Clinical and experimental aspects of corneal transplantation

Clinical and Experimental Aspects of Corneal Transplantation KerynAnne Williams and DouglasJohn Coster

orneal transplantation is both widely practised and frequently misunderstood. Somewhere between 50,000 to 100,000 corneal grafts are performed around the world each year and the procedure is generally acknowledged to be a highly successful therapeutic option. However, the common belief that all patients who are blind as a result of corneal disease can be cured by corneal transplantation and that corneal grafts are virtually always successful is incorrect. Some people with corneal opacities may gain no visual benefit from corneal transplantation, whereas others who might be helped are not given the opportunity of a graft because of a chronic shortage of donor material. The most troublesome misconception is that because the cornea is an immunologically privileged site, corneal grafts never undergo rejection. In fact, irreversible immunologic rejection is the major cause of corneal graft failure. The cornea is the transparent surface at the front of the eye through which light passes on its way to the retina. The major refracting element of the ocular optical system, it contributes over 80% of the converging lens power of the eye. Maintenance of good vision requires that the cornea be intact, crystal clear, of undisturbed ultrastructure, and possessed of a ve~'y regular anterior surface. A variety of disease processes (including infection and traumatic damage) may scar the cornea, rendering it opaque to the passage of light, or may cause corneal perforation. Dystrophic conditions of the cornea can result in corneal thinning or may produce oedema (often accompanied by pain) or irregularities of corneal shape and surface architecture, all of which can reduce visual acuity. Some infants are born with congenital corneal opacities or abnormalities that are often bilateral. Corneal transplantation is a

C

From the Departmozt ~ Ophthabnolog)~, Flinders University of South Australia, BedfordPark, Australia. Supported in part by the National Health & Medical Research Council and the Ophthalmic ResearchInstitute ofAustralia. Address reprint ~quests to Keryn Anne Williams, PAD, Department of Ophthalmology,,Flinders Medical Centre,BedfordPark, SA 5042, Australia. Copyright © 1993 by W.B. Saunders Cbmpa~, 0955-470X/93/0701-000455.00/0

44

well-established surgical treatment for the restoration of vision in such cases. The World Health Authority has estimated that 10 to 15 million people worldwide are blind 1 and that a further 42 million are affected by severe visual loss. 2 Blindness is associated with enormous personal and economic cost to those concerned. Cataract is the major cause of blindness, with diseases of the cornea second. 2 Although the majority of those blind from corneal disease live in developing countries, 3 the problem is by no means restricted to these geographic locations. However, the major indications for graft do vary considerably from place to place. In Australia, with a population of approximately 17 million people, about 1,500 corneal grafts are performed each year (data from the Australian Corneal Graft Registry). The rate is higher in America, with 30,000 to 40,000 grafts being reported per year in a population of about 215 million, 4'5 and lower in the United Kingdom, with 1,500 to 2,000 grafts reported per year in a population of about 60 million. 6 The two major factors that influence the number of corneal grafts performed are the availability of donor corneas for transplantation and the relative proportions of different indications for graft within the potential recipient pool. The chronic shortage of donor corneas in most countries is the primalT factor limiting the number of people who can benefit from the procedure.

Brief Historical Overview Rycroft 7 and Mannis and Naachmer 8 have provided detailed and fascinating historical reviews of the surgical procedure of corneal transplantation, from the early suggestions of de Quengsy and Erasmus Darwin in the last decade of the eighteenth century, through the first partially successful experimental allografts in the gazelle and the rabbit described by Bigger in 1837, to the first successful human allograft reported in 1906 by Zirm. It is of interest that early attempts at corneal xenografts usually failed and that interest in this type of graft has only recently been rekindled. The impact of improved surgical technology and instrumentation on the practice of

Transplantation Reviews, I.bl 7, No 1 (Januao,), 1993:pp 44-64

Corneal Transplantation

corneal transplantation has been reviewed by Forstot and Kaufman. 9 Successful outcomes have been influenced as much by the development of the operating microscope, modern ophthalmic sutures of high tensile strength, and microinstruments, as by developments in the technology of storage of donor corneas and the advent ofimmunosuppressive drugs.

Relevant Anatomic and Physiological Considerations The h u m a n cornea m is bounded anteriorly by a nonkeratinised epithelium, 5 to 6 cells thick. The basal epithelial cells are attached to a 60- to 65-nm thick basement membrane and the anterior surface is in contact with the tear film. Small numbers of corneal Langerhans cells I1 are found in the basal layers of the epithelium, their numbers increasing towards the limbus, which is the junctional area between the transparent cornea and the opaque sclera. Under the basement membrane lies Bowman's membrane, an acellular collagenous layer found only in primates. The corneal stroma, which forms 90% of the bulk of the cornea, is composed of a highly ordered array of collagen layed down in lamellae running parallel to the corneal surface, interspersed with keratocytes (corneal fibroblasticlike cells believed to be responsible for producing the stromal matrix) and a few macrophages and interstitial dendritic cells. Beneath the stroma lies Descemet's membrane. The cornea is limited on its poste-

Figure 1. Silver-stained fiatmount of human corneal endothelial cells counterstained with haematoxylin. Note the enlargement of some cells in the monolayer.

45

riot surface by the corneal endothelium, which is in contact with the aqueous humour. In histological flat-mount preparations, the endothelium appears as a monolayer of hexagonal cells with a "cobblestone" appearance (Fig 1). In humans, the central cornea is approximately 0.56- to 0.57-mm thick. Despite its straightforward anatomy, the cornea is by no means physiologically inert. The endothelium is particularly important to corneal function 9d2-14 in that it contains a leaky, sodium-potassium ATPase-dependent electrolyte pump responsible for the active transport of ions across the posterior endothelial cell membrane and for the concomitant passive flow of water from the stroma to the anterior chamber. The stroma is thus maintained in a state of relative dehydration. Loss of the p u m p function results in the stroma becoming oedematous: because corneal clarity depends on the maintenance of the ordered structure of the stromal lamellae, 12,t5 an oedematous cornea becomes opaque. The corneal endothelium seems to be derived from neural crest and is a mesothelium. It shares only a name with vascular endothelium. In humans, corneal endothelial cells have been shown to have a somewhat limited capacity for mitosis, repair of the monolayer occurring by a process of enlargement and sliding of existing cells to cover the defect. 14 However, it is interesting that human corneal endothelial cells may be induced to undergo mitosis in vitro. 16 A typical adult endothelial cell density is of

46

Williams and Coster

Clinical Aspects of Corneal Transplantation

cornea

The Corneal Graft Procedure

:~. damaged area

), diseased cornea removed

replaced by donor cornea

Figure 2. Diagrammatic representation of the procedure of penetrating corneal transplantation seen ill crosssection. The damaged section of the recipient's cornea is removed and replaced by normal donor cornea. the order of 2,500 to 3,000 cells/mm2; a density of approximately 500 cells/mm 2 is believed to be sufficient to maintain corneal deturgescence and hence clarity) 3 Endothelial cell loss may occur as a result of disease or surgery, and is a major underlying reason for corneal transplantation in many recipients.

