ACTA OPHTHALMOLOGICA
Vol. 70 (1992) Suppl. 202
CLINICAL MEASURES TO PROMOTE CORNEAL EPITHELIAL HEALING Roswell R. Pfister The Eye Research Laboratories, Brookwood Medical Center, Birmingham, Alabama, USA
Abstract. It is necessary to know the specific pathobiology of a persistent epithelial defect to determine the strategy to be employed to assist in its repair. Lid position and function must be normal and any deficiency in the quantity and quality of the tears enhanced by tear preparations and closure of the lacrimal canaliculi. Adverse drug effects must be eliminated. Multiple corneal punctures and excision of reduplicated basal lamina have virtually eliminated the problem of recurrent corneal erosions. Control of any inflammatory process also speeds healing. Vitamin supplements, especially A, reverse defects associated with xerophthalmalia. In any of these diseases, mechanical treatments consisting of soft contact lenses for persistent epithelial defects and collagen shields for the delivery of antibiotics or steoids to the eye may be employed. Tarsorraphy relieves the problem of persistent epithelial defects in neurotrophic keratitis and a variety of other conditions characterized by persistent surface breakdown. Preliminary data from open label studies of epidermal grcwth factors and fibronectin are encouraging but not yet conclusive.
dence of regrowth, further evaluation and intervention is indicated. A complete ocular examination, with attention to all relevant factors, is required to establish the cause(s) of the problem. Only then can a measured plan for treatment be initiated. The following constitutes the authors’ approach and treatment of these vexing problems.
Drug toxicity Topical drugs can not only treat serious eye disease but can, under certain circumstances, be the cause. In each eyedrop formulation there is one or more active ingredients and a variety of additional compounds to stabilize the active drug or preserve the preparation from microbial contamination. When solutions are used frequently, or for a prolonged period, some of the constitu-
Key words: epithelium healing. Table 1.
Introduction Epithelium normally regrows over the surface of an abraded cornea in a relatively rapid and predictable manner (Fig. 1A and B). However, there are numerous conditions and events which might either interfere with, or create non-healing epithelial defects. (Table 1). Such a defect is considered persistent when cells fail to show the expected rate of healing for the time course involved (Fig. 2). If 2 to 4 days pass without evi-
Conditions which might either interfere with, or create nonhealing epithelial defects. Tear film abnormalities Intrinsic epithelial disorders Basement membrane disorders Lid abnormalities - exposure Metabolic disturbances Iatrogenic Trauma Neurogenic Infectious Inflammation Nutrition Immune disease
73
Roswell R. Pfister
Fig. 1. A.
Fig. 1. Epithelial cells regrow over an abraded surface (A), extending out broad lamellipodia and fine filopodia over the basement membrane at the advancing edge and (B), using the adhesion plaques on the underside of the filopodia to maintain firm adhesion and assist the foreward movement of the cell by contraction
of the contained intracellular actin-myocin network.
tients can create further complications, making it difficult to separate the primary disease process from the iatrogenic effects of the medications used. Idiosyncratic reactions or chronic use of drugs, particularly topical anesthetics, has been repeatedly documented to cause and maintain epithelial defects and even scarring and vascularization (Pfister & Burstein, 1976). The reason for this effect is that topical anesthetics probably disrupt actin assembly in the epithelial cells, which is essential for normal wound healing (Higbee & Hazlett 1984). Scanning electron microscopy of the surface of corneal epithelium, after instillation of most
74
antibiotics, showed little effect on the plasma cell membrane. Pilocarpine caused only mildly to moderately disruptive effects in the membrane, but severe damage occurred when preservatives such as benzalkonium chloride were employed (Pfister & Burstein, 1976) (Fig. 3). Benzalkonium chloride is also injurious to regenerating epithelium; there is loss of tight adherence of the leading edge, loss of membrane activity, and peeling back of the advancing layer (Fig. 4). The first step in the treatment of all persistent epithelial defects of the cornea is to list all of the topical medications and eliminate all but the necessary drugs. Review the formulations of
Clinical measures to promote corneal epithelial healing
Fig. I . B.
these essential drugs and substitute other equivalent formulations without epithelial toxic components or, better yet, have your pharmacist compound the drug in sterile water or saline depending on its osmolarity. In many cases the treatment of choice is to remove all medications from topical use. Removal of the offending compound might be all that is required to eliminate the epithelial defect.
Fig. 2. Initial epithelial regrowths of 12 mm corneal injuries are compared. (a) Abrasion. (b) Keratectomy. (c) 1 N NaOH injury (d) keratectomy alkali-injury. The rate of epithelial regrowth of abrasions over basement membrane is twice that of keratectomy, presumably because of the very irregular keratectomy base. Arrest and reversal of epithelial regrowth at 84 hours occurs in each alkali burn regardless of size of the defect. Circle, mean; brackets, standard deviation (from Pfister R R (1976): Exp Eye Res 23: 519).
0
1
2
3 4 5 6 7 1 Epithelialregrowth (days)
'
75
Roswell R. Pfister
Fig. 3. Thirty minutes after two drops of a 0.01 Vo benzalkonium chloride solution applied to the eye, most of the top layer of cells are desquamating. Insets, Severe degenerative membrane changes are notable in these dying cells (from Pfister R R & Burstein N (1976): Invest Ophthalmol 15: 246).
Mechanical protection Exposure keratitis
- eyelid dysfunction
It is important not to overlook eyelid dysfunction as a cause of epithelial defects or abnormalities. In the presence of a brief or self-limited ocular exposure, temporary closure of the eyelid with patch, or tape applied directly to the eyelid, might prevent the emergence of a defect. When the periorbital skin is damaged or the lids unable to close, a temporary moist chamber can be created by swim goggles or by covering the orbital opening with plastic wrap adhered to the orbital rim with an ointment. Temporary or permanent trasorraphy is indicated when corneal exposure is prolonged, such as in lagophthalmos from seventh nerve palsy or the neurotrophic problems associated with herpes zoster ophthalmicus. A variety of other trophic and devitalized ulcers are also likely to respond to a one or two pillar tar-
76
sorrhaphy. Scarring of the eyelid resulting in lagophthalmos usually responds to a pinch graft to lengthen the lid. Trichiasis must be eliminated by cryoablation, electrolysis or, in the case of entropion, marginal eyelid rotation. Laxness of the canthal ligaments holding the tarsus of the upper eyelid results in the floppy eyelid syndrome. Nocturnal exposure in these obese patients is treatable by taping at night or tightening the upper lid by wedge resection of tarsus. Poor lid-globe congruity, occasioned by a perilimbal elevation (i.e. muscle surgery, episcleritis, etc)'might lead to a localized dessicated area known as a dellen. Temporary patching and steroid drops usually diminish the elevation, thereby eliminating the condition.
