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Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Can this metabolite interfere with fluoresceintrace r studies? Tr Japan 113. Am Ophthalmol Soc LXXXI:125, 1983. 7. Grotte D, Mattox V, and Brubaker RF: Fluorescent, physiolog- ical, and pharmacokinetic properties of fluorescein glucuronide. References Exp Res 40:23, 1985. 8. McLaren JW and Brubaker RF: Fluorescein and fluorescein 1. Cunha-Vaz JG and Maurice DM: The active transport of fluo- glucuronide in ocular fluorophotometry. ARVO Abstracts. Invest rescein by the retinal vessels and the retina. J Physiol 191:467, Ophthalmol Vis Sci 25(Suppl):106, 1984. 1967. 9. Matsunaga I, Nagataki S, and Tamura Z: Synthesis of fluorescein 2. Cunha-Vaz JG and Maurice DM: Fluorescein dynamics in the monoglucuronide. Chem Pharmacol Bull 32:2832, 1984. eye. Doc Ophthalmol 26:61, 1969. 10. Nagataki S and Matsunaga I: Binding of fluoresceinmonogluc - uronide to human serum albumin. Invest Ophthalmol Vis Sci, 3. Stone RA and Wilson CM: Fluorescein transport in the anterior 26:1175, 1985. uvea. Invest Ophthalmol Vis Sci 22:303, 1982. 11. Kaiser RJ and Maurice DM: The diffusion of fluoresceini n the 4. Chen SC, Nakamura H, and Tamura Z: Studies on the metab- . Exp Eye Res 3:156, 1964. olites of fluoresceini n rabbit and human urine. Chem Pharmacol 12. Maurice DM: Drug exchanges between the blood and vitreous. Bull 28:1403, 1980. In The Blood-Retinal Barriers, Cunha-Vaz JG, editor. New York, 5. Chen SC, Nakamura H, and Tamura Z: Determination of flu- Plenum Press, 1980, pp. 165-178. orescein and fluorescein monoglucuronide excreted in urine. 13. Kitano S and Nagataki S: Fluorescein monoglucuronide in the Chem Pharmacol Bull 28:2812, 1980. rabbit vitreous. ARVO Abstracts. Invest Ophthalmol Vis Sci 6. Grotte D, Mattox V, and Brubaker RF: Fluorescein glucuronide: 26(Suppl):107, 1985.

The Effect of Collagen Cross-Linkage Inhibitors on Rabbit After Radial Keratotomy

Massimo Dusin, Chi-Wong You, Tarsuo Yamaguchi, Marguerite D. McDonald, and Herbert E. Kaufman

The inhibition of collagen cross-linkage by /?-aminopropioni- In either case, any condition that delays or reduces trile (BAPN) and D-penicillamine was tested in an attempt healing of the radial cuts may, at the same time, allow to enhance the myopic correction achieved by radial kera- the intraocular pressure (IOP) to "work" on the totomy. Two groups of six rabbits each underwent radial ker- for a longer time, possibly enhancing the optical effect. atotomy in both ; one eye of each rabbit was treated with Additionally, the postoperative regression in correction BAPN (33% in white petrolatum) or D-penicillamine (1.5 M observed after radial keratotomy, which has been at- in sterile water) three times a day for 6 weeks, while the other eye received only vehicle. In spite of previous reports of pos- tributed to the scarring process in the cornea, could itive results with BAPN, neither BAPN nor penicillamine also be reduced by an agent that affects wound healing. was found to affect keratometry readings or corneal topog- Topical corticosteroids appear to reduce postoperative raphy after radial keratotomy. Our results suggest that factors regression in rabbits, but the mechanism has not yet other than collagen cross-linkage exert major influences in been explained. determining the outcome of this refractive procedure. Invest The purpose of this study was to ascertain if the Ophthalmol Vis Sci 27:1001-1005, 1986. inhibition of collagen cross-linkage is an effective means of reducing wound healing and enhancing the The corneal healing processes that follow radial ker- correction achieved by radial keratotomy. atotomy are the same as those that occur after any Materials and Methods. Animal care and treatment nonperforating wound to the cornea, and involve both in this investigation were in compliance with the epithelial and stromal repair. It has been assumed that ARVO Resolution on the Use of Animals in Research. there is a relationship between wound healing after ra- The methods described in previous publications2'3 were dial keratotomy and the optical effect and predictability followed as precisely as possible, in the attempt to re- of the surgical procedure. The effectiveness of radial produce their results. Two groups of six New Zealand keratotomy is thought to derive from a weakening and albino rabbits (2.5-3 kg body weight) each received consequent bulging of the peripheral cornea that flat- radial keratotomy in both eyes. One eye of each rabbit tens the central optical zone.' Whether the bulging ef- was treated topically with drug, either 33% BAPN in fect in the periphery is a result of force exerted by the white petrolatum or 1.5 M D-penicillamine in sterile intraocular pressure on the weakened cornea, or merely water, three times a day for 6 weeks. The fellow eye a concomitant of the processes of wound healing, is received only vehicle on the same schedule. Two ad- not yet clear. ditional control rabbits, with no surgery, received the