Corneal grafts may be either full-thickness (penetrating keratoplasty) or partial-thickness (lamellar keratoplasty). The procedure for penetrating keratoplasty is shown diagrammatically in Fig 2: a section of damaged or diseased cornea (usually circular in shape) is removed and replaced by normal cornea. Clinical corneal grafts are typically 7.0 to 8.0 m m in diameter but may be any size. The graft is anchored with interrupted or running sutures (or a combination of both), usually of 10-0 nylon, that may stay in place for 12 months or more postoperatively (Fig 3). Most grafts are penetrating because of the need to replace the corneal endothelium. Transplantation of the cornea is thus, at least in penetrating keratoplasty, the transplantation of the corneal endothelium. Unfortunately, corneal endothelium is also one of the primary targets for graft rejection.

Donor Corneas and Eye Banking Although corneas are sometimes harvested from multiorgan donors immediately after death, the majority of all corneas used for transplantation are taken from cadaveric donors within 12 (and prefera-

Figure 3. Successful penetrating corneal transplant. Note the 10-0 nylon suture with the knot at 12 o'clock.

Corneal Tramplantation

bly within 6) hours of death. The stringent restrictions on donor age and general health that are in place for essential organ donors are less important for corneal donors, so that, although younger donors may represent the ideal, corneas taken from quite elderly people can prove perfectly satisfactory, provided the corneal endothelium is healthy and the endothelial cell count high. Contraindications to donation vary from one country to another, but they usually include some or all of: seropositivity for the human immunodeficiency virus (H1V) or the hepatitis B or C viruses, rabies, any neurological disorder with an actual or possible viral etiology, a history of previous eye disease or surgery, death from unknown causes, haematologic malignancies and other neoplasias with metastasis, unstable diabetes, jaundice, and septicaemia. The transmission of rabies, Creutzfeldt-Jakob disease, hepatitis B virus, and a variety of bacterial and fungal organisms from infected donor corneas, reviewed by O'Day "~and by Feigenbaum and Pepose] 7 can pose a threat to the life of the recipient. Screening for hepatitis B and (more recently) hepatitis C and for H1V is performed by most eye banks, and is usually performed on donor cadaveric serum rather than on ocular fluids. ~8 Hepatitis B surface antigen has been detected in ocular washings and emulsified corneal tissue from corneal donors, 19and two cases of transmission to corneal graft recipients have recently been reported. 5 O f seven cases in which a cornea from an H1V-positive individual has been inadvertently transplanted, none of the recipients has thus

T a b l e

47

far seroconverted. 2°,21 However, HIV has been recovered from the tears, ~2conjunctiva, 23corneas 24 (including after corneal preservation25), and retinae ='6 of infected individuals. Eye banks routinely discard corneas from any hepatitis B-, C-, or HIV-seropositive donor. Donor corneas are usually placed in some form of storage system before being released for transplantation (Table 1). The simplest system is the moist pot, in which an enucleated globe is stored on a moistened swab in a sealed container at 4°C for up to 24 hoursS This straightforward (if antediluvian) method is seldom used by modern eye banks and the majority of corneas, excised from the globe as corneoscleral preparations, are placed into either short-, intermediate-, or long-term storage? 8 Corneas may be preserved for a m a x i m u m of 4 days (short-term storage) at 4°C by submersion in McCarey-Kaufman (M-K) medium, -~!~-31for 10 to 12 days at 4°C by submersion in one or other of the intermediate-term storage solutions, which include K-Sol (Clico, Inc, Bellvue, WA), 32 J M solution (Kaken Pharmaceutical Co, Hongo, Tokyo, Japan), 33 CSM (Aurora Biologicals Ltd, Williamsville, NY), 34 Dexsol (Chiron Ophthalmics Inc, Irvine, CA),35 and Optisol (Chiron Ophthalmics Inc), 36 or for 28 days at 31 ° to 34°C 37-4° by suspension in organ-culture medium. A small number of corneas are cryopreserved, 41 which allows almost indefinite corneal storage (long-term storage). However, cryopreservation has proved technically difficult and somewhat cumbersome, and is seldom used.

1. Comparison of Storage Systems for Donor Corneas

Storage Variable Medium (ReferenceNo.) Moist pot 28 M-K medium 3° K-Sol 33

JM 34 CSM 35

DexsoP~ Optisol ~7 CPTES 44 Organ culture (USA) 39 Organ culture (Europe) 41 Cryopreservation42 *Molecularweight 40 Kd. ]'Molecularweight 70 Kd. SMolecularweight 500 Kd.

Maximum Time (da~s) <1 <4 < 14 < 14 < 14 < 14 < 14 < 14 < 28 < 28 indefinite

Colloid OsmoticAgent

Temperature (degreesG') 4 4 4 4 4 4 4 4 34 31 - 196

Dextran (wt / vol)

Chondroitin Sulphate (wt/ vol)