Soft contact lens and collagen shields The use of collagen shields after acute epithelial defects is controversial. One animal study showed
Clinical measures to promote corneal epithelial healing
Fig. 4. Thirty minutes after 0.01 Qa benzalkonium chloride was applied to actively migrating corneal epithelial cells. Advancing layer of cells lose their locomotory activity (ruffles and filopodia) showing extensive membrane degeneration. The top cell peeled back, exposing the underlying cell, which also shows some sepapation of leading edge (from Pfister R R & Burstein N (1976): Invest Ophthalmol 15: 246).
that after simple abrasion the epithelial defects closed 8 hours earlier than controls, but a second investigation showed no difference. Collagen shields might not enjoy any advantage over other forms of treatment for abrasion apart from the possible advantage of greater comfort, in some patients, and the ability to deliver high concentrations of drugs to the eye from a shield previously soaked in antibiotic. The mainstay of treatment of persistent epithelial defects, not of an exposure type, is the therapeutic soft contact lens. In a series of 22 persistent epithelial defects occurring after penetrating keratoplasty, 16 healed with the continuous use of a B + L 04 therapeutic soft contact lens. Groden et al. (1989). Although any properly fit extended wear soft contact lens could be used, the author favors the disposable variety because it offers the advantage of removal and replace-
ment at the time of office visits. In the same study, collagen shields, derived from porcine scleral collagen, did not heal any of 7 eyes with persistent epithelial defects; but of these patients 5 of 6 subsequently healed under a soft contact lens.
Tear deficiency The quantity of the tear film layer is also critically important to the health and integrity of the epithelial layer. The aqueous layer composes the bulk of the thickness of the precorneal tear film (about 7 pm), laying between the superficial lipid layer and the deeper mucin on the epithelial cells. Between blinks the aqueous component thins from evaporation as well as flow into the nasolacrimal passages. As the aqueous portion thins, the superficial lipid layer approaches the 77
Roswell R. Pfister
deepest mucin layer. If the aqueous component of the tear film is not replenished by blinking, lipid contamination of the mucin coacervate, adjacent to the surface cells, creates a localized hydrophobic epithelium. If such a dry area persists, partial or full-thickness epithelial breakdown may occur (Fig. 5). The effects of aqueous tear deficiency, as occurs in keratoconjunctivitis sicca, include surface drying, punctate epithelial staining, mucus plaques, and nonhealing epithelial defects. The importance of the superficial lipid layer rests largely on its ability to retard the evaporation of tears (by a factor of 10). Lipid deficiencies occur when there is destruction of the meibomian glands, as in lid resections, after alkali injuries, after diseases producing scarring, accutane therapy and in severe and recalcitrant blepharitis (Mathers et al., 1991; Mathers et al., 1991). Although not proved at this time it is possible that the lipid abnormalities in blepharitis might be improved by lid scrubs, warm compresses and antibiotics. When accutane therapy causes meibomian gland abnormalities, symptoms prompt cessation of drug therapy. Mucin is the spreading agent of the tears produced from conjunctival goblet cells. Vitamin A in the diet is required to maintain normal epithelial tissues and their appendages, in particular, goblet cells. Primary vitamin A deficiency is a serious problem in many third world countries whee there is an insuffient dietary source. In industrialized nations the deficiency is usually a part of another systemic disease such as malabsorption syndromes, alcoholism and eating disorders. Vitamin A deficiency reduces mucin production through the loss of conjunctival goblet cells. When weaning guinea pigs were fed vitamin A-deficient diets, goblet cells disappeared from the conjunctiva in 7 weeks (Pfister, 1978) (Fig. 6). Keratinization of the ocular surface, loss of surface microprojections, epithelial defects, corneal ulceration, and keratomalacia are common in the case of chronic vitamin A deficiency. Restitution of vitamin A to the diet is curative, with a complete return of goblet cells. Goblet cell deficiencies also occur in a variety of unrelated conditions including erythema multiforme, pemphigoid, alkali burns, and even keratoconjunctivitis sicca. In most of these conditions ocular irritation caused by poor wetting 78
of the ocular surface is a lifelong condition. Not uncommonly, corneal epithelial defects, ulceration, and vascularization occur. Mild cases of dry eye are successfully treated with artificaial tears used every hour and ointment at bedtime. Non-preserved preparations are preferable, especially if epithelial disturbances already exist. Preparations with higher viscosity are preferred by patients with the driest eyes. If three consecutive schirmer I testings on different appointment days give values of 3-5 mm of wetting, permanent occlusion of the lower lacrimal punctae is indicated. If testing shows 0-2 mm wetting then closure of upper and lower punctae is reasonable. The occlusive effect of temporary collagen plugs, as a trial prior to permanent closure, has been seriously questioned (Groden & Forstot, 1991). Using sophisticated radiosintigraphy the 0.4 collagen plug impeded the flow of dye from the eye by an average of only 16.5 Yo. Until further work supports the use of such plugs, the physician must exercise caution in interpreting the results of the procedure. Glasses with side panels which create higher humidity atmospheres around the eyes are useful for the most severe sufferers. Filamentary keratitis, almost always associated with dry eye, usually responds to mechanical removal of the filament with a jeweler forcep and restitution of tears. Occasional sufferers might require a soft contact lens to prevent formation of the filaments.
Autoimmune disease Although a variety of autoimmune diseases can result in corneal epithelial defects, only two will be considered here. Persistent epithelial defects are especially common in Sjogrens syndrome where the progression to ulceration and perforation might occur rapidly. The infiltrate present around the ulcer is disproportionately less than usually expected in the presence of bacterial disease. Measures to improve the tear film form the first line of defence, followed by soft lens therapy. The combination of conservative treatments, aided by immunosuppression, results in a high rate of success (Pfister & Murphy, 1980). Mooren’s ulcer. is characterized by a peripheral corneal infiltrate which ulcerates, producing a typical central overhanging edge extending circumferentially and later centrally. Unilateral in-
Clinical measures to promote corneal epithelial healing
Fig. 5. (A) Epithelial irregularity of two dry spot areas, arrows, is evident on an otherwise smooth corneal surface. (B) This dry spot consisted of a cluster of five corneal epithelial cells separating from contiguous surface, (C) Separating epithelial cells in a dry spot show loss of surface microprojections and irregularity and disruption of plasma membrane. Retraction fibrils, arrowheads, and discrete anterior cellular edges mark disrupted attachments with adjacent cells. PD, thinest pachymetry depth. (from Pfister R R & Renner M E (1977): Invest Ophthalmol 16: 1025).