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Table 1. Keratometry readings (spherical equivalent) at different times after radial keratotomy

Treated eye Untreated eye Rabbit number Preoperative 2 weeks 6 weeks 4 months Preoperative 2 weeks 6 weeks 4 months

BAPN 3041 49.25 43.62 43.12 41.62 49.00 43.62 42.12 41.50 3044 50.00 42.75 42.75 42.50 49.87 44.50 43.00 42.00 3051 48.25 43.50 43.12 41.50 48.75 43.00 40.50 39.37 2686 49.50 45.25 43.00 41.75 49.00 45.12 43.75 41.75 3201 48.50 44.00 43.62 43.00 49.00 44.25 42.25 42.50 2690 47.50 42.62 42.50 41.75 47.50 41.75 42.00 41.87 3053* 47.50 45.50 44.25 43.62 47.62 45.00 44.75 43.75 3055* 49.00 45.75 45.50 45.00 48.75 45.50 44.25 44.12 Penacillamine 2572 50.00 44.25 44.12 41.75 50.50 46.87 44.87 42.25 2573 47.00 41.37 40.87 40.12 47.12 42.25 41.87 41.25 2577 50.00 46.37 44.37 44.00 50.25 46.37 44.00 43.87 2680 49.75 43.62 41.87 40.62 50.25 43.12 43.50 41.75 2684 48.62 42.75 40.62 39.62 48.50 44.25 42.00 41.00 2685 47.00 39.75 42.25 40.37 47.00 39.87 41.62 40.00 2575* 48.75 48.50 47.25 46.00 49.00 48.87 46.50 46.00 2682* 47.50 46.25 45.75 44.00 48.00 47.37 45.00 44.12

• Controls—no surgery.