-5.0% T40* -3.5% T70t -1.0% T40 2.5% T40 --5.0% T500~

--2.50% -1.35% 1.35% 2.50% 2.50% 1.50% --

- -

- -

48

Williams and Coster

M-K medium and all of the intermediate-term storage media contain colloid osmotic agents, generally dextran, chondroitin sulphate, or a combination of the two, to maintain corneal dehydration during extended storage at 4°C. All conventional storage media are based on standard tissue-culture media and are designed to reflect the solute concentrations of the extracellular environment. Experimental work is in progress on the use of intracellular-type solutions for the hypothermic storage of corneas 42-44but information on graft outcome (in experimental animals or in humans) after corneal storage in such solutions has yet to be published. Organ-culture systems, first developed in Minneapolis, MN, 37,38 and later modified in Scandinavia ~9 and the Netherlands, 4° are now widely used in Britain 45 and in Europe. 46 A newly described method in which corneas are cultured at an air-liquid interface 47 shows promise, but corneas stored in this manner have not yet been tested clinically. Corneal cryopreservation has been extensively reviewed by Taylor. 28 The successful transplantation of cryopreserved rabbit and human corneas on any scale was first described in 19644~,49 in studies in which the anterior chambers of enucleated globes were first irrigated with a cryoprotectant solution containing dimethyl sulfoxide, glycerol, and plasma, and then the whole eyes immersed in cryoprotectant and frozen to -79°C. Subsequent studies have used excised corneoscleral preparations 41'5° and corneas are generally frozen in a dimethyl sulfoxide-containing cryoprotectant to - 196°C, although experimental work exploring the possibility of using warmer storage temperatures has recently been reported. 51 Current research is focussing on the use of alternative cryoprotectants, 52 on methods of assessing endothelial cell viability after cryopreservation, 53 and on attempts to produce corneal vitrification. 54 For virtually any form of corneal preservation other than the simple moist pot, access to storage solutions and to some laboratory equipment is required. Furthermore, although M-K medium is relatively inexpensive, the cost and lack of availability of some other commercial storage media and ancillary equipment tend to preclude the use of intermediateand long-term preservation systems in many eye banks outside of the developed countries. Uniquely local solutions to the problems of corneal storage have evolved: in India, for example, some enucleated globes are stored for short periods by immersion in

glycerol (R.P. Dhanda, personal communication, March 1992) or even honey.

Indications for Corneal Transplantation The indications for keratoplasty vary, depending on geographic location and local patterns of practice. In Australia, the most common indications for penetrating keratoplasty are keratoconus (Fig 4), pseudophakic bullous keratopathy (corneal decompensation associated with the removal of a cataract and intraocular lens implantation), and failed previous graft (Table 2). In the United States the most common indication is pseudophakic bullous keratopathy, 55,56 whereas in a large single-centre cohort reported from the Netherlands, the most common indication for graft was herpetic keratitis. 57Although few largescale reports are available from developing countries, corneal opacities resulting from the sequelae of trauma and ocular infection are likely to be the leading indications for graft.

F i g u r e 4. Keratoconus. The cornea is avascular but protrudes forward into a cone. As the disease progresses, the irregular astigmatism associated with the highlyirregular anterior refracting surface becomes increasingly difficult to correct with a contact lens and corneal transplantation may be advised.

Corneal Tramplantation

Table 2. Indications for Penetrating Corneal Transplantation Within Australia, 1985 to 199 t

D#ease /Problem Keratoconus Bullous keratopathy pseudophakic aphakic unspecified Failed previous graft Corneal scars and opacities Corneal dystrophy Fuchs' dystrophy Other Corneal ulcers perforations Other Herpetic infection active HSV* HZO~ Miscellaneous:) Not recorded

Subtotal

Total

Percentage (%)

1,071 850

31 25

478

14

364 234

11 7

119

3

104

3

159 81

6 2

577 165 108

170 64 82 37 97 7

Note: Data are from 3,460 grafts collectedthrough the Australian Corneal Graft Registry. *Herpes simplexvirus. tHerpes zoster ophthalmicus. ~:Includesdeformitiesand degenerations, 54; interstitial and other keratitis and corneal abscesses, 54; ocular trauma, 13; congenital disorders, 12; astigmatism not associated with keratoconus, 12: iridocorneoendothelial (ICE) syndrome, 4; acute bacterial infection, 3; keratoconjunctivitis, 2; scleral disorders. 2; autoimmune disease, 2; benign neoplasia, 1. Corneal Graft Outcome Blindness is not generally a life-threatening disorder, so that outcome studies for corneal transplantation, unlike those for essential organ grafts, need take no particular account of recipient survival. The important determinants of corneal graft outcome primarily are graft survival, improvement in visual acuity, relief of pain, structural repair of a damaged globe, and (occasionally) better cosmesis. Because the opportunities for improvement centre largely around the first two, most outcome studies deal with graft survival and with postoperative visual acuity. Graft survival depends largely on the indication for transplantation. First grafts for keratoconus, in particular, do extremely well: Kaplan-Meier probabilities of graft survival of over 95% at 2 to 5 years are by no means uncommon. 57-6°The importance of keratoconus, aside from the fact that it is a frequent indication for graft in developed countries, lies with the clues it provides to the vexed question of the so-called "immunologic privilege" enjoyed by the cornea. 61 Corneal privilege does exist, in the sense

49

that penetrating corneal grafts of moderate diameter placed into normal eyes of experimental animals are seldom rejected, at least in the continuing absence of any untoward stimulus. However, corneal grafts are not placed clinically into normal eyes and the clinical experience is that some grafts fail from irreversible immunologic rejection. Patients with keratoconus and a few other noninflammatory dystrophic conditions of the cornea, such as Fuchs' dystrophy, 6°'a5are exceptions and fare well after transplantation. It seems that in these relatively fortunate recipients corneal privilege is maintained. The normal cornea is to all intents and purposes avascular. The rich network of capillaries that supplies the sclera stops at the limbus. However, corneal neovascularization commonly occurs after corneal infection or any inflammatory stimulus affecting the anterior segment, and many patients who present for corneal transplantation have vascularized corneas and a history of anterior segment inflammation. Patients with keratoconus and some corneal dystrophies, by contrast, usually retain avascular corneas in quiet eyes. It has long been recognized that vascularization 57'62-64and inflammation 6°,65before or at the time of graft exert a negative influence on the outcome of corneal transplantation in univariate analyses, although the importance of the former is not always strong in multivariate analysis. 6°,66It seems probable that the relatively poor outcome of grafts performed for indications such as herpetic keratitis, 67-69in which survival at 5 years may decrease to 50% in some studies, may be caused in part by the confounding effects of vascularization and inflammation. However, preexisting vascularization has not always been found to be a risk factor in these graft recipients 7° and alternative interpretations will be discussed subsequently in this review. There is general agreement that herpetic recurrences after transplantation are associated with a particularly poor prognosis. 6° Other risk factors for corneal graft failure include: graft diameter, with most 6°,63 but not all 64 studies reporting poorer survival with increasing graft size and one study noting decreased smwival of very small grafts6°; a history of one or more failed grafts in the ipsilateral eye, 60,71 although in one report, second grafts fared as well as did first grafts59; and the presence of some types of intraocular lenses in the grafted eye, 6° most probably because of accelerated loss of endothelial cells consequent on the insertion or the continuing presence of these lenses. 72