79
Roswell R. Pfister
Fig. 6. (A) Control cornea shows smooth, continuous sheet of flat polygonal epithelial cells. (B) Seven-week vitamin A-defecient cornea. Entire surface layer of cells shows extensive separation and desquamation from epithelial sheet. (C). Control conjunctiva. Numerous goblet cells, arrowheads, in various stages of maturation, stud the surfaces of palpebral and cul-de-sac conjunctiva. (D) Six-week vitamin A-deficient conjunctiva. Keratinized epithelial cells show extensive separations and desquamatiion from surface epithelial sheet. Total absence of goblet cells is a striking finding (from Pfister R R & Renner M E (1978): Invest Ophthalmol 17: 874).
volvement is most common in the elderly and is more responsive to topical steroids and coniunctival resection. Bilateral disease in the youngmen, especially those occurring simultaneously, are more aggressive, requiring immunosuppression for control (Brown & Mondino, 1984; Foster, 1985).
80
BASEMENT MEMBRANE The attachment of the epithelium to its underlying basement membrane is crucially important to its integrity. Basement membrane is a secretion product of the basal corneal epithelial cells, requiring about 6 weeks to reform after its destruc-
Clinical measures to promote corneal epithelial healing
tion Khodadoust (1968). During this interval the epithelium adheres poorly to the underlying stroma except at its leading edge. In contrast, epithelium that has migrated over preformed basement membrane tightly adheres over its entirety in 6 days. In either case strong adherence is correlated with the presence of many electron densities (hemidesmosomes) along the basal cell membrane (see: PBBllysaho et al., 1992; Cipson et al., 1992). Damage to the basement membrane and its disappearance or failure to regenerate appears to constitute the major reason for poor adherence of the epithelium in many diseases. A variety of diseases are rooted in basement membrane disorders. In a biopsy specimen examined by electron microscopy, traumatic recurrent corneal erosion syndrome showed the absence or discontinuity of basement membrane in the affected area. Goldman (1969). A technique of multiple, non-perforating punctures made one-half stromal thickness, performed in the area of erosions, has virtually eliminated the disease (McLean et al., 1986). In addition, metaherpetic erosions are frequently correlated with therapeutic caustic scrubs of the cornea that damage basement membrane (Kaufman, 1964). Dystrophic processes, characterized by corneal erosions, such as anterior membrane dystrophy, represent multilamination of the basement membrane. Scraping the corneal surface to remove the epithelium and redundant layers of basement membrane often eliminates the erosions. If erosions persist then multiple punctures of the stroma are indicated. The shield lesion of vernal catarrh also represents an erosive phenomenon consequent t o the abrasive effect of upper lid vegetations and severe mast cell degranulation. Diabetics are subject to epithelial erosions, especially after surgery, though evidence to implicate an abnormally thickened basement membrane is not convincing (Taylor & Kinsey, 1981). Substantial damage to the basement membrane has also been demonstrated in patients with keratoconus who had been treated by thermokeratoplasty (Fogle, 1977). Only in the epithelial recovery after an alkali burn does this correlation between basement membrane injury and recurrent erosion break down. Disappearance of the basement membrane after the burn is correlated with loss of progression of epithelial migration, but this reversal in epithelial movement occurs even af6
ter an alkali burn in a cornea previously subjected to a lamellar keratectomy (Pfister, 1976) (Table 1).
Role of inflammation Large numbers of polymorphonuclear leukocytes in corneal tissues are likely to encourage the development or persistence of epithelial defects as well as ulceration. Corneas with epithelial defects cultured in vitro showed significant inhibition of epithelial healing when the perfusate contained either viable stimulated PMN or a PMN lysate (Wagoner, 1984). Drugs directed against the respective infectious entity usually eliminates the agent and thereby terminates the inflammatory component inhibitory to healing. Topical sodium citrate decreases corneal ulceration by inhibiting neutrophils in alkali-injured animal eyes (Pfister et al. 1982). Its utility in the human awaits the results of an ongoing clinical trial. For these reasons it is important to control the inflammatory response to protect the stroma as a suitable substrate for epithelial regrowth.
Influence of perilimbal epithelial loss The size and specific involvement of the epithelial defect influences the course and the permanence of epithelial recovery. In the absence of basement membrane or stromal injury, epithelial defects that do not exceed the limits of the cornea usually heal without incident. When epithelial defects destroy most of the limbal palisades of Vogt, epithelium from the conjunctiva must repopulate the cornea. Conjunctival epithelium has substantially different biochemistry and morphology compared to corneal epithelium. A classic example of damage to the perilimbal conjunctiva resulting in poor reepithelization is the alkali-injured eye. To treat this condition it has been shown that superficial keratectomy followed by transplantation of healthy conjunctiva onto the alkali-injured eye improved the vision and stabilized the ocular surface (Thoft, 1979). In these studies, seven of 10 eyes had improved sight, but all showed an improved and more stable ocular surface. The discovery of a discrete group of stem cells in the depths of a narrow zone of perilimbal conjunctival epithelium has given credence to the 81
Roswell R. Pfister
concept of conjunctival transplantation and led to newer surgical approaches to corneal reepithelialization. Evidence that these cells are progenitors of corneal epithelium includes reaction to a specific monoclonal antibody (Zieske & Bukusoglu, 1990), the presence of a specific 64 K keratin, and an enhanced growth curve compared to corneal or conjunctival epithelium. Excision of all limbal epithelium from a normal eye leads to poor epithelialization and corneal vascularization; a sign that a critical element has been removed from the generative source of corneal epithelial cells. These basic data give substance to current recommendations of limbal conjunctival autograft from the uninjured eye to the injured eye for those patients who have sustained extensive unilateral stem cell injury (Kenyon, 1989).
Inhibition of polymorphonuclear leukocytes by cit rate Severe alkali burns present a very complex process of healing including an initial phase of normal epithelial healing followed by abrupt loss of adhesion of the leading edge and roll-back of the epithelial sheet. The subsequent influx of neutrophils leads to ulceration and perforation of the eye (Pfister, 1976). A new and clinically untried idea is the inhibition of polymorphonuclear leukocytes (PMNs) in situ by topical citrate. Sodium citrate prevents the accumulation, phagocytosis, and subsequent release of degradative enzymes and free oxygen radicals from PMNs. Animal and laboratory studies with citrate have shown it to be a chelator of extracellular Ca2+. Calcium is probably necessary for the activation of the PMNs, acting as an important intracellular second messenger. Inhibition of the PMNs through calcium depletion may be caused by interference with calcium-calmodulin modulated microfilament or microtubule interfaces in the plasma membrane (Pfister et al., 1984). Hence, topical treatment with citrate may bring the entire set of PMN activities to a halt.
Fibronectin as an enhancer of epithelial healing Fibronectin has been implicated as a key element in wound healing for its involvement in cell82
to-cell and cell-to-matrix adhesion and cell spreading. Eye trauma causes exudation of large proteins such as fibrinogen which reach the bare basement membrane where polymerization and deposition take place. In vitro studies of rabbit corneas show accelerated epithelial healing in the presence of fibronectin (Nishida et al., 1983). Fibronectin eye drops have been used to treat a variety of conditions with persistent epithelial defects. Epithelial defects occurring in herpetic keratitis, trophic corneal ulcers and after cataract surgery have responded to fibronectin drops when used in an open labeled study (Nishida et al., 1985; Nishida et al., 1983; Nishida et al., 1987). An alternative explanation is suggested by an animal study showing that albumin eye drops were as effective as fibronectin in the treatment of persistent epithelial defects (Boisjoly et al., 1987). This suggests that whatever favorable effect noted might be a nonspecific response.