same topical treatment with each drug and vehicle to Results. There was no significant difference in my- detect toxicity. Preoperatively, slit-lamp examination, opic correction between treated and control eyes (Ta- keratometry, corneal topographic analysis by Sun bles 1, 2) at any time interval after surgery, for either photokeratoscope, and ultrasonic pachymetry were drug. Although the difference was not statistically sig- performed. nificant, the mean correction achieved at 4 months in The surgical procedure was the same as was used in the penicillamine group was slightly larger than in the the previous studies2'3—eight radial incisions with a control group: 7.64 D ± 1.32 D for the treated eyes, central optical zone 3 mm in diameter. Ultrasonic pa- and 7.25 D ± 1.03 D for the control eyes. However, chymeter (Kremer corneometer; Accutome, Frazer, in the BAPN group, the treated eyes showed even less PA) readings were taken at a speed of 1640 m/sec, in correction than the controls: 6.81 D ± 0.98 D and 7.35 the center of the optical zone and paracentrally at 3, D ± 1.27 D, respectively. was an- 6, 9, and 12 o'clock. The cuts were made by a Beaver alyzed as described previously.4 The mean radii of the (Waltham, MA) No. 76 A microblade. Kogan and inner three rings were averaged; results were substan- Katzen2 used a metal blade; Moorhead et al3 did not tially equivalent to the keratometry readings (Tables specify the type of blade used. The depth of the cuts 3,4). was more than 90% of the corneal thickness, as de- Except for a prolonged blepharitis in all eyes treated scribed by Kogan and Katzen.2 The depth was deter- with BAPN, no clinical abnormalities were seen. mined by adjusting the blade to the thinnest pachy- Whether this finding was the result of mechanical meter reading minus 0.02 mm, as described by Moor- abrasion by the BAPN crystals or a toxic phenomenon head et al.3 The cuts extended peripherally from the was not clear. central optical zone to 1 mm from the limbus. No an- In the control rabbits, which had no surgery, some terior chamber perforation occurred. corneal flattening was seen with time, as a result of the Slit-lamp examination was performed at 14 days and normal aging process, but the net flattening was con- 1, 2, 3, and 4 months after surgery. Keratometry and siderably less than that seen in the operated eyes (Tables corneal topographic analysis by Sun photokeratoscope 1-4). No difference in flattening was seen between the were performed at 14 days, 6 weeks, and 4 months control eyes that received the drug and the control eyes after surgery, in a masked fashion. Maximal care was that received only vehicle. taken to align the cornea with either the keratometer Discussion. The mechanism by which radial kera- or the photokeratoscope. The rabbits were immobilized totomy induces a modification of the corneal shape is in a specially designed box supported by a tilting base. not clear. Some of the problems that remain include In all cases the surgery and measurements were per- a lack of predictability and postsurgical regression, both formed with the center of the considered as the of which are related to healing of the radial incisions. center of the cornea. Therefore, considerable attention has been paid to us-

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Table 2. Myopic correction in diopters (D) at different times after radial keratotomy

Treated eye Untreated eye

Rabbit number 2 weeks 6 weeks 4 months 2 weeks 6 weeks 4 months

BAPN 3041 5.62 6.12 7.62 5.37 6.87 7.50 3044 7.25 7.25 7.50 5.37 6.87 7.87 3051 4.75 5.12 6.75 5.75 8.25 9.37 2686 4.25 6.50 7.75 3.87 5.25 7.25 3201 4.50 4.87 5.50 4.75 6.75 6.50 2690 4.87 5.00 5.75 5.75 5.50 5.62 Average 5.20 5.81 6.81 5.14 6.58 7.35 ±SD ±1.10 ±0.96 ±0.98 ±0.72 ±1.09 ±1.27 3053* 2.00 3.25 3.87 2.62 2.87 3.87 3055* 3.25 3.50 4.00 3.25 4.50 4.62 Penicillamine 2572 5.75 5.87 8.25 3.62 5.62 8.25 2573 5.62 6.12 6.87 4.87 5.25 5.87 2577 3.62 5.62 6.00 3.87 6.25 6.37 2680 6.12 7.87 9.12 7.12 6.75 8.50 2684 5.87 8.00 9.00 4.25 6.50 7.50 2685 7.25 4.75 6.62 7.12 5.37 7.00 Average 5.70 6.37 7.64 5.14 5.96 7.25 ±SD ±1.18 ±1.30 ±1.32 ±1.59 ±0.63 ±1.03 2575* 0.25 1.50 2.75 0.12 2.50 3.00 2682 1.25 1.75 3.50 0.62 3.00 3.87

• Controls—no surgery.

ing drugs that have the potential to interfere with While epithelial repair is completed in a few hours wound healing, on the premise that a delay in wound after corneal injury in both human and animal models, healing or the synthesis of abnormal collagen may en- the stromal phase plays a more important role and hance the refractive effect. takes much longer. The healing of the stroma consists

