Background
A corneal ulcer is defined as a disruption of the epithelial layer with involvement of the corneal stroma. This condition is associated with inflammation, either sterile or infectious.
The primary purpose of this article is to highlight the pathogenesis of noninfectious stromal ulceration. The infective causes and mechanisms of autoimmune ulcerative keratitis, particularly peripheral, are not included within this article.
Pathophysiology
An understanding of the pathophysiology of sterile corneal ulceration requires a review of the processes involved in epithelial and stromal wound healing, as well as an examination of the role of precorneal tear film, corneal nerves, proteolytic enzymes, and cytokines.
Epithelial wound healing
Corneal ulceration always begins with an epithelial defect. A persistent epithelial defect allows the corneal stroma to be exposed to the external environment and permits the process of stromal degradation.
Within minutes after a small corneal epithelial injury, cells at the edge of the abrasion begin to migrate centripetally to cover the defect rapidly at a rate of 60-80 µm/h. A longer delay of 4-5 hours is seen in larger defects. This delay is required for preparatory cellular changes prior to rapid cell movement.
Epithelial cells adjacent to the area of the defect flatten, lose their hemidesmosome attachments, and migrate on transient focal contact zones that are formed between cytoplasmic actin filaments and extracellular matrix proteins. Vinculin, a plasma protein, links fibers to talin, which is a cell membrane protein. It, in turn, is linked to integrin. Contraction of actin fibers pulls the cell body forward. Vinculin, integrin, fibronectin, fibrinogen, and fibrin are formed continuously and cleaved to allow for cell migration. Plasmin is the protease responsible for cleaving fibrinogen and fibrin at these focal contact zones.
The basement membrane is also important for epithelial migration, and abnormalities in basement membrane structure, whether due to trauma (eg, recurrent erosion syndrome) or dystrophy (eg, basement membrane dystrophy), can lead to persistence of corneal epithelial defects and stromal ulceration.
After 24-30 hours, mitosis begins to restore epithelial cell population. Basal and limbal stem cells contribute to mitosis. A sufficient supply of progenitor stem cells to facilitate epithelial cell proliferation is important for the cornea. A deficiency of limbal stem cells, from either disease (eg, aniridia) or trauma (eg, chemical burn), can preclude adequate epithelial wound healing.
Stromal wound healing
Stromal wound healing occurs via stromal keratocyte migration, proliferation, and deposition of extracellular matrix molecules, including collagen (specifically type III), adhesion proteins (eg, fibronectin, laminin), and glycosaminoglycans. These processes are facilitated by a phenotypic change among quiescent keratocytes to become active myofibroblasts, a task mediated by transforming growth factor-beta (of presumptive epithelial origin).
Stromal necrosis and degradation
The corneal wound repair process is intricately linked to a complex inflammatory response that must be precisely regulated to ensure proper healing.
Invasion of monocytes/macrophages is critical in wound healing; however, in the corneal stroma, excessive infiltration of monocytes/macrophages is considered to be unfavorable because they secrete matrix metalloproteinases (MMPs) and other proteins undesirable for tissue healing. Numerous cytokines and growth factors that are up-regulated in corneal cells further contribute to tissue inflammation.
Matrix metalloproteinases (MMPs) are a group of structurally related endopeptidases that require a metal cofactor. The main function of metalloproteinases is to degrade extracellular matrix and basement membrane components. MMP-2 and MMP-9 are known as gelatinases and are involved in cleaving collagen types IV, V, VII, and X, as well as fibronectin, laminin, elastin, and gelatins. MMP-1 (neutrophil collagenase) and MMP-8 (fibroblast or keratocyte collagenase) are involved in cleaving collagen types I, II, and III.
Barely detected in an unwounded cornea, MMPs are strongly induced during wound healing. Metalloproteinases are secreted as proenzymes by neutrophils infiltrating the wound, injured epithelial cells, and keratocytes. They are activated by proteolytic cleavage of the N-terminal region in the extracellular compartment. In vivo, tissue inhibitors of metalloproteinases (TIMPs) inhibit collagenase activity by blocking activation of MMPs. TIMPs represent a multigene family that includes at least 4 members.
