PREVENTION O F I M M U N E GRAFT R E J E C T I O N AFTER CORNEAL TRANSPLANTATION J A M E S C. F L E M I N G , M.D.,

F R A N C I S R. R E I D , M.D., Memphis,

Immune graft rejection is an important cause of corneal transplant failure. Graft loss from the immune reaction is 9 to 14%.1,% The use of corticosteroids to sup­ press the immune graft reaction has re­ duced graft rejection to its present rate. Corticosteroids expose the patient to a number of adverse side effects, including corticosteroid-induced glaucoma. Fluorometholone has a low incidence of corticosteroid glaucoma. 3 - 5 We report herein the effectiveness of topical fluorometholone to topical dexamethasone phosphate and prednisolone acetate in the suppres­ sion of immune allograft rejection after corneal transplantation. M A T E R I A L AND M E T H O D S

The experimental model used in this study has been previously described. 6 - 9 Six millimeter penetrating keratoplasties were performed on New Zealand white rabbits weighing 2 to 4 kg (4.4 to 8.8 lbs). Two animals were operated on at the same time, each serving as a host for the corneal transplant obtained from the other. Preoperatively, each animal re­ ceived three drops of 10% phenylephrine HC1 and 1% cyclopentolate HC1. Anes­ thesia was induced by intramuscular xylazine and ketamine. Topical anesthe-

From the University of Tennessee Center for the Health Sciences, Department of Ophthalmology, Memphis, Tennessee. This study was supported in part by the Medical Research Service of the Veterans Administration, Allergan Pharmaceuticals, and by Public Health Service research grant RR-05423 from the University of Tennessee. Reprint requests to Thomas O. Wood, M.D., 66 North Pauline, Memphis, TN 38103.

AND T H O M A S O. W O O D ,

M.D.

Tennessee

sia was also obtained by using proparacaine 0.5%. The penetrating keratoplasties were performed under the operating micro­ scope. A 6-mm trephine was used to cut a groove to approximately three-fourths depth of the rabbit cornea. The anterior chamber was then entered with a razor blade knife. Heparin sodium 1:10,000 was instilled topically in the anterior chamber to prevent aqueous clotting. The corneal section was completed with cor­ neal scissors. The corneal button from the first rabbit was then transferred into the trephined cornea of the second rabbit, and the second animal's cornea to the first. The grafts were then secured with a running 7-0 black silk suture. Postoperatively, all rabbits received atropine 1%, one drop in the operated eye twice daily; and polymyxin B sulfate, neomycin, and bacitracin, one drop three times daily. The animals were observed for vascularization of the host cornea. When this new vessel growth reached the corneal button, the silk sutures were re­ moved. This occurred within ten to 14 days. The corneal sutures were removed under the operating microscope by using topical anesthesia, proparacaine 0.5%. After suture removal, the topical atropine and antibiotic solutions were discontin­ ued. Approximately 16 days after transplant, animals underwent skin grafting. General anesthesia was induced with intramuscu­ lar xylazine and ketamine. In each of the host-donor pairs, a 2.5 x 2.5-cm (1 x 1-inch) section of skin was marked on the

AMERICAN JOURNAL O F OPHTHALMOLOGY 88:97-101, 1979

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abdomen. Once dissected free, the skin was exchanged and sewn subcutaneously in the area of the skin donor site. On the day of skin grafting, all rabbits with clear grafts were divided randomly into four drug groups. Group 1 received polyvinyl alcohol and served as the con­ trol, Group 2 received dexamethasone phosphate, Group 3 received fluorometholone, and Group 4 received prednisolone acetate. Initially all corticosteroid treated groups received commercially available strength drops: dexamethasone phos­ phate 0.1%, fluorometholone 0.1%, and prednisolone acetate 1.0%. After seven days, three rabbits died in different corti­ costeroid groups. These deaths were probably caused by systemic corticoster­ oid toxicity. Because the rabbit has an enormous mucous membrane surface to absorb corticosteroids when applied topi­ cally, the concentration of all corticoster­ oid drops was decreased to one-fourth the original strength: dexamethasone phos­ phate 0.025%, fluorometholone 0.025%, and prednisolone acetate 0.25%. These concentrations were maintained through­ out the remainder of the study. The dose regimen for all four groups was the same. The operated eye of each animal received one drop of the appropri­ ate medication three times a day. The treatment in each group began at the time of skin grafting. The study was continued for four weeks (Fig.l). Examination of the corneal transplants was performed with a hand­ held slit lamp at two-day intervals. The corneal reaction was graded on an empiri­ cal basis from 0 to 4 + . A reading of 0 indicated no reaction with the corneal transplant being faintly outlined in the host cornea; 1+ was used when the graft was well outlined and easily seen; 2 + corresponded to slight haziness of the donor button with a clear host cornea (rejection lines and limbal injection usu-

JULY, 1979

2 4 6 8 10 12 14 16 18 20 22 24 26 28 CONTROL

X

X X X X X

DEXAMETHASONE

XX

0 0 0 0 0 0 0 0

X

FLUOROMETHOLONE

X

PREDNISOLONE ACETATE

X

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Fig. 1 (Fleming, Reid, and Wood). X-rabbits that show rejection. O-rabbits without rejection. The numbers indicate days after skin transplantation.