Growth factors Epidermal growth factor (EGF) enhances the rate of healing and induces hyperplasia in corneal epithelium (Cohen, 1984; Ho, 1974). EGF stimulated complete epithelial healing after alkaliinjury in two studies, but in each instance recurrent erosions reestablished the defect (Eiferman, 1987; Singh & Foster, 1987). Although no beneficial effect on epithelial migration could be shown by topical administration of mouse EGF after penetrating keratoplasty in the human, epithelial defects healed in half the time over epikeratophakia lenticules using human EGF. Eiferman (1985). The latter study is of special importance, since persistent epithelial defects do occur after epikeratophakia, sometimes leading to ulceration and requiring replacement of the lenticule. Growth factors may find a more favored place in the acceleration of wound strength (Woost, 1985) by the mechanism of enhancement of macromolecular synthesis.
References Boisjoly H, Beaulieu A, Giasson M & Menard C (1987): The effect of fibronectin compared to albumin on rabbit epithelial wound healing. Invest Ophthalmol Vis Sci (Suppl) 28: 52.
Clinical measures to Promote corneal epithelial healing ~~
Brown S I & Mondino B J (1984): Therapy of Mooren’s ulcer. Am J Ophthalmol 98: 1-6. Cohen S (1984): Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. Am J Ophthalmol 98: 411-415.
Eiferman R, Brightwell J , Rowsey J, et al. (1985): Acceleration of corneal epithelial resurfacing and keratocyte migration in primate epikeratophakia by biosynthetic human EGF. Invest Ophthalmol Vis Sci (Suppl) 26. Eiferman R & Schultz G S (1987): Treatment of alkali burns in rabbits with epidermal growth factor. Invest Ophthalmol Vis Sci (Suppl) 28: 52. Foster C S (1985): Systemic immunosuppressive therapy for progressive bilateral Moorens ulcer. Ophthalmology 92: 1436-1439. Fogle J A, Kenyon K R & Stark W J (1977): Damage to epithelial basement membrane by thermokeratoplasty. Am J Ophthalmol 83: 392-401. Goldman J N, Dohlman C H & Kravitt B A (1969): The basement membrane of the human cornea in recurrent epithelial erosion syndrome, Trans Am Acad Ophthalmol Otolaryngol 73: 471-481. Groden L T, White W & Updegraff S (1989): Porcine collagen corneal shield treatment of persistent epithelial defects following penetrating keratoplasty. Invest Ophthalmol Vis Sci (Suppl) 30: 340. Higbee R G & Hazlett L D (1984): Topical ocular anesthetics: effect on corneal cytoskeleton. Invest Ophthalmol Vis Sci (Suppl) 25: 321. Ho P C, Davis W H, Elliott J H & Cohen S P (1974): Kinetics of corneal epithelial regeneration and epidermal growth factor. Invest Ophthalmol Vis Sci 13: 804-809.
Kaufman H E (1964): Epithelial erosion syndrome: metaherpetic keratitis. Am J Ophthalmol57: 983987.
Kenyon K R, Tseng S C (1989): Limbal autograft transplantation for ocular surface disorders. Ophthalmology 96: 709. Khodadoust A A, Silverstein A M, Kenyon K R & Dowling J E (1968): Adhesion of regenerating corneal epithelium: the role of basement membrane. Am J Ophthalmol 65: 339-348. Kinoshita S, Friend J & Thoft R A (1984): Ocular surface epithelial regeneration and disease. Int Ophthalmol Clin 24: 169. Mathers W D, Shields B S, Sachdev M S , Petroll M W & Jester J V (1991): Meibomian gland dysfunction in chronic blepharitis. Cornea 10: 286-
rent erosion: treatment by anterior stromal puncture. Ophthalmol 93: 784-788. Nishida T, Nakagawa S, Awata T et al. (1983): Fibronectin promotes epithelial migration of cultured rabbit cornea in situ. J Cell Biol 97: 1653. Nishida T, Jakagawa S & Manabe R (1985): Clinical evaluation of fibronectin eyedrops on epithelial disorders after herpetic keratitis. Ophthalmology 92: 213-216.
Nishida T, Yagi J, Fukuda M, Kusube T & Otori T (1987): Spontaneous persistent epithelial defects after cataract surgery. Cornea 6: 32-37. Pfister R R (1976): The alkali burned cornea. I. Epithelial and stromal repair. Exp Eye Res 23: 5 19-535.
Pfister R R (1978): The corneal and conjunctival surface in vitamin A deficiency: a scanning electron microscope study. Invest Ophthalmol Vis 17: 874883.
Pfister R R, Haddox J D, Dodson R W & Deshazo W J (1984): Polymorphonuclear leukocytic inhibition by citrate, other metal chelators, and trifluoperazine. Invest Ophthalmol Vis Sci 25: 955-970. Pfister R R, Paterson C A & Hayes S A (1982): The efficacy of sodium citrate in the treatment of severe alkali burns on the eye is influenced by the route of administration. Cornea 1: 205-21 1. Pfister R R & Burstein N (1976): The effects of ophthalmic drugs, vehicles, and preservatives on corneal epithelium: a scanning electron microscope study. Invest Ophthalmol Vis Sci 15: 246-259. Pfister R R & Murphy G E (1980): Corneal ulceration and perforation associated with Sjogrens syndrome. Arch Ophthalmol 98: 89-94. Singh G & Foster C S (1987): Epidermal growth factor in alkali-burned corneal epithelial wound healing. Am J Ophthalmol 103: 802-807. Taylor H R & Kinsey R A (1981): Corneal epithelial basement membrane changes in diabetes. Invest Ophthalmol Vis Sci 20: 548-553. Thoft R A (1979): Conjunctival transplantation. Ophthalmology 86: 1084. Wagoner M D, Kenyon K R, Gipson I K et al. (1984): Polymorphonuclear neutrophils delay corneal epithelial wound healing in vitro. Invest Ophthalmol Vis Sci 25: 1217-1221. Zieske J D & Bukusoglu H (1990): Numbers of limbal basal cells expressing a 50-kD antigen increase after wounding. Invest Ophthalmol Vis Sci (Suppl) 31: 538.
290.