A relatively higher degree of collagenolysis relative to synthesis is thought to result in degradation, progressive corneal thinning, and, hence, ulceration of the corneal stroma. Up-regulation of MMPs leads to an imbalance between MMPs and TIMPs, leading to the pathology. MMPs are induced at the transcriptional level by various cytokines and growth factors, such as interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), and transforming growth factor-beta (TGF-beta).
Synthetic inhibitors of mammalian metalloproteinase (SIMP) have been shown to effectively inhibit corneal ulceration when started earlier in treatment, as well as in established ulcers. [1] . Many matrix metalloproteinase inhibitors (MMPIs) have been studied; however, selective MMPIs to target corneal MMPs are being sought.
Extracellular matrix metalloproteinase inducer (EMMPRIN), or CD147, is a cell membrane glycoprotein enriched on epithelial cells during corneal wound healing. It has been shown that it is up-regulated on epithelial cells by EGF and TGF-beta. This, in turn, induces fibroblasts, by direct interaction, to increase their own level of EMMPRIN, leading to induction of MMP. Inhibition of EMMPRIN may represent a promising future therapeutic strategy in situations of excess extracellular matrix degradation associated with chronic wound healing. [2]
Since all metalloproteinase enzymes require metal cofactors Ca2+ and Zn2+, such chelating agents as ethylenediaminetetraacetic acid (EDTA), acetylcysteine, and penicillamine inhibit collagenase activity; however, these agents have been found to have limited efficacy in vivo. Topical 3% N-acetylcysteine has shown a beneficial effect on corneal wound healing. Tetracyclines also possess anticollagenolytic activity by chelating metal cations. Other possible mechanisms of action include inhibition of gene expression of neutrophil collagenase and epithelial gelatinase, inhibition of alpha1-antitrypsin degeneration, and scavenging of reactive oxygen species. [3]
As a result of collagen breakdown, tripeptide products of collagen are released. These are chemotactic for neutrophils, which migrate into the injured tissue where they release additional MMPs as well as superoxide radicals. These agents potentiate further collagenolytic action and corneal degradation. Superoxide dismutase (SOD) enzymatically reduces the superoxide radical to hydrogen peroxide, thus effectively eliminating highly reactive oxygen metabolites before any further damage. Studies have shown a beneficial effect of lecithinated SOD, which is retained on the ocular surface longer than native SOD when applied as an eye drop solution. [4]
In cells along the leading edge of the wound, there is a specific activation of the Ser/Thr kinase, Cdk5. Cdk5 activity limits the accumulation of active Src. Active Src promotes epithelial cell migration. However, excessive Src activity can also cause degradation of E-cadherin and a complete loss of cell-cell adhesion, so its activity and localization must be stringently controlled. Topical application of a Cdk5 inhibitor, olomoucine, increases the rate of debridement wound closure without causing appreciable dissociation or detachment of epithelial cells. [5]
Matrix regenerating agent, also known as ReGeneraTing Agent (RGTA), includes polymers that mimic extracellular matrix proteins, mainly heparin sulphates and act as a scaffold. RGTA also provides proteolytic defense for epithelial cells as they migrate and proliferate during corneal wound healing. RGTA eye drops have shown benefits in patients with persistent epithelial defects as well as neurotrophic keratopathy. [6]
The role of corneal nerves
The cornea is densely innervated by fibers of the ophthalmic division of the trigeminal nerve and sympathetic nerve fibers from the superior cervical ganglion. Corneal nerves provide important protective and trophic functions, and interruption of corneal innervation may result in altered epithelial morphology and function, poor tear film, and delayed wound healing. Decreased corneal sensation from denervation can result in stromal ulceration and perforation. These ulcers result from decreased metabolic and mitotic rates in the corneal epithelium and reduced acetylcholine, choline acetyltransferase, and substance P concentrations.
In 1954, the classic experiment by Sigelman et al demonstrated that ocular surface changes associated with neurotropic keratitis in denervated animals persist despite tarsorrhaphy, suggesting a trophic effect of the corneal nerves. [7] Evidence suggests that sensory neuron loss leads to a severe depletion of acetylcholine in an otherwise acetylcholine rich tissue, resulting in a relative decrease in epithelial cell growth.