ally was first a 2 + reaction); 3 + reaction had moderate corneal transplant edema with partial vascularization of the central button; and 4 + represented 360 degrees of corneal vascularization with complete opacification of the donor corneal tissue. At the termination of the experiment, all eyes that received a corneal transplant were enucleated and placed in 10% for­ malin. Representative eyes from each drug group and all eyes that showed re­ jection were selected for histopathologic examination in a double-masked fashion. RESULTS

Rejection clinically was diagnosed on the day an endothelial rejection line was noted. Rejection, as noted in the control group, first began as an endothelial line that moved across the posterior surface of the cornea (Fig. 2). Progression of vascu­ larization of the graft was simultaneously noted. As rejection progressed, the graft slowly opacified (Fig. 3). The graft rejection in the corticosteroid groups when it did occur, underwent an attenuated course with slow movement of the endothelial line and no true opacifica-

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Ill—

Fig. 2 (Fleming, Reid, and Wood). Corneal graft with rejection line.

Fig. 4 (Fleming, Reid, and Wood). Clear corneal graft.

tion of the graft. Grafts that did not show rejection in the corticosteroid treated group also underwent characteristic changes. The vessels at the graft margin attenuated and in most grafts these ves­ sels appeared bloodless. The grafts them­ selves remained clear (Fig. 4). Of the nine members in Group 1 (con­ trol group), treated with polyvinyl alco­ hol, eight had rejection (Fig. 1). Graft rejection occurred from day 6 to day 24 after skin transplantation. These results are similar to other published data. 7 1 0 One rabbit did not show rejection. During and after suture removal this animal never developed vessel growth to the graft, which is necessary for the immune reaction to occur. 10 ' 11

In Group 2, treated with dexamethasone phosphate, one of eight rabbits de­ veloped an endothelial rejection line (Fig. 1). This animal developed a cyst in the wound edge and the rejection line appeared adjacent to the cyst. The rejec­ tion line appeared on day 2 1 . In Group 3, the fluorometholone treat­ ed group, one of nine rabbits developed an endothelial rejection line on day 26 (Fig. 1). On day 21 a second animal in the group developed an epithelial rejec­ tion line that did not progess. Histologic examination showed no rejection in this second animal. In Group 4, treated with prednisolone acetate, one of ten rabbits had a horse­ shoe-shaped endothelial rejection line that did not change. The other nine rabbits in the group maintained clear cor­ neal grafts and showed no evidence of rejection. The mean rejection day in Group 1 (control group) was day 13. In contrast, rejection when it did occur in Groups 2 to 4 (corticosteroid-treated groups), occurred on or after day 21 (Fig. 4). All eyes that showed rejection and a random selec­ tion of eyes from the nonrejection groups were examined histopathologically in a double-masked fashion. The histologic diagnosis confirmed our clinical diagno­ sis.

Fig. 3 (Fleming, Reid, and Wood). Vascularized and opaque corneal graft.

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AMERICAN JOURNAL OF OPHTHALMOLOGY DISCUSSION

The logistics of rabbit corneal trans­ plantation with consistently clear grafts have been developed by many investiga­ tors.6""9 Maumenee 1 1 has shown that after obtaining a clear graft, further sensitization of the host animal by transplantation of skin from the donor animal causes an opacification of the corneal graft within two to four weeks. He also cited that blood vessels of the recipient animal must grow into the donor material before the transplant can affect an immune response. Clinically, this immune graft rejection can be suppressed by corticosteroids. However, the long term use of topical corticosteroids is often necessary, and chronic use of topical corticosteroids ex­ acerbates or produces certain pathologic states. Posterior subcapsular cataract for­ mation may occur, 1 2 - 1 4 the virulence of ocular viral infections and the incidence of ocular bacteria and fungal infections is increased, 15 and corticosteroid-induced glaucoma may occur. 16,17 Recent data comparing topical dexamethasone, betamethasone, prednisolone, and fluorometholone, showed that fluorometholone is least likely to raise intrao­ cular pressure. 3 ' 4 ' 18 To the best of our knowledge no information has been pub­ lished concerning the effectiveness of fluorometholone in the suppression of the host vs graft immune reaction. Fluorometholone is a synthetic hor­ mone related to the glucocorticoids. The main clinical usefulness of fluorometh­ olone is its anti-inflammatory effect.3'4 Bio-availability of fluorometholone in the cornea and anterior chamber after topical administration has been shown. 5 In Group 1 (control group), all but one rabbit underwent transplant rejection, and this one animal never developed ves­ sel ingrowth to the corneal graft. This reaffirms the finding of Maumenee, 1 1 Polack, 10 and others that in this experimen­

