering in normal and cold swollen rabbit corneas and their structural implications. Journal of Physiology 233(3): 589–612.

Fini, M. E. (1999). Keratocyte and fibroblast phenotypes in the repairing cornea. Progress in Retinal and Eye Research 18(4): 529–551.

Guerriero, E., Chen, J., Sado, Y., et al. (2007). Loss of alpha3(IV) collagen expression associated with corneal keratocyte activation.

Investigative Ophthalmology and Visual Science 48(2): 627–635. Hay, E. D. and Revel, J. P. (1969). Fine structure of the developing avian

cornea. Monographs in Developmental Biology 1: 1–144.

Jester, J. V. (2008). Corneal crystallins and the development of cellular transparency. Seminars in Cell and Developmental Biology 19(2): 82–93.

Maurice, D. M. (1957). The structure and transparency of the cornea.

Journal of Physiology 136(2): 263–286.

McCally, R. L. and Farrell, R. A. (1982). Structural implications of small-angle light scattering from cornea. Experimental Eye Research 34(1): 99–113.

Meek, K. M., Leonard, D. W., Connon, C. J., Dennis, S., and Khan, S. (2003). Transparency, swelling and scarring in the corneal stroma.

Eye (London, England) 17(8): 927–936.

Meek, K. M. and Quantock, A. J. (2001). The use of X-ray scattering techniques to determine corneal ultrastructure. Progress in Retinal and Eye Research 20(1): 95–137.

Morishige, N., Petroll, W. M., Nishida, T., Kenney, M. C., and Jester, J. V. (2006). Noninvasive corneal stromal collagen imaging using two- photon-generated second-harmonic signals. Journal of Cataract and Refractive Surgery 32(11): 1784–1791.