Corneal Wound Healing And The Role Of Growth Factors

Vol.18, No. 9

September 1996

V

Continuing Education Article

FOCAL POINT ★ Growth factors have exciting potential for use in clinical treatment of uncomplicated and complicated corneal wounds.

KEY FACTS ■ Each layer of the cornea (epithelium, stroma, and endothelium) heals by different mechanisms and is consequently affected by growth factors in different ways. ■ Because it is avascular, growth factors reach the cornea by means of tears, aqueous humor, and limbic vessels. ■ Epidermal growth factor, which helps regulate epithelial cell turnover, is a potent stimulant of wound healing for all corneal layers. ■ Transforming growth factor β stimulates collagen synthesis in other tissues, but its role in healing of the corneal stroma is unclear.

Corneal Wound Healing and the Role of Growth Factors Louisiana State University

Allison Swank, DVM Giselle Hosgood, BVSc, MS, FACVSc

W

ound healing involves many well-characterized cellular activities, including migration, mitosis, and synthesis of extracellular matrix components. Regulation of wound healing has recently been examined at the molecular level. Peptide growth factors, which are produced locally and systemically, act to coordinate and regulate the wound healing process in all tissues of the body, including the cornea. The healing of the cornea is unique because the tissue is avascular. Stimulation and regulation of healing rely on peptide growth factors that reach the wound through the tears and limbic vessels. Clinical trials involving humans and dogs have studied the effect of exogenous growth factors on corneal wounds that heal normally as well as those with impaired healing.1,2 The growth factors evaluated in clinical and experimental studies include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor α (TGF-α), and transforming growth factor β (TGF-β).1–11

ANATOMY OF THE CORNEA The cornea is the clear, transparent anterior portion of the fibrous tunic of the eye; the anatomy of the cornea is similar for all mammalian species (Figure 1). The cornea consists of three distinct layers: the anterior layer or epithelium (0.08 mm in dogs and cats), the intermediate layer or stroma (0.5 to 0.6 mm in dogs and cats), and the posterior layer or endothelium (one cell-layer thick).12 Familiarity with corneal anatomy is important in understanding the mechanism of healing and the effect of the various growth factors on healing. The corneal epithelium is an organized, layered structure. Its superficial portion consists of 4 to 10 layers of squamous cells, below which are 2 to 4 layers of polyhedral (or wing) cells. The innermost structure is a basal columnar layer12; this layer produces the underlying basement membrane and the hemidesmosomes (plaques and connecting fibrils), which attach the epithelium to the stroma. Without the basement membrane, the epithelium can be removed easily from the stroma; this occurs in animals with diseases of the basement mem-