50

Williams and Coster

Risk factors of disputed relevance include donor age, with some studies reporting decreased graft survival with increasing donor age 63,6. and others being unable to show any particular effect/°'73 In view of the evidence that corneal endothelial cell counts decrease with increasing age, a negative influence of increasing age might well be expected. The failure to show this effect in some instances is possibly a reflection of the substantial reserve capacity of the normal endothelium. ~4 The modern statistical tools of multivariate analysis 74 are increasingly being applied to corneal transplantation, especially to registry data, 6° in an attempt to isolate independent risk factors for graft failure from among the plethora of potential factors that have been identified in univariate analyses. It will be clear from the preceding discussion that the results of corneal transplantation vary considerably, depending on the indications for transplantation and the state of the recipient cornea at the time of graft. Kaplan-Meier graft smwival for some of the patient subgroups described previously can decrease to less than 50% at 3 years and, as in other forms of transplantation, the centre effect also influences outcome. 6° The upshot of this situation (in which graft outcome can be predicted with a fair degree of accuracy before the operation) is that where donor

corneas are scarce, available donor material is frequently given preferentially to recipients in whom the chances of success are high. Such patients include those suffering from keratoconus, which is often bilateral, or one of the corneal dystrophies. The excellent results obtained for these patients help to fuel the mistaken belief that corneal transplantation is invariably successful. Corneal grafts can fail (Fig 5) for a number of reasons, and failure is indeed often multifactorial. However, irreversible immunologic rejection (Fig 6) is almost invariably the most c o m m o n cause of graft loss. :~9'~;°It accounts for 42% of such cases in the Australian database, with the sequelae of uncontrolled increased intraocular pressure, herpetic recurrences, and other infective complications accounting for a further 25%. 6o Our most recent analysis of 2,248 patients followed up for as long as 5 years, suggests that one in every five patients in this multicentre cohort will experience at least one rejection episode in the postoperative period. Two thirds of these will be reversible. Many recipients of corneal grafts expect an improvement in their visual acuity after transplantation. Plainly, the first requirement in such cases is that the graft survive. However, a secondary requirement is that a regular anterior refracting surface be

Figure 5. Failed corneal graft. The central cornea is oedematous and cloudy.

Cbrneal Tramplantation

51

Figure 6. Slit-lamp view of rejecting corneal graft. A rejection line, visible in the bottom left-hand corner, comprises a wave of leucocytes that will move across the cornea, killing underlying cells of the cornea in their wake. The cornea thickens and clouds behind the advancing line because the endothelial monolayer has been damaged. achieved in the graft. Irregular astigmatism is a major cause of poor postoperative acuity in otherwise successful corneal transplants. 75'76 The Australian data suggest that approximately 90% to 40% of all recipients will suffer from five or more dioptres of irregular astigmatism after corneal transplantation 6°,76 and many of these will require refractive surgery to reduce their astigmatism. Comorbidities in the grafted eye, sometimes unrecognized before the operation, provide a third reason for disappointing postoperative acuity. 77 Such comorbidities, which include cystoid macular oedema, amblyopia, and retinal detachment, may affect up to 40% of some cohortsfl ~ Visual acuity is usually measured with the standard Snellen chart, best-corrected with the patient wearing any prescribed spectacles or contact lens. At least half of all recipients require some correction after graft. 76In a study of patients receiving transplantations for keratoconus, Kirkness et al reported that 91% of the cohort eventually achieved a corrected acuity of 6/12 or better. 59 In the Australian register, 85% of patients receiving grafts for keratoconus have achieved 6/18 or better, whereas 56% of the total

cohort with penetrating corneal grafts have achieved a best-corrected Snellen acuity of 6/18 or better. Graft failure is now the major cause of poor acuity. In a recent study, TM we examined the patients' own perceptions of the outcome of their corneal graft after follow-up for at least 2 years. Not unexpectedly, dissatisfaction was associated with graft failure but also in some instances with contact lens wear even though the level of corrected Snellen acuity achieved was ve W good. Interestingly, satisfaction was positively associated with achievement of a better level of acuity in the grafted eye than in the contralateral eye, as well as with graft clarity. These data suggest that from the viewpoint of the recipient, achievement of an excellent optical result in the grafted eye is not necessarily the most important determinant of perceived success.

I m m u n o l o g i c a l A s p e c t s of Corneal Transplantation Animal Models Many immunologic factors influencing corneal transplantation have been established in experimental

52

H'illia~r~rand Coster

systems and a brief description of the more commonly used models is warranted. Animal models of corneal transplantation may be broadly divided into heterotopic and orthotopic models. Heterotopic grafts are usually performed in the mouse, either by insertion of full-thickness corneas into thoracic wall, 79 abdominal pockets, 8° or into the subcapsular renal space. 81 A difficulty with all such models is that graft survival must largely be assessed by end-point histology, although some assessment of outcome can be made by direct visual inspection of the graft at the end of the experiment. In addition, at least in grafts under the renal capsule, corneal endothelium fails to survive, even in isografts, a20rthotopic corneal transplantation has been reported in the rat, ~3,84 rabbit, ~5,~6 cat, 87 monkey, ~8 and mouse. 89 Rotational autografts have been used for some purposes but most studies, especially those designed to investigate the immune response to a graft, have used allografts or, less frequently, xenografts. 9° Various other models have been described, for example, those in which corneas are placed in intracorneal lamellar pockets, 9~,9ebut most interest has centred around the use of full-thickness, orthotopic grafts, which are the most clinically relevant. The choice of model depends largely on two factors: (1) whether an inbred or an outbred model is required, and (2) whether corneal endothelial cell repair by mitosis is acceptable, given the experimental aims. Mitotic repair in vivo is extensive and rapid in the rat, ~3'94 occurs to a limited extent in the rabbit, 95 and probably occurs rarely, if at all, in the adult cat, 96 or in humansJ 4 This is particularly important, for example, when rejection is being studied or corneal viability after experimental storage is being assessed, because repair may occur by mitosis of host rather than graft endothelial cells and the former may then migrate over the graft. Additional factors of importance may relate to similarities (or disparities) in size, anatomy, physiology, and rejection processes between humans and the various experimental species. Overall size and general anatomy will influence the technical ease with which a corneal graft may be performed. The recently described procedure oforthotopic transplantation of the cornea in the inbred mouse must certainly be applauded as a technical tour-de-force, but reported technical failure rates are rather high. 97 This is not surprising, considering the very small size of the mouse eye. However, the murine model is particularly appealing because of the vast range of biological reagents, probes, and strains available in