Depletion of substance P associated with sensory denervation can lead to changes associated with neurotrophic keratitis by affecting both epithelial cell and keratocyte migration. Substance P binds to the high-affinity neurokinin-1 receptor (NK-1R). It has been reported that substance P administered with insulinlike growth factor 1 (IGF-1) or EGF synergistically facilitates corneal epithelial migration and adhesion. Nakamura and coworkers (1999) determined that only the four-amino-acid sequence (FGLM) from the C terminal of substance P is necessary. [8] In addition, synthetic peptide containing four amino acids from C terminal of IGF-1 was found to have synergistic effect on corneal epithelial cell migration. This finding has implications for the clinical use of topically applied neuropeptides, since full-length peptides are more readily degraded and inactivated by peptidases and are associated with more side effects. [9]
Restoration of corneal sensation with corneal neurotization, a peripheral sensory nerve graft, is a recent and novel surgical technique to treat neurotrophic keratopathy. Corneal neurotization could be direct which involves transferring ipsilateral or contralateral supraorbital and supratrochlear ophthalmic branches of the trigeminal nerve or indirect with sural nerve transfer. The physiology of reinnervation likely occurs from a combination of direct sprouting from proximal nerve endings and the release of neurotrophic factors. [10]
The role of the precorneal tear film in ulceration
The exposure of the bare corneal stroma to its environment secondary to deficient or impaired epithelial wound healing is thought to contribute to stromal degradation through environmental factors, cytokines, lytic enzymes, and neutrophils in the tear film. Direct neutrophil adhesion to the corneal stroma theoretically allows hydrolytic and collagenolytic enzymes, including MMP-8 (neutrophil collagenase), to contribute to the degradation of the corneal stromal extracellular matrix.
Dohlman et al (1969) and subsequently Kenyon et al (1979) demonstrated that a glued on methylacrylate lens applied to a rabbit alkali burn model of corneal ulceration protected the stroma from collagenolysis by neutrophils and injured epithelial cells. [11, 12] Keratocyte fibroblasts also may contribute to this milieu. The prevention of neutrophil infiltration and the promotion of epithelialization are thought to be at least some of the mechanisms responsible for the beneficial effect of amniotic membrane graft use in preventing stromal ulceration. [13]
In addition, cytokines, such as hepatocyte growth factor (HGF), keratocyte growth factor (KGF), and EGF, are produced by the lacrimal gland and, thus, are present in tears. HGF is up-regulated in response to corneal injury in parallel with increased aqueous tear production. In the wounded cornea, these cytokines may play an important role in regulating epithelial healing. Inflammatory cytokines, including IL-1alpha, are detectable in normal human tears and may be important in causing further degradation of the corneal stroma, either directly by inducing keratocyte apoptosis or by recruiting inflammatory cells via their chemotactic properties.
Also, an irregular tear film and a decreased tear film breakup time over the area of the bare stroma can cause a dellen effect that may contribute to an unfavorable cellular environment for the viability and proliferation of stromal keratocytes.
The role of cytokines
The complex autocrine and paracrine functions of the cytokines involved in the interactions between the corneal epithelium and stromal keratocytes are important in achieving the appropriate responses to corneal wound healing. While their precise triggers and interactions are still being elucidated, cytokines can induce and mediate many of the fundamental steps involved in wound healing.
Epithelial cell migration, proliferation, and differentiation are influenced by the stromal keratocyte cytokines, KGF and HGF. These cytokines are modulated further in vivo by the effects of other cytokines and truncated receptors of these molecules.
In what is likely to be merely the tip of the iceberg with respect to the understanding of cytokine-cytokine interactions, both KGF and HGF mRNA production are altered by the fibroblast cytokines, EGF, TGF-alpha, PDGF, and IL-1. In addition, EGF, PDGF, IL-1alpha, IL-6, and TNF at low concentrations appear to enhance fibronectin (FN)-induced epithelial cell migration.
