Effects of Ultraviolet Radiation on Corneal Epithelial Metabolism

Invest. Ophthalmol. Visual Sci. 694 Reports July 1978

model. Further in vivo evaluation of this regimen in animals and man seems warranted. Effects of ultraviolet radiation on corneal From the Department of Ophthalmology (Dr. Haft, epithelial metabolism. GARY N. FOULKS, Dr. Friedman) and the Department of Medicine, Divi- JUDITH FRIEND, AND RICHARD A. THOFT. sion of Infectious Diseases (Dr. Walsh, Dr. Miller), Montefiore Hospital and Medical Center, Albert Ein- Acute exposure to ultraviolet light of 257 nm wavelength stein College of Medicine, and the Department of produced a photokeratitis associated with characteristic Pharmacy (Dr. Loran), Montefiore Hospital, Bronx, metabolic alterations of corneal epithelial metabolism in N. Y. This work was supported in part (Dr. Walsh) by a rabbits. Significant increases in corneal hydration oc- U. S. Public Health Service Training Grant T01 AI curred simultaneously with decreased corneal epithelial 0040-05 from the National Institute of Allergy and Infec- glycogen content, but adenosine triphosphate content tious Diseases and by the National Eye Institute, grant and enzyme activity of epithelial extracts were not af- R01 EY 00613-05. Submitted for publication Jan. 11, fected despite clinical and histological damage to the 1978. Present address of Dr. Friedman: Department of corneal epithelium. The pattern and time course of ul- Ophthalmology, Mt. Sinai School of Medicine, New traviolet damage to the cornea are distinctly different York, N. Y. Reprint request: Dr. Michael H. Miller, from those of other forms of trauma. Department of Medicine, Division of Infectious Dis- The destructive effects of ultraviolet (UV) light eases, Montefiore Hospital and Medical Center, 111 on the cornea have long been attributed to damage East 210th St., Bronx, N. Y. 10467. to nucleic acids and proteins in the epithelium.1' 2 Key words: 5-fluorocytosine, fungal endophthalmitis, Whether or not there are primary effects on the penetration, aqueous, vitreous, serum, pharmacoki- processes responsible for energy transformation netics, subconjunctival has not been investigated, although such effects have been found in all other forms of corneal in- REFERENCES jury which have been studied to date.3' 4 1. Edwards, J. E., Foos, R. Y., Montgomerie, J. Z., Therefore, in an attempt to investigate whether and Guze, L. B.: Ocular manifestations of Candida there is any alteration of the energy transformation septicemia: review of seventy-six cases of hemato- pathway after injury believed to affect primarily genous Candida endophthalmitis, Medicine 53:47, structural proteins, some of the metabolites and 1974. enzymes responsible for energy transformation in 2. Hiles, D. A., and Font, R. L.: Bilateral intraocular the corneal epithelium have been measured after cryptococcosis with unilateral spontaneous regres- far-ultraviolet radiation (UV-B). These are corre- sion: report of a case and review of the literature, Am J. Ophthalmol. 65:98, 1968. lated with evolution of the clinical changes, as well 3. Green, W. R., Bennett, J. E., and Goos, R. D.: as with changes in corneal hydration. Ocular penetration of amphotericin B. A report of Methods laboratory studies and a case report of postsurgical Preparation of animals. Adult rabbits weighing Cephalosporium endophthalmitis, Arch. Ophthal- 2 to 3 kg were exposed to the radiation of a 30-watt mol. 73:769, 1965. germicidal UV lamp at a distance of 10 cm from 4. Bennett, J. E.: Drugs five years later: flucytosine, the anterior corneal surface for 15 min per eye. Ann. Intern. Med. 86:319, 1977. We immobilized the animals but avoided in- 5. Davson, H.: Physiology of the Ocular and Cerebro- strumentation or medication of the lids or cornea. spinal Fluids, Boston, 1956, Little, Brown & Co. 6. Kaspar, R. L., and Drutz, D. J.: Rapid, simple The emission spectrum of the GE30T8 lamp bioassay for 5-fluorocytosine in the presence of am- source used is predominantly 257 nm, with lesser photericin B, Antimicrob. Agents Chemother. 7: emissions at 310 nm and 360 nm (Fig. 1). The 7 462, 1975. calculated exposure at 257 nm was 1.48 X 10 ergs 1 7. Wagner, J. G.: Fundamentals of Clinical Pharma- (70 to 80 times the photokeratitis threshold ), and cokinetics, Hamilton, 111., 1975, Drug Intelligence exposure at all other wavelengths was below Publications, Inc. threshold. At 0, 4, 7, 24, 48 and 72 hr after expo- 8. Garrod, L. P., Lambert, H. P., and O'Grady, sure, rabbits were killed by an overdose of sodium F.: Antibiotic and Chemotherapy, London, 1973, pentobarbital, and tissue samples were prepared Churchill Livingstone. as described below. 9. Richards, A. B., Jones, B. R., Whitwell, J., and Clayton, Y. M.: Corneal and intra-ocular infection Histological studies. Histological examination by Candida albicans treated with 5-fluorocytosine, was performed at 24 hr after exposure. The whole Trans. Ophthalmol. Soc. U.K. 89:867, 1969. excised cornea was fixed in 4% buffered glutaral- 10. Jones, B. R.: Principles in the management of dehyde. Scanning electron microscopy was per- oculomycosis, Am. J. Ophthalmol. 79:719, 1975. formed by standard techniques on the epithelial