Table 3. Corneal topography: keratometry readings (spherical equivalent) at different times after radial keratotomy

Treated eye Untreated eye Rabbit number Preoperalive 2 weeks 6 weeks 4 months Preoperative 2 weeks 6 weeks 4 months

BAPN 3041 49.3 43.4 43.2 41.7 49.0 43.8 42.0 41.5 3044 50.0 42.7 42.6 42.5 49.9 44.4 43.2 42.2 3051 48.3 43.6 43.2 41.5 48.6 42.9 40.6 39.5 2686 49.8 45.4 43.1 41.9 49.3 45.5 43.6 41.7 3201 48.6 44.1 43.7 43.2 48.8 44.0 42.0 42.5 2690 47.5 42.4 42.2 41.5 47.4 41.8 42.1 41.9 3053* 47.7 45.6 44.3 43.8 47.7 45.1 44.9 43.9 3055 49.5 46.2 45.9 44.9 48.6 45.8 45.0 44.7 Penacillamine 2572 49.7 44.3 44.0 41.9 50.1 46.0 44.9 42.3 2573 47.2 41.5 41.0 40.2 47.3 42.4 42.0 41.1 2577 49.9 46.4 44.5 44.0 50.1 46.3 44.1 43.9 2680 49.7 43.7 41.9 40.5 50.1 43.3 43.4 41.8 2684 48.5 42.6 40.5 39.6 48.3 44.6 42.3 40.5 2685 47.2 39.8 42.3 40.3 47.1 39.9 41.4 39.8 2575* 48.4 48.3 47.1 46.0 48.2 48.1 46.9 46.3 2682* 47.7 46.3 45.8 44.5 47.4 46.5 45.9 44.2

Controls—no surgery.

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Table 4. Corneal topography: myopic correction in diopters (D) at different times after radial keratotomy Treated eye Untreated eye

Rabbit number 2 weeks 6 weeks 4 months 2 weeks 6 weeks 4 months BAPN 3041 5.9 6.1 7.6 5.2 7.0 7.5 3044 7.3 7.4 7.5 5.5 6.7 7.7 3051 4.7 5.1 6.8 5.7 8.0 9.1 2686 4.4 6.7 7.9 3.8 5.7 7.6 3201 4.5 4.9 5.4 4.8 6.8 6.3 2690 5.1 5.3 6.0 5.6 5.3 5.5 3053* 2.1 3.3 3.9 2.6 2.8 3.8 3055* 3.3 3.6 4.6 2.8 3.6 3.9 Penicillamine 2572 5.4 5.7 8.8 4.1 5.2 7.8 2573 5.7 6.2 7.0 4.9 5.3 6.2 2577 3.5 5.4 5.9 3.8 6.0 6.2 2680 6.0 7.8 9.2 6.8 6.7 8.3 2684 5.9 8.0 8.9 3.7 6.0 7.8 2685 7.4 4.9 6.9 7.2 5.7 7.3 2575* 0.1 1.3 2.4 0.1 1.3 2.9 2682* 1.4 1.9 3.2 0.9 1.5 3.2

* Controls—no surgery.