Mathers W D, Shields B S, Sachdev M S, Petrol1 W M & Jester J V (1991): Meibomian gland morphology and tear osmolarity: changes with accutane therapy. Cornea 10: 286-290. McLean E N, MacRae S M & Rich L J (1986): Recur-
Author’s address: Roswell R. Pfister The Eye Research Laboratories Brookwood Medical Center Birmingham, A1 35209, USA
83
Vol. 70 (1992) Suppl. 202
CLINICAL MEASURES TO PROMOTE CORNEAL EPITHELIAL HEALING Roswell R. Pfister The Eye Research Laboratories, Brookwood Medical Center, Birmingham, Alabama, USA
Abstract. It is necessary to know the specific pathobiology of a persistent epithelial defect to determine the strategy to be employed to assist in its repair. Lid position and function must be normal and any deficiency in the quantity and quality of the tears enhanced by tear preparations and closure of the lacrimal canaliculi. Adverse drug effects must be eliminated. Multiple corneal punctures and excision of reduplicated basal lamina have virtually eliminated the problem of recurrent corneal erosions. Control of any inflammatory process also speeds healing. Vitamin supplements, especially A, reverse defects associated with xerophthalmalia. In any of these diseases, mechanical treatments consisting of soft contact lenses for persistent epithelial defects and collagen shields for the delivery of antibiotics or steoids to the eye may be employed. Tarsorraphy relieves the problem of persistent epithelial defects in neurotrophic keratitis and a variety of other conditions characterized by persistent surface breakdown. Preliminary data from open label studies of epidermal grcwth factors and fibronectin are encouraging but not yet conclusive.
dence of regrowth, further evaluation and intervention is indicated. A complete ocular examination, with attention to all relevant factors, is required to establish the cause(s) of the problem. Only then can a measured plan for treatment be initiated. The following constitutes the authors’ approach and treatment of these vexing problems.
Drug toxicity Topical drugs can not only treat serious eye disease but can, under certain circumstances, be the cause. In each eyedrop formulation there is one or more active ingredients and a variety of additional compounds to stabilize the active drug or preserve the preparation from microbial contamination. When solutions are used frequently, or for a prolonged period, some of the constitu-
Key words: epithelium healing. Table 1.
Introduction Epithelium normally regrows over the surface of an abraded cornea in a relatively rapid and predictable manner (Fig. 1A and B). However, there are numerous conditions and events which might either interfere with, or create non-healing epithelial defects. (Table 1). Such a defect is considered persistent when cells fail to show the expected rate of healing for the time course involved (Fig. 2). If 2 to 4 days pass without evi-
Conditions which might either interfere with, or create nonhealing epithelial defects. Tear film abnormalities Intrinsic epithelial disorders Basement membrane disorders Lid abnormalities - exposure Metabolic disturbances Iatrogenic Trauma Neurogenic Infectious Inflammation Nutrition Immune disease
73
Roswell R. Pfister
Fig. 1. A.
Fig. 1. Epithelial cells regrow over an abraded surface (A), extending out broad lamellipodia and fine filopodia over the basement membrane at the advancing edge and (B), using the adhesion plaques on the underside of the filopodia to maintain firm adhesion and assist the foreward movement of the cell by contraction
of the contained intracellular actin-myocin network.
tients can create further complications, making it difficult to separate the primary disease process from the iatrogenic effects of the medications used. Idiosyncratic reactions or chronic use of drugs, particularly topical anesthetics, has been repeatedly documented to cause and maintain epithelial defects and even scarring and vascularization (Pfister & Burstein, 1976). The reason for this effect is that topical anesthetics probably disrupt actin assembly in the epithelial cells, which is essential for normal wound healing (Higbee & Hazlett 1984). Scanning electron microscopy of the surface of corneal epithelium, after instillation of most
74
antibiotics, showed little effect on the plasma cell membrane. Pilocarpine caused only mildly to moderately disruptive effects in the membrane, but severe damage occurred when preservatives such as benzalkonium chloride were employed (Pfister & Burstein, 1976) (Fig. 3). Benzalkonium chloride is also injurious to regenerating epithelium; there is loss of tight adherence of the leading edge, loss of membrane activity, and peeling back of the advancing layer (Fig. 4). The first step in the treatment of all persistent epithelial defects of the cornea is to list all of the topical medications and eliminate all but the necessary drugs. Review the formulations of
Clinical measures to promote corneal epithelial healing
Fig. I . B.
these essential drugs and substitute other equivalent formulations without epithelial toxic components or, better yet, have your pharmacist compound the drug in sterile water or saline depending on its osmolarity. In many cases the treatment of choice is to remove all medications from topical use. Removal of the offending compound might be all that is required to eliminate the epithelial defect.
Fig. 2. Initial epithelial regrowths of 12 mm corneal injuries are compared. (a) Abrasion. (b) Keratectomy. (c) 1 N NaOH injury (d) keratectomy alkali-injury. The rate of epithelial regrowth of abrasions over basement membrane is twice that of keratectomy, presumably because of the very irregular keratectomy base. Arrest and reversal of epithelial regrowth at 84 hours occurs in each alkali burn regardless of size of the defect. Circle, mean; brackets, standard deviation (from Pfister R R (1976): Exp Eye Res 23: 519).
0
1
2
3 4 5 6 7 1 Epithelialregrowth (days)
'
75
Roswell R. Pfister
Fig. 3. Thirty minutes after two drops of a 0.01 Vo benzalkonium chloride solution applied to the eye, most of the top layer of cells are desquamating. Insets, Severe degenerative membrane changes are notable in these dying cells (from Pfister R R & Burstein N (1976): Invest Ophthalmol 15: 246).
Mechanical protection Exposure keratitis
- eyelid dysfunction
It is important not to overlook eyelid dysfunction as a cause of epithelial defects or abnormalities. In the presence of a brief or self-limited ocular exposure, temporary closure of the eyelid with patch, or tape applied directly to the eyelid, might prevent the emergence of a defect. When the periorbital skin is damaged or the lids unable to close, a temporary moist chamber can be created by swim goggles or by covering the orbital opening with plastic wrap adhered to the orbital rim with an ointment. Temporary or permanent trasorraphy is indicated when corneal exposure is prolonged, such as in lagophthalmos from seventh nerve palsy or the neurotrophic problems associated with herpes zoster ophthalmicus. A variety of other trophic and devitalized ulcers are also likely to respond to a one or two pillar tar-
76
sorrhaphy. Scarring of the eyelid resulting in lagophthalmos usually responds to a pinch graft to lengthen the lid. Trichiasis must be eliminated by cryoablation, electrolysis or, in the case of entropion, marginal eyelid rotation. Laxness of the canthal ligaments holding the tarsus of the upper eyelid results in the floppy eyelid syndrome. Nocturnal exposure in these obese patients is treatable by taping at night or tightening the upper lid by wedge resection of tarsus. Poor lid-globe congruity, occasioned by a perilimbal elevation (i.e. muscle surgery, episcleritis, etc)'might lead to a localized dessicated area known as a dellen. Temporary patching and steroid drops usually diminish the elevation, thereby eliminating the condition.