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HEALING OF THE brane. 12,13 Precorneal tear EPITHELIUM film provides a smooth optiEpithelial wounds of the cal surface.12 The outermost cornea heal by reepithelialcells of the epithelium have ization; this process involves small villous projections that migration of cells, mitosis, anchor the tear film to the and cell differentiation cornea.12,13 (Table I). The epithelial The stroma contains ficells at the wound edge bebroblasts, keratocytes, colgin to migrate within hours l a gen, and extracellular of injury. 12 The epithelial matrix. Collagen fibrils are cells that are located behind arranged in an orderly manthe migrating cells begin ner and are spaced evenly in dividing within 24 to 36 the stroma; the even spacing hours.12 The epithelial cells of the collagen fibrils allows that cover the defect differfor transparency of the corentiate to form a stratified nea. Glycosaminoglycans in squamous layer. 12 Corneal the extracellular matrix help epithelium can be commaintain the orderly arpletely replaced in 2 weeks.14 rangement of collagen fibrils If removed, the basement in the cornea. Keratocytes membrane may take weeks can synthesize collagen, glyor months to regenerate; cosaminoglycans, and the until regeneration occurs, mucoprotein of the extracelthe epithelium can be relular matrix. Keratocytes also transform into fibro- Figure 1—Anatomy of the normal cornea. Exocrine and au- moved easily from the strocytes during the initial tocrine regulation of epithelial cell turnover are shown. (EGF ma.12 The importance of the phase of healing. Lympho- = epithelial growth factor, TGF-α = transforming growth fac- basement membrane in epithelial healing becomes obcytes, macrophages, and tor α.) vious in animals with epneutrophils are occasionally ithelial basement membrane present in the extracellular dystrophies, which show delayed and compromised matrix.12,13 healing. The endothelium is a single layer of flat, hexagonal mesothelial cells. The lateral surfaces of these cells inHEALING OF THE STROMA terdigitate; hence, they are not true endothelial cells.13 Uncomplicated wounds of the stroma heal without Between the stroma and endothelial cells is Descemet’s reliance on a blood supply. Leukocytes and keratocytes membrane, a modified basement membrane of corneal proliferate and migrate to the defect by means of the endothelium that, in adult dogs, is roughly four times tear film and limbic vessels12 (Table I). Macrophages reas thick as the endothelium12,13 (Figure 1). Descemet’s move cellular debris. Lost collagen fibrils are temporarimembrane is strong and highly elastic. The endothelily replaced by a fibrin matrix. During the 2 weeks after um is metabolically active and contains many mitoinjury, the fibroblasts proliferate and rapidly synthesize chondria.12,13 collagen and other components of the extracellular maThe epithelium (acting as an external barrier) and trix. Collagen fibrils in the regenerating stroma are disendothelium (acting as a Na +-K +-ATPase pump as organized, and corneal transparency is decreased. Over well as an internal barrier) control the water content time, scar density decreases.12 of the cornea and thus maintain its transparency. A change in the water content would alter the orderly HEALING OF THE ENDOTHELIUM arrangement of the collagen fibrils, thus affecting Unlike the epithelial and stromal cells of the cornea, corneal transparency. Decreased integrity of the epthe endothelial cells in most species do not actively diithelium or endothelium can result in corneal edema; vide but enlarge and migrate to re-form a functional edema disrupts the orderly arrangement of cells and monolayer after injury12 (Table I). The loss of endothecollagen fibers, thereby causing cloudiness of the lium during corneal injury in human, cats, and dogs cornea. STROMA ■ ENDOTHELIUM ■ NORMAL CORNEAL ANATOMY

The Compendium September 1996

results in thinning and spreading of existing cells.15 If loss of endothelium precludes reestablishment of a functional monolayer, a corresponding focal area of corneal edema results.12,15

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adhesion of new corneal epithelium.1,6,17 Several studies of the Corneal Layer Characteristics of Healing effects of EGF in rabbits have demonstrated that Epithelium Migration of epithelium to defect EGF accelerates healing Mitosis of cells to retain number of cells and increases tensile Differentiation of new cells strength (i.e., the amount of tension applied perStroma Secretion of fibrin matrix Migration of fibroblasts into defect CHARACTERISTICS OF pendicular to the wound Mitosis of fibroblasts COMMONLY STUDIED before it tears apart) of Collagen secretion GROWTH FACTORS experimentally induced Reorganization of collagen fibrils Epidermal Growth corneal epithelial ulFactor cers and stromal inciEndothelium Enlargement of cells at edge of defect Epidermal growth facsions3,7,16,17 (Tables II and Migration of defect margin cells into wound tor is the most frequently III). Studies have also Thinning of existing cells investigated growth factor shown that EGF is effecNo occurrence of mitosis for corneal wound healtive for treatment of nating. EGF and TGF-α are structurally similar and have urally occurring nonhealing or recurrent corneal eromany similar effects (Tables II and III). Epithelial cells, sion.1,2 Cells must be exposed to EGF for at least 5 to 6 hours for EGF to impact DNA synthesis and cell stromal fibroblasts, and endothelial cells express EGF proliferation and enhance subsequent healing of the receptors.14 Experimental intraperitoneal injection of 125 I-labeled EGF in rats showed selective increases of racornea.3,17 dioactivity in the cornea and epidermis, thus suggesting Platelet-Derived Growth Factor an affinity of EGF in these tissues.9 Epidermal growth factor increases RNA, DNA, proMany types of cells release or secrete PDGF. Such tein synthesis, and mitosis in corneal epithelium and cells include platelets at the site of injury as well as stromal fibroblasts; this factor is also chemotactic for macrophages, fibroblasts, vascular smooth muscle epithelial and stromal cells.16 EGF may enhance synthecells, and vascular endothelial cells.11,17 In vitro, PDGF sis of structural and attachment proteins (including stimulates synthesis of fibronectin, hyaluronic acid, colfibronectin); these proteins promote migration and lagenase (which is important in remodeling of fibrous TABLE I Characteristics of Normal Healing of the Corneal Layers