Downloaded from iovs.arvojournals.org on 10/02/2021 Volume 17 Number 7 Reports 695

35 -,

To 30- \ 2759 \

25-

20-

10-

n 250 300 350 400 450 500 550 600 Wavelength (nm)

Fig. 1. Germicidal lamp spectral distribution (for 1-watt input to lamp typical of G15T8, G25T8, G30T8, G8T5, and G4T4/1).

surface, with critical-point CO2 drying and 6-phosphate dehydrogenase (G-6-PDH), 6-phos- platinum coating.5 Transmission electron micros- phogluconic dehydrogenase (6-PGDH), lactate copy was performed on Epon-embedded sections dehydrogenase (LDH), malate dehydrogenase of the cornea. (MDH), and aldolase (ALD) were measured by Preparation of tissue samples and biochemical enzymatic optical tests according to the method of studies. Epithelium was removed by scraping with Bergmeyer.6a Phosphorylase (P-ase) measurement a No. 15 blade and immediately frozen in liquid was by the enzymatic optical method of Jones and nitrogen, followed by lyophilization, weighing, Wright.(ib Activities were determined in Interna- and storage at —70° C until extraction. For tional Units per gram of protein at 24° C. Protein adenosine triphosphate (ATP) analysis the corneal determination was by the Lowry method.(ic Sta- epithelium was frozen in situ with a stainless steel tistical significance was determined by the Stu- confonner cooled in liquid nitrogen, followed by dent's t test.3 lyophilization of the enucleated globe and scraping Results. The radiation exposure invariably pro- of the freeze-dried epithelium. Corneal epithe- duced a clinical photokeratitis, first manifest at lium and stromal hydrations were determined about 8 hr after irradiation as mild conjunctival separately by weighing the tissue before and after hyperemia and fine granularity of the corneal epi- lyophilization and are expressed as milligrams of thelium. Keratitis was maximal at 24 to 48 hr, with H2O per milligram diy weight. stippling and erosion of the corneal surface but Glycogen was measured after hydrolysis by the without sloughing of the epithelium. Mild sub- hexokinase method as described by Reim et al.6 epithelial haze was discernible at 24 and 48 hr. By Values are expressed in micromoles glucose units 72 hr the surface appeared lustrous, with almost per gram dry weight tissue. complete disappearance of the subepithelial haze. ATP was measured by the firefly luminescence Scanning and transmission electron microscopy test after perchloric acid extraction and is ex- showed that the pathologic changes were limited pressed as micromoles ATP per gram dry weight. to the epithelium. Inflammatory cells were rarely Epithelial enzyme activities were measured seen. after glass-to-glass homogenization of the lyophil- Changes in corneal hydration as a function of ized tissue in phosphate buffer at pH 7.6 on ice, time are illustrated in Table I. Progressive in- followed by repeated freeze-thawing. Glucose- crease in hydration parallels the clinical keratitis,

Downloaded from iovs.arvojournals.org on 10/02/2021 Invest. Ophthalmol. Visual Sci. 696 Reports July 1978

Table I. Corneal hydration following ultraviolet irradiation Time after Epithelial hydration Stromal hydration exposure (hr) (mg H2O/mg dry wt.) p value (mg H-2O/mg dry wt.) p value

Control (6) 5.06 ± 0.92 3.51 ± 0.02 4(4) 5.30 ± 0.29 >0.05 3.64 ± 0.04 <0.01 7(4) 5.52 ± 1.06 >0.05 3.88 ± 0.10 <0.01 24(6) 7.22 ± 3.43 >0.05 4.17 ± 0.5 <0.01 40(3) 6.85 ± 0.44 >0.05 4.67 ± 1.01 <0.01 48(4) 6.37 ± 2.30 >0.05 4.69 ± 0.94 <0.01

Numbers in parentheses indicate the sample size. The values shown are averages ± S.E.M. The p values given compare a value to the comparable control value by the Student t test. Stromal values were significant (p < 0.01). Epithelial hydration differences are not statistically significant (p > 0.05).