the species and the wealth of knowledge of the murine major histocompatibility complex (MHC). Because of the relatively large size of the rat lens in relation to the whole eye, corneal transplantation in the rat is complicated by the likelihood of accidental damage to the lens and subsequent cataract formation. Corneal transplantation in the cat is technically demanding because the cornea is extremely floppy and sutures "cheese-wire" readily. In addition, there are ethical dilemmas to be faced by those who seek to use this pet species. However, corneal grafts are sometimes performed in cats in situations where a nonreplicative corneal endothelium is mandatory. For experimental studies of the immunology of corneal graft rejection or of alternative regimens of immunosuppression for corneal grafts, the corneal privilege of the normal eye must be overcome to ensure rejection in the first place. Various stratagems are used to ensure rejection will occur in corneal allografts, including transplantation into prevascularized eyes, 86,98,99 and the transfer of donorstrain skin to an animal already bearing a corneal graft, a6 There is strong evidence that the privilege enjoyed by a corneal graft transplanted into a normal eye represents an afferent, rather than an efferent block, and once sensitization has occurred, rejection rapidly follows, a6 Despite a recent revival of interest in corneal xenografts, l°° it is probably fair to conclude that allotransplantation in the inbred rat (Fig 7) is now the model of choice for many studies of the basic biology of corneal graft rejection (despite the caveat that rat endothelium divides readily in vivo) and that the rabbit is the favoured species when an outbred model is required or when regimens of immunosuppression are being examined.

Expression of MHC, ABO, and Adhesion Molecules in the Cornea M H C class I antigens are constitutively expressed on corneal epithelium and keratocytes in humans l°M°4 and the rat, 1°5 are probably expressed on at least some normal adult rat endothelial cells, 1°6 but may be absent or weakly expressed on h u m a n corneal endothelial cells. 101,10_~,104However, isolated reports of localization on h u m a n endothelial cells do exist 1°a,1°6 and endothelia from human infants under 2 years of age are reportedly quite clearly M H C class I antigenpositive. 102 In normal human, 101-1(14guinea pig, 107,108and rat 105 corneas, M H C class II antigens are localised primarily on Langerhans or dendritic cells, most of

Corneal Transplantation

53

F i g u r e 7. Successful orthotopic corneal isograft in the rat. An interrupted suture pattern has been used. The cornea is avascular; the vessels visible are within the iris.

which are found in the basal epithelium. The corneas of normal h u m a n infants contain more epithelial Langerhans cells than do adult corneas. 1°9 Substantial species variations have also been observed] I with the mouse, for example, showing a paucity of corneal Langerhans cells 79,110c o m p a r e d with the human. Expression of M H C antigens can be induced or upregulated in the cornea as in other tissues by in vitro culture and under the influence of inflammatory cytokines. Thus, interferon g a m m a has been shown to induce expression of class II antigens on cultured h u m a n corneal endothelial, 1~1 stromal,11 i,l 1.~ and epithelial cells 112and on rabbit endothelial cells, tt3 Rejecting h u m a n corneal allografts exhibit expression of both class I and II antigens on endothelium, stromal cells, and basal epithelial cells, 114 whereas herpes simplex virus infection seems to induce class II antigens on cells in the corneal stroma but not on endothelial cells, l°s'll4 It is not clear whether the increased numbers of M H C class II antigen-positive cells found in the s t r o m a of rejected or HSV-infected corneas represent upregulation of expression on keratocytes or an accumulation of class II antigenpositive dendritic cells in these corneas, but the latter is the more likely explanation. Vascularized human, 115,116 rabbit, 9~ and rat ~7 corneas have been shown to contain significantly more dendritic cells in

the central stroma than do normal corneas; such infiltrates seem to be sessile. A B O blood group antigens have been reported to be present by absorption-elution studies in h u m a n cornea 11~ but absent from endothelium.~t9 The potential role of adhesion molecules and homing receptors in controlling the influx of leucocytes into the eye is currently engendering some interest. Intercellular adhesion molecule-1 (ICAM-1) has recently been shown to be expressed on cultured h u m a n corneal endothelial cells; upregulation on endothelial and stromal cells was observed after incubation of whole corneas in vitro with interferon g a m m a , t u m o u r necrosis factor-co or interleukin113.12<~,t21 Interestingly, normal mouse cornea does not express ICAM-1, although this antigen can be induced on corneal epithelium and keratocytes but not on endothelium by overnight incubation in supernatant from Concanavalin A - t r e a t e d murine spleen cells. ~1 The expression of VLA proteins in the cornea has been examined and one study found h u m a n corneal epithelium to be positive for VLA-2-, -3, -4, -5, and -6 proteins, whereas keratocytes expressed the common V I A b e t a chain and VLA- 1, -2, -3, -4, -5, and -6 proteins were localized to endothelium? 22 O t h e r investigators have reported VIA-1 to be expressed on epithelium as well as VLA-2 to -6.123

54

Williams and Coster

The Effect of HLA and ABO Matching on Human Corneal Graft Survival Matching for MHC class I antigens has been shown in a small number of studies to improve corneal graft smMval in high-risk cases, 63,6~,124-126 although there are reports to the contrary. 127 The situation with respect to MHC class II antigens is even less clear, with some investigators reporting (or at least suggesting) a beneficial effect of matching for DR antigens I%'12a129 and others showing no positive influence?~3Forthcoming reports on the influence of HLA matching on corneal graft smMval from The United Kingdom Transplant Service and from the organizers of a multicentre trial in the United States are being awaited udth some interest. There have been some recent suggestions, not to our knowledge as yet published, that matching for ABO blood group antigens in corneal transplantation may improve graft survival in high-risk cases, but again, evidence to the contrary also exists. 63'~24'1:3t~There is one report that group A donor-group O recipient combinations do well postgraftY

correlation between rejection rates of corneas and livers in any of these strain combinations and the investigators suggested that the survival of corneal grafts in the rat may be controlled by immune response genes, not all of which are linked to the rat MHC. The response to various major and minor MHC differences was further dissected using congenic strains and it was concluded that individual class I and class II differences were of no more importance than minor incompatibilities. These fascinating and somewhat surprising data, recently extended by Katami in a detailed review) 36 suggest an unexpected level of complexity in the immune response to a corneal graft. Confirmatory evidence has recently been provided by Nicholls et al, 137 who have emphasized the likely importance of minor histocompatibility antigen disparities, and have suggested that cornea-specific alloantigens may play a role in corneal graft rejection. Establishing the theoretical basis for any sensible policy of antigen matching in human corneal transplantation may be more difficult than was previously anticipated.