Soft contact lens and collagen shields The use of collagen shields after acute epithelial defects is controversial. One animal study showed
Clinical measures to promote corneal epithelial healing
Fig. 4. Thirty minutes after 0.01 Qa benzalkonium chloride was applied to actively migrating corneal epithelial cells. Advancing layer of cells lose their locomotory activity (ruffles and filopodia) showing extensive membrane degeneration. The top cell peeled back, exposing the underlying cell, which also shows some sepapation of leading edge (from Pfister R R & Burstein N (1976): Invest Ophthalmol 15: 246).
that after simple abrasion the epithelial defects closed 8 hours earlier than controls, but a second investigation showed no difference. Collagen shields might not enjoy any advantage over other forms of treatment for abrasion apart from the possible advantage of greater comfort, in some patients, and the ability to deliver high concentrations of drugs to the eye from a shield previously soaked in antibiotic. The mainstay of treatment of persistent epithelial defects, not of an exposure type, is the therapeutic soft contact lens. In a series of 22 persistent epithelial defects occurring after penetrating keratoplasty, 16 healed with the continuous use of a B + L 04 therapeutic soft contact lens. Groden et al. (1989). Although any properly fit extended wear soft contact lens could be used, the author favors the disposable variety because it offers the advantage of removal and replace-
ment at the time of office visits. In the same study, collagen shields, derived from porcine scleral collagen, did not heal any of 7 eyes with persistent epithelial defects; but of these patients 5 of 6 subsequently healed under a soft contact lens.
Tear deficiency The quantity of the tear film layer is also critically important to the health and integrity of the epithelial layer. The aqueous layer composes the bulk of the thickness of the precorneal tear film (about 7 pm), laying between the superficial lipid layer and the deeper mucin on the epithelial cells. Between blinks the aqueous component thins from evaporation as well as flow into the nasolacrimal passages. As the aqueous portion thins, the superficial lipid layer approaches the 77
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deepest mucin layer. If the aqueous component of the tear film is not replenished by blinking, lipid contamination of the mucin coacervate, adjacent to the surface cells, creates a localized hydrophobic epithelium. If such a dry area persists, partial or full-thickness epithelial breakdown may occur (Fig. 5). The effects of aqueous tear deficiency, as occurs in keratoconjunctivitis sicca, include surface drying, punctate epithelial staining, mucus plaques, and nonhealing epithelial defects. The importance of the superficial lipid layer rests largely on its ability to retard the evaporation of tears (by a factor of 10). Lipid deficiencies occur when there is destruction of the meibomian glands, as in lid resections, after alkali injuries, after diseases producing scarring, accutane therapy and in severe and recalcitrant blepharitis (Mathers et al., 1991; Mathers et al., 1991). Although not proved at this time it is possible that the lipid abnormalities in blepharitis might be improved by lid scrubs, warm compresses and antibiotics. When accutane therapy causes meibomian gland abnormalities, symptoms prompt cessation of drug therapy. Mucin is the spreading agent of the tears produced from conjunctival goblet cells. Vitamin A in the diet is required to maintain normal epithelial tissues and their appendages, in particular, goblet cells. Primary vitamin A deficiency is a serious problem in many third world countries whee there is an insuffient dietary source. In industrialized nations the deficiency is usually a part of another systemic disease such as malabsorption syndromes, alcoholism and eating disorders. Vitamin A deficiency reduces mucin production through the loss of conjunctival goblet cells. When weaning guinea pigs were fed vitamin A-deficient diets, goblet cells disappeared from the conjunctiva in 7 weeks (Pfister, 1978) (Fig. 6). Keratinization of the ocular surface, loss of surface microprojections, epithelial defects, corneal ulceration, and keratomalacia are common in the case of chronic vitamin A deficiency. Restitution of vitamin A to the diet is curative, with a complete return of goblet cells. Goblet cell deficiencies also occur in a variety of unrelated conditions including erythema multiforme, pemphigoid, alkali burns, and even keratoconjunctivitis sicca. In most of these conditions ocular irritation caused by poor wetting 78
of the ocular surface is a lifelong condition. Not uncommonly, corneal epithelial defects, ulceration, and vascularization occur. Mild cases of dry eye are successfully treated with artificaial tears used every hour and ointment at bedtime. Non-preserved preparations are preferable, especially if epithelial disturbances already exist. Preparations with higher viscosity are preferred by patients with the driest eyes. If three consecutive schirmer I testings on different appointment days give values of 3-5 mm of wetting, permanent occlusion of the lower lacrimal punctae is indicated. If testing shows 0-2 mm wetting then closure of upper and lower punctae is reasonable. The occlusive effect of temporary collagen plugs, as a trial prior to permanent closure, has been seriously questioned (Groden & Forstot, 1991). Using sophisticated radiosintigraphy the 0.4 collagen plug impeded the flow of dye from the eye by an average of only 16.5 Yo. Until further work supports the use of such plugs, the physician must exercise caution in interpreting the results of the procedure. Glasses with side panels which create higher humidity atmospheres around the eyes are useful for the most severe sufferers. Filamentary keratitis, almost always associated with dry eye, usually responds to mechanical removal of the filament with a jeweler forcep and restitution of tears. Occasional sufferers might require a soft contact lens to prevent formation of the filaments.
Autoimmune disease Although a variety of autoimmune diseases can result in corneal epithelial defects, only two will be considered here. Persistent epithelial defects are especially common in Sjogrens syndrome where the progression to ulceration and perforation might occur rapidly. The infiltrate present around the ulcer is disproportionately less than usually expected in the presence of bacterial disease. Measures to improve the tear film form the first line of defence, followed by soft lens therapy. The combination of conservative treatments, aided by immunosuppression, results in a high rate of success (Pfister & Murphy, 1980). Mooren’s ulcer. is characterized by a peripheral corneal infiltrate which ulcerates, producing a typical central overhanging edge extending circumferentially and later centrally. Unilateral in-
Clinical measures to promote corneal epithelial healing
Fig. 5. (A) Epithelial irregularity of two dry spot areas, arrows, is evident on an otherwise smooth corneal surface. (B) This dry spot consisted of a cluster of five corneal epithelial cells separating from contiguous surface, (C) Separating epithelial cells in a dry spot show loss of surface microprojections and irregularity and disruption of plasma membrane. Retraction fibrils, arrowheads, and discrete anterior cellular edges mark disrupted attachments with adjacent cells. PD, thinest pachymetry depth. (from Pfister R R & Renner M E (1977): Invest Ophthalmol 16: 1025).