TABLE II Summary of Growth Factor Characteristics16 Growth Factor EGF

Molecular Weight (kDa) 6

Peptide Structure

Growth Factors in Family

Location

Monomer

EGF, TGF-α, and other growth factors

Nearly all body fluids, platelets

PDGF and vascular endothelial growth factor

Endothelial cells, platelets, macrophages, fibroblasts

PDGF

28–35

Dimer, A and B chains

TGF-α

5–20

Monomer

TGF-β

25

Dimer of two identical chains

Macrophages, eosinophils, keratinocytes TGF-β1–5; only TGF-β1–3 are found in mammalian cells

Macrophages, lymphocytes, fibroblasts, bone cells, keratinocytes, platelets

EGF = epidermal growth factor, PDGF = platelet-derived growth factor, TGF = transforming growth factor.

NORMAL CORNEAL HEALING ■ GROWTH FACTOR CHARACTERISTICS

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TABLE III Summary of Effects of Growth Factors on Experimental Corneal Wound Healing Growth Factor

Target Corneal Layer

EGF

Epithelium

Present in tears; plays a role in normal epithelial cell turnover Increases epithelial cell proliferation (in vitro) Increases epithelial cell migration (in vitro) Increases epithelial cell adhesion (in vitro) Stimulates cell differentiation (in vivo)

Stroma

Increases number of fibroblasts but has no effect on collagen synthesis (in vitro) Increases wound strength (in vivo)

Endothelium

Increases endothelial cell mitosis (in vivo) Increases endothelial cell density (in vivo) Increases percentage of hexagonal, functional cells (in vivo)

PDGF

Stroma

Increases wound strength (in vivo)

TGF-α

Epithelium

Present in tears and aqueous humor Can stimulate healing, but precise role in healing is unclear (in vivo)

Endothelium

Present in aqueous humor Concentration increases after injury, but precise role in healing is unclear

Epithelium

Decreases epithelial proliferation (in vitro) Has no effect on epithelial cell migration or adhesion alone but antagonizes effect of EGF (in vitro)

Stroma

Increases number of fibroblasts (chemotactic) (in vitro) Increases collagen synthesis (in vitro)

TGF-β

Effect

EGF = epidermal growth factor, PDGF = platelet-derived growth factor, TGF = transforming growth factor.

scar tissue), and other growth factors for corneal wound healing (including PDGF itself )17 (Tables II and III). PDGF may act synergistically with other growth factors during healing of wounds of the cornea and the dermis.11 PDGF receptors are present on corneal epithelial cells, stromal fibroblasts, and endothelial cells.11

Transforming Growth Factor α Transforming growth factor α can stimulate corneal wound healing, but its absence (studied in transgenic mice) does not impair corneal wound healing18 (Tables II and III). A large percentage of transgenic mice that were incapable of producing TGF-α displayed ocular abnormalities of variable incidence and degree, thereby suggesting that TGF-α production is necessary for normal development of the eye.19 Because both EGF and TGF-α can stimulate corneal wound healing alone, it is possible that redundancy has developed in the EGF–TGF-α system, perhaps as a protective adaptation.18

Transforming Growth Factor β Transforming growth factor β can stimulate chemotaxis of inflammatory cells (fibroblasts, macrophages, and monocytes) and synthesis of the extracellular corneal matrix in vitro.16,17,19 TGF-β stimulates production of fibronectin,5,8 stimulates synthesis of collagen (much more potently than EGF),14 and enhances proliferation of stromal fibroblasts.5,19 All three of these actions make TGF-β likely to be useful in healing of the corneal stroma. Immunohistochemical techniques demonstrated the presence of TGF-β in corneal fibroblasts and macrophages after epithelial wounding, thereby suggesting that TGF-β also participates in healing of corneal epithelial wounds6 (Tables II and III). Transforming growth factor β inhibits proliferation of corneal epithelial cells in vitro.6,17 This factor has also been shown to antagonize the stimulatory action of EGF, possibly by downregulation of the EGF receptor. Thus, TGF-β may act as a modulator of EGF effects in

EXPERIMENTAL CORNEAL WOUND HEALING ■ TGF-α ■ TGF-β

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maintaining corneal epithelium.6 Exogenous TGF-β promotes scar formation in wounds; diseases of chronic, progressive scarring with obliteration of normal tissue (e.g., progressive proliferative vitreoretinopathy and hepatic cirrhosis) have been associated with an increased concentration of TGF-β.19 Whether this will impact the clinical use of TGF-β has not been established.