Relevance of MHC and Other Antigens in Experimental Corneal Transplantation

Macroscopic, Histological, and Phenotypic Correlates of Graft Rejection

In models of heterotopic corneal cell transfer and whole cornea transplantation in the rat, Treseler et a1131'132 examined the generation ofcytotoxic effector cells across different MHC antigen barriers. They concluded that effector cells were most easily generated when donor and recipient were disparate for both class I and II antigens and that although a major part of the response generated was directed against class I antigens, class II antigen-positive cells were contributing to the immunogenicity of the grafts. Niederkorn et a1133showed that orthotopie rat grafts transplanted across an MHC class II antigen barrier alone were rejected in sensitized but not unsensitized animals. They suggested that the transience of MHC class II antigen expression on epithelium tbllowing transplantation in the rat is a major determinant of the rejection process. These workers also examined the fate of corneas transplanted across a single class I locus barrier: all preimmunized and a proportion of naive animals rejected their grafts. 134 Katami et al studied the rejection of orthotopic grafts in 28 fully allogeneic and 2 partially allogeneic rat donor-recipient strain combinations/4,135 In the fully allogeneic combinations, three patterns of rejection were observed: acute rejection, delayed rejection, and prolonged survival. There was no particular

Maumenee l~a has described the onset of a typical clinical corneal allograft rejection: the anterior segment becomes slightly inflamed, cells may be visible in the anterior chamber and small keratic precipitates (local accumulations of cells) appear on the endothelium. A rejection line often appears and moves across the graft. Such lines represent waves of leucocytes that move across the cornea, destroying the underlying cells so that the cornea decompensates in the wake of the advancing edge. ~:~jEpithelial, stromal, and endothelial rejection are all wellrecognized entities 1:~9-142but tend to merge into one another once rejection is well established. Graft rejection in experimental animals mimics to varying degrees, the macroscopic pattern observed in clinical grafts, depending on the species. Thus, rejection processes in the rabbit, for example, seem very similar to those observed clinically but rejection lines are seldom if ever observed in the rat, and orthotopic rat corneal grafts that can be shown by pachymetry to be quite oedematous do not always appear particularly opaque. Histological studies of the cells present in rejecting grafts have mostly been performed in experimental animals. In the rabbit, the leucocytic infiltrate within the stroma is heterogeneous, containing lymphocytes and blasts together with some macro-

Cbrneal Transplantation

phages, plasma cells, and polymorphonuclear granulocytes. 139,m Immunohistochemical analysis of unmodified, rejecting rabbit grafts has shown that about half the leucocytes accumulating within the graft are T cells, two thirds carry MHC class II determinants and about one fifth carry myeloid markers. 143 Kinetic studies of the cells invading the anterior chamber over a 10-day period showed a similar pattern. In the rat the graft infiltrate is similarly heterogeneous, containing neutrophils, lymphocytes, and macrophages) 44 Both CD4- and CD8positive cells have been observed in particular donorrecipient strain combinations, 145q47 with CD4 cells predominating early in rejection, 145 and most T cells bearing the interleukin-2 receptor. 147 Upregulation of MHC class II antigens on endothelium in rejecting rat 147 and rabbit t43'148 grafts, presumably as a result of local cytokine release, may fuel the rejection process. As an interesting aside, there has been one report ~49 of acute corneal graft rejection 10 years after transplantation for keratoconus, in a patient undergoing treatment with interferon alfa-2 for hairy cell leukaemia. The rejection episode was reversed with topical corticosteroids. A simplistic explanation for the onset of rejection in this case would seem to be an interferon alfa-2-induced upregulation of expression of donor-type MHC class I antigens 15° on cells within the cornea, providing additional targets for host effector cells. Steroids have been shown to downregulate MHC class II antigens 151 but, of course, also exert a plethora of effects on inflammatory cells. The number of reports describing the cells present in human grafts during rejection is limited, largely because serial biopsies are never performed on corneal grafts and grafts are seldom removed at the time of rejection. In consequence, most human corneal grafts examined have represented late or burnt-out rejection. Several studies have shown an increased number of dendritic cells 9a H4 in sections of rejected human allografts.

Immunologic Monitoring in Corneal Transplantation The subject of immunologic monitoring in corneal transplantation is easily dealt with: little has been done, perhaps because the fate of the graft can so easily be followed by direct visual inspection, because forewarning of impending rejection is frequently given by a sudden, slight spike of inflammation in the anterior segment of the grafted eye, or simply be-

55

cause such studies are notoriously difficult. Young and Stark 152followed up the development oflymphocytotoxic antibodies against a random panel of target cells over a 2-year period in 10 patients before and following corneal transplantation. They also monitored changes in the proportions of their peripheral blood mononuclear cells. Six developed cytotoxic antibodies after corneal transplantation but there was no correlation between the presence of such antibodies and graft outcome. There was a suggestion that patients with increased levels of circulating CDS-positive cells in the periphery after rejection episodes may have fared well.Jager et aP 53 followed up 55 patients with penetrating corneal grafts and showed that the presence of cell-mediated reactivity and antibodies to a corneal component in a substantial proportion of patients before graft bore no relationship to subsequent graft outcome.