JULY, 1979

tal model, "vascularization is a requisite for the immune reaction." 1 0 The graft rejection in the remaining control animals followed the sequence of ciliary flush, vascular engorgement, endothelial rejection line, graft edema, and graft vascularization. The day of rejection was uniformly noted to coincide with the appearance of a rejection line. This served as a firm end point. Anticipation of rejec­ tion by the degree of vascular changes at the corneoscleral limbus could have yielded less objective results. In those corticosteroid-treated rabbits that rejected the cornea, the rejection line was just as obvious as in the control group. The difference between control and corticosteroid rejection was one of intensity. Our data show that fluorometholone, dexamethasone phosphate, and predniso­ lone acetate had similar therapeutic ef­ fects and fluorometholone was as effec­ tive as dexamethasone and prednisolone in the suppression of immune graft rejec­ tion. The treatment of graft rejection after it had occurred was not evaluated. Although the clinical and histologic features of immune graft rejection in the rabbit and human are similar, the experi­ mental model used in this study may not parallel spontaneous human graft rejec­ tion. SUMMARY

The effectiveness of fluorometholone was compared to dexamethasone phos­ phate and prednisolone acetate in pre­ venting the immune corneal graft reaction in rabbits. Clear corneal grafts were ob­ tained. Rejection was induced after skin from the corneal donor animal was graft­ ed subcutaneously in the host animal and the animals were randomized into four treatment groups. Rejection occurred in eight of nine rabbits in the control group; one of eight in the dexamethasone phos-

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phate group; one of eight in the fluor­ ometholone group; and one of ten in the prednisolone acetate group. Histologic examination confirmed the above find­ ings. In this animal study fluorometholone prevented immune graft rejection in a percentage similar to that of prednisolone acetate and dexamethasone phosphate.

ACKNOWLEDGMENT

John K. Duckworth, M.D., Department of Pathol­ ogy, Methodist Hospital, Memphis, Tennessee, per­ formed the histopathologic examination of all tissue specimens in this study.

REFERENCES 1. Polack, F. M.: Clinical and pathologic aspects of the corneal graft reaction. Trans. Am. Acad. Ophthalmol. Otolaryngol. 77:418, 1973. 2. Fine, M.: Clinical and pathologic aspects of the corneal graft reaction (Discussion). Trans. Am. Acad. Ophthalmol. Otolarnygol. 77:430, 1973. 3. Buch, H. E., and Ellis, R. A.: Clinical studies with a new steroid. Fluorometholone. Ann. Oph­ thalmol. 7:937, 1975. 4. Fairbairn, W. D., and Thorson, J. C : Fluor­ ometholone. Anti-inflammatory and intraocular pressure effects. Arch. Ophthalmol. 86:138, 1971. 5. Kupferman, A., and Leibowitz, H. M.: Penetra­ tion of fluorometholone into the cornea and aqueous humor. Arch. Ophthalmol. 93:425, 1975. 6. Lorenzetti, D. W. C , and Kaufman, H. E.:

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Experimental production of graft reactions with suppression by topical corticosteroids. Arch. Oph­ thalmol. 76:274-281, 1966. 7. Polack, F. M.: Inhibition of immune corneal graft rejection by azathioprine. Arch. Ophthalmol. 74:683, 1965. 8. Wind, A. E., and Wood, T. O.: Effects of corti­ costeroids on corneal thickness following penetrat­ ing keratoplasty. Am. J. Ophthalmol. 71:1018, 1971. 9. Khodadoust, A. A.: Penetrating keratoplasty in the rabbit. Am. J. Ophthalmol. 66:899, 1968. 10. Polack, F . M.: Histopathological and histochemical alterations in early stages of corneal graft rejection. J. Exp. Med. 116:709, 1962. 11. Maumenee, A. E.: The influence of donorrecipient sensitization on corneal grafts. Am. J. Ophthalmol. 34 (pt. 2):142, 1951. 12. Polack, F. M.: Editorials on recent advances. Corneal transplantation. Invest. Ophthalmol. 12:85, 1973. 13. Oglesby, R. B., Black, R. L., von Sallmann, L., and Bunim, J. J.: Cataracts in rheumatoid arthritis patients treated with corticosteroids. Arch. Ophthal­ mol. 66:519, 1961. 14. Wood, T. O., Waltman, S. R., and Kaufman, H. E.: Steroid cataracts following penetrating kera­ toplasty. Ann. Ophthalmol. 3:496, 1971. 15. McLean, J. M.: Oculomycosis. Am. J. Oph­ thalmol. 56:537, 1963. 16. Leopold, I. H.: The steroid shield in ophthal­ mology. Trans. Am. Acad. Ophthalmol. Otolaryngol. 71:272, 1967. 17. Becker, B., and Mills, D. W.: Corticosteroids and intraocular pressure. Arch. Ophthalmol. 70:500, 1963. 18. Marum, E.: Evaluation of a new topical ster­ oid, fluorometholone, in the postoperative course of the cataract operation. Ann. Ophthalmol. 7:139, 1975.

Prevention of immune graft rejection after corneal transplantation.

PREVENTION O F I M M U N E GRAFT R E J E C T I O N AFTER CORNEAL TRANSPLANTATION J A M E S C. F L E M I N G , M.D., F R A N C I S R. R E I D , M.D.,...
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