During infectious ulceration of the corneal stroma, enzymes that degrade the ECM of the stroma are released by both host cells and the infecting bacteria. Furthermore, pseudomonal elastase degrades collagen directly as well as promotes collagen degradation by keratocytes, in part, via activation of pro-MMPs.^[186] Thus, there appear to be at least three different pathways for the degradation of stromal collagen fibers in individuals with infectious corneal ulceration: (1) direct degradation by bacterial collagenase, (2) degradation by MMPs released from keratocytes (or myofibroblasts) activated by bacterial factors such as elastase, or (3) activation by infiltrated inflammatory cells.

Cytokines and growth factors

Both keratocytes and infiltrated cells, such as lymphocytes, neutrophils, and macrophages, secrete cytokines or growth factors and modulate behaviors of cells in the healing corneal stroma. Each cytokine or growth factor activates signal transduction pathways that regulate the expression of specific genes that contribute to the inflammatory response. Targeting of such regulation at the ligand or signaling level may provide new strategies for treatment of wound-related pathology. TGF-β is thought to play a key role in healing of the corneal stroma.^[187],[188] It is expressed by both epithelial cells and stromal cells (keratocytes or scleral fibroblasts) as well as by inflammatory cells that activate stromal cells and promote their transdifferentiation into myofibroblasts. Myofibroblasts contribute not only to wound repair but also to post-injury stromal scarring in the cornea and sclera as a result of the overproduction of matrix components. Blockade of TGF-β signaling effectively reduces the fibrogenic reaction and consequent scarring and opacification in a mouse model of corneal alkali burn.^[187],[188]

The proinflammatory cytokine tumor necrosis factor (TNF)-α is also up-regulated in response to injury.^[189] TNF-α induces various effects in the cornea under pathological conditions such as injury, allergy, and infection.^[190–193] However, the complete loss of TNF-α in the cornea of knockout mice results in enhancement of post-alkali burn inflammation, suggesting that the role of TNF-α in the cornea might depend on the specific condition.^[194]

Neovascularization in the corneal stroma

The cornea is an avascular tissue and must remain transparent in order to refract light properly. Neovascularization in the cornea resulting from inflammation associated with various conditions such as trauma, microbial infection, or alkali burn or from limbal stem cell deficiency can thus impair vision. Cytokines and growth factors orchestrate cell behavior associated with the development of corneal neovascularization. Vascular endothelial growth factor (VEGF) and TGF-β thus contribute to injury-induced neovascularization, with ECM components supporting the growth of new vessels. These factors are up-regulated in the corneal stroma (in both inflammatory cells and resident cells) during wound healing or inflammatory disorders.^[195]

Development of the Anterior Eye Segment

Characterization of the development of ocular tissues during embryogenesis is important for understanding the pathogenesis of congenital anomalies of the cornea and anterior eye segment (Fig. 1.14).^[196],[197] Morphogenesis of the eye is achieved by cell lineages of various origins including the surface and neural ectoderm during embryonic development. Epithelial cells of the cornea are derived from the epidermal ectoderm, whereas keratocytes, scleral fibroblasts, and endothelial cells are of neural crest (neuroectodermal) origin. The surface ectoderm above the neuronal optic cup invaginates to form the crystalline lens. After the lens vesicle has separated from the surface ectoderm, the epithelium on the immature lens differentiates into the corneal epithelium. Neural crest-derived mesenchymal cells migrate in the space between the lens and primitive corneal epithelium and develop into the corneal stroma, endothelium, iris, and trabecular meshwork. Many anomalies of the anterior eye segment result from impaired differentiation of these neural crest-derived tissues.

The surface ectoderm above the optic cup invaginates during the fifth week of gestation in humans, and the primitive corneal epithelium develops junctional complexes by the sixth week. Most scleral fibroblasts differentiate from neural crest cells that surround the optic cup during the sixth week. Mesodermal cells also contribute to development of the sclera and the extraocular muscles. The neural crest cell-derived mesenchyme migrates into the space between the primitive corneal epithelium and lens vesicle in three waves during the seventhh week. The first wave of migration results in formation of the corneal endothelium and trabecular endothelium; the second wave of cells differentiates into keratocytes; and the third wave gives rise to the iris. During the eighth week, the keratocytes form five to eight layers of collagen lamellae and the corneal endothelium starts to form Descemet's membrane. Defects in the migration of neural crest-derived mesenchymal cells are responsible for anomalies of the cornea and anterior eye segment including Peters’ anomaly. Several genes, including those encoding TGF-β2 and the transcription factor FOXC, have been implicated in the differentiation of neural crest cells into the primitive corneal stroma in mice.^[198]

The spaces among collagen fibers become occupied by proteoglycans that are formed as a result of the binding of glycosaminoglycan chains to previously accumulated core proteins. Even by the sixth month of gestation, the cornea is still not fully mature. The epithelium has only three or four layers of cells, and keratan sulfate proteoglycans continue to accumulate. During the seventh month, however, the cornea is well developed, with the epithelium consisting of four or five layers with readily recognizable basal, wing, and superficial cells. The stroma is also almost fully developed at this time, with the accumulation of keratan sulfate proteoglycans among collagen fibers being virtually complete. Hyaluronan is a major glycosaminoglycan in the corneal stroma during the early stages of embryonic development, but its abundance declines concomitantly with the increase in that of keratan sulfate, chondroitin sulfate, and dermatan sulfate, giving rise to a glycosaminoglycan composition similar to that of the adult stroma.^[199]

Recent advances in transgenic and gene knockout technologies in mice have provided important insight into the role of specific genes in the development and homeostasis of corneal tissue as well as into congenital anomalies in humans.^[199],[200] Interpretation of such studies also depends on an understanding of the normal process of eye development in the mouse (see Fig. 1.14). The surface ectoderm invaginates into the optic cup at embryonic day (E) 10.5 in mice. At E12.5, the primitive lens has already separated from the surface ectoderm, which will become the corneal epithelium, and the neural crest-derived mesenchyme has begun to migrate into the space between the primitive corneal epithelium and lens. In contrast to the human embryo, the neural crest-derived cells migrate into this space in a single wave. At E14.5, the embryo has already developed the epithelium, stroma, and endothelium of the cornea, and at E18.5 the corneal stroma has increased in thickness as a result of the synthesis of matrix macromolecules. The upper and lower eyelids fuse to each other between E14.5 and E16.5; the eyelids separate and the eyes reopen after birth, and the corneal epithelium then undergoes final maturation.