The Role of Accessory Cells in Corneal Graft Rejection The distribution of Langerhans cells in the corneal epithelium of various species, including humans, rat, guinea pig, mouse, and chick, has been well documented. Lt,t54-156 The number of such cells in the epithelium depends on species u and age. 1°9,157,158 Rare Langerhans or dendritic cells are found in the normal human, 115,t59 rabbit, 98 and rat 117 corneal stroma, with nmnbers increasing toward the limbus. Dendritic cells are also present in human conjunctiva. m~ The complement of dendritic cells in the cornea has been shown to increase substantially after ocular inflammation and corneal vascularization, 98,115,117,155,161bacterial infection, t6~ and corneal transplantation, 163 and to decrease after systemic or topical t r e a t m e n t with corticosteroids.t55 The role of dendritic cells in corneal graft rejection has received a great deal of attention. Donor corneal dendritic cells carried as stimulator or passenger cells within a graft may be partly responsible for triggering the afferent arm of the immune response. Certainly in some model systems, donor corneas in which the numbers of dendritic cells have been artificially increased before transplantation exhibit increased immunogenicity after transplantion. 9a~61'j62 In both the mouse and the rat, the presence of passenger dendritic cells within a cornea induces delayed-type hypersensitivity (DTH)-type responsiveness and the generation of cytotoxic effector cells in the recipient, whereas grafts lacking such passenger cells induce cytotoxic effector cells only. t64'165

56

Williams and Coster

Efforts to reduce the immunogenicity of a donor cornea by reducing the number of passenger cells transferred within the graft have met with some success in experimental models. Thus, prolongation of murine corneal graft survival has been observed after culture of donor corneas in high concentrations of oxygen,a° and also following removal of the corneal epithelium (which contains the majority of the corneal dendritic cell load in this species) or the soaking of the cornea in anti-MHC class II antibody plus complement before graft. 166 Rat corneal graft survival has been prolonged after hyperthermic preservation of the donor corneas for 1 week at 4°C before graft. 135 UV-B irradiation of the donor cornea has prolonged graft survival in the m o u s e , 167'168 rabbit, 169-171and rat. 135In the mouse, the transplantation of UV-B-treated corneas rendered the recipients anergic to subsequent grafts. 16~ The likelihood (or otherwise) of some of these approaches ever being able to be modified for use in the human cornea has been reviewed elsewhere. Ir-~ However, one simple approach deserves further comment. Corneal dendritic cells do not seem to survive for more than 1 to 2 weeks in the conventional corneal organ culture systems already used in many eye banks for the preservation of human corneas for transplantation, I7~,174 and there is at least one report that cultured corneas show better survival after transplantation than do corneas stored by other means. 63 The importance of host-derived, rather than donor dendritic cells in corneal transplantation has also been generating some interest recently. We have provided evidence that the accumulation of dendritic cells of recipient origin in the bed of the graft is associated with a significantly increased risk of rejection of human corneal grafts. 175 At least in the rat, such cells, isolated from vascularized, disaggregated corneas, can be shown to possess antigen-presenting cell function in vitro, j7~ We9~,117 and other investigatorsi.39,165 have suggest ed that indirect antigen processing may occur after corneal transplantation, although it is clear that damage to the graft from effector cells so generated should only occur in situations of at least partial MHC compatibility. I17 The implication is that partial matching for MHC class II antigens may not necessarily improve corneal graft survival. However, irrespective of the mechanisms involved, there is experimental evidence that strategies designed to reduce the numbers or function of cells in the bed of the graft can prolong corneal graft survival. 169

Immunosuppression for Corneal Transplantation Maumenee 13ghas pointed out that for any immunosuppression to be effective in human grafts, the rejection process needs to be halted before irreparable damage has been done to the corneal endothelium, because the essentially nonreplieative nature of human endothelium severely limits the potential for repair. Clinical corneal grafts are usually immunosuppressed with topical glucocorticosteroids. 177 Because the biological activity of these 21-carbon steroid molecules depends on the presence of an hydroxyl group at the carbon-ll position, l l-keto steroids such as cortisone and prednisone, which need to be converted in vivo by the liver to the active 11-13hydroxyl derivatives cortisol and prednisolone, are inappropriate for topical administration. 17~In consequence, all ophthalmic preparations designed for topical application, such as dexamethasone, prednisolone acetate or phosphate, or fluorometholone acetate, are 11-13-hydroxyl steroids. Topical steroids are efficiently absorbed across the cornea 179and are potent anti-inflammatory and immunosuppressive agents for the prevention and reversal of corneal graft rejection. Inmmnosuppressive regimens vary fi'om centre to centre, but a common regimen 177 might involve the topical administration of 0.5% wt/vol prednisolone phosphate to the graft four times daily for 100 days, reducing to once daily for a further 9 to 12 months until well after the graft sutures have been removed at, typically, 1 year postoperatively. Ophthalmologists are usually justifiably circumspect about administering systemic immunosuppressive drugs to patients with corneal grafts, but regimens of systemic therapy may occasionally be warranted in patients who are at high risk of rejection and whose quality of life is extremely poor because of their blindness. Starting at the time of transplantation, we have used a combination of oral Cyclosporin A and azathioprine, together with topical corticosteroid, I8° in patients who are likely to reject their grafts and who have been fully informed and carefully counselled about the risks associated with systemic immunosuppression. Recipients with corneal graft rejection require additional immunosuppression. At the Flinders Medical Centre, patients suffering from rejection episodes are generally admitted to hospital and treated with hourly instillation of 1% prednisolone acetate to the graft until the episode has been reversed. However, there is no doubt that a proportion of rejection

6brneal Tramplantation

episodes cannot be reversed with such a protocol. A regimen of a single pulse of 500 mg intravenous methyl prednisolone together with topical corticosteroid has recently been reported to reverse corneal graft rejection in 8 of 10 patients, l~l This perhaps presages an increased willingness by ophthalmologists to consider the use of systemic immunosuppression, albeit for a brief period. Cyclosporin A has been shown to prolong corneal graft survival in a dose-dependent fashion when administered to rabbits either systemically99,1~2-1~4by retrobulbar 185,mGor subconjunctiva1187,18S injection, or topically.99'm7-193However, a consistent finding in the early studies was that grafts tended to reject after the drug was discontinued. Furthermore, Cyclosporin A was shown to be somewhat less effective in delaying corneal graft rejection in the rabbit than topical steroid, la6,m2 Topical steroid also seemed more effective than Cyclosporin A in delaying corneal neovascularization and in reducing anterior segment inflammation in both the rabbit 18
57