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Fig. 6. (A) Control cornea shows smooth, continuous sheet of flat polygonal epithelial cells. (B) Seven-week vitamin A-defecient cornea. Entire surface layer of cells shows extensive separation and desquamation from epithelial sheet. (C). Control conjunctiva. Numerous goblet cells, arrowheads, in various stages of maturation, stud the surfaces of palpebral and cul-de-sac conjunctiva. (D) Six-week vitamin A-deficient conjunctiva. Keratinized epithelial cells show extensive separations and desquamatiion from surface epithelial sheet. Total absence of goblet cells is a striking finding (from Pfister R R & Renner M E (1978): Invest Ophthalmol 17: 874).
volvement is most common in the elderly and is more responsive to topical steroids and coniunctival resection. Bilateral disease in the youngmen, especially those occurring simultaneously, are more aggressive, requiring immunosuppression for control (Brown & Mondino, 1984; Foster, 1985).
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BASEMENT MEMBRANE The attachment of the epithelium to its underlying basement membrane is crucially important to its integrity. Basement membrane is a secretion product of the basal corneal epithelial cells, requiring about 6 weeks to reform after its destruc-
Clinical measures to promote corneal epithelial healing
tion Khodadoust (1968). During this interval the epithelium adheres poorly to the underlying stroma except at its leading edge. In contrast, epithelium that has migrated over preformed basement membrane tightly adheres over its entirety in 6 days. In either case strong adherence is correlated with the presence of many electron densities (hemidesmosomes) along the basal cell membrane (see: PBBllysaho et al., 1992; Cipson et al., 1992). Damage to the basement membrane and its disappearance or failure to regenerate appears to constitute the major reason for poor adherence of the epithelium in many diseases. A variety of diseases are rooted in basement membrane disorders. In a biopsy specimen examined by electron microscopy, traumatic recurrent corneal erosion syndrome showed the absence or discontinuity of basement membrane in the affected area. Goldman (1969). A technique of multiple, non-perforating punctures made one-half stromal thickness, performed in the area of erosions, has virtually eliminated the disease (McLean et al., 1986). In addition, metaherpetic erosions are frequently correlated with therapeutic caustic scrubs of the cornea that damage basement membrane (Kaufman, 1964). Dystrophic processes, characterized by corneal erosions, such as anterior membrane dystrophy, represent multilamination of the basement membrane. Scraping the corneal surface to remove the epithelium and redundant layers of basement membrane often eliminates the erosions. If erosions persist then multiple punctures of the stroma are indicated. The shield lesion of vernal catarrh also represents an erosive phenomenon consequent t o the abrasive effect of upper lid vegetations and severe mast cell degranulation. Diabetics are subject to epithelial erosions, especially after surgery, though evidence to implicate an abnormally thickened basement membrane is not convincing (Taylor & Kinsey, 1981). Substantial damage to the basement membrane has also been demonstrated in patients with keratoconus who had been treated by thermokeratoplasty (Fogle, 1977). Only in the epithelial recovery after an alkali burn does this correlation between basement membrane injury and recurrent erosion break down. Disappearance of the basement membrane after the burn is correlated with loss of progression of epithelial migration, but this reversal in epithelial movement occurs even af6
ter an alkali burn in a cornea previously subjected to a lamellar keratectomy (Pfister, 1976) (Table 1).
Role of inflammation Large numbers of polymorphonuclear leukocytes in corneal tissues are likely to encourage the development or persistence of epithelial defects as well as ulceration. Corneas with epithelial defects cultured in vitro showed significant inhibition of epithelial healing when the perfusate contained either viable stimulated PMN or a PMN lysate (Wagoner, 1984). Drugs directed against the respective infectious entity usually eliminates the agent and thereby terminates the inflammatory component inhibitory to healing. Topical sodium citrate decreases corneal ulceration by inhibiting neutrophils in alkali-injured animal eyes (Pfister et al. 1982). Its utility in the human awaits the results of an ongoing clinical trial. For these reasons it is important to control the inflammatory response to protect the stroma as a suitable substrate for epithelial regrowth.
Influence of perilimbal epithelial loss The size and specific involvement of the epithelial defect influences the course and the permanence of epithelial recovery. In the absence of basement membrane or stromal injury, epithelial defects that do not exceed the limits of the cornea usually heal without incident. When epithelial defects destroy most of the limbal palisades of Vogt, epithelium from the conjunctiva must repopulate the cornea. Conjunctival epithelium has substantially different biochemistry and morphology compared to corneal epithelium. A classic example of damage to the perilimbal conjunctiva resulting in poor reepithelization is the alkali-injured eye. To treat this condition it has been shown that superficial keratectomy followed by transplantation of healthy conjunctiva onto the alkali-injured eye improved the vision and stabilized the ocular surface (Thoft, 1979). In these studies, seven of 10 eyes had improved sight, but all showed an improved and more stable ocular surface. The discovery of a discrete group of stem cells in the depths of a narrow zone of perilimbal conjunctival epithelium has given credence to the 81
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concept of conjunctival transplantation and led to newer surgical approaches to corneal reepithelialization. Evidence that these cells are progenitors of corneal epithelium includes reaction to a specific monoclonal antibody (Zieske & Bukusoglu, 1990), the presence of a specific 64 K keratin, and an enhanced growth curve compared to corneal or conjunctival epithelium. Excision of all limbal epithelium from a normal eye leads to poor epithelialization and corneal vascularization; a sign that a critical element has been removed from the generative source of corneal epithelial cells. These basic data give substance to current recommendations of limbal conjunctival autograft from the uninjured eye to the injured eye for those patients who have sustained extensive unilateral stem cell injury (Kenyon, 1989).
Inhibition of polymorphonuclear leukocytes by cit rate Severe alkali burns present a very complex process of healing including an initial phase of normal epithelial healing followed by abrupt loss of adhesion of the leading edge and roll-back of the epithelial sheet. The subsequent influx of neutrophils leads to ulceration and perforation of the eye (Pfister, 1976). A new and clinically untried idea is the inhibition of polymorphonuclear leukocytes (PMNs) in situ by topical citrate. Sodium citrate prevents the accumulation, phagocytosis, and subsequent release of degradative enzymes and free oxygen radicals from PMNs. Animal and laboratory studies with citrate have shown it to be a chelator of extracellular Ca2+. Calcium is probably necessary for the activation of the PMNs, acting as an important intracellular second messenger. Inhibition of the PMNs through calcium depletion may be caused by interference with calcium-calmodulin modulated microfilament or microtubule interfaces in the plasma membrane (Pfister et al., 1984). Hence, topical treatment with citrate may bring the entire set of PMN activities to a halt.
Fibronectin as an enhancer of epithelial healing Fibronectin has been implicated as a key element in wound healing for its involvement in cell82
to-cell and cell-to-matrix adhesion and cell spreading. Eye trauma causes exudation of large proteins such as fibrinogen which reach the bare basement membrane where polymerization and deposition take place. In vitro studies of rabbit corneas show accelerated epithelial healing in the presence of fibronectin (Nishida et al., 1983). Fibronectin eye drops have been used to treat a variety of conditions with persistent epithelial defects. Epithelial defects occurring in herpetic keratitis, trophic corneal ulcers and after cataract surgery have responded to fibronectin drops when used in an open labeled study (Nishida et al., 1985; Nishida et al., 1983; Nishida et al., 1987). An alternative explanation is suggested by an animal study showing that albumin eye drops were as effective as fibronectin in the treatment of persistent epithelial defects (Boisjoly et al., 1987). This suggests that whatever favorable effect noted might be a nonspecific response.