lial and endothelial cells can also synthesize EGF; as a result, epithelial cells can govern their own turnover (and wound healing) via an autocrine pathway. 16 Corneal epithelial cells, fibroblasts, and endothelial cells contain EGF receptors.18 The impaired corneal wound healing that occurs in tear deficiency syndromes in humans has been attributed to lack of EGF and other factors.3

THE ROLE OF FIBRONECTIN AND GROWTH FACTORS Both TGF-β and EGF increase fibronectin synthesis in vitro.5,6 Platelet-derived growth factor also upregulates fibronectin synthesis in vitro.20 Fibronectin is produced by stromal fibroblasts only during wound healing; it is not present in healthy cornea. Fibronectin is a multifunctional extracellular matrix glycoprotein that stimulates cell adhesion, cell migration, and protein synthesis in many cell types.17 Fibronectin stimulates fibroblast migration chemotactically, thus promoting stromal wound healing. Exogenous application of fibronectin has been shown to stimulate wound healing.5 Thus, EGF and TGF-β are likely to be helpful in healing of the corneal epithelium and stroma through increased cell proliferation and synthesis of collagen and fibronectin.5 In vivo studies, however, currently do not support this theory. Further study should clarify the relationship between TGF-β and EGF.

USE OF GROWTH FACTORS The application of growth factors on corneal cells in vitro and in experimentally induced corneal wounds has greatly elucidated the role of growth factors in normal corneal wound healing. The following discussion of the experimental and clinical roles of EGF, TGF-β, and PDGF in corneal wound healing is intended to reveal the potential use of these growth factors for clinical treatment of uncomplicated and complicated corneal wound healing by means of exogenous application.

GROWTH FACTORS IN TEARS AND AQUEOUS HUMOR The normal cornea is continuously exposed to TGFα and EGF.1,16–18 The concentration of EGF in tear film varies among species (0.7 to 8.6 ng/ml in humans).9 The lacrimal gland is the primary source of EGF in tears; mRNA for EGF and TGF-α is present in this gland.18 Chemical irritants increase the amount of EGF released, but the concentration of EGF decreases as a result of the increase in tear fluid production. It is postulated that increased EGF release during irritantinduced tearing ensures its availability to EGF receptors, even during periods of increased tearing.10 EGF seems to be necessary for the integrity of the normal corneal surface and normal cell turnover because levels of EGF in tears are decreased in humans with ocular surface disease.16 TGF-α is also present in aqueous humor. Normal corneal epithelial cell turnover is governed by EGF through exocrine and autocrine pathways (Figure 1). Exocrine stimulus is that which comes from external sources, whereas autocrine stimulus is released by and affects functioning of the same cell.17 The EGF in tears stimulates corneal epithelial cell migration and mitosis in an exocrine manner. Normal corneal epithe-

Experimental Healing of Corneal Epithelium In vitro, TGF-β decreased proliferation of corneal epithelial cells (measured by incorporation of thymidine into epithelial cells) in rabbits, whereas EGF increased proliferation.6 TGF-β had no effect on the number of cells attached to a fibronectin matrix, whereas EGF increased the number of attached cells. Transforming growth factor β had no effect on epithelial migration, but EGF increased such migration. Interestingly, TGF-β antagonized the effect of EGF in all of these situations. Therefore, it seems that TGF-β modulates the effect of EGF, possibly as a mechanism to prevent excessive cellular proliferation6 and scarring. In an experiment with rabbits, EGF derived from mice at concentrations of 0.05 mg/ml, 0.5 mg/ml, and 2 mg/ml was applied topically every 4 hours until epithelial wounds of the cornea had healed.9 The healing rate with this factor was 33% greater than that without growth factor. No dose-response relationship for mouse-derived EGF and corneal reepithelialization was established because of the wide dose range used. When the investigators applied mouse-derived EGF (0.5 mg/ml) 16 times daily, healing was enhanced 110% over the rate noted with application of this dose four times daily. The investigators found a negative effect of the too-frequent topical application, however: Application 16 times daily caused microtrauma, although no detrimental or toxic effect of the pharmacologic agent was noted.9 Clinical Healing of Corneal Epithelium The effect of EGF (2 mg/ml) on naturally occurring

FIBRONECTIN SYNTHESIS ■ GROWTH FACTORS ■ TEARS

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corneal abrasions, superficial epithelial lesions, infected corneal ulcers, corneal epithelial burns (physical or chemical), and surgery for conjunctival-corneal tumors in humans has been investigated.2 Two drops of EGF solution were applied to the affected eye every hour for the first 5 hours, then every 4 hours until healing of the cornea was achieved. If only one eye was diseased, treatment-induced healing rates were compared with the naturally occurring epithelial regeneration established for a period of hours or days before EGF treatment was initiated. If lesions were present in both eyes, one eye was used as a control. EGF increased the rate of corneal reepithelialization by a mean of 28.6%.