sporin A might show little, if any, therapeutic advantage over topical steroids for the prevention of corneal graft rejection. Topical preparations of Cyclosporin A in castor oil are available in Europe for clinical use in high-risk cases on a restricted basis, but few clinical accounts are available. Mayer and Casey 199 reported a series of 10 patients treated with topical Cyclosporin A (in addition to topical steroid) for up to 6 months postoperatively; six grafts failed, three from rejection, and some epithelial toxicity attributable to the Cyclosporin A was observed. Belin et al -~°°described a group of 11 patients treated with a combination of topical steroid and Cyclosporin A; 10 grafts were surviving at follow-up times ranging from 6 to 24 months. Whole blood levels of 14 to 64 ng/mL of Cyclosporin A were measured in these patients, all of whom developed transient epithelial keratitis. Hill has examined the efficacy of systemic Cyclosporin A in high-risk clinical corneal transplantation2 < He reported improved graft survival in a cohort of patients receiving systemic Cyclosporin A, systemic steroids, and topical steroid when compared with cohorts receiving either systemic plus topical steroid or topical steroid only. The drugs were initially administered for periods of up to 1 year. Some side effects were observed but the regimen was generally well-tolerated. For the forseeable future, the cautious use of systemic rather than topical Cyclosporin A may represent the most appropriate way to administer this drug in corneal transplantation. There have as yet been few reports of the use of the newer generation of chemical immunosuppressants in corneal transplantation. FK506, another water-insoluble agent, has been shown to prolong rabbit corneal graft survivaFU2when administered by twice-weekly subconjunctival injection of an aqueous suspension (0.1 mg/kg). Remarkably, it was as effective in prolonging the survival of second grafts as of first grafts and very little, if any, ocular toxicity of FK506 was observed. Antibody therapy is not used routinely in clinical corneal transplantation but is an area of active research. Systemic administration of heterologous antilymphocyte serum or globulin can prolong the smwival of penetrating corneal allografts in the rabbit, 2°3-9°5 although rejection occurs once therapy ceases. ~°5 Subconjunctival or topical administration of antilymphocyte serum has essentially no effect. 2°5,-9~6More recently, it has been reported that the intracameral injection of one or more mouse

58

Williams and Coster

monoclonal antibodies to human T-cell determinants (combinations of complement-fixing antiCD2, -CD3 and -CD6 antibodies), directly into the anterior chamber of patients undergoing corneal graft rejection, can favourably modify the course of rejection. 9°7,2°8 The interpretation of outcome in the longer term was made difficult in these studies 2°7 because of additional immunosuppression with steroids, but interestingly, no side effects attributable to the intracameral injections were observed. We have subsequently been able to show the reversal of corneal graft rejection episodes in the rabbit following two intracameral injections of monoclonal antibodies to rabbit T-cell or myeloid antigens. ~43'2°9Not all animals responded and a self-limiting fibrinous reaction was observed in the anterior chambers of about half the treated animals. There is increasing interest in the use of brief courses of monoclonal antibodies as prophylactic agents to delay or prevent corneal graft rejection. Perioperative treatment with an antibody to ICAM-1 has been shown to delay the influx of inflammatory cells into murine corneas grafted into the subcapsular renal space. ~1 Intraperitoneal injections of monoclonal antibody to CD4 but not to CDS, administered on four occasions, have been reported to prolong orthotopic corneal graft survival across a major MHC mismatch in a small number of adult thymectomised mice. 97 Any prolongation of survival observed in this study was somewhat marginal, but the value of antibody therapy has recently been confirmed by Ayliffe et al, 21° who have shown delayed rejection of orthotopic corneal grafts in euthymic rats that had been chronically depleted of CD4positive cells by multiple intraperitoneal injections of monoclonal antibody before graft. A small number of grafts survived indefinitely. There have been several interesting reports of the use ofimmunotoxins as immunosuppressive drugs in experimental corneal transplantation. Shirao et al ~ll examined the efficacy of a conjugate of ricin A chain and an F(ab').~ fragment of a routine monoclonal antibody with specificity for rabbit T cells in preventing corneal allograft rejection in the rabbit. The conjugate was ineffective in prolonging graft survival when administered either subconjunctivally or intravenously, possibly because administration was delayed until the seventh postoperative day. In another approach, Herbort et a1212tested the immunosuppressive capacity of a recombinant chimaeric product composed of human interleukin-2 fused to a modified pseudomonas exotoxin that lacked its cell recog-

nition domain. In an orthotopic rat model of penetrating corneal transplantation in which Cyclosporin A was administered for the first twelve postoperative days, subsequent treatment with the immunotoxin improved graft outcome when compared with controls. However, the majority of animals exhibited failed grafts at 6 weeks. A variety of other immunosuppressive regimens have been examined in experimental animals. Ayliffe et al -~1~have recently shown active enhancement of rat corneal grafts with a single, donor-strain preoperative blood transfusion; interestingly, the effect was apparent in some but not all strain combinations. We have shown that urocanic acid, which appears to reduce accessory cell function, 214can prolong corneal graft survival in the rabbit, el5 and other investigators have suggested that a combination of topical cyclophosphamide and methotrexate is also effective.216

The Artificial Cornea There is currently no such thing as an artificial cornea, although this is an area of active research. In some desperate cases a keratoprosthesis may be implanted into the eye, but in most instances a corneal graft will be performed using material from a human donor. The development of an artificial cornea would obviate many of the problems relating to donor supply and corneal graft rejection that currently act to limit success of clinical corneal transplantation. However, all attempts at maintaining full-thickness artificial grafts in situ have thus far failed, the major problem being breakdown of the graft-host interface.

Conclusion Corneal transplantation is a successful procedure but there is room for substantial improvement in both donor cornea supply and in graft outcome. The supply of donor corneas needs to be increased so that more people may be offered the opportunity of a graft. The visual outcome following corneal transplantation needs to be improved so that the maximum potential benefit can be gained by all recipients of corneal grafts. Most importantly, there is a need for better immunosuppression or for other ways of modifying the immune response to a corneal graft, so that graft failures caused by irreversible rejection can be prevented or at least limited in extent. It seems likely that more people are seriously disadvantaged for want of a functioning corneal graft than for any

Cbrneal Transplantation

other currently practised form of organ transplantation.

Acknowledgment We thank all the contributors to the Australian Corneal Graft RegisnT, Glenn Boucher for help with the photographs, and Wendy Laffer for editorial assistance. We are grateful to Professor Heddy Zola for critical comments on the manuscript.

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