essentially of removal of the damaged epithelium and Moreover, once the epithelium has covered the corneal fibers by proteolytic enzymes released by polymorpho- wounds, it could be difficult for the drug to penetrate nuclear leukocytes that infiltrate the wound, followed to the underlying stroma where the newly synthesized by replacement with new keratocytes and fibers. In this collagen is forming. It may be that BAPN is locally process, inactive stellate keratocytes transform into catabolized by monoamine oxidase (MAO), which is elongated proliferating fibroblasts, which begin to syn- present in the ocular tissues of the albino rabbits.7 thesize procollagen.5 The procollagen is secreted into Fleisher et al8 could not reproduce antifibrotic effects the extracellular space, where intra- and extracellular in albino rabbits by systemic administration of BAPN, cross-linkages are formed.6 These covalent bonds are and they explained these results in terms of the high created as the result of the oxidative deamination of serum level of MAO in albino rabbits. either lysyl or hydroxylysyl residues to the correspond- The other collagen cross-linkage inhibitor tested in ing reactive aldehydes, which can either react with this study, D-penicillamine, is characterized by the similar groups or with the terminal groups of lysine or presence of a sulfhydryl group in the molecule, which hydroxylysine on adjacent collagen molecules. The can chelate various ions, among which are Ca+2 and enzyme, lysyl-oxidase, is responsible for this reaction Cu+2. The sulfhydryl group is also responsible for the in the presence of several cofactors, among which is cross-linkage inhibition by binding the free aldehyde Cu+2. The definitive cross-linked collagen is in a stable radicals of procollagen and preventing them from tak- equilibrium in which slow degradation by endogenous ing part in the cross-linkage.9 At low concentrations, collagenase is balanced by replacement with newly the blockage of the aldehyde groups is the principal synthesized collagen. mechanism by which penicillamine inhibits collagen BAPN has been reported to be an effective antifi- cross-linkage, but at high concentrations the chelation brotic agent in various kinds of models; in particular, of Cu+2, an important cofactor of the lysyl-oxidase en- it has been shown to reduce the tensile strength of newly zyme, must be considered. The antifibrotic property synthesized collagen after corneal wounds, and to en- of penicillamine has been tested in various animal hance the effect of radial keratotomy.2 3 Its effect is the models, and has recently been shown to reduce post- result of a potent inhibition of the enzyme lysyl-oxi- traumatic vitreal fibrosis in rabbits. We prepared a 1.5 dase; this inhibition prevents the newly synthesized M collyrium of penicillamine in sterile water to facil- collagen from assuming the definitive cross-linked itate the release of the drug from the vehicle. The lack form. of significant results in our model may have been a It is not clear why our results do not reproduce those result of a number of factors, including low concen- published previously, but some possibilities may be tration of the drug, low frequency of administration, considered. Little is known about the interaction be- and/or low penetration into the corneal stroma. It is tween BAPN and the petrolatum vehicle, which could also interesting to note that, in the 1970s, penicillamine retain the molecules of the drug in its gel structure. was shown to inhibit collagenase activity by chelating