Experimental Healing of Corneal Stroma Growth factors that affect cell migration, proliferation, and synthesis of collagen and other extracellular matrix components are likely to impact healing of the stroma.5 EGF and PDGF accelerate healing and increase tensile strength of corneal wounds involving the epithelium and stroma.6 TGF-β is likely to cause the greatest increase in collagen synthesis based on results of tests on dermal wound healing15,16 and in vitro studies of corneal fibroblasts. 15,16 Stromal fibroblasts treated with 0.1 to 10 ng/ml of TGF-β increased collagen production 220% over controls, whereas fibroblast cultures treated with EGF (0.1 to 10 ng/ml) increased collagen production 40% over controls.5 In addition, TGF-β is a potent chemotactic agent for fibroblasts and increases cell number and cell productivity.15,16 In contrast, EGF predominantly increases the number of fibroblasts rather than the rate of collagen synthesis in the stromal wound.18 In an in vivo experiment in rabbits, topical PDGF (204 µg/ml three times daily for 7 days) increased the bursting strength of sutured, 95%-depth corneal lacerations 67% over saline-treated controls (360 mm Hg versus 210 mm Hg).11 Histopathologic examination revealed an increased number of fibroblasts and amount of types III and IV collagen at the wound edge of PDGF-treated corneas.11 In the same rabbits, PDGF administered three times daily for 17 days to penetrating keratoplasty lesions that were 6 mm in diameter increased bursting strength 47% over saline-treated controls (1042 mm Hg versus 707 mm Hg).11 Similarly, EGF increases strength of corneal epithelial and stromal wounds. Full-thickness, 6-mm surgical incisions in rabbit corneas treated with topical EGF (0.5 mg/ml) three times daily for 5 days had a tensile strength 17 times greater than that of saline-treated controls.8 Also, sutured, 7-mm perforating corneal incisions in rabbits treated with recombinant human EGF (0.02 mg/ml, 50 µl topically twice daily) had greater

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tensile strength than those of saline-treated control corneas without a decrease in ductility (i.e., the ability to be fashioned into a new form; this characteristic is a protective mechanism against rupture).7 Thus, EGF seems beneficial in corneal stromal wound healing by increasing tensile strength without decreasing ductility.7 Other studies have demonstrated the beneficial effects of EGF, TGF-β, and PDGF on corneal wound healing.15,16,19

Experimental Healing of Corneal Endothelium The corneal endothelium of cats, similar to that of humans, undergoes limited mitosis in response to injury. Aqueous humor in both species contains low levels of growth factors; cats have only 7 ng/ml of TGF-α.4 In this species, the concentration of TGF-α increases 14-fold within 2 hours of injury to the corneal endothelium and returns to base levels within 24 hours;20 this occurrence suggests that TGF-α plays a role in endothelial wound healing. Epidermal growth factor is a potent mitogen that binds to endothelial cells in vitro, thereby stimulating endothelial cell regeneration.4 Because the thickness and transparency of the cornea depend on a functional endothelium, the ability to induce endothelial regeneration in vivo would be invaluable. Intracameral (anterior chamber) injection of EGF (10 µg, 0.1 ml) in the eyes of a cat after transcorneal cryoinjury caused no significant difference in endothelial cell density, corneal thickness, endothelial cell structure, or rate of endothelial healing over saline-treated controls.4 Untreated and treated endothelial layers healed by enlargement and migration of adjacent cells rather than by regeneration and mitosis. It was postulated that, because turnover of aqueous humor occurred quickly (within approximately 2 hours), the duration of exposure of endothelial cells was insufficient to influence endothelial healing.4 EGF injected into the anterior chamber of the rabbits’ eyes had a half-life of only 0.6 hours.18 It has been shown that cells must be continuously exposed to EGF during at least a 4-hour period for this growth factor to have a mitogenic influence.3 In addition, the in vivo concentration of EGF may have been insufficient to bind to and stimulate endothelial cells.3 When EGF–sodium hyaluronidase (10 to 100 µg/ml) was injected into the anterior chamber of feline eyes that had endothelial surface injury (caused by application of a plastic-tipped cannula), the rate of endothelial wound closure was significantly greater than that found in sodium hyaluronidase–treated controls.18 EGF delivered in sodium hyaluronate increased endothelial cell mitosis, cell density, and percentage of hexagonal cells.21 Endothelial dysfunction is more closely related