Table II. Glycogen content of epithelium after UV irradiation Time after exposure Glycogen content (hr) (fjunol glucoseIgm dry wt.) p value

Control 260 ± 24 (4) 4 7 17 222 ± 85 (4) >0.05 24 163 ± 10 (11) <0.0005 48 273 ± 33 (4) >0.05 72 308 ± 17 (4) <0.025

Values are expressed as ±S.E.M., with the number of determinations in parentheses. The p values given compare the glycogen level to the control glycogen level, by the Student t test.

with maximal hydration of both epithelium and swelling implicates increased leakage through the stroma at 24 hr although only the stromal values damaged surface layer as the mechanism respon- are statistically significant. sible for increased stromal hydration. In experi- Changes in the epithelial glycogen are illus- mental animals, epithelial damage or removal in- trated in Table II. Maximum reduction of glyco- variably results in marked stromal swelling, with gen occurred at 24 hr, but levels had returned to hydration values even exceeding those reported normal and above by 48 and 72 hr. Again, the here.7 There may have been an increase in maximum change appeared at times of peak pho- epithelial hydration also, but the large variability tokeratitis. ATP levels were not affected. of the epithelial values, reflecting our attempts to The enzymatic activities measured were normal avoid manipulation of the epithelium prior to at 4, 7, 17, 24 and 48 hr. LDH activity showed a biochemical determinations, make this uncertain. slight but insignificant decrease at 24 hr and The drop in epithelial glycogen content, al- G-6-PDH, 6-PGDH, MDH, ALD, and P-ase though consistent with the fact that even minimal were comparable to control values at all times. trauma can decrease glycogen stores,3 is unique in Discussion. Clinically and ultrastructurally, the its time course. Glycogen in other instances de- damaging effect of UV irradiation in these experi- creases rapidly, whereas in the UV-damaged epi- ments was confined to the anterior portion of the thelium the fall is modest until 24 hr after irradia- cornea, primarily to the epithelial layer. There tion, coincident with the clinical appearance of were, however, significant changes in stromal hy- maximal keratitis. dration. Minimal changes are found at 4 hr, with a Glycogen depletion can be caused by increased maximum hydration at 24 to 48 hr, commensurate use of glucose and glycogen or by decreased with the maximal clinical effect. Although func- glycogen synthesis. Net glycogen catabolism could tional damage to the endothelium has not been occur if energy needs outstripped the available ruled out as a cause for this swelling, the normal glucose flux. The slow decrease in glycogen over electron microscopic appearance of that layer, in 24 hr may be a direct effect on glycogen synthesis, addition to the anterior stromal location of the rather than an increased demand for energy seen edema fluid, make entrance of the fluid from the after other forms of injury. In direct injury to the anterior route most likely. The coincidence of epithelium a rapid fall in glycogen is characteristic maximal epithelial keratitis with maximal stromal and parallels an increased epithelial hydration,

Downloaded from iovs.arvojournals.org on 10/02/2021 Volume 17 Number 7 Reports 697