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Ca2+ in alkali-burned corneas;10 if penicillamine in- From the Lions Eye Research Laboratories, LSU Eye Center, Lou- hibits both the synthesis and the catabolism of collagen, isiana State University Medical Center School of Medicine, New Or- leans, Louisiana. Dr. Yau is presently affiliated with the Department the net balance of its effect may be minimal. of Ophthalmology, Mackay Memorial Hospital, Taipei, Taiwan. It could be argued that, if the incisions are not deep Supported in part by USPHS Grants EY04082, EYO3635, EY02580, enough, the wound healing process will be limited, and and EY02377, from the National Eye Institute, National Institutes drugs will exert little effect on the results. However, it of Health, Bethesda, Maryland. Presented in part at the Association is clear from the postoperative K readings in this study for Research in Vision and Ophthalmology Meeting, Sarasota, Florida May 7, 1985. Submitted for publication: July 17, 1985. Reprint re- that the cuts were of maximal depth, providing ade- quests: Herbert E. Kaufman, MD, LSU Eye Center, 136 South Roman quate scope for observing the effects of drug activity, Street, New Orleans, LA 70112. if any. The mechanism by which radial cuts modify corneal References shape is not completely understood, and there is no 1. Fyodorov SN and Durnev VV: Operation of dosaged dissection evidence that wound healing is the main factor in de- of corneal circular ligament in cases of of mild degree. termining the effect of radial keratotomy. Jester et al" Ann Ophthalmol 11:1885, 1979. showed that doubling the number of incisions from 8 2. Kogan LL and Katzen L: Enhancement of radial keratotomy by to 16 did not affect either the corneal flattening or the chemical inhibition of collagen cross-linkages: a preliminary re- amount of regression after radial keratotomy in non- port. Ann Ophthalmol 15:842, 1983. 3. Moorhead LC, Carroll J, Constance G, Jenkins DE, and Ar- human primate eyes. If wound healing were a major meniades CD: Effects of topical treatment with Beta-aminopro- determinant in the outcome of this surgery, twice as pionitrile after radial keratotomy. Arch Ophthalmol 102:304, many wounds should produce some difference in post- 1984. operative results. 4. KJyce SD: Computer-assisted corneal topography. High-reso- lution graphic presentation and analysis of keratoscopy. Invest However, it is still difficult to explain why our find- Ophthalmol Vis Sci 25:1426, 1985. ings do not show the significant enhancement of sur- 5. Fuller GC: Perspectives for the use of collagen synthesis inhibitors gical results obtained with drug treatment in previous as antifibrotic agents. J Med Chem 24:651, 1981. studies. The conflicting results obtained with BAPN 6. Siegel RC and Martin GR: Enzymatic synthesis of lysine-derived and the negative result obtained with D-penicillamine aldehydes and the production of cross-linked components. J Biol Chem 245:1653, 1970. seem to suggest that the inhibition of collagen cross- 7. Waltman S and Sears M: Catechol-O-methyl transferase and linkage may not be a satisfactory approach to con- monoamine oxidase activity in the ocular tissues of the albino trolling the correction achieved by radial keratotomy. rabbits. Invest Ophthalmol 3:601, 1964. Further studies with larger numbers of animals and a 8. Fleisher JH, Speer D, Brendel K, and Chvapil M: Effect of par- standardized surgical technique are necessary to con- gyline on the metabolism of Beta-aminopropionitrile (BAPN) by rabbits. Toxicol Appl Pharmacol 47:61, 1979. firm the results reported here, and future investigations 9. Nimni ME: Penicillamine and collagen metabolism. Scand J in primate eyes may be useful to demonstrate possible Rheumatol(Suppl)28:71, 1979. effects of such drugs in a model more similar to human 10. Francois J, Cambie E, Feher J, and Van Den Eekhout E: Col- eyes. lagenase inhibitors (penicillamine). Ann Ophthalmol 5:391, 1973. 11. Jester JV, Steel D, Salz J, Miyashiro J, Rife L, Schanzlin DJ, Key words: radial keratotomy, collagen cross-linkage, /3-ami- and Smith RE: Radial keratotomy in non-human primate eyes. nopropionitrile, D-penicillamine, rabbit, cornea Am J Ophthalmol 92:153, 1981.

Differential Protein Synthesis in Steroid-Treated Ocular Surface Epithelium

B. D. Srinivasan, P. S. Kulkarni, and S. P. Bhar

Topical prednisolone and cortexolone, a known glucocorticoid weights in the migrating conjunctiva! epithelium, while treat- receptor antagonist, differentially affected the synthesis of ment with prednisolone plus cortexolone resulted in a pattern proteins in normal corneal epithelium and migrating con- of protein bands which resembled the saline-treated control. junctiva! epithelium after complete corneal deepithelialization, Crude extracts of prednisolone-treated migrating epithelium

as measured by 35s-methionine incorporation and SDS- also enhanced the inhibition of phospholipase A2 activity, PAGE (sodium dodecyl sulfate-polyacrylamide gel) electro- and this prednisolone-induced inhibition was reversed by cor- phoresis. Application of either prednisolone or cortexolone texolone. Invest Ophthalmol Vis Sci 27:1005-1009, 1986 to corneal epithelium resulted in similar protein synthesis patterns, each showing two new protein bands of about 15K and 32K. Cortexolone, but not prednisolone, initiated the ap- Several commercially available corticosteroid prep- pearance of several protein bands of different molecular arations, such as Pred Forte (1 % prednisolone acetate;

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