STROMAL HEALING ■ RABBITS ■ CORNEAL ENDOTHELIUM

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to changes in cell size and shape than to changes in cell density.21 This study demonstrated that EGF stimulated early restoration of cell size and shape in feline corneal endothelium. Thus, EGF may increase endothelial healing rates and promote endothelial regeneration if it remains in the anterior chamber for a prolonged period. The concentration of EGF and maintenance of this concentration in aqueous humor is influenced by the vehicle in which EGF is delivered; saline and methylcellulose do not maintain an adequate concentration of EGF in the aqueous humor for delivery of EGF to the endothelium.18 Sodium hyaluronidase, however, maintains EGF concentration for an adequate period for effective delivery of EGF to the endothelium.21 Increasing the frequency of intracameral injections is not a practical method of maintaining an adequate concentration of EGF in the aqueous humor.

PERSISTENT CORNEAL EROSION Persistent corneal erosion is common in dogs21–24; this disorder can result from a primary disease of the basement membrane of the epithelium or endothelium, or it can be secondary to other types of ocular disease. Persistent corneal erosion has also been reported to occur in horses.25 Epithelial basement membrane disease that contributes to persistent corneal erosion has been previously termed indolent ulcer disease, rodent ulcer disease, and boxer ulcer disease. 21 Debridement and topical antibiotic administration with or without contact lens placement is the traditional treatment for persistent corneal erosion associated with primary disease of the epithelial basement membrane.21 Superficial keratectomy has been effective in resolving corneal erosion but can cause permanent scarring.21 More recently, superficial stromal keratotomy (punctate or linear) has been effective in healing erosions.22 This procedure is usually done with topical anesthesia, and it rarely causes gross corneal scars. The tiny scars that develop staple the epithelium to the stroma.22 It is possible that growth factors (possibly EGF) are elaborated at these sites. Hyperosmotic agents, because they tend to be ineffective and can be irritating, are not recommended.21 Topical fibronectin, although not commercially available, has been helpful in treating some forms of chronic corneal lesions in humans.26 Autogenous serum provides a ready source of fibronectin.21 Use of exogenous growth factors to treat persistent corneal erosion in its early phases may be a promising alternative or adjunct to standard therapy. EGF stimulates the synthesis of fibronectin and other structural or attachment proteins that promote migration and adhe-

sion of healing epithelium. EGF would be beneficial in healing corneal erosion secondary to a disease characterized by abnormal epithelial adhesion. Clinical results in dogs support this assertion.1 In dogs, a topical, biosynthesized EGF (100 µg/ml) that was highly homologous to human EGF was applied four times daily to erosions that had persisted for 4 weeks or longer or had recurred.1 Eight of 10 cases of erosion that were treated with EGF healed within 2 weeks, whereas only 2 of 10 cases of placebo-treated erosion healed within 2 weeks.1 One of the dogs in this study developed diffuse corneal edema and anterior uveitis during treatment with EGF. The investigators proposed that the antigenicity of the EGF may have caused cell-mediated immunity, 1 although endothelial dystrophy may have been the primary cause of the edema and secondary erosion. Despite this complication, the investigators concluded that topical EGF was effective for treating persistent corneal erosions.1

CONCLUSIONS Rapid, efficient wound healing of the cornea is essential to preserve vision. Endogenous growth factors, which are present in the tears, aqueous humor, and limbic vessels, are intimately involved in regulation of corneal wound healing. Growth factors govern chemotaxis of inflammatory cells, mitosis, migration, and cell differentiation as well as production and regulation of other growth factors. Because each layer of the cornea heals by a different mechanism, further research is necessary to determine which growth factors are most beneficial for each layer. No in vivo studies have been done to determine the effect of TGF-β on corneal stromal wounds to validate the in vitro finding that this growth factor significantly increases collagen synthesis. Further research is also necessary to elucidate whether TGF-β has an adverse impact on epithelial wound healing. Exogenous growth factors can increase the rate of wound healing and promote the early return of strength in the cornea. Topical growth factors have been evaluated in experimentally induced epithelial ulcers, stromal incisions (lacerations), and endothelial surface injury and cryoinjury. Precocious establishment of strength and integrity of the cornea may allow the early removal of corneal sutures, thereby decreasing such suture-related complications as excessive scarring and irritation.1 In addition, growth factors promote mitosis during endothelial healing, which normally heals by thinning and spreading of existing cells. Mitosis of endothelial cells may ensure a more functional layer of cells and prevent corneal edema.