Growth factors Epidermal growth factor (EGF) enhances the rate of healing and induces hyperplasia in corneal epithelium (Cohen, 1984; Ho, 1974). EGF stimulated complete epithelial healing after alkaliinjury in two studies, but in each instance recurrent erosions reestablished the defect (Eiferman, 1987; Singh & Foster, 1987). Although no beneficial effect on epithelial migration could be shown by topical administration of mouse EGF after penetrating keratoplasty in the human, epithelial defects healed in half the time over epikeratophakia lenticules using human EGF. Eiferman (1985). The latter study is of special importance, since persistent epithelial defects do occur after epikeratophakia, sometimes leading to ulceration and requiring replacement of the lenticule. Growth factors may find a more favored place in the acceleration of wound strength (Woost, 1985) by the mechanism of enhancement of macromolecular synthesis.
References Boisjoly H, Beaulieu A, Giasson M & Menard C (1987): The effect of fibronectin compared to albumin on rabbit epithelial wound healing. Invest Ophthalmol Vis Sci (Suppl) 28: 52.
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Brown S I & Mondino B J (1984): Therapy of Mooren’s ulcer. Am J Ophthalmol 98: 1-6. Cohen S (1984): Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. Am J Ophthalmol 98: 411-415.
Eiferman R, Brightwell J , Rowsey J, et al. (1985): Acceleration of corneal epithelial resurfacing and keratocyte migration in primate epikeratophakia by biosynthetic human EGF. Invest Ophthalmol Vis Sci (Suppl) 26. Eiferman R & Schultz G S (1987): Treatment of alkali burns in rabbits with epidermal growth factor. Invest Ophthalmol Vis Sci (Suppl) 28: 52. Foster C S (1985): Systemic immunosuppressive therapy for progressive bilateral Moorens ulcer. Ophthalmology 92: 1436-1439. Fogle J A, Kenyon K R & Stark W J (1977): Damage to epithelial basement membrane by thermokeratoplasty. Am J Ophthalmol 83: 392-401. Goldman J N, Dohlman C H & Kravitt B A (1969): The basement membrane of the human cornea in recurrent epithelial erosion syndrome, Trans Am Acad Ophthalmol Otolaryngol 73: 471-481. Groden L T, White W & Updegraff S (1989): Porcine collagen corneal shield treatment of persistent epithelial defects following penetrating keratoplasty. Invest Ophthalmol Vis Sci (Suppl) 30: 340. Higbee R G & Hazlett L D (1984): Topical ocular anesthetics: effect on corneal cytoskeleton. Invest Ophthalmol Vis Sci (Suppl) 25: 321. Ho P C, Davis W H, Elliott J H & Cohen S P (1974): Kinetics of corneal epithelial regeneration and epidermal growth factor. Invest Ophthalmol Vis Sci 13: 804-809.
Kaufman H E (1964): Epithelial erosion syndrome: metaherpetic keratitis. Am J Ophthalmol57: 983987.
Kenyon K R, Tseng S C (1989): Limbal autograft transplantation for ocular surface disorders. Ophthalmology 96: 709. Khodadoust A A, Silverstein A M, Kenyon K R & Dowling J E (1968): Adhesion of regenerating corneal epithelium: the role of basement membrane. Am J Ophthalmol 65: 339-348. Kinoshita S, Friend J & Thoft R A (1984): Ocular surface epithelial regeneration and disease. Int Ophthalmol Clin 24: 169. Mathers W D, Shields B S, Sachdev M S , Petroll M W & Jester J V (1991): Meibomian gland dysfunction in chronic blepharitis. Cornea 10: 286-
rent erosion: treatment by anterior stromal puncture. Ophthalmol 93: 784-788. Nishida T, Nakagawa S, Awata T et al. (1983): Fibronectin promotes epithelial migration of cultured rabbit cornea in situ. J Cell Biol 97: 1653. Nishida T, Jakagawa S & Manabe R (1985): Clinical evaluation of fibronectin eyedrops on epithelial disorders after herpetic keratitis. Ophthalmology 92: 213-216.
Nishida T, Yagi J, Fukuda M, Kusube T & Otori T (1987): Spontaneous persistent epithelial defects after cataract surgery. Cornea 6: 32-37. Pfister R R (1976): The alkali burned cornea. I. Epithelial and stromal repair. Exp Eye Res 23: 5 19-535.
Pfister R R (1978): The corneal and conjunctival surface in vitamin A deficiency: a scanning electron microscope study. Invest Ophthalmol Vis 17: 874883.
Pfister R R, Haddox J D, Dodson R W & Deshazo W J (1984): Polymorphonuclear leukocytic inhibition by citrate, other metal chelators, and trifluoperazine. Invest Ophthalmol Vis Sci 25: 955-970. Pfister R R, Paterson C A & Hayes S A (1982): The efficacy of sodium citrate in the treatment of severe alkali burns on the eye is influenced by the route of administration. Cornea 1: 205-21 1. Pfister R R & Burstein N (1976): The effects of ophthalmic drugs, vehicles, and preservatives on corneal epithelium: a scanning electron microscope study. Invest Ophthalmol Vis Sci 15: 246-259. Pfister R R & Murphy G E (1980): Corneal ulceration and perforation associated with Sjogrens syndrome. Arch Ophthalmol 98: 89-94. Singh G & Foster C S (1987): Epidermal growth factor in alkali-burned corneal epithelial wound healing. Am J Ophthalmol 103: 802-807. Taylor H R & Kinsey R A (1981): Corneal epithelial basement membrane changes in diabetes. Invest Ophthalmol Vis Sci 20: 548-553. Thoft R A (1979): Conjunctival transplantation. Ophthalmology 86: 1084. Wagoner M D, Kenyon K R, Gipson I K et al. (1984): Polymorphonuclear neutrophils delay corneal epithelial wound healing in vitro. Invest Ophthalmol Vis Sci 25: 1217-1221. Zieske J D & Bukusoglu H (1990): Numbers of limbal basal cells expressing a 50-kD antigen increase after wounding. Invest Ophthalmol Vis Sci (Suppl) 31: 538.
290.
Mathers W D, Shields B S, Sachdev M S, Petrol1 W M & Jester J V (1991): Meibomian gland morphology and tear osmolarity: changes with accutane therapy. Cornea 10: 286-290. McLean E N, MacRae S M & Rich L J (1986): Recur-
Author’s address: Roswell R. Pfister The Eye Research Laboratories Brookwood Medical Center Birmingham, A1 35209, USA
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