with probable increased rates of cellular cation ex- metabolism of the cornea, INVEST. OPHTHALMOL. change. 1:178, 1962. The curious phenomenon of the latent period 5. Hayes, T. L.: Scanning electron microscope tech- between exposure and clinical manifestation of in- niques in biology. In Roehler, J. K., editor: Ad- vanced Techniques in Biological Electron Micros- jury remains to be explained. The mobilization of copy, New York, 1973, Springer-Verlag. epithelial cellular glycogen and increase in cellular 6. Reim, M., Foerster, K. H., and Cattepoel, H.: hydration are also rather slow following UV expo- Some criteria of the metabolism of the donor cornea. sure. This may indicate that the effects of the In Proceedings of the 21st International Congress of radiation on the pathways responsible for energy Ophthalmology, Amsterdam, 1970, Excerpta transformation are secondary to cellular structural Medica Foundation, p. 728. and enzymatic protein changes. Possibly, as in 6a. Bergmeyer, H. U., editor: Methods of Enzymatic other cells, damage to structural membranes oc- Analysis, New York, 1974, Academic Press, Inc. curs through peroxide formation, with subsequent 6b. Jones T. H. D., and Wright, B. E.: Partial purifi- lipid peroxidation and cellular permeability cation of glycogen phosphorylase from Dictystelium changes.8' 9 The delay in onset could also be ex- discoideum, J. Bacteriol. 104:754, 1970. plained by an effect of UV on RNA and DNA, with 6c. Lowry, O. H., Rosebrough, N. S., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin delayed transcription and translation to protein phenol reagent, J. Biol. Chem. 193:265, 1951. synthesis or frank inactivation by nucleotide di- 7. Thoft, R. A., Friend, J., and Dohlman, C. H.: merization or crosslinkage.10' " Corneal glucose concentration, Arch. Ophthalmol. Regardless of the molecular mechanism in- 85:467, 1971. volved, the effects noted in experimental UV 8. Johnson, B. E., and Daniels, F.: Lysosomes and the keratitis can be accounted for by changes observed reactions of skin to ultraviolet light, J. Invest. Der- in the epithelial layer. These differences in the matol. 53:85, 1969. time course of the response to UV injury and to 9. Lavelle, F., Michelson, A. M., and Dimitrijevie, L.: other forms of epithelial stress3 imply that epithe- Biological protection by superoxide dismutase, Biochem. Biophys. Res. Commun. 55:350, 1973. lial response to insult may differ depending on the 10. Murphy, T. M.: Nucleic acids: interaction with solar form of stress applied. UV radiation, Curr. Top. Radiat. Res. 10:199, 1975. We thank Henry D. Perry, M.D., and Ralph C. 11. Atsusa, J., et al.: Modification of the effect of UV Eagle, M.D., for performing and interpreting the elec- irradiation by nutritive conditions on induced for- tron microscopy. mation of catechol oxygenase in Pseudomonas ef- fusa, Int. J. Radiat. Biol. 14:517, 1968. From the Department of Cornea Research, Eye Re- search Institute of Retina Foundation, and the Cornea Service, Massachusetts Eye and Ear Infirmary, Boston, Mass. This work was supported in part by Biomedical Research Support Grant RR05527, Eye Research Core Increased saccadic latencies in amblyopic Services Support Grant P30EY-01784, Research Fellow- eyes. KENNETH J. CIUFFREDA,* ROBERT V. ship No. EY-05132 (Dr. Foulks) and Research Grant KENYON, AND LAWRENCE STARK. EY-01830, all from the National Eye Institute, National Institutes of Health; and in part by the Massachusetts Increased saccadic latencies were measured in the Lions Eye Research Fund, Inc. Submitted for publica- amblyopic eyes of subjects having amblyopia without tion Nov. 18, 1977. Reprint requests: Gary N. Foulks, strabismus, constant strabismus amblyopia, and inter- M.D., Eye Research Institute of Retina Foundation, 20 mittent strabismus. The subjects tracked a small, bright Staniford St., Boston, Mass. 02114. spot of light moving with random, horizontal step dis- placements of 0.25 to 8.5 degrees over the central retina. Key words: corneal epithelium, ultraviolet radiation, Normal saccadic latencies were generally found during photokeratitis monocular tracking with the nonamblyopic eye as well as during binocular tracking. Studies of eye-hand reaction time in amblyopic eyes have shown delays to occur over REFERENCES the central retina; our new finding establishes this for 1. Cogan, D., and Kinsey, V. E.: Action spectrum of saccadic initiation. Normal trajectories found for all keratitis produced by ultraviolet radiation, Arch. tracking saccades, normal saccadic latencies measured Ophthalmol. 35:670, 1946. when the nonamblyopic eye was utilized for tracking, 2. Zuclich, J.: Ocular effects of ultraviolet laser radia- and synchronous movement of the eyes under all test tion, SAM-TR-74-32, USAF School of Aerospace conditions point to a sensory rather than motor basis Medicine, Brook AFB, Texas, 1974. underlying these delays. Our results are inter})reted in 3. Thoft, R. A., and Friend, J.: Biochemical aspects of terms of a processing delay in the sensory pathways lead- contact lens wear, Am. J. Ophthalmol. 80:139, 1975. ing from the central region of the amblyopic eye to those 4. Kinoshita, J.: Some aspects of the carbohydrate centers involved in saccadic initiation.

Downloaded from iovs.arvojournals.org on 10/02/2021