ENDOTHELIAL DYSFUNCTION ■ KERATECTOMY ■ KERATOTOMY

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There has been no report in the veterinary literature of clinical use of growth factors in the past few years, perhaps because these products are not freely available commercially. However, it is important for the clinician to understand the physiology of corneal wound healing. If exogenous growth factors become available in the future, an appreciation of the rationale for use of exogenous growth factors could potentially enhance manipulation of corneal wound healing.

About the Authors When this article was submitted for publication, Dr. Swank was affiliated with the Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana. She currently works for Florida Veterinary Services Emergency Clinic in Tampa, Florida. Dr. Hosgood, who is a Diplomate of the American College of Veterinary Surgeons, is affiliated with the Department of Small Animal Clinical Sciences at Louisiana State University.

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corneal stromal incisions. Exp Eye Res 40:47–60, 1985. 9. Elliott J: Epidermal growth factor: In vivo ocular studies. Trans Am Ophthalmol Soc LXCVIII:629–656, 1980. 10. Van Setten B: Epidermal growth factor in human tear fluid: Increased release but decreased concentrations during reflex tearing. Curr Eye Res 9:79–83, 1990. 11. Murali S, Hardten D, DeMartelaere S, et al: Effect of topically administered platelet-derived growth factor on corneal wound strength. Curr Eye Res 13:857–862, 1990. 12. Slatter DH: Cornea and sclera, in Fundamentals of Veterinary Ophthalmology. Philadelphia, WB Saunders Co, 1990, pp 257–303. 13. Samuelson DA: Ophthalmic embryology and anatomy, in Gelatt KN: Veterinary Ophthalmology. Philadelphia, Lea & Febiger, 1991, pp 3–123. 14. Cenedella RI, Fleschner CR: Kinetics of corneal epithelium turnover in vivo: Studies of lovastatin. Invest Ophthalmol Vis Sci 31:1957–1962, 1990. 15. Whitley RD: Canine cornea, in Gelatt KN (ed): Veterinary Ophthalmology. Philadelphia, Lea & Febiger, 1991, pp 307–356. 16. Schultz G, Chegini N, Grant M, et al: Effects of growth factors on corneal wound healing. Acta Ophthalmol 70[Suppl 202]:60–66, 1992. 17. Schultz G, Khaw P, Oxford K, et al: Growth factors and ocular wound healing. Eye 8:184–187, 1994. 18. Bennett N, Schultz G: Growth factors and wound healing: Biochemical properties of growth factors and their receptors. Am J Surg 165:728–736, 1993. 19. Tripathi B, Kwait P, Tripathi R: Corneal growth factors: A new generation of ophthalmic pharmaceuticals. Cornea 9:2–9, 1990. 20. Raphael B, Kerr N, Shimizu R, et al: Enhanced healing of cat corneal endothelial wounds by epidermal growth factor. Invest Ophthalmol Vis Sci 34:2305–2312, 1993. 21. Kirschner S: Persistent corneal ulcers: What to do when ulcers won’t heal. Vet Clin North Am Small Anim Pract 20: 627–642, 1990. 22. Munger RI, Champagne ES: Multiple superficial punctate laceratotomies for the treatment of recurrent erosions in dogs. Trans Am Coll Vet Ophthalmol 18:103–115, 1987. 23. Gelatt KN, Samuelson DA: Recurrent corneal erosions and epithelial dystrophy in the boxer dog. JAAHA 18:453–460, 1982. 24. Kirshner SE, Niyo Y, Betts DM: Idiopathic persistent corneal erosions: Clinical and pathological findings in 18 dogs. JAAHA 25:84–90, 1989. 25. Cooley P, Wyman M: Indolent-like corneal ulcer in 3 horses. JAVMA 188:295–297, 1986. 26. Nishida T, Ohashi Y, Awata T, et al: Fibronectin: A new therapy for corneal trophic ulcer. Arch Ophthalmol 101: 1046–1048, 1983.