Not to be eclipsed by stromal influences, epithelial cells modulate important keratocyte responses to epithelial cell injury. Keratocyte wound healing processes, including MMP production and regulation, HGF and KGF production, and keratocyte apoptosis, are mediated via various cytokines, including stimulators like IL-1 and soluble Fas ligand and major inhibitor TGF-beta2.
Beyond keratocyte cell death caused by mechanical injury or necrosis associated with neutrophil infiltration, IL-1– and Fas ligand–mediated apoptosis is an important stromal response to epithelial injury. Since both of these cytokines can be produced by keratocytes, autocrine modulation of these responses may occur. IL-1 and PDGF also regulate MMP expression in stromal keratocytes.
Autologous serum and umbilical cord serum harbor many growth factors and neuropeptides like EGF, TGF-beta, vitamin A, fibronectin, substance P, IGF-1, NGF, and other cytokines. Treatment with autologous serum and umbilical cord serum eye drops seem promising for the restoration of the ocular surface epithelial integrity in patients with persistent epithelial defect, chemical burns, and severe dry eye syndrome.
Platelets are storage pools of growth factors, including platelet-derived growth factors, TGF-beta, EGF, FGF, IGF- I, and VEGF. Autologous platelet-rich plasma has a large quantity of growth factors that have been found to promote the healing of dormant corneal ulcers and dry eye syndrome. Platelet-rich plasma has shown a good safety profile and may have better efficacy than autologous serum. [14]
Platelet-activating factor (PAF) is a potent bioactive lipid that is generated in the cornea after injury. PAF is a receptor-mediated strong inflammatory mediator, a chemotactic to inflammatory polymorphonuclear leukocytes, and an inducer of several proteases that degrade the extracellular matrix. Corneal epithelial cells, keratocytes, and endothelial cells express the PAF receptor, and, in corneal epithelial cells, injury up-regulates PAF receptor gene expression. The role of PAF receptor antagonists in preventing corneal injury is under investigation.
Plasminogen is synthesized in the cornea and can be activated to plasmin by a plasminogen activator. This synthesis is stimulated by IL-1alpha and IL-1beta. In turn, plasmin is able to activate latent collagenase. Studies have demonstrated that uPA (urokinase plasminogen activator), but not tPA (tissue plasminogen activator), is induced in the migrating epithelial cells during corneal epithelial wound healing. Amiloride, a specific uPA inhibitor, effectively decreases uPA activity in the cornea as well as in the tear fluid and favorably affects corneal healing.
Amniotic membrane extract eye drops (AMEED) can be utilized for tissue healing in severe cases of ocular damage. AMEED contains several diverse growth factors including EGF, HGF, bFGF, protease inhibitors, and the HC-HA/PTX3 complex. Several studies have demonstrated the efficacy of AMEED therapy to improve ocular surface burns, ulcerations, and epithelial damage and may have potential use in treating limbal stem cell deficiency. [15]
Thymosin beta-4 is a water-soluble polypeptide that interferes with NF-κB signaling pathways to promote corneal wound healing and to decrease inflammation. Studies have shown the promising ability of Tβ4 as an anti-inflammatory agent to promote epithelial repair. [16]
Nerve growth factor NGF may improve corneal nerve sensitivity via the release of several neuropeptides in addition to its trophic effect. NGF directly promotes proliferation and differentiation of epithelial cells and indirectly by increasing substance P and other peptides. Recombinant human nerve growth factor (rhNGF), has recently been FDA approved in the U.S. as a therapy for neurotrophic keratitis. [17]
Tofacitinib citrate inhibits the Janus kinase (JAK) pathway, which is critical for immune cell activation, proinflammatory cytokine production, and cytokine signaling, hence reducing the inflammatory processes that lead to corneal ulcerations. [18]
Cytokines and trophic factors from the corneal nerves, tear film, conjunctiva, conjunctival vessels, endothelium, and anterior chamber may have important modulating effects on corneal epithelial and stromal healing responses and, thus, corneal ulceration.
Epidemiology
Frequency
United States
The incidence rate depends on the etiology of the corneal ulcer.
Mortality/Morbidity
Corneal scarring, decreased vision, neovascularization, perforation, and blindness are associated with this condition.
Sex
Because of an increased incidence of injuries, this condition may be seen more frequently